The Malarial Carbamoyl Phosphate Synthetase II Gene As a Target for Dnazyme Therapy

The Malarial Carbamoyl Phosphate Synthetase II Gene as a Target for DNAzyme Therapy

By Marilyn Katrib

A thesis presented for the degree of Doctor of Philosophy. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia. May, 2007.

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Surname or Family name: Katrib

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Title: The Malarial Carbamoyl Phosphate Synthetase II Gene as a Target for DNAzyme Therapy

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Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels.

The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present.

Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial.

Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin-conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.

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TABLE OF CONTENTS

Table of Contents ...... i List of Figures...... viii List of Tables ...... x Acknowledgments ...... xi Conference Abstracts, Publications and Gene Sequences...... xii List of Abbreviations ...... xiii Abstract...... xiv

1 Introduction ...... 2 1.1 General Introduction ...... 2 1.1.1 Classification of Malaria...... 2 1.1.2 Lifecycle of Human Malarial Parasites...... 2 1.1.2.1 The Asexual Stage within the Human Host ...... 3 1.1.2.2 The Sexual Stage within the Insect Vector ...... 3 1.1.3 Clinical Manifestations of Malaria ...... 5 1.1.3.1 Uncomplicated Malaria...... 5 1.1.3.2 Severe Malaria ...... 5 1.1.4 The History of Malaria...... 6 1.1.4.1 Ancient History ...... 6 1.1.4.2 Modern History ...... 6 1.1.4.3 The Eradication Program ...... 7 1.2 The Current Global Situation...... 8 1.2.1 The Incidence of Global Malaria ...... 8 1.2.2 Current Control of Malaria...... 10 1.2.2.1 Chemotherapy ...... 10 1.2.2.2 Alternative Control Strategies...... 12 1.3 Molecular Biology of Plasmodium...... 14 1.3.1 The Plasmodium Genome...... 14 1.3.1.1 The Plasmodium Sequencing Projects...... 14 1.3.1.2 Plasmodium Genome Characteristics ...... 15 1.3.2 The Plasmodium Transcriptome ...... 16

1.3.3 The Plasmodium Proteome ...... 17 1.4 General Metabolism and Transport ...... 18 1.5 Pyrimidine Biosynthesis in Plasmodium falciparum...... 20 1.5.1 The de novo Pyrimidine Biosynthetic Pathway ...... 21 1.5.2 Carbamoyl Phosphate Synthetase...... 23 1.5.2.1 Types and Characteristics of CPS...... 23 1.5.2.2 The Functional Domains of CPSII...... 23 1.5.2.3 The Structural Organisation of CPSII...... 26 1.5.3 The P. falciparum CPSII Gene and Predicted Protein Sequence ...... 28 1.6 Insertions within Plasmodium Proteins ...... 30 1.7 Antisense Therapy ...... 34 1.7.1 Advantages of Antisense Therapy ...... 35 1.7.2 Mechanism of Action of Antisense Molecules...... 36 1.7.2.1 Mechanism of Action of Antisense Oligodeoxynucleotides...... 36 1.7.2.2 Mechanism of Action of Nucleic Acid Enzymes...... 38 1.7.2.3 Mechanism of Action of RNAi...... 40 1.7.3 Antisense Accessibility ...... 41 1.7.4 Biological Stability and Modifications ...... 43 1.7.5 Bioavailability and Delivery Systems...... 45 1.7.6 Biodistribution, Pharmacokinetics and Toxicity...... 47 1.7.7 Biological Activity of the 10-23 DNAzyme ...... 48 1.8 Application of Antisense Therapy to Malaria...... 51 1.9 Experimental Background ...... 52 1.10 Aims...... 54

2 Materials and Methods ...... 56 2.1 Materials ...... 56 2.1.1 General Reagents...... 56 2.1.2 Radio-chemicals...... 56 2.1.3 Oligonucleotides ...... 57 2.1.4 Enzymes and Reaction Kits ...... 57 2.1.5 Parasite Species...... 58 2.1.6 Bacterial Strains and Helper Phage...... 58 2.2 General Methods...... 58

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2.2.1 Sterilisation and Containment...... 58 2.2.2 Disposal of Waste ...... 59 2.2.3 Standard Stock Solutions ...... 59 2.2.4 Dialysis Tubing Preparation...... 60 2.2.5 Autoradiography ...... 60 2.2.6 Polyacrylamide Gel Electrophoresis...... 60 2.2.7 Growth and Maintenance of E. coli Cultures...... 60 2.2.7.1 Media ...... 60 2.2.7.2 Glycerol Stocks...... 61 2.2.7.3 Inoculation ...... 61 2.2.8 Growth and Maintenance of P. falciparum Cultures ...... 61 2.2.8.1 Media and Culture Maintenance ...... 61 2.2.8.2 Analysis of Parasitaemia...... 62 2.2.8.3 Synchronisation of Ring-stage Parasites...... 62 2.2.8.4 Liquid Nitrogen Storage...... 62 2.3 General DNA Procedures ...... 63 2.3.1 Phenol:chloroform Extractions ...... 63 2.3.2 Ethanol Precipitations...... 63 2.3.3 Restriction Enzyme Digests...... 64 2.3.4 Agarose Gel Electrophoresis...... 64 2.3.4.1 Quantitative Agarose Gel Electrophoresis...... 64 2.3.4.2 Preparative Agarose Gel Electrophoresis...... 64 2.3.5 Estimation of DNA Concentration and Size ...... 65 2.3.6 Small-scale Plasmid Preparations ...... 65 2.3.7 Large-scale Plasmid Preparations ...... 65 2.3.8 Caesium Chloride Equilibrium Density Centrifugation...... 66 2.3.9 Double-stranded Plasmid DNA Preparations for Sequencing ...... 66 2.3.10 Single-stranded Plasmid DNA Preparations for Sequencing...... 67 2.4 Isolation and Characterisation of CPSII Genes from Plasmodium...... 67 2.4.1 Isolation of Plasmodium Genomic DNA...... 67 2.4.1.1 Isolation of P. falciparum Genomic DNA ...... 67 2.4.1.2 Isolation of P. berghei and P. chabaudi Genomic DNA ...... 67 2.4.1.3 Isolation of P. vivax Genomic DNA ...... 68 2.4.2 Isolation of CPSII Gene Fragment Clones...... 68

2.4.2.1 PCR with Degenerate Primers ...... 68 2.4.2.2 PCR Product Preparation ...... 69 2.4.2.3 PCR Product Ligations...... 69 2.4.2.4 Preparation of Competent Cells and Transformations...... 69 2.4.2.5 Detection of Recombinant Clones ...... 70 2.4.3 Plasmodium CPSII Sequence Analysis...... 70 2.4.3.1 Generation of Nested Deletions ...... 70 2.4.3.2 Automated Sequencing using Big Dye Chemistry ...... 71 2.4.3.3 Bioinformatic Analysis of Sequencing Data...... 71 2.4.3.3.1 Sequence Contigs and Alignments ...... 71 2.4.3.3.2 Hydrophobicity Profiles...... 72 2.4.3.3.3 CPSII Structural Analysis ...... 72 2.5 Design and Selection of DNAzymes Targeting PbCPSII Insertion II...... 73 2.5.1 DNAzyme Site Selection ...... 73 2.5.1.1 Transcription Template Preparation...... 73 2.5.1.1.1 Linearised Clone DNA...... 73 2.5.1.1.2 PCR Product DNA...... 73 2.5.1.2 In Vitro Transcription...... 74 2.5.1.3 Multiplex DNAzyme Cleavage Reaction...... 74 2.5.1.3.1 Primer Extension...... 74 2.5.1.3.2 Cycle Sequencing...... 75 2.5.1.3.3 Product Visualisation ...... 75 2.5.1.4 Individual Cleavage Assays...... 75 2.5.1.5 Computer Predicted Site Selection...... 76 2.5.2 DNAzyme Cleavage and Kinetics ...... 76 2.5.2.1 Single Turnover Cleavage...... 76 2.5.2.2 Analysis of Single Turnover Kinetics Data ...... 76 2.5.2.3 Multiple Turnover Cleavage ...... 77 2.5.2.4 Analysis of Multiple Turnover Kinetics Data...... 77 2.6 Plasmodium Inhibition Studies...... 77 2.6.1 Inhibition of P. berghei In Vivo ...... 77 2.6.1.1 Animal Trial Experimental Design ...... 77 2.6.1.2 MD14 Preparation and Storage...... 78 2.6.1.3 MD14 Toxicity Test...... 78

2.6.1.4 MD14 Drug Administration...... 78 2.6.1.5 Analysis of Parasitaemia...... 78 2.6.1.6 Analysis of Organs...... 79 2.6.2 Inhibition of P. falciparum In Vitro ...... 79 2.6.2.1 DNAzyme Stability Studies in Human Serum...... 79 2.6.2.2 Parasite Inhibition Bioassays...... 79 2.6.2.3 Analysis by Microscopic Visualisation...... 80 2.6.2.4 Analysis by Flow Cytometry ...... 80 2.6.2.5 Examination of DNAzyme Uptake by Confocal Laser Scanning Microscopy ...... 81

3 Isolation of the CPSII Gene from Five Species of Plasmodium...... 84 3.1 Introduction and Aims ...... 84 3.2 Results...... 85 3.2.1 Isolation of P. berghei and P. chabaudi CPSII Genes...... 85 3.2.1.1 Stage 1: Isolation of Insertion I...... 85 3.2.1.2 Stage 2: Isolation of Insertion II ...... 86 3.2.1.3 Stage 3: Isolation of Core CPSII Regions...... 94 3.2.2 Isolation of the P. vivax CPSII Gene ...... 95 3.2.2.1 Stage 1: Isolation of the Core CPSII Region ...... 95 3.2.2.2 Stage 2: Isolation of the Insertion II Region ...... 98 3.2.2.3 Stage 3: Isolation of the Insertion I Region ...... 99 3.2.3 Identification of the P. yoelii CPSII Gene ...... 100 3.2.3.1 Data-mining ...... 100 3.2.4 Sequence Analysis Results...... 100 3.2.4.1 Nucleotide Conservation...... 100 3.2.4.2 Amino Acid Conservation ...... 101 3.2.4.3 Hydrophilicity ...... 108 3.2.4.4 Compositional Analysis...... 110 3.2.4.5 Structural Analysis...... 110 3.3 Discussion...... 113

4 Selection and Cleavage Activities of DNAzymes Targeting PbCPSII ...... 120 4.1 Introduction...... 120 4.1.1 The 10-23 DNAzyme...... 120 4.1.2 The DNAzyme Kinetic Pathway...... 120 4.1.3 Single Turnover Kinetics ...... 121 4.1.4 Multiple Turnover Kinetics...... 122 4.2 Experimental Background and Aims...... 122 4.3 Results...... 123 4.3.1 DNAzyme Selection...... 123 4.3.1.1 Design of New DNAzymes...... 123 4.3.1.2 DNAzyme Site Accessibility ...... 123 4.3.1.2.1 Preparation of the Long RNA Substrate ...... 123 4.3.1.2.2 The Multiplex Cleavage Assay ...... 127 4.3.1.2.3 Individual Cleavage...... 129 4.3.1.2.4 Computer Predicted Site Selection ...... 132 4.3.1.3 Summary of DNAzyme Selection...... 134 4.3.2 DNAzyme Cleavage Kinetics ...... 135 4.3.2.1 Cleavage of the Long Transcribed Substrate ...... 136 4.3.2.1.1 Single Turnover Kinetics ...... 136 4.3.2.1.2 Multiple Turnover Kinetics...... 137 4.3.2.2 Cleavage of Short Synthetic Substrates ...... 140 4.3.2.2.1 Single Turnover Kinetics ...... 140 4.3.2.2.2 Multiple Turnover Kinetics...... 141 4.4 Discussion...... 145 4.4.1 DNAzyme Selection...... 145 4.4.2 DNAzyme Kinetics ...... 147

5 Plasmodium Inhibition Studies ...... 152 5.1 Experimental Background and Aims...... 152 5.2 Results...... 153 5.2.1 Inhibition of P. berghei In Vivo ...... 153 5.2.2 Investigation into Low Levels of DNAzyme Inhibition ...... 155 5.2.2.1 DNAzyme Integrity...... 155 5.2.2.2 Selective Uptake of DNAzymes into Parasitised RBCs ...... 157

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5.2.3 Optimising DNAzyme Delivery ...... 162 5.2.3.1 Liposomal Delivery Methods...... 162 5.2.3.2 Methylpyrroporphyrin Conjugates...... 163 5.2.3.2.1 Uptake of MPP DNAzymes into P. falciparum Infected RBCs 165 5.2.3.2.2 P. falciparum Inhibition Bioassays with MPP DNAzymes...... 172 5.3 Discussion...... 174 5.3.1 DNAzyme Suppression of P. berghei In Vivo ...... 174 5.3.2 DNAzyme Efficacy against P. falciparum In Vitro...... 177

6 Conclusions and Future Directions...... 185

Appendix...... 190 Appendix 1...... 190 Appendix 2...... 191 Appendix 3...... 192 Appendix 4...... 193

References ...... 200

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LIST OF FIGURES Figure 1.1 Lifecycle of the human Plasmodium parasites...... 4 Figure 1.2 Global distribution of malaria transmission risk...... 9 Figure 1.3 World-wide drug resistance to P. falciparum...... 9 Figure 1.4 A metabolic overview of P. falciparum...... 19 Figure 1.5 A schematic representation of the pyrimidine metabolic pathway of P. falciparum...... 22 Figure 1.6 A schematic representation of CPSII functionality...... 25 Figure 1.7 The organisation of CPSII in different organisms...... 27 Figure 1.8 A schematic representation of PfCPSII...... 30 Figure 1.9 Mechanisms of inhibition of translation through antisense technology...... 37 Figure 1.10 A schematic representation of the 10-23 DNAzyme...... 40 Figure 1.11 The structure of the 3'-3' internucleotide linkage inversion...... 45 Figure 3.1 Amino acid alignment of variable region of MSP-1 from P. berghei strains...... 86 Figure 3.2 Overview of the relative primer sites in the CPSII gene from P. berghei and P. chabaudi...... 87 Figure 3.3 Agarose gel electrophoresis of P. berghei CPSII insertion II PCR products...... 91 Figure 3.4 Agarose gel electrophoresis of P. chabaudi CPSII insertion II PCR products...... 92 Figure 3.5 Uni-directional deletions of the P. berghei CPSII insertion II gene fragment...... 93 Figure 3.6 Agarose gel electrophoresis of P. chabaudi and P. berghei CPSII core region PCR products...... 94 Figure 3.7 Overview of the relative primer sites of the CPSII gene from P. vivax...... 95 Figure 3.8 Agarose gel electrophoresis of the core CPSII PCR products from P. vivax...... 97 Figure 3.9 Agarose gel electrophoresis of the P. vivax CPSII insertion II PCR product...... 98 Figure 3.10 Agarose gel electrophoresis of the P. vivax CPSII insertion I PCR product...... 99 Figure 3.11 Amino acid alignment of the Plasmodium CPSII proteins...... 106 Figure 3.12 Recurrence profiles of the Plasmodium CPSII proteins...... 109

Figure 3.13 Amino acid composition of Plasmodium CPSII insertions compared to core regions...... 111 Figure 3.14 Structural characterisation of Plasmodium CPSII...... 112 Figure 4.1 Minimal kinetic pathway of the DNAzyme cleavage reaction...... 121 Figure 4.2 Transcription products encompassing PbCPSII insertion II...... 126 Figure 4.3 The 1600nt DNAzyme substrate...... 126 Figure 4.4 The radio-labelled 1600nt substrate...... 127 Figure 4.5 A schematic representation of the multiplex cleavage assay...... 128 Figure 4.6 The PbCPSII multiplex cleavage assay...... 130 Figure 4.7 Individual cleavage reactions of the PbCPSII DNAzymes...... 131 Figure 4.8 Diagrammatic representation of RNA secondary structure...... 132 Figure 4.9 Single turnover cleavage of the in vitro transcribed long substrate...... 138 Figure 4.10 Multiple turnover cleavage of the in vitro transcribed long substrate...... 139 Figure 4.11 Single turnover cleavage of the short synthetic substrate...... 142 Figure 4.12 Multiple turnover cleavage of the short synthetic substrate...... 143 Figure 4.13 A representative modified Eadie-Hofstee plot for cleavage of the short substrate...... 144 Figure 5.1 Inhibition of P. berghei in vivo by MD14...... 154 Figure 5.2 Integrity of MD14 DNAzymes from different batches...... 156 Figure 5.3 Stability of modified MD14 in 10% human serum...... 156 Figure 5.4 Confocal microscopy of DNAzyme uptake into P. falciparum infected RBCs...... 158 Figure 5.5 P. falciparum 24h bioassays with M5L...... 160 Figure 5.6 Methylpyrroporphyrin conjugates...... 164 Figure 5.7 Confocal microscopy of DNAzyme uptake into trophozoite-stage P. falciparum infected RBCs...... 166 Figure 5.8 Flow cytometry histograms of DHE stained P. falciparum rings...... 168 Figure 5.9 Flow cytometry histograms of DHE stained P. falciparum rings incubated with MPP-M5L-FITC...... 169 Figure 5.10 Flow cytometry histograms of DHE stained P. falciparum trophozoites..170 Figure 5.11 Flow cytometry histograms of DHE stained P. falciparum trophozoites incubated with MPP-M5L-FITC...... 171 Figure 5.12 Inhibition of P. falciparum in vitro cultures by MPP-DNAzymes...... 173

LIST OF TABLES

Table 1.1 Current antimalarial drugs...... 13 Table 1.2 Inserts of P. falciparum enzymes...... 32 Table 1.3 Efficacy of DNAzymes targeting diseases in vitro...... 49 Table 1.4 Efficacy of DNAzymes targeting diseases in vivo...... 50 Table 3.1 CPSII PCR products from P. berghei and P. chabaudi...... 89 Table 3.2 CPSII PCR products from P. vivax...... 96 Table 3.3 A/T content of insertion I, insertion II and core CPSII gene sequences...... 100 Table 3.4 Nucleotide identity across Plasmodium CPSII genes...... 102 Table 3.5 Amino acid identity across Plasmodium CPSII proteins...... 106 Table 3.6 Amino acid content across the different Plasmodium CPSII proteins...... 107 Table 4.1 DNAzymes designed against PbCPSII insertion II...... 124 Table 4.2 Cleavage efficiencies of PbCPSII DNAzymes...... 133 Table 4.3 Kinetic constants for the cleavage of in vitro transcribed long substrate. ....140 Table 4.4 Kinetic constants for the cleavage of short synthetic substrate...... 144 Table 5.1 Transfection reagent mediated delivery of M5L...... 163

ACKNOWLEDGMENTS

First and foremost, my deepest gratitude goes to my supervisor Tom Stewart. Your enthusiasm, love of science and teaching, have been an inspiration to all who have had the good fortune to work with you. Thank you for being my mentor and for always believing in me, right from the start when I was a work experience student. I am also enormously grateful for your encouragement, support and understanding during my illness and the delay of this thesis. You can finally enjoy your retirement now!

To the School of Biotechnology & Biomolecular Sciences, UNSW, for the excellent opportunities and friendships I have made over the years. My appreciation extends to a number of academics that provided me with endless help and advice including Wendy Glenn, Alan Wilton, Annette Gero, Louise Lutze-Mann, Barry Milborrow, George Mendz and Paul March. In particular, Bill O’Sullivan, who has virtually been a second supervisor from beginning to end. Thank you to all the past members of the molecular parasitology lab and shark bay who provided me with sound grounding in molecular parasitology and a fun work environment especially Simone, Lolina, Ed and Collette. In addition, I wish to thank Rick Cavicchioli and members of the extremophiles lab for their understanding, motivation and support! Special thanks must go to Lily, my co-lab manager and coffee buddy.

Credit must go to our external collaborators Johnson & Johnson Research for financial backing and to Dr Sun, for allowing me to conduct work in his laboratory. Special thanks go to the parasitology crew at UTS, especially Nick, Kate, Mike, Nigel and David, who encouraged and supported the completion of this thesis, which was independent of them.

I have been blessed with many wonderful friends that have shared in the laughter and the tears and helped me through the dark times when I wanted to give up. Thanks especially to Cath, Jos, Harvey, Alan, Anne, Heather, Yael, Pete and Neil for their support, consistent weekly get- togethers and many fun trips to Smiths Lake. To Alexi, your enthusiasm and soul-searching discussions have been wonderful! Thanks also to my old school pal Teresa for the collaboration, advice, cheery optimism and persistent 25-year friendship. A special thanks to my great friend Kirsten who never failed in her support even when she was half way across the world in Germany! You are a true friend.

To my extended family, the Darsa’s, especially Uncle Mohamed for always being interested in my work and teaching me from a very young age to question and seek knowledge. Sandra and Joe, you and your family have provided me with immeasurable love, happiness and have kept me grounded. Thanks also for cracking the whip from time to time! Sana, Tony and Stewart, your encouragement, prayers, and religious discussions have given me great comfort. Tania, thanks for all the laughs and distracting piano lessons…..

Finally, this thesis is dedicated to my wonderful family. I have driven you all insane at one stage or another for which I’m truly sorry! Mum and Dad, you have always provided a strong family unit filled with love, generosity, honesty and loyalty. I am forever grateful for teaching me the most important lessons in life. To my brothers, Jay, George and Esty, thanks for the financial support, holidays, laptop and pep talks at 3am! Mandy and Wendy (Irene and Kylie), my sisters, you are the best! Thanks for looking after me, and providing me with unlimited hugs, hilarious shopping expeditions, clothes, jewellery and make-up. Thanks to my Aunty Violette and Grandmother Genevieve, whose daily prayers have undoubtedly helped me finish.

“For with God, nothing is impossible” Luke 1:37

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CONFERENCE ABSTRACTS, PUBLICATIONS AND GENE SEQUENCES

Abstracts:

Katrib, M., Davies, N. P., and Stewart, T. S (2000). Unique Insert Sequences Within Carbamoyl Phosphate Synthetase II Gene From P. berghei and P. chabaudi. Molecular Approaches to Malaria, Lorne, Australia.

Katrib, M. and Stewart, T. S. (2001). Analogous Insert Sequences of the Carbamoyl Phosphate Synthetase II Gene in Four Different Species of Plasmodium. Annual Scientific Meeting of the Australian Society for Parasitology, Palm Cove, Australia

Publications:

Alessi, E., Katrib, M., Mattei, D., Stewart, T., and Pizzi, E. (Submitted to BMC Evolutionary Biology 22nd December 2006) Plasmodium specific domains: a pilot study of inserts from carbamoyl phosphate synthetase II and GSK3-related kinase.

Katrib, M., Sun, L. Q., Gerlach, W. L., and Stewart, T. S. (In Preparation). In vitro and in vivo targeting of the P. berghei Carbamoyl Phosphate Synthetase II mRNA by DNAzymes.

Katrib, M., Rede, T. and Stewart, T. S. (In Preparation). Transport and efficacy of porphyrin-conjugated DNAzymes against P. falciparum CPSII.

Genome Sequences:

Plasmodium berghei K173 carbamoyl phosphate synthetase II gene, partial cds. Genbank Accession number: AF286897

Plasmodium chabaudi adami carbamoyl phosphate synthetase II gene, partial cds. Genbank Accession number: AF286898

Plasmodium vivax carbamoyl phosphate synthetase II (CPSII) gene, partial cds. Genbank Accession number: AF327646

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LIST OF ABBREVIATIONS

A adenosine ADP adenosine diphosphate AMP adenosine monophosphate AS ODN antisense oligodeoxynucleotides ATP adenosine triphosphate bp base pairs C cytosine CAD multifunctional polypeptide having carbamoyl phosphate synthetase II (CPSII, C), aspartate transcarbamoylase (ATCase, A) and dihydroorotase (DHOase, D) activities CPS carbamoyl phosphate synthetase CPSII carbamoyl phosphate synthetase II DHE dihydroethidium DNAzyme deoxyribozyme dNTPS deoxynucleotide triphosphates EDTA ethylene diamine tetraacetic acid EtBr ethidium bromide FITC fluorescein isothiocyanate G guanosine GAT glutamine amidotransferase domain IPTG isopropyl-β-D-galactopyranoside kb kilo base pairs MPP methylpyrroporphyrin NPPs new permeability pathways nt nucleotides ORF open reading frame OD optical density ODN oligodeoxynucleotides PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PbCPSII Plasmodium berghei carbamoyl phosphate synthetase II PcCPSII Plasmodium chabaudi carbamoyl phosphate synthetase II PCR polymerase chain reaction PfCPSII Plasmodium falciparum carbamoyl phosphate synthetase II PvCPSII Plasmodium vivax carbamoyl phosphate synthetase II PyCPSII Plasmodium yoelii carbamoyl phosphate synthetase II RBC red blood cell RNase ribonuclease SDS sodium dodecyl sulphate T thymidine TEMED N,N,N',N'-tetramethylethylenediamine Tris tris-(hydroxymethyl) methane X-gal 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside WHO World Health Organisation

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ABSTRACT

Today, malaria remains the biggest killer of the third world, infecting over 500 million people a year, over a million of which will die, despite intensive research efforts. An effective strategy to combat the causative agent of human malaria, Plasmodium falciparum is necessary. Biochemical and genetic studies have revealed fundamental differences between parasite and host that can be exploited for rational drug design. In particular, the de novo pyrimidine biosynthetic pathway of P. falciparum is an ideal target as the parasite is unable to salvage pre-formed pyrimidines and must rely on de novo synthesis, where as the human host is able to salvage or synthesise its own pyrimidines and the human red blood cell itself has no requirement for pyrimidines. Carbamoyl phosphate synthetase II, or CPSII, is the first and rate-limiting enzyme of the de novo pyrimidine biosynthetic pathway of P. falciparum and catalyses the conversion of L-glutamine, bicarbonate and two molecules of ATP to carbamoyl phosphate. Unlike the human counterpart, the gene encoding P. falciparum CPSII is unusually high in A/T content (76%) and contains two extensive, unique insertion sequences of approximately 700bp and 1800bp that exist within the mature transcript. These insertions are unique to P. falciparum CPSII and are absent in CPSII genes from other organisms isolated to date. These unusual features of P. falciparum CPSII at the gene level make it amenable for targeting by antisense therapies.

Antisense therapy, and in particular DNAzymes, have emerged as an important tool in the control of gene expression and as therapeutic agents because of their ease of synthesis, stability and ability to interact directly and specifically with mRNA targets. Previous studies have demonstrated that exogenous delivery of antisense, ribozymes and DNAzymes, targeting the junction of insertion II of P. falciparum CPSII, effectively inhibited the growth of laboratory cultures of P. falciparum at sub-micro molar levels.

In order to design DNAzymes to be tested in a rodent malaria system, the CPSII gene sequence had to be determined from the rodent malaria parasites P. berghei and P. chabaudi. Additionally, the CPSII gene was isolated from P. vivax, the parasite responsible for the majority of human malaria morbidity, in the hope of designing a synergistic DNAzyme in the future. The Plasmodium CPSII genes were isolated from

P. vivax, P. berghei and P. chabaudi using a PCR based approach. The sequence of the CPSII gene was also identified from the P. yoelii genome database to further characterise the nature of the insertions.

The CPSII genes from all five Plasmodium species examined in this thesis contain insertions of similar size and nature and are located in the same position as seen in P. falciparum CPSII. Analysis of the insertion regions indicates that, although they are poorly conserved with respect to amino acid identity, they are all highly hydrophilic, highly recurrent and low-complexity in nature. Despite having poor general homology, the insertions contain highly conserved elements. The insertions of malarial CPSII are rapidly evolving, yet they are maintained across the five species of Plasmodium, indicating the essential role they play in the function of the enzyme and hence survival of the parasite.

Thirty-one new DNAzymes were designed targeting insertion II of P. berghei CPSII, including regions of the gene targeted previously in P. falciparum. A thorough approach was undertaken to investigate new accessible sites within a 1600nt in vitro transcribed mRNA fragment and included: a multiplex reaction; single turnover cleavage of each individual DNAzyme; as well computer-predicted RNA secondary structure. Of the thirty-one DNAzymes designed and tested, seventeen showed detectable cleavage products by single turnover analysis. Of these, four showed significant cleavage activity against the 1600nt transcript, with the DNAzyme MD14 cleaving greater than half the target transcript within the first five minutes. These DNAzymes were then further characterised for kinetic behaviour on the in vitro transcribed substrate as well as short, synthetic RNA substrates. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Under these conditions, MD14 showed only a slight reduction in parasitaemia of infected mice. The final stage of the project involved investigation into DNAzyme uptake into malaria infected red blood cells and more efficient DNAzyme delivery methods in order to improve DNAzyme efficacy.

CHAPTER 1

Introduction Chapter 1: Introduction 2

1 INTRODUCTION

1.1 General Introduction

Malaria is by far the most important tropical parasitic disease and has long been a source of human affliction throughout the history of mankind. Today, malaria remains the third biggest killer of known infectious diseases worldwide. The first serious attempt by the World Health Organisation (WHO) in 1955 to control the disease was unsuccessful with the emergence of widespread drug-resistant strains of the parasite and insecticide-resistance of the mosquito vector. The resurgence of malaria in recent years led to a renewed effort to control the disease and was termed the ‘roll back malaria’ program. At present, the stated aims are unlikely to be fulfilled.

The advent of rapid developments in recombinant DNA technology, molecular genetics and immunology has led to a more sophisticated strategy for malaria control. In particular, the completion of the genome sequencing projects of several species of malaria along with the human host and mosquito vector, offer new hope for the future. Understanding the parasite’s basic biology and the vector-host-parasite relationship will greatly facilitate the development of control strategies, efficacious vaccines and improved chemotherapeutics by rational drug design.

1.1.1 Classification of Malaria Malaria is caused by protozoan parasites of the genus Plasmodium. Plasmodium parasites are classed within the phylum Apicomplexa, order Haemosporidiidea, subclass Coccidiida and family of Plasmodiidae. The genus Plasmodium contains 172 species that infect birds, reptiles and mammals. There are four species of Plasmodium that cause human malaria, P. falciparum, P. vivax, P. ovale and P. malariae.

1.1.2 Lifecycle of Human Malarial Parasites The lifecycle of the human malarial parasite is complex and is essentially similar for all species (Figure 1.1). It consists of two distinct stages: the asexual stage and the sexual stage. The asexual stage occurs in the vertebrate or human host, which includes the exo-erythrocytic (Figure 1.1A) and erythrocytic cycles (Figure 1.1B). All the clinical

Chapter 1: Introduction 3

symptoms and pathogenic manifestations are associated with the parasite entering the erythrocytic cycle (Wyler, 1993). The sexual stage occurs within the female Anopheles mosquito, which then transmits the parasites to humans (Figure 1.1C).

1.1.2.1 The Asexual Stage within the Human Host Infection begins when sporozoites are injected into the blood stream of the human host by the Anopheles mosquito (Figure 1.1 step 1). The sporozoites arrive at the liver where they penetrate the hepatocytes (Figure 1.1 step 2). The sporozoites then trigger the production of hepatic schizonts (Figure 1.1 step 3), which rupture the host cell releasing 30 000-40 000 merozoites into the blood stream (Figure 1.1 step 4) that invade the surrounding erythrocytes (Figure 1.1 step 5). In P. falciparum and P. malariae, this trigger of schizogony occurs after a short period of 9-16 days whereas in P. vivax and P. ovale, there can be an incubation period of up to 10 months. P. vivax also has a preference for invading reticulocytes whereas P. falciparum can invade erythrocytes of all ages (Hadley, 1986). Invasion of erythrocytes by malarial parasites is also dependant on binding of parasite proteins to erythrocyte receptors. P. vivax also differs from P. falciparum, in that it requires interaction with the Duffy blood group antigen and cannot invade Duffy-negative human erythrocytes.

Within the red blood cell (RBC), the parasite develops through a number of morphologically distinct stages, from ring-stage to trophozoite and finally to the multinucleated schizont. The RBC then ruptures, releasing 6-32 merozoites into the blood stream that can participate in another 36-49 hour cycle of infection (Figure 1.1 step 6). The clinical manifestations of the disease commence within 2-3 days after the initial erythrocytic invasion. At some stage during the erythrocytic cycle, schizogony can lead to the differentiation into the sexual forms: micro-gametocytes (male) and macro-gametocytes (female) (Figure 1.1 step 7) (Wyler, 1993). Gametocytes have no direct detrimental activity on the host, but remain within the circulation and are ingested by the mosquito during a blood meal.

1.1.2.2 The Sexual Stage within the Insect Vector Within minutes of ingestion of an infected blood meal (Figure 1.1 step 8), the gametocytes differentiate into gametes in a process called exflagelation and emerge from erythrocytes (Figure 1.1 step 9). Within the midgut of the mosquito, fertilisation

Chapter 1: Introduction 4

proceeds within 1 hour and the resulting worm-like zygote develops into a motile ookinete (Figure 1.1 step 10). After traversing the peri trophic matrix, the ookinete cross the midgut epithelium and lodge beneath the basal lamina facing the mosquito body cavity (Ghosh et al., 2000). During the next 10-15 days, it differentiates into a mature oocyst (Figure 1.1 step 11). Within the oocyst, sporogony (multiple fission of a zygote) occurs in which thousands of elongated sporozoites are produced. When the oocyst ruptures (Fig 1.1 step 12), the sporozoites travel to and penetrate the salivary glands where they mature and reside in vacuoles until the mosquito takes its next blood meal, thereby maintaining the transmission of the disease from host to host.

Figure 1.1 Lifecycle of the human Plasmodium parasites. (A) Represents the exo-erythrocytic cycle. (B) Represents the parasite cycle within the red blood cell or erythrocyte. (C) Represents the sporogenic cycle or the sexual cycle within the mosquito. Reproduced with permission from The Centers for Disease Control and Prevention, Atlanta, USA (CDC, 2006b).

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1.1.3 Clinical Manifestations of Malaria 1.1.3.1 Uncomplicated Malaria Following the infective bite of the Anopheles mosquito, an incubation period of 7 to 30 days occurs before the first symptoms appear. The primary attack is characterised by periodic bouts of fever, typically lasting 8-12 hours. The fevers occur every second day with ‘tertian’ malaria (P. falciparum, P. vivax and P. ovale) and every third day with ‘quartan’ malaria (P. malariae). The most common symptoms of malaria include a combination of the following: fatigue, headache, nausea, vomiting or mild diarrhoea and muscular pain. An elevated body temperature, perspiration, and an enlarged spleen often accompany these symptoms. In P. falciparum infections, additional symptoms include mild jaundice, enlargement of the liver and an increased respiratory rate. These characteristic symptoms of malaria are a result of the parasite development in erythrocytes, and the consequent rupture of these cells by merozoite liberation. In particular, fever occurs as parasite-derived molecules activate inflammatory cells such as macrophages, resulting in the secretion of pro-inflammatory cytokines including powerful endogenous pyrogens such as interleukin-1 (IL-1) and tumour necrosis factor- α (TNF-α) (Hisaeda et al., 2005).

1.1.3.2 Severe Malaria P. falciparum has a feature, which distinguishes it from the other three human parasites and is thought to be a contributing factor in the often-fatal pathology of this species. Namely, the mature blood stages of the parasite disappear from the peripheral circulation and instead adhere to the endothelial lining of postcapillary venules of several organs such as the heart, lung, small intestine, spleen, skeletal muscles, brain and placenta causing severe complications (Pongponratn et al., 1991). The manifestations of severe malaria include cerebral malaria, severe anaemia, hemoglobinuria, pulmonary oedema, abnormalities in blood coagulation and thrombocytopenia, cardiovascular shock, acute kidney failure, metabolic acidosis and hypoglycaemia. Fatality often follows cerebral malaria, due to the occlusion of brain micro-vessels sequestered with infected erythrocytes (20% fatality rate) (Miller et al., 2002).

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The clinical pattern of severe malaria differs significantly between non-immune adults and semi-immune children. The most common presentations of severe malaria in African children are severe anaemia, cerebral malaria and respiratory distress (Schellenberg et al., 1999). Severe malaria anaemia includes direct RBC lysis by the parasites, indirect RBC lysis by the immune system and bone-marrow suppression (Ekvall, 2003). Evidence suggests that the disease can impair intellectual development (Fernando et al., 2003). In addition, cerebral malaria can result in persisting developmental abnormalities (Carter et al., 2005a; Carter et al., 2005b).

1.1.4 The History of Malaria 1.1.4.1 Ancient History Of all afflictions throughout the history of mankind, malaria has been responsible for the greatest toll on human life. What is now known as malaria, existed throughout ancient times. Accounts of malaria date back over 4000 years and malarial antigens have been detected in Egyptian mummies from 3200 BC (Sherman, 1998). Superstitious and irrational explanations for this disease, led to the Latin name ‘mal’aria for evil emanations or bad air, common to swamps (WHO, 1992).

Ancient medical treatments ranged from magical practices to herbal remedies. In China 168 BC, the Qinhaosu plant (Artemisia annua) was described as an antimalarial remedy (Hien et al., 1993). The active ingredient of Qinhaosu was isolated by Chinese scientists in 1971 and is known as artemisinin and is today a very potent and effective antimalarial drug. An Indian chief in South America brought another remedy for the ‘fevers’ to the attention of the Jesuit missionary Juan Lopez in 1500 AD. This remedy was derived from the bark of the Cinchona tree and contained quinine, the basis of many antimalarial derivatives still in use today.

1.1.4.2 Modern History The most significant events in the study of malaria occurred towards the end of the nineteenth century. Laveran, a French army doctor in Algeria, discovered in 1880 the malarial parasite in the RBCs of an infected man and was awarded the Nobel Prize for his discovery (Smith et al., 1985). In 1886, Golgi, an Italian neurophysiologist, established that there were at least two forms of the disease differing in onset of fever

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which coincided with the rupturing of the blood cell and release of the merozoites. In 1897, Sir Ronald Ross, working in Secunderabad, India, was first to discover the developing form in the gut of the mosquito and was awarded the Nobel Prize in 1902 for his discovery (Manson, 2002). In 1899 Grassi and co-workers demonstrated the complete sporogenic cycle of P. falciparum, P. vivax and P. malariae by feeding Anopheles mosquitoes on malaria patients which then were used to infect two volunteers, both of whom developed malaria. These discoveries led to the initiation of measures designed for the control and treatment of malaria.

1.1.4.3 The Eradication Program During the early part of the twentieth century, much effort to control the transmission of malaria was devoted to eradicating mosquitoes with the use of larvicides. However, after the malaria epidemics of the First World War and the difficulties faced in securing supplies of quinine, research was initiated into possible alternative synthetic drugs (Bruce-Chwatt, 1985). This resulted in the development of a wide range of antimalarials: pamaquine (1924), mepacrine (1930), chloroquine (1934), proguanil (1945), amodiaquine (1946), primaquine (1950) and pyrimethamine (1951). Synthetic insecticides such as dichloro-diphenyl-trichloroethane (DTT) were developed prior to the start of the Second World War (WWII) and raised the inspiring possibility of total eradication of malaria from the planet. However, in the Asia Pacific area during WWII, malaria caused more illness and deaths among military forces than war casualties.

In 1955, the WHO launched the Global Eradication Program (GEP) at the 8th World Health Assembly in Mexico City. Eradication efforts focused on the use of residual insecticides, antimalarial drug treatment and surveillance. Between 1957 and 1970, malaria was eliminated from many parts of the world and over 727 million people were liberated from malaria. However, some nations were completely left out of the eradication program, such as most of sub-Saharan Africa (CDC, 2006a). Furthermore, long-term maintenance of the GEP was unfeasible and the epidemics of the disease returned to areas where it had previously been eradicated, resulting in the failure of the GEP. Since then, there is evidence that the burden of malaria increased in the 1990’s in terms of the proportion of population at risk, severity of infection and deaths (WHO, 2005). Malaria re-emerged in several countries and in sub-Saharan Africa, malaria- related child mortality is estimated to have increased by up to two-fold during the

Chapter 1: Introduction 8

1980’s and early 1990’s. Furthermore, drug-resistant strains of P. falciparum and P. vivax have emerged around the world.

1.2 The Current Global Situation

1.2.1 The Incidence of Global Malaria Malaria remains a major cause of morbidity and mortality. In 1998, the ‘roll back malaria’ (RBM) initiative was launched by the WHO, United Nations Children’s Fund (UNICEF), United Nations Development Program (UNDP) and the World Bank. Its aim was a coordinated international approach to fighting malaria and to halving the malaria burden by 2010 by the prompt use of effective drugs, insecticide treated materials, intermittent preventive treatment in pregnancy and emergency and epidemic preparedness and response. Although the WHO claim that it is not yet possible to determine how effective RBM has been in lessening the global burden of malaria (WHO, 2005), reports suggest that the aim of halving the malaria burden by 2010 not only seems unlikely, but that the malaria morbidity and mortality have increased since the implementation of the RBM campaign (Yamey, 2004).

As of 2005, the WHO estimates 107 countries and territories, inhabited by 3.2 billion people, are at risk of malaria transmission (Figure 1.2). Every year there are at least 300-500 million clinical cases reported, resulting in an estimated 1.5–2.7 million deaths (Korenromp, 2004) where over 80% of deaths occur in sub-Saharan Africa (WHO, 2005). Of the annual deaths that occur in African, the majority are children under the age of five (Snow et al., 2005). Furthermore, the disease costs Africa about US$12 billion every year (Greenwood et al., 2005).

Malaria is also a significant indirect cause of death. Infection in pregnancy can cause severe maternal anaemia, low birth weight and premature delivery resulting in up to 200 000 African infant deaths annually (ter Kuile et al., 2004). In addition, children infected with malaria are more susceptible to other childhood diseases such as respiratory disease (Molineaux, 1997).

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Figure 1.2 Global distribution of malaria transmission risk. Reproduced with permission from the Rollback Malaria Partnership (WHO, 2005).

Figure 1.3 World-wide drug resistance to P. falciparum. Data from studies in sentinel sites, up to 2004 (WHO, 2005). Reproduced with permission from the Rollback Malaria Partnership.

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P. falciparum causes most of the severe, potentially fatal malaria accounting for over 95% of deaths and is most prevalent in sub-Saharan Africa (WHO, 2005). A further 7% are caused by either P. vivax or mixed infections of both (WHO, 2005). Cases of P. ovale and P. malariae infections are less frequent and do not often lead to a fatal condition (Bruce-Chwatt, 1985). Although P. vivax is rarely fatal, it is the most frequent cause of debilitating human malaria. It is commonly found in Asia, the Americas, Europe and North Africa. It is the most prevalent species of human malaria because of the population densities in these regions, particularly in Asia. Persistent P. vivax parasites can remain in the liver for up to 5 years after elimination of erythrocytic stages, resulting in great socio-economic burden to humanity. The disease is thought to be responsible for an estimated annual reduction of 1.3% in economic growth in affected countries (Bloom et al., 1998; Sachs, 2001).

1.2.2 Current Control of Malaria 1.2.2.1 Chemotherapy Current chemotherapeutic treatment of malaria still relies heavily on the antimalarials developed in the early 20th century. Most drugs used in the treatment of malaria today are active against the blood stages of the parasite and can be classed into four groups. The 4-amino quinolines and aryl amino alcohols include chloroquine, quinine, amodiaquine, mefloquine (Lariam®) and halofantrine. The 4-amino quinolines are known to interfere with the detoxification of free haem, which is generated through the degradation of haemoglobin (Ginsburg et al., 1999; Sanchez et al., 2000). In addition, primaquine is active against the dormant liver stages of Plasmodium. The second class of antimalarials is the antifolates that are often used in combinations such as sulfadoxine-pyrimethamine (Fanisdar®) and atovaquone-proguanil (Malarone®). Pyrimethamine and proguanil are inhibitors of dihydrofolate reductase (DHFR), whilst sulfadoxine is an inhibitor of dihydropteroate synthetase, an additional enzyme of folate metabolism. The third class of antimalarials is the artemisinin derivatives, which include artemisinin, artemether, arteether and artesunate. This class of antimalarials is thought to be active against the sexual stage gametocytes. The last class of drugs that exhibit antimalarial properties are antibiotics such as rifampicin, doxycycline, clindamycin and azithromycin that target the plastid-like organelle or apicoplast.

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Initially, the most effective antimalarial was chloroquine. However, by 1970 resistance to chloroquine became global in distribution (Figure 1.3). Despite this, it remains the first-line treatment of malaria. A survey across Africa revealed that chloroquine is still being used in up to 84% of malaria infections, even where the drug is ineffective (Breman et al., 2004). Resistance to sulfadoxine-pyrimethamine has occurred in most countries where it was used in place of chloroquine (Figure 1.3) (Greenwood et al., 2005). In Thailand, resistance to these antimalarials appeared within a year of their use. Quinine resistance appears sporadically, and a moderate risk of treatment failure appears to be limited to some regions of Southeast Asia and New Guinea (Zalis et al., 1998). Resistance to mefloquine and atovaquone predominate in South East Asia where this drug is more commonly used.

One class of drugs that have not, as yet, seen the emergence of drug-resistance are the artemisinin derivatives. Artemisinin derivatives are effective due to the rapid clearance of parasites as well as reducing overall transmission of the disease by decreasing the viability of gametocytes. Although the artemisinin derivatives act faster than any other antimalarials with an approximate parasite and fever-clearance time of 32 hours in contrast to the 2-3 days required by conventional antimalarials, they are up to twenty times more costly than conventional therapies and are also cleared rapidly from the plasma (Mutabingwa, 2005). Hence, parasite recrudescence is common in short course treatments when not combined with other, longer lasting drugs (Talisuna et al., 2004). Furthermore, the safety of artemisinin in pregnant women has been questioned (WHO, 2003).

Combination therapies are now becoming more common and are recommended by WHO as the treatment policy for P. falciparum malaria in all countries experiencing resistance. The combination of sulfadoxine-pyrimethamine/amodiaquine (Table 1.1, (4)) is efficacious in Africa where there is moderate resistance to these drugs (Staedke et al., 2004). The most highly efficacious and widely promoted combination therapies are the artemisinin-based combination therapies (ACTs) (Table 1.1). Since 2001, 42 malaria-endemic countries have adopted ACTs. Of these, 23 are in Africa although only 9 countries were actually implementing ACTs as of 2004 (WHO, 2005). The major drawback to large-scale use of ACTs is the high cost, which has also encouraged the use of counterfeit ACTs. Although the current choice of ACTs is restricted, the

Chapter 1: Introduction 12

future is encouraging with several new combinations at various stages of development (Greenwood et al., 2005).

Recently, the Medicines for Malaria Venture (MMV) has been established with the ultimate goal of registering drugs with a suitable product profile for uncomplicated malaria. These include efficacy against drug resistant strains, cure within three days, low toxicity especially in children and pregnancy, low risk of emergence of resistance, adeptness in formulation and packaging, and a low cost of goods (Biagini et al., 2005). In an effort to improve drug development, a three-pronged approach is being taken that includes the re-design of existing drugs, novel use of older drugs and identifying novel parasite-specific targets by an improved understanding of the parasites biology.

A wide range of new antimalarial drugs are in various stages of clinical development. Chloroproguanil-dapsone (Lapdap ™), targeting DHFR and dyhydropteroate synthase, is currently under development as a replacement for sulfadoxine-pyrimethamine and was shown to be effective against sulfadoxine-pyrimethamine resistant parasites in clinical trials (Mutabingwa et al., 2001). More recently, Chloroproguanil-dapsone- artesuntate has been assessed in successful phase II trials in adults and children in Gambia and Malawi (Biagini et al., 2005). Other antimalarial drugs in clinical development include: synthetic peroxide OZ277; improved diamidine DB289, artemether combined with lumefantrine (Paediatric Coartem™), pyronaridine- artesunate, dihydroartemesinin-peperaquine (Artekin™), AQ-13 aminoquinoline, isoquine, pyridones (reviewed in (Biagini et al., 2005)); tafenoquine, pyronaridine and fosimidomycin (reviewed in (Wiesner et al., 2003)).

1.2.2.2 Alternative Control Strategies The control of malaria has traditionally relied on a two-pronged approach: vector control and chemotherapy. It is now apparent that the most effective strategy to manage the disease will involve a combination of chemotherapy, vector control and immunisation.

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NON-ARTEMISININ COMBINATIONS (1) Quinine/sulfadoxine-pyrimethamine Used effectively in Europe and Asia. Cost and side-effects make treatment inappropriate in Africa.

(2) Quinine/doxycycline Similar to (1). Mainly used in Thailand where there is resistance to sulfadoxine-pyrimethamine.

(3) Sulfadoxine-pyrimethamine/chloroquine Used in some African countries but not where resistance to both drugs is high.

(4) Sulfadoxine-pyrimethamine/amodiaquine More effective than (3) where amodiaquine resistance is low.

ARTEMISININ-BASED COMBINATION THERAPIES (ACTs) (5) Artemether-lumefantrine Currently the only internationally licensed co-formulation ACT. Available in Asia and Africa.

(6) Artesunate/amodiaquine Currently co-packaged. Adopted as policy by some African countries. Effective where amodiaquine resistance is low.

(7) Dihydroartemisinin-piperaquine Co-formulated drug used widely in Asia.

(8) Artesunate/mefloquine Main antimalarial drug policy in much of southeast Asia. Too expensive for Africa.

(9) Artesunate/sulfadoxine-pyrimethamine Used in some Asian countries. Ineffective where amodiaquine resistance has failed.

(10) Dihydroartemisinin-napthoquine- New formulation used in China and Vietnam. Early reports are trimethoprim encouraging.

Table 1.1 Current antimalarial drugs. Examples of currently used combinations of antimalarial drugs (Greenwood et al., 2005).

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In areas of high endemicity, the use of vector control measures such as insecticide treated nets, have been highly successful in reducing morbidity and mortality of malaria (Gardiner et al., 2005). However, their relatively high costs, poor distribution and availability (Lindblade et al., 2004), and the potential problem of insecticide resistance occurring, may affect their long-term effectiveness. Environmental management of the mosquito vector has shown some success in reducing populations, but requires a detailed knowledge of the behaviour and distribution of local species of Anopheles mosquitoes (Greenwood et al., 2005). The completion of the Anopheles sequencing project has increased interest in vector control and will lead to a better understanding of insecticide resistance as well as identification of new targets for insecticides.

It is now accepted that an effective vaccine is also necessary to control the spread and burden of the disease. Support for the notion that an effective vaccine against malaria is achievable is based on the fact that individuals acquire natural immunity when exposed to the disease. Secondly, it has been shown that protection can be induced against experimental infections in animals and human volunteers (Gardiner et al., 2005). Furthermore, passive transfer of immunoglobulins from immune people has also been shown to be protective. However, the development of an effective vaccine has been greatly hindered by factors such as the complex parasite lifecycle, the large repertoire of antigenic variants and the ability of the parasite to undergo rapid antigenic switching. Despite this, the pool of candidate antigens is rapidly increasing with the advent of improved genomic and proteomic information.

1.3 Molecular Biology of Plasmodium

1.3.1 The Plasmodium Genome 1.3.1.1 The Plasmodium Sequencing Projects In 1996, a consortium of funding agencies, genome centres, and malaria investigators was formed to sequence the genome of P. falciparum 3D7 clone (Butler, 1997; Hoffman et al., 1997). A strategy was adopted whereby individual chromosomes were resolved by pulse-field gel electrophoresis, delegated to three different genome centres and subjected to shotgun sequencing. In 2002, the complete genome sequence of P. falciparum (Gardner et al., 2002) and the rodent malaria P. yoelii (Carlton et al., 2002)

Chapter 1: Introduction 15

were published. In 2005, the complete genome sequences of two additional rodent malaria species, P. berghei and P. chabaudi were published (Hall et al., 2005b). Several other species of Plasmodium have now been sequenced including that of P. knowlesi, P. reichenowi and P. gallinaceum. Comparative studies of the completed genomes have already provided valuable insight into the biology of the parasite including evolutionary biology, drug and vaccine candidate validation and choice of suitable animal models for in vivo testing. Further, the complete sequence of the Homo sapien genome, along with that of the Anopheles mosquito vector, is also available (Lander et al., 2001; McPherson et al., 2001; Venter et al., 2001). The completion of the parasite, host, and vector genomes will allow further insight into the relationship of the species and should greatly facilitate the discovery of rational drug and vaccine targets for the control of human malaria.

1.3.1.2 Plasmodium Genome Characteristics The P. falciparum genome is composed of 22.8 megabases (Mb) spread amongst 14 chromosomes that vary between 0.643-3.92Mb each (Gardner et al., 2002). The A/T content of the genome is approximately 80% in coding regions and up to 90% in introns and intergenic regions, and is the most A/T-rich genome sequenced to date. Comparable A/T biases are thus far, only observed in the eukaryote Dictyostelium discoideum and the bacterial pathogen Borrelia burgdorferi (Fraser et al., 1997; Glockner et al., 2002).

Approximately 5300 protein-encoding genes were identified resulting in a gene density of one gene every 4.338 kilobases (kb). The mean length of a P. falciparum gene was found to be 2.3kb, which is substantially larger than in other organisms (Gardner et al., 2002). Furthermore, a relatively high proportion of genes (15.5%) were found to be greater than 4kb. Introns were predicted in 54% of genes.

The nuclear genome was found to contain a full set of transfer RNA (tRNA) ligase genes and 43 tRNAs to bind all codons except Cys coding tRNAs, TGT and TGC. Unlike other eukaryotes, the P. falciparum genome encodes several, single 18S, 5.8S and 28S ribosomal RNA (rRNA) units on different chromosomes. The sequences coding each unit are different and are developmentally regulated and expressed at different stages of the lifecycle (Li et al., 1994; Waters, 1994). Putative centromere

Chapter 1: Introduction 16

sequences have been found in 11 of the 14 chromosomes by sequence comparison with Saccharomyces pombe.

In general terms, the Plasmodium genomes sequenced thus far are similar to that of P. falciparum and are all haploid and between 22 and 26Mb. All are distributed amongst 14 linear chromosomes with a size range of 0.5-3Mb. The A/T content for all species is high (approximately 80%) except for P. vivax which has an average A/T content of 60% (Carlton, 2003). However, P. vivax telomeric regions appear to be more A/T-rich than other regions when compared to the relatively uniform distribution of G/C bases in the P. falciparum genome (Carlton, 2003). Each species of Plasmodium seems to have 5000-6000 predicted genes of which 60% are orthologous among species (Hall et al., 2005a). Gene synteny or location, order and fine-scale organisation of genes seem to be well conserved over large regions amongst Plasmodia (Janse et al., 1994; Carlton et al., 1998; Carlton et al., 1999; van Lin et al., 2000; Tchavtchitch et al., 2001). Most variation within the chromosomes occur in the subtelomeric regions, where many of the genes unique to each species, such as genes involved in antigenic variation, can be found (Gardner et al., 2002; Hall et al., 2005a).

Genome comparisons, transcription analysis, gene silencing and knockout studies along with proteomics, have revealed a plethora of genes and their associated proteins involved in a range of parasite activities. In broad terms, most of the studies have focused on invasion, infection and immune evasion of all stages of the parasite lifecycle, along with DNA replication and repair, the apicoplast, transport and secretory pathways, and metabolism.

1.3.2 The Plasmodium Transcriptome Comparative genomics of the Plasmodium sequences have already begun to provide exciting data in the areas of antigenic variation, gene expression and regulation and evolutionary studies. Much information has resulted from two groups conducting studies on the transcriptomes. Le Roch and colleagues conducted studies on much of the complex lifecycle taking RNA from sporozoites, merozoites and gametocytes (Le Roch et al., 2002; Le Roch et al., 2003; Le Roch et al., 2004; Daily et al., 2005). Bozdech and co-workers on the other hand, made a detailed analysis on the RNA of the

Chapter 1: Introduction 17

asexual stage of infection that is responsible for symptoms of the disease (Bozdech et al., 2003a; Bozdech et al., 2003b). Both groups found that apart from housekeeping transcripts, a certain number of upregulated transcripts were shared between stages. Le Roche and co-workers confirmed that 25% of the genome was up-regulated in gametocytes and of the 1489 genes found to be regulated in erythrocytic stages, 746 of them were also regulated in gametocytes and sporozoites, respectively. Bozdech and colleagues also found that the majority of the genome is actively transcribed during the intra-erythrocytic stage including 75% of proteins thought to be specific for the sporozoite, gametocyte and gamete stages.

A detailed examination of transcription profiles in the erythrocytic stage of life cycle revealed that only a minority of genes (20%) were constantly expressed (Bozdech et al., 2003a; Bozdech et al., 2003b). The general finding was that 300 genes were found to be transcribed following invasion of RBCs, followed by 950 genes transcribed in the ring-trophozoite, 1050 genes transcribed in the trophozoite-schizont stage and approximately 550 genes transcribed in mid-late schizogony. Also reported is the transcriptional regulation of a wide range of individual proteins involved in processes such as transcription, translation, glycolytic pathway, synthesis of RNA and DNA, the TCA cycle, proteosome and plastid associated genes.

Scientists have begun to utilize the Anopheles and Plasmodium genomes in combination. Subtractive hybridisation methods have been used to characterise the parasite transcripts and mosquito genes induced by the parasite (Abraham et al., 2004; Srinivasan et al., 2004). As a result of these studies, many novel genes have been identified such as death genes and candidate antigens for transmission-blocking vaccines.

1.3.3 The Plasmodium Proteome Of the 5268 predicted proteins of the P. falciparum genome, approximately 60% have not been identified and appear to be unique to this organism, reflecting the evolutionary distance from sequenced eukaryotes (Gardner et al., 2002). In contrast, 5% of proteins had sequence similarity to other organisms with 1.3% of P. falciparum genes involved

Chapter 1: Introduction 18

in adhesion or invasion of host cells, and a further 3.9% involved in evasion of the host immune system.

Published high-throughput proteomic analysis has been carried out on the gametocytes and sporozoites of P. falciparum (Florens et al., 2002; Lasonder et al., 2002) and a similar study has been carried out on P. berghei (Khan et al., 2004). These studies identified >2500 proteins, hundreds of which were stage-specific. Furthermore, the stage-specific proteins from each species such as those involved in recognition, motility and invasion, were in good agreement with each other. Both proteomes also contained many common, conserved proteins such as those involved in the apicoplast, mitochondria and metabolism.

It is beyond the scope of this thesis to review the numerous and detailed proteomic studies of Plasmodium and the reader is directed to several reviews (Carlton et al., 2002; Janssen et al., 2004; Rasti et al., 2004; Blair et al., 2005; Cowman et al., 2005; Gelhaus et al., 2005; Hall et al., 2005b; Khan et al., 2005; Gilson et al., 2006; Kats et al., 2006; Kooij et al., 2006).

1.4 General Metabolism and Transport

In the past, metabolic studies have been restricted to the intra-erythrocytic stage of the parasite life cycle owing to the difficulties in obtaining sufficient material from other stages. Analysis of the P. falciparum genome has lead to a global view of the metabolism of the parasite regardless of life-cycle stage (Figure 1.4). Much information has been gleaned of the enzymes that characterise these pathways (Gardner et al., 2002; Becker et al., 2004; Ralph et al., 2004; van Dooren et al., 2006).

The complete catalogue of identified transporters is presented in Figure 1.4, and resembles those of obligate intracellular prokaryotic parasites (Gardner et al., 2002). Of particular interest are the new permeability pathways or NPPs. Following infection by the malaria parasite (12-16h after invasion), there is a substantial increase in the permeability of the host erythrocyte membrane due to the appearance of the NPPs. These pathways are thought to provide the major route of entry of at least some essential nutrients required by the parasite and they regulate the efflux from the infected

Chapter 1: Introduction 19

cell of various metabolic wastes (Becker et al., 2004). The transport properties of the NPPs have been characterized and are broadly anion selective, but with a significant permeability to both organic and inorganic cations (Ginsburg et al., 1985).

Figure 1.4 A metabolic overview of P. falciparum. Metabolic pathways and transporters represented, as elucidated after completion of the P. falciparum genome project (Gardner et al., 2002). Reproduced with permission.

Chapter 1: Introduction 20

1.5 Pyrimidine Biosynthesis in Plasmodium falciparum

Upon red cell invasion, P. falciparum develops and replicates up to 36 fold within a 48h period. During this process, a vast amount of compounds are needed, of which nucleic acid precursors are a major component. The parasite achieves this in part, by substantially up-regulating the expression of enzymes involved in pyrimidine biosynthesis and purine salvage (Gero et al., 1990). It has long been known that the malaria parasite is unable to salvage preformed pyrimidines and must therefore rely on the synthesis of pyrimidines de novo (Gutteridge et al., 1970; Reyes et al., 1982; Seymour et al., 1994). In fact, many parasitic protozoa are completely dependant on de novo biosynthesis for their pyrimidine requirements because they lack the relevant enzymes involved in salvage, notably, thymidine kinase, uridine-cytidine kinase, deoxycytidine kinase and cytidine kinase (Hill et al., 1981; Hammond et al., 1982; Reyes et al., 1982; Krungkrai et al., 1989). Uridine and thymidine can enter the RBC, but due to the absence of thymidine kinase, these nucleosides cannot be further metabolised (Jarvis et al., 1983). The genes encoding these enzymes have not been reported since the completion of the genome sequence.

This contrasts markedly with the human host where both de novo synthesis and the salvage of preformed nucleosides are significant. However, the human RBC lacks the ability to synthesis pyrimidines and relies completely on the salvage of preformed pyrimidine nucleosides (Gero et al., 1990), thereby making the pyrimidine biosynthesis pathway an ideal target for antimalarial therapies. In addition, total inhibition of the human pyrimidine biosynthesis de novo, as in the case of hereditary orotic aciduria, can be alleviated by administration of oral uridine (Kelly et al., 1978).

In past studies, the presence of the enzymes of the pyrimidine biosynthesis pathway of P. falciparum have been confirmed (Sherman, 1979; Reyes et al., 1982; Gero et al., 1984; Gero et al., 1990). Some of the genes and corresponding enzymes involved in the pathway have been cloned and characterised in our laboratory. They include those encoding P. falciparum carbamoyl phosphate synthetase II (PfCPSII) (Flores et al., 1994), CTP synthetase (PfCTPase) (Hendriks et al., 1994; , 1998; Yuan et al., 2005), phosphoribosylpyrophosphate synthetase (PfPRPPS) (Grover, 1998) and aspartate transcarbamoylase (PfATCase) (Hillier, 2001). More recently, the presence of the

Chapter 1: Introduction 21

genes of the pyrimidine biosynthetic pathway has been confirmed in the P. falciparum genome (Gardner et al., 2002).

1.5.1 The de novo Pyrimidine Biosynthetic Pathway Carbamoyl phosphate synthetase II (CPSII, EC 6.3.5.5) catalyses the first and rate limiting step of the pathway and is responsible for the conversion of L-glutamine, two molecules of ATP and bicarbonate to carbamoyl phosphate (Figure 1.5). Aspartate transcarbamoylase (ATCase, EC 2.1.3.2), the second enzyme of the pathway, catalyses the condensation of aspartate and carbamoyl phosphate to yield carbamoyl aspartate and phosphate. In the next step, removal of a water molecule from carbamoyl aspartate by dihydroorotase (DHOase, EC 3.5.2.3), closes the pyrimidine ring and dihydroorotate is produced, which is the oxidised to orotate by dihydroorotate dehydrogenase (DHODase, EC 1.3.3.1). This step plays an important role of supplying electrons to coenzyme Q in the electron transport chain. Phosphoribosyl pyrophosphate synthetase (PRPPS, EC 2.7.6.1) catalyses the conversion of ribose-5'-phosphate to phosphoribosylpyrophosphate (PRPP), which acts as a precursor for the conversion of orotate to orotidine 5'-phosphate by orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10). PRPP is also a precursor for purine and amino acid synthesis and plays an important regulatory role in many biosynthetic pathways (Bentsen et al., 1996). Orotidine 5'-phosphate is decarboxylated by orotidine-5'-decarboxylase (ODCase, EC 4.1.1.23) to yield UMP (Jones, 1980). The subsequent phosphorylation of UMP to UDP and UTP occurs via cytidylate kinase (CK, EC 2.7.4.14) and nucleoside diphosphate kinase (NDPK, EC 2.7.4.6). Cytidine triphosphate synthetase (CTPase, EC 6.3.4.2) catalyses the synthesis of cytidine nucleotides from UTP in the presence of glutamine and ATP. CTPase can be considered the last enzyme in the pyrimidine biosynthetic pathway and is the major branch point in nucleotide metabolism and membrane phospholipid synthesis (Yang et al., 1995).

Various compounds have been tested for their ability to inhibit in vitro growth of P. falciparum by interfering with nucleotide metabolism. In one of the largest studies to date, Queen and colleagues tested sixty-four purine and pyrimidine analogs known to inhibit one or more steps of nucleotide synthesis (Queen et al., 1990). Twenty-two of the sixty-four compounds tested reduced P. falciparum growth by 50% at 50μM or less.

Chapter 1: Introduction 22

Half of these compounds were confirmed to inhibit pyrimidine biosynthesis de novo with a third acting directly on the CPSII, the control point of the pathway.

L-Glutamine CPSII

L-Aspartate + Carbamoyl-P ATCase

N-Carbamoyl-L-Aspartate DHOase

L-Dihydroorotate DHODase

Orotate PRPPS Ribose 5'- phosphate PRPP OPRTase Orotidine-5'-P (OMP) ODCase

UMP CK

UDP NDPK

UTP CTPase

CTP

Figure 1.5 A schematic representation of the pyrimidine metabolic pathway of P. falciparum. The enzymes of each step of the pathway are highlighted in red.

Chapter 1: Introduction 23

1.5.2 Carbamoyl Phosphate Synthetase 1.5.2.1 Types and Characteristics of CPS The common role of carbamoyl phosphate in pyrimidine and arginine synthesis, presents biological systems with the problem of how to control the pathways separately according to their different and varying needs for pyrimidines and arginine. Prokaryotic and eukaryotic systems diverge quite distinctly in this respect. In most bacteria, carbamoyl phosphate is synthesised for both the arginine and pyrimidine pathways by a single enzyme, CPS. The proteobacteria studied use one CPS enzyme for both arginine and pyrimidine biosynthesis, while gram-positive bacteria have two CPS enzymes, which are separately regulated for arginine, and pyrimidine biosynthesis (Lawson et al., 1996).

Eukaryotes on the other hand, generally have two different synthetases: one specific for the arginine pathway (CPSI or III) and another for the pyrimidine pathway (CPSII) (Jones, 1980). CPSII, which is cytoplasmic, is usually glutamine-dependent and is allosterically controlled by pyrimidines (Simmer et al., 1990). Recent evidence indicates mammalian CPSII (or CAD) is located in the cytoplasm of resting cells, but when cells proliferate, a fraction of the cytosolic CAD is translocated to the nucleus and activated by phosphorylation (Sigoillot et al., 2005).

In apicomplexan protozoans, it appears that there is only the one CPS enzyme, the pyrimidine-specific CPSII (Aoki et al., 1994; Flores et al., 1994; Chansiri et al., 1995; Nara et al., 1998; Gao et al., 1999; Fox et al., 2003). The single CPS present in parasitic protozoans branch deepest within the eukaryotic tree and suggest that the CPS duplication evolved after their divergence (Lawson et al., 1996).

1.5.2.2 The Functional Domains of CPSII The glutamine-dependent synthesis of carbamoyl phosphate requires a series of four reactions. These steps are catalysed by two distinct domains of CPSII, namely, a typically 40-42kDa glutamine amidotransferase domain (GAT) and a 118-120kDa synthetase domain (CPS) (Figure1.6A). The GAT domain is composed of a putative structural domain (PSD) on the amino terminal end linked to the glutaminase (GLNase) subdomain. In E. coli, the PSD is thought to be involved in subunit interactions

Chapter 1: Introduction 24

between the GLNase and CPS subdomains (Rubino et al., 1987). The GLNase subdomain is homologous to the trp-G-type amidotransferases and contains catalytic residues for the hydrolysis of glutamine.

The significantly larger CPS domain consists of two synthetase subdomains, CPS.A and CPS.B, with individual ATP binding sites that are functionally equivalent (Guy et al., 1996). There is significant internal similarity within the CPS domains, which are thought to have arisen as a result of a gene duplication of an ancient kinase gene (Nyunoya et al., 1983). Each CPS subdomain is composed of three further subdomains (A1, A2, A3 and B1, B2, B3) (Figure 1.6A). The middle 25kDa subdomains, A2 and B2, are responsible for ATP binding (Simmer et al., 1990). These catalytic subdomains have high catalytic activity that is suppressed by interactions with the 11kDa A1 or B1 subdomains (Guy et al., 1997). The 20kDa A3 and in particular, B3, are thought to be involved in regulation of activity of the enzyme (Guy et al., 1997).

The first reaction involves binding of glutamine and subsequent hydrolysis by the GAT domain, followed by transfer of the resulting amide group to the CPS domain (Figure 1.6B). The CPS subdomains are responsible for the following three reactions, which include: phosphorylation of bicarbonate to carboxyphosphate; nucleophilic attack of the carboxyphosphate by ammonia to carbamate; and the phosphorylation of carbamate to carbamoyl phosphate (Anderson et al., 1966). The first bicarbonate phosphorylation step is carried out by CPS.A and the second phosphorylation of carbamate is carried out by CPS.B (Post et al., 1990).

Chapter 1: Introduction 25

GAT domain CPS domain

PSD GLNase CPS.A CPS.B

A1 A2 A3 B1 B2 B3

B. CPS.A Carboxyphosphate Bicarbonate

O MgATP O O MgADP C C P OH O- OH O O- O-

Pi NH3 Glutamine

CPS.B O O O MgATP . C P C - - H2N O O MgADP H2N O O-

Carbamoyl phosphate Carbamate

Figure 1.6 A schematic representation of CPSII functionality. (A) The CPSII functional domains. (B) The four chemical steps of the synthesis of carbamoyl phosphate by CPSII. The GAT domain carries out the first step, highlighted in red. The CPS domain carries out the subsequent three steps, highlighted in blue. The CPS subdomains, CPS.A and CPS.B, each catalyse an ATP-dependant reaction.

Chapter 1: Introduction 26

1.5.2.3 The Structural Organisation of CPSII The amino acid sequences of the GAT and CPS domains are highly conserved across most species. In contrast to the highly conserved nature of the sequence, the structural organisation of the CPS enzymes is highly variable amongst different organisms (Figure 1.7). Eubacteria, archaeabacteria and plants express a monofunctional GAT subunit (carA), as well as a monofunctional CPS subunit (carB) (Jones, 1980; Zhou et al., 2000). All bacteria studied thus far contain a heterodimeric form of the enzyme that contains a 40kDa carA subunit and a 120kD carB subunit. The heterodimeric CPS can be encoded by genes that are separately transcribed, co-transcribed, or even on separate chromosomes (Werner et al., 1985; Kwon et al., 1994; Lawson et al., 1995).

Lawson and co-workers proposed that there was a fusion between the GAT and CPS domains to form a monomeric CPS in the eukaryotes after the divergence of archaea (Lawson et al., 1996). This is evident in the parasitic protozoans such as P. falciparum, Leishmania mexicana, Toxoplasma gondii, Babesia bovis and Trypanosoma cruzii, where CPSII is a bifunctional protein composed of a N-terminal GAT domain fused to a C-terminal CPS domain (Nara et al., 2000; Fox et al., 2003). Typically, parasitic CPSII enzymes contain regions of extra amino acids not found in the consensus sequence of other organisms. The significance of these insertions is discussed in subsequent sections.

Later in evolution, another fusion occurred with the second and third enzymes of the pyrimidine pathway (Figure 1.7). In S. cerevisiae, CPSII (URA2) is part of a bifunctional polypeptide of CPSII and ATCase, whose domains are separated by an inactive DHOase domain homologous to functional DHOases coded for on a different chromosome (Souciet et al., 1989). The mammalian pyrimidine-specific mammalian CPSII is part of a 243kDa multifunctional CAD complex, comprising a single polypeptide encoding the first three enzymes of the pathway (CPSII, DHOase and ATCase) fused via linkers of varying lengths (Shoaf et al., 1973; Mori et al., 1975; Coleman et al., 1977; Davidson et al., 1993).

Chapter 1: Introduction 27

GAT CPS DHOase ATCase Prokaryotes, Archaea & Plants

Protozoan parasites

Saccharomyces cerevisae

Mammalian CAD

Figure 1.7 The organisation of CPSII in different organisms. A schematic representation of the organisation of the pyrimidine-specific CPS from prokaryotes to higher eukaryotes.

A general observation of enzymes that catalyse successive reactions in a metabolic pathway, as far as molecular evolution is concerned, is that in prokaryotes these enzymes are independent but are associated into multi-functional proteins. In higher organisms, successive enzymes of a pathway often function as multi-enzyme complexes as in the case with pyrimidine biosynthesis. The clustering of enzymes catalysing a reaction sequence is advantageous as side reactions are minimized as substrates are channelled from one catalytic site to the next, preventing dilution of intermediate products.

The X-ray crystal structure of CPS from E. coli has identified an intermolecular tunnel of 100Å in length that connects the glutamine-binding site within the GAT subunit with the two phosphorylation sites within the CPS subunit (Thoden et al., 1997; Thoden et al., 1999). The ammonia generated by the GAT subunit travels the molecular tunnel

Chapter 1: Introduction 28

leading to the first ATP binding site where it converts carboxy phosphate to carbamate. This region is referred to as the ‘ammonia tunnel’ and is thought to be formed by residues in the GAT domain interacting with residues in the amino end of the CPS.A subdomain (Thoden et al., 2002). Carbamate then shuttles to the second ATP binding site via the ‘carbamate tunnel’ to generate carbamoyl phosphate. The channelling of ammonia and carbamate through the interior of the protein has been proposed as a mechanism to deliver these two labile and potentially toxic intermediates, from one active site to the next (Holden et al., 1998; Raushel et al., 1998; Raushel et al., 1999).

Much less is known about the more integrated enzymatic CPSII systems in yeast and mammals. However, channelling of substrates from one enzyme active site to the next is thought to occur in both these systems (Irvine et al., 1997; Serre et al., 1998; Serre et al., 1999). Irvine and co-workers proposed that reciprocal allostery occurs between the active sites of ATCase and CPSII in the mammalian CAD complex (Irvine et al., 1997). Binding of aspartate to the active site of ATCase causes a conformational change at the active site of CPSII, which protects it from inhibition by its product, carbamoyl phosphate. Reciprocally, the substrate for CPSII increases the affinity of carbamoyl phosphate and aspartate for the active site of ATCase. Irvine and co-workers state that reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesized and dedicated to the next step in pyrimidine biosynthesis (Irvine et al., 1997).

Noteworthy, is the channelling of carbamoyl phosphate observed in arginine synthesis between CPS and ornithine transcarbamoylase of Pyrococcus furiosus and the physical interaction between these enzymes (Massant et al., 2002; Massant et al., 2005). Furthermore, channelling is also observed in the fifth and sixth enzymes of pyrimidine biosynthesis. In higher eukaryotes, OPRTase and OMP decarboxylase activities have fused to form a bifunctional protein referred to as UMP synthase (Jones, 1980; Jacquet et al., 1988; Yablonski et al., 1996)

1.5.3 The P. falciparum CPSII Gene and Predicted Protein Sequence The PfCPSII gene encodes an unusually large CPSII enzyme, the largest characterised to date. Notably, the 7.2kb PfCPSII gene is 450bp longer than the mammalian CAD gene sequence. The 7.2kb of coding sequence codes for 2391 amino acid residues

Chapter 1: Introduction 29

resulting in an estimated 275kDa protein. This is principally due to the presence of two relatively large insertion sequences that do not disrupt the open reading frame (ORF) and are translated to be 232 and 603 amino acids respectively (Flores et al., 1994). To date, the 603 amino acid insertion remains the second largest isolated in a gene encoding a Plasmodium enzyme.

The PfCPSII gene exists as a single copy and there is no evidence of linkage to the subsequent enzymes in the pyrimidine biosynthetic pathway. The base composition of the gene is characteristic of P. falciparum DNA, having a 76.3% A/T-rich coding region while the 5' and 3' untranslated regions are 83.9% and 88.4% A/T-rich respectively, making the average A/T composition 80.9% for the 8.9kb sequence (Flores et al., 1994). The gene exhibits the same unusual codon bias, such as A or T occurring most frequently in the first or third position, as was originally reported in other P. falciparum genes (Saul et al., 1988).

Alignment of PfCPSII with other CPSII enzymes reveals extensive conservation of the protein, with the exception of these insertions that are unique to the parasite protein. In the past, the various subdomain boundaries were predicted based on homology studies (Simmer et al., 1990; Flores et al., 1994). The GLNase subdomain was predicted to start at His483 and end at Asp670. The CPS.A and CPS.B subdomains were also well conserved and were predicted to start at Lys691 and end at Met1253, and Cys1857 to Val2391 respectively. Conserved residues implicated in previous studies to play important roles in the GLNase and synthetase catalytic mechanisms were located in PfCPSII (Flores et al., 1994).

The insertions in PfCPSII are precisely positioned in the junctions of the well- conserved domains (Figure 1.8). The first (insertion I) is located at the precise boundary between the two subdomains (PSD and GLNase) of the GAT domain, and the second (insertion II), is located between the two halves of the synthetase domains (CPS.A and CPS.B) (Figure 1.8).

Northern analysis and reverse transcription of PfCPSII mRNA revealed the same sized products as those expected from genomic DNA, supporting the presence of both insert regions at the mRNA level (Flores et al., 1994). The nucleotide sequences of both

Chapter 1: Introduction 30

insertions do not possess the notable features of P. falciparum introns. Their A/T content is typical of coding regions, 79% for insertion I and 77% for insertion II. Introns are bound by consensus donor and acceptor splice sites, which were not detected around the boundaries of the insert regions.

Interestingly, CPSII from parasites B. bovis and T. gondii also contain insertion sequences in the linker region of the GAT domain and within the linker of the CPS domain region (Chansiri et al., 1995; Fox et al., 2003). However, these insertions are relatively short stretches of amino acids when compared to the vast insertions of P. falciparum (Appendix 4).

Orthodox CPSII

PSD GLNase CPS.A CPS.B

Ins I Ins II

GAT domain CPS domain

P. falciparum CPSII

Figure 1.8 A schematic representation of PfCPSII. A schematic comparison of the domain structure of other CPSII (orthodox) with the enlargened PfCPSII. The enlargened PfCPSII gene has two insertion sequences of 232 and 603 amino acids respectively, dividing the two conventional subdomains of GAT and CPS domains (Flores et al., 1994).

1.6 Insertions within Plasmodium Proteins

The insertions of PfCPSII are not unusual in the Plasmodium genome, and occur in high frequency in a range of proteins that include those involved in pathogenesis, immune evasion and housekeeping enzymes. Some examples of insertions found in enzymes of

Chapter 1: Introduction 31

P. falciparum are listed in Table 1.2. These insertions are not found in homologous proteins from other genera, making these Plasmodium proteins larger than their counterparts. The majority of these unusual sequences seem to be located between catalytic domains with the exception of some small inserts. In contrast, PfCTP synthetase has a large insert of 220 amino acids located within a catalytic domain (Hendriks et al., 1994).

The number of estimated proteins in Plasmodium is comparable to that of the yeast S. cerevisiae. However, the genome of P. falciparum is considerably larger and this may be partly attributed to the common occurrence of insertions in P. falciparum proteins. A comparison of orthologous proteins revealed Plasmodium proteins can be up to 50% longer than yeast proteins due to the abundance of insertions (Aravind et al., 2003). In a study of insertions and deletions across 136 bacterial and protozoan genomes, P. falciparum had by far, an extremely high amount of insert-containing proteins attaining almost 30% of total proteins, when compared to human homologs (Cherkasov et al., 2006). The number of expected insertions, 300, was significantly lower than the observed number of insertions, 1310, when proteins were compared to human homologues.

Plasmodium-specific insertions are often composed of low entropy or low-complexity regions (LCRs) due to the nature of their amino acid composition. Low complexity regions in protein sequences are characterized by a limited set of amino acids, often including repeats of one or more residues (Wootton, 1994). Almost 90% of P. falciparum proteins contain at least one LCR. Plasmodium insertions of low- complexity are characterised by a limited, highly recurrent and predominantly hydrophilic composition of amino acids that have a rapidly evolving nature (Pizzi et al., 2000; , 2001; Xue et al., 2003). The hydrophilic amino acids that dominate are usually asparagine, aspartic acid, glutamine and serine. In the case of gamma glutamyl cysteine synthetase (γ-GCS), the first insertion encodes 33% asparagine and glutamine; the second insertion, 30% asparagine and lysine and the third insert, 32% asparagine and serine (Luersen et al., 1999). The asparagine content alone for insert 1 and 2 of P. falciparum protein kinase 1, are 28% and 20% respectively (Kappes et al., 1995).

Chapter 1: Introduction 32

P. falciparum Gene Insert Regions References (amino acids) CPSII 232 and 603 (Flores et al., 1994) CTPase 55 and 210 (Hendriks et al., 1994) OPRTase 20 and 66 (Krungkrai et al., 2004) RNA polymerase I 435, 168, 556 and (Fox et al., 1993) 679 RNA polymerase II 128, 181 and 259 (Li et al., 1989) RNA polymerase III 113, 597 and 191 (Li et al., 1991) Dihydroorotate dehydrogenase 42 (LeBlanc et al., 1993) DNA polymerase α 50, 91, 33 and 49 (White et al., 1993) Protein kinase-1 178 and 330 (Kappes et al., 1995) ATPase 3 300, 142, 459, 242 (Rozmajzl et al., 2001) Lactate dehydrogenase 1, 5 (Bzik et al., 1993) Serine/Threonine phosphatase-α 8, 14, 6, 6,8, (Li et al., 1998) Subtilisin-like protease-1 21, 50, 5, 9, 16, 6, 8, (Jean et al., 2005) 9 and 2 Glutathione reductase 13 and 33 (Farber et al., 1996) Glucose-6-phosphate 58 (O'Brien et al., 1994) dehydrogenase-6- phosphogluconolactonase Dihydrofolate reductase - 6, 23 and 37 (Bzik et al., 1987) thymidylate synthase Gamma-glutamylcysteine synthetase 94, 108, 239 (Luersen et al., 1999) S-adenosylmethionine 39, 147, 196 and 274 (Birkholtz et al., 2004) decarboxylase/ornithine decarboxylase Telomerase reverse-transcriptase Regions tallying up (Figueiredo et al., to 850 2005)

Table 1.2 Inserts of P. falciparum enzymes.

Chapter 1: Introduction 33

The parasite-specific insertions often form large, non-globular regions that are postulated to be extruded from the globular protein core due to their hydrophilic, low- complexity nature (Gardner et al., 1998). Structural evidence indicates that non- globular regions exist as solvent-exposed, disordered coils (Huntley et al., 2002). Crystallographic evidence has confirmed the predicted surface location of the parasite- specific inserts of P. falciparum dihydrofolate reductase-thymidylate synthase (DHFR- TS) (Yuvaniyama et al., 2003).

For both the low-complexity and complex classes of insertions, the function is largely unknown. However, it would seem likely that considering the ubiquitous nature of these insert sequences and the large size of some, they have a common biologically significant role. The importance of the insertions is also emphasised by their occurrence in the same genes of different Plasmodium species. For example, the inserts of P. falciparum glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase also occur in P. falciparum, P. yoelii, P. chabaudi and P. knowlesi and suggest the insertion event took place in a common ancestor regardless of the variation in size and sequence (Clarke et al., 2003).

Some malaria vaccine researchers have speculated that insertions play a role in antigenic diversification or immune response (Anders, 1986; Schofield, 1991; Ferreira et al., 2003; Hughes, 2004; Cortes, 2005; Verstrepen et al., 2005). This seems unlikely to be the main function, as the inserts appear to be structurally unconstrained and non- immunogenic (Birkholtz et al., 2004). In addition, it simply cannot explain the function of insertions in some of the enzymes of Plasmodium (Table 1.2). It has also been suggested that the insertions are spacers between protein structural elements (Huntley et al., 2000) or that they may even be spliced during protein maturation (Pizzi et al., 2001). Currently, there is no evidence supporting the latter suggestion. In addition, co- workers have demonstrated by immunofluorescence and western blot, that the insertions of PfCTPase are present in the mature enzyme within the parasite (Yuan et al., 2005).

The limited scientific studies aimed at elucidating the function of insertions, have mainly focused on the housekeeping enzymes of Plasmodium. Gilberger and colleagues revealed that parasite-specific insertions in glutathione reductase of P. falciparum are involved in folding, stability and substrate binding of the enzyme

Chapter 1: Introduction 34

(Gilberger et al., 2000). Clarke and co-workers found that deletion of the inserts of P. berghei glucose-6-phosphate dehydrogenase, abolished enzyme activity (Clarke et al., 2003). New evidence shows that the parasite-specific insertions of S- adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) are essential for protein-protein interactions and in turn, enzyme activity (Birkholtz et al., 2004). In addition, Jean and co-workers showed, by mutational studies, that the majority of high complexity insertions in a subtilisin-like protease-1 are responsible for enzyme maturation and activity (Jean et al., 2005). In the crystallography study of PfDHFR-TS, the two insertions were shown to play a major role in stabilising the interdomain interactions (Yuvaniyama et al., 2003).

In summary, relatively few studies have addressed the function of Plasmodium-specific insertions experimentally, but those that have, have focused on insertions within enzymes. These studies have shown that they are of highly significant biological importance for enzyme activity and stability. When coupled with the fact that these insertions do not exist in homologues from other genera, namely human, these insertions become extremely attractive targets for chemotherapeutic control of malaria.

In the past, the PfCPSII insertion II region has proven to be an effective locus for targeting by antisense therapy (Flores et al., 1997; Flores et al., 1999). The remainder of this chapter will explore in more detail, antisense therapy in general and in relation to treatment of P. falciparum.

1.7 Antisense Therapy

Nucleic acid therapy involves the use of nucleic acids to manipulate or disrupt gene expression. All methods rely on nucleotide sequence specificity but differ in where and how they affect the process of genetic flow, either by affecting the target gene itself or the mRNA derived from that gene.

The anti-mRNA approach can be further classified into antisense and non-antisense strategies. The use of "decoy" molecules that destabilise the mRNA is one such example of the latter approach, and works by attracting mRNA stabilising proteins therefore inducing instability and ultimately destruction of the message (Beelman et al.,

Chapter 1: Introduction 35

1995). The term ‘antisense therapy’ encompasses several types of complementary single-stranded nucleic acids that have the ability to alter gene-expression by binding to, and consequently interfering with, the function of a target mRNA transcript. Antisense strategies are based on three different types of nucleic acid molecules that are being investigated to either understand the function of target genes or as potential therapeutics. These include: antisense oligodeoxynucleotides (AS ODNs); nucleic acid enzymes such as ribozymes and deoxyribozymes (DNA enzymes or DNAzymes); and RNA interference (RNAi) molecules. The advantages, application and limitations of these molecules are effectively the same and are discussed generally below.

1.7.1 Advantages of Antisense Therapy In principle, there are a number of advantages in using antisense therapy as opposed to the more conventional antimalarial therapies. Antisense therapy relies on the well- characterised Watson-Crick concept of hybridisation. The specificity of this approach is based on the assumption that any sequence longer than a minimal number of nucleotides (20nt) occurs only once in a given genome (Dallas et al., 2006). This specificity of binding is well understood and is one of the principal arguments for the use of such molecules. In general, conventional therapeutic agents act post- translationally by interfering with protein function. A major disadvantage to this approach is that point mutations in the genetic code can rapidly confer drug resistance. Theoretically, more sequence mutations may be tolerated by nucleic acid agents and therefore, resistance due to point mutations are less likely to develop.

The key mechanism of action of most conventionally produced pharmaceuticals relies on the predicted binding affinity and interaction with other cellular constituents, which are predominantly proteins. Although there have been major advances in understanding protein-ligand interactions and molecular modelling of the binding sites of proteins, it remains difficult to design specific therapeutics. There are several reasons for this, mainly their often large molecular size and tendency to bind other naturally occurring molecules that have similar structure to the target molecule. Such non-specific interactions produce side effects and require elaborate and time-consuming studies on toxicity. The size of antisense molecules are relatively small which is an added advantage in terms of production as well as minimisation of non-specific interactions.

Chapter 1: Introduction 36

Therapeutic nucleic acids can also be readily designed and synthesised almost immediately once the target gene sequence is known, eliminating the time-consuming and laborious methods of protein characterisation.

1.7.2 Mechanism of Action of Antisense Molecules All antisense molecules share a common step in hybridising to a target RNA, usually mRNA, by Watson-Crick base-pairing. The formation of this antisense:RNA complex can alter the expression of the transcript through a number of mechanisms, often depending on the type of antisense molecule it is, along with associated modifications and the location at which binding occurs. Furthermore, this process can occur at any point between conclusion of the transcript and initiation of translation or even possibly during translation (Jen et al., 2000).

1.7.2.1 Mechanism of Action of Antisense Oligodeoxynucleotides AS ODNs are short sequences of single-stranded DNA that are typically between 18- 25nt in length. The two main mechanisms of inhibition associated with this type of antisense occur in the cytoplasm and affect translation of the target mRNA by either steric hindrance or RNase H mediated cleavage (Figure 1.9A). Initially it was believed that the major mechanism of antisense action involved translational arrest by steric hindrance of the ribosomal machinery at the 5' cap site or translation initiation regions (Paterson et al., 1977). However, it was found that this was a minor mechanism and was used only by a few types of antisense molecules. It is also now known that AS ODNs can no longer block the ribosomal translation complex, once assembled. The major route of antisense action and the most widely understood mechanism involves RNase H mediated cleavage of the target mRNA (Agrawal et al., 1990). Cellular RNase H is an endonuclease that requires an RNA-DNA heteroduplex as a substrate in order to hydrolyse the RNA strand. However, any modification to the deoxy sugar moiety at the 2' position prevents RNase H activity. Modified nucleic acid derivatives such as morpholinos, locked nucleic acids and peptide nucleic acids are thought to elicit function by steric hindrance of the ribosomal assembly (Figure 1.9A) (Dallas et al., 2006).

Chapter 1: Introduction 37

A. Antisense Oligonucleotides

mRNA Antisense ODN

mRNA

RNase H

mRNA Modified Antisense ODN

B. Nucleic Acid Enzyme

mRNA Ribozyme / DNAzyme

C. RNA interference

Long dsRNA

DICER

siRNA

mRNA

RISC complex

Figure 1.9 Mechanisms of inhibition of translation through antisense technology. (A) An antisense oligonucleotide hybridises to target mRNA that is then either degraded by RNase H or blocks translation by steric hindrance. (B) Nucleic acid enzymes cleave the target mRNA directly due to their catalytic activities. (C) Long, double-stranded RNA is processed to siRNA by DICER. The siRNA uses the RISC complex to degrade the target mRNA. Adapted from (Dallas et al., 2006).

Chapter 1: Introduction 38

The AS ODN can also enter the nucleus and regulate mRNA maturation by inhibition of the 5' cap formation (Kurreck, 2003). Splicing inhibition in the nucleus can also occur, whereby binding of the AS ODN to splice donor or splice acceptor sites, can lead to interference with mRNA maturation and in turn, function (Lu et al., 2003). In addition, RNase H degradation can also be activated in the nucleus.

1.7.2.2 Mechanism of Action of Nucleic Acid Enzymes Ribozymes, and DNAzymes, are single-stranded nucleic acids that contain a conserved sequence that causes the cleavage of the target RNA upon binding (Figure 1.9B). Both nucleic acid enzymes have three basic components: (i) a highly conserved nucleotide catalytic domain; (ii) base-pairing arms flanking the catalytic domain for binding to the target RNA; and (iii) a recognition cleavage site in the target sequence. However, as their names indicate, DNAzymes are composed of DNA whilst ribozymes are composed of RNA.

The five naturally occurring classes of ribozymes include hairpin, hammerhead, group I intron, ribonuclease P and hepatitis delta virus ribozyme. Among these, the two most widely studied, due to their small size and versatility, are the hammerhead and hairpin ribozymes. The hammerhead ribozyme is the most widely used ribozyme in biotechnology and was first isolated from viroid RNAs that undergo site-specific self- cleavage as part of their replication process.

The ribozyme and substrate RNA associate through base-pairing of complementary sequences surrounding the catalytic centre to form a complex. This results in a secondary structure formation necessary for cleavage. In the presence of divalent metal cations such as Mg2+, the substrate is cleaved 3' to the recognition sequence, generating two products with 2'-3' cyclic phosphate and a 5'-OH termini. The hammerhead ribozyme cleaves the target RNA 5' to the triplet NUX, where N is any base and X is any base except ‘G’ (Zoumadakis et al., 1995). After cleavage of the target message, the ribozyme may be released and can participate in further reactions, hence acting catalytically (Haseloff et al., 1988).

Unlike ribozymes, the DNAzymes have not been observed in nature, but instead have been isolated by many rounds of in vitro selection (Breaker et al., 1995; Santoro et al.,

Chapter 1: Introduction 39

1997). Two different catalytic motifs (8-17, 10-23) were originally found via in vitro selection. Many DNAzymes with a broad range of activities have since been selected by this method. The enzymatic activities of these include: cleavage of RNA or DNA; RNA ligation; RNA branch formation; porphyrin metallation; DNA phosphorylation, ligation, adenylation and deglycosylation (reviewed in (Peracchi, 2005) and (Joyce, 2004)).

The RNA-cleaving DNAzyme has the ability to cleave target RNA molecules under physiological conditions in a manner similar to ribozymes, provided there is a purine- pyrimidine dinucleotide in the sequence of the target RNA (Santoro et al., 1997). This can be accomplished at very high kinetic efficiency, with rates equal to or greater than that of ribozymes and endo-ribonucleases (Santoro et al., 1997). The activity of the DNAzyme is characterised by progression through multiple rounds of mRNA substrate binding, cleavage and release of products (Stage-Zimmermann et al., 1998).

Like the hammerhead ribozyme, the 10-23 DNAzyme is composed of a central core (15nt) responsible for catalytic activity and is flanked by recognition or substrate- binding arms (5-9nt) (Figure 1.10). Binding occurs by Watson-Crick hybridisation via the recognition arms and, subsequently, cleavage can take place between any unpaired purine-paired pyrimidine releasing two products with 2'-3' cyclic phosphate and a 5'-OH termini (Figure 1.10) (Santoro et al., 1998). Since the isolation of the original 10-23 DNAzymes, more DNAzymes have been produced that enable targeting to all possible dinucleotide sequences (Cruz et al., 2004). In general, the mechanism of action of the 10-23 DNAzyme is poorly understood. To date, two published crystal structures of the enzyme-substrate complex have been reported but both complexes rearranged in the crystal to form non-functional structures (Nowakowski et al., 1999; Nowakowski et al., 2000).

The mode of action of the 10-23 DNAzyme combines the advantageous features of both ribozymes and antisense DNA. As well as possessing catalytic activity and acting via steric hindrance, DNAzymes have the potential to effect RNase H mediated cleavage associated with DNA-RNA hybrids. DNAzymes are significantly more stable than ribozymes due to the deoxyribose backbone and are relatively inexpensive and easy to synthesise. The DNAzyme also has the added advantage of functioning against a wider

Chapter 1: Introduction 40

selection of cleavage sites in a chosen target. Further, Kurreck and co-workers demonstrated that DNAzymes were more potent in a comparative study between hammerhead ribozymes and DNAzymes (Kurreck et al., 2002).

5' 3' RY Target RNA

R recognition arm DNAzyme A15 G1 G14 G2

C13 C3 catalytic core A12 T4

A11 A5

C10 G6 R= Purine (A or G) A9 T8 C7 Y= Pyrimidine (C or U)

Figure 1.10 A schematic representation of the 10-23 DNAzyme. Shown is the conserved catalytic motif and cleavage specificity of the 10-23 DNAzyme (Joyce, 2001).

1.7.2.3 Mechanism of Action of RNAi RNAi was originally discovered as a naturally occurring pathway in plants (Jorgensen, 1990) and invertebrates (Fire et al., 1998). When long, double-stranded RNA is introduced into some organisms, it is processed by the cytoplasmic RNase III enzyme Dicer into 21-23nt small interfering RNAs (siRNAs). SiRNAs are then incorporated into the multi-component RNA-induced silencing complex (RISC), which unwinds the duplex and uses the antisense strand as a guide to seek and degrade the homologous mRNA. The RISC complex continues to degrade homologous mRNAs, amplifying the silencing effect (Dykxhoorn et al., 2003; Dorsett et al., 2004; Hannon et al., 2004; Dykxhoorn et al., 2005). The use of RNAi to down-regulate gene expression in mammals was unsuccessful due primarily to non-specific inhibition. It has now been shown that use of short synthetic siRNAs and short hairpin RNAs (shRNAs) circumvents this, and specific down-regulation of target mammalian mRNAs is

Chapter 1: Introduction 41

achievable (Brummelkamp et al., 2002; Paul et al., 2002). Subsequently, non-specific effects of siRNA have been described (Jackson et al., 2003). Furthermore, it has been shown that siRNAs can non-specifically activate components of the interferon pathway (Moss et al., 2003; Sledz et al., 2003).

1.7.3 Antisense Accessibility Designing AS ODNs, ribozymes and DNAzymes that are effective poses a formidable technical challenge. Binding and cleavage are inhibited by regions of internal base- pairing in the secondary structure of the message. For some RNA molecules, tertiary structure and protein binding in vivo can also hinder binding and cleavage abilities. It is therefore, generally accepted that effective antisense design depends on accurate prediction of the secondary structure of RNA (Vickers et al., 2000; Andronescu et al., 2005). The precise prediction of structural features of the message and binding with other cellular constituents in vivo remains difficult to determine. There are a variety of strategies aimed at detecting accessible sites in target RNAs.

The translation initiation site is often chosen as a target. This is because translation requires the binding of proteins in this region, and hence is thought to be generally free of secondary structure (Kozak, 1991; , 1994). However, these regions often share homology with other transcripts from the same organism and can, in fact, result in reduced specificity (Sohail et al., 2000b).

Two of the conventional approaches taken are sequence-walking and computer folding. Sequence walking involves the synthesis of many AS ODNs targeting a particular transcript and testing them for in vitro inhibition of cell cultures. Such an extensive screen is expensive, time-consuming and laborious. In addition, only 2-5% of AS ODNs on average are generally found to be good antisense reagents (Sohail et al., 2000b). An example is provided by Peyman and colleagues, who found that only one AS ODN of a hundred tested, could significantly reduce the cytopathic effects of HSV- 1 virus in culture (Peyman et al., 1995).

Computer-predicted mRNA folding programs such as mFold (Zuker, 1989; , 2003) are available that theoretically predict folding based on the lowest energy of conformation

Chapter 1: Introduction 42

of the target molecule, hence allowing single-stranded regions to be detected. Another computer algorithm is the sFold program that predicts only the best secondary structure of the target transcript (Ding et al., 2004). More recently, new software in conjunction with mFold, Target-Finder, has been developed to facilitate target site selection based on the method of mRNA accessible site tagging (Bo et al., 2005). However, success with these programs has been limited and may be due to the fact that often, and especially for longer molecules, a number of conformations have similar predicted energies. In addition, these programs do not take into account the variables present inside the cell. Therefore, trial and error laboratory testing is required to assess the performance of AS molecules.

A number of experimental techniques have been developed which appear to be much more reliable at predicting mRNA accessibility. All approaches are based on experimental detection of AS ODN binding to the target RNA. Two such examples include the use of S1 nuclease or RNase H to empirically determine cleavage site accessibility (Pavlakis et al., 1980; Knapp, 1989). A mixture of AS ODNs are incubated with cell extracts or transcripts and then exposed to RNase H. It is the sensitivity of this enzyme to heteroduplex formation that indicates mRNA accessibility. Cairns and co-workers have taken a similar approach by designing 80 DNAzymes targeting a human papilloma virus transcript (Cairns et al., 1999). The DNAzymes were incubated with the target and cleavage sites mapped by primer extension. Others have used the single-strand specific ribonuclease T1 to footprint these regions in target RNAs (Gewirtz, 1997). A more thorough approach has been taken involving the use of oligonucleotide scanning arrays (Sohail et al., 2000a). Combinatorial synthesis involves a large number of AS ODNs attached to a solid support, such as a glass slide, to which radio-labelled transcript is hybridised. In this way, AS ODNs that bind the target transcript are identified. A recent technique has involved use of quantum dot- conjugated probes in fluorescent hybridisation studies to detect accessible regions of target RNAs (Bakalova et al., 2005). Further, molecules such as green fluorescent protein (t Hoen et al., 2002) and cyan fluorescent protein (Guapillo et al., 2006) have been used in reporter assays to determine the in vivo effect of AS molecules.

The use of various combinations of the above methods appears to be the most reliable way of determining regions of mRNA accessibility. In the case of the catalytic

Chapter 1: Introduction 43

antisense molecules, ribozymes and DNAzymes, a measure of cleavage ability is a necessary step in determining which are efficient molecules.

1.7.4 Biological Stability and Modifications Biological instability is a major factor to consider when delivering both RNA and DNA oligonucleotides to cells. Unmodified AS ODNs are rapidly degraded in biological fluids by both endo- and exo-nucleases (Wickstrom, 1986). Due to this instability in tissue culture, the first generation of chemically modified AS ODNs, the phosphorothioates, were designed (Agrawal et al., 1988; Matsukura et al., 1989) and have since been widely used because of their nuclease resistance. However, their non- sequence specific effects have also been widely reported in a range of different cell types. These effects are thought to involve hybridisation to non-target RNAs and sequence-independent interference with key structural proteins, enzymes, receptors or transcription factors due to the negatively charged nature of the phosphorothioate molecules (Zon, 1995).

To further enhance nuclease resistance and increase binding affinity, the second generation of AS modifications, the 2'-alkyl modifications of the ribose, were developed. The 2'-O-methyl and the 2'-O-methoxyethyl are the two most widely studied second generation AS ODNs (Crooke, 2004). However, these modifications do not support RNase H-mediated cleavage and instead relied on other antisense mechanisms such as steric hindrance (Figure 1.9A).

The third generation of modifications utilised to improve stability and efficacy of AS molecules involved modifications to the furanose ring of the nucleotide and the most studied of which include the peptide nucleic acid (PNA), locked nucleic acid (LNA) and phosphoroamidate morpholino oligomer (PMO) (Kurreck, 2003). Interestingly, PNA can also affect gene function by hybridising to double-stranded DNA (Nielsen, 2004).

Due to their unstable RNA backbone, ribozyme modifications have been extensively studied. These modifications usually involved substitution of the 2'-hydroxyl moiety with other moieties such as 2'-deoxy, 2'-O-methyl, 2'-amino or 2'-fluoro derivatives. However, these modifications result in reduced or eliminated catalytic activity in certain

Chapter 1: Introduction 44

conserved positions. Therefore, the most effective ribozymes are chimeric, with modifications incorporated into the hybridisation arms and unpaired nucleotides of the catalytic domain (Sun et al., 2000).

Only a small number of modifications have been investigated in DNAzyme suppression studies. The most common modification used successfully to stabilise DNAzymes and allow them to retain catalytic activity, is the introduction of a single 3'-terminal nucleotide (usually T) inversion by a 3'-3' inter-nucleotide linkage (Figure 1.11). This modification renders protection from nucleases to the vulnerable 3' end of the DNAzyme. Incubation of a DNAzyme with the 3' inversion in 100% human serum resulted in a half-life of 24h at 37°C compared to a half-life of 2h of an unmodified molecule (Sun et al., 1999). Reports have shown that inclusion of LNA monomers into the binding arms of DNAzymes allows enhanced stability against nucleases as well as cleavage activities (Schubert et al., 2003; Fahmy et al., 2004; Schubert et al., 2004; Fluiter et al., 2005). In addition, conjugate DNAzymes with polyamines and peptides have shown enhanced activity and stability (Kubo et al., 2005).

A number of antisense modifications that have different mechanism of action have been employed to enhance the antisense effect. One such example is the use of acridine-AS ODNs that use the intercalating property of acridine to improve the binding energy of hybridisation to the target (Helene et al., 1985). Chemical or photochemically active groups have been linked to AS ODNs. Activation of such groups can effectively modify the target mRNA and hence interfere with transcript function. Kean and co- workers showed sequence specific inhibition of rabbit β-globin mRNA translation by photochemical cross-linking of psoralen-derivatised AS ODNs (Kean et al., 1988). Other groups have used different agents such as phenanthrolines and porphyrins linked to AS ODNs to produce site-specific modification of the target nucleic acids (Le Doan et al., 1990; Mastruzzo et al., 1994; Ma et al., 2000).

Chapter 1: Introduction 45

Figure 1.11 The structure of the 3'-3' internucleotide linkage inversion. The left panel depicts the normal 5'-3' internucleotide linkage and the right panel a 3'-3' internucleotide linkage inversion (Sun et al., 2000). Reproduced with permission.

1.7.5 Bioavailability and Delivery Systems One of the major limitations for the use of antisense nucleic acid molecules in a pharmaceutical role is delivering them to their site of action, the RNA in the cytoplasm or nucleus. Although unmodified ribozymes can be synthesised inside the cell via vector-mediated transcription, modified ribozymes, DNAzymes and AS ODNs, are generally poorly taken up by cells and require an efficient delivery method to transport them across the cell membrane. AS ODNs are thought to enter cells by a combination of fluid-phase (pinocytosis), adsorptive and receptor-mediated endocytosis. The exact mechanism of uptake is dependant on a number of factors such as AS ODN chemistry, length, conformation, concentration; cell type, cell cycle stage, degree of cell differentiation, passage number and culture conditions or cellular environment (Akhtar et al., 2000). After internalisation, the majority of the ODN is compartmentalised in endosome/lysosome, golgi complex and the endoplasmic reticulum. Micro-injection of ODNs into the cytoplasm of cells, by-passing the endocytic pathway, leads to accumulation in the nucleus (Leonetti et al., 1991). Therefore, an efficient delivery method that transports ODNs into the cytoplasm of a cell should allow cytosol-to

Chapter 1: Introduction 46

nuclear migration to proceed. A number of strategies have been devised with varying success.

Mechanical techniques like electroporation and microinjection are useful for in vitro studies but are not usually amenable to in vivo delivery. Chemical delivery methods such as the incorporation of AS ODNs into liposomes, have been tested expansively in both in vitro and in vivo systems. Liposomes are composed of an aqueous compartment enclosed by a phospholipid bilayer. Cationic liposomes and lipids form stable complexes with the negatively charged ODNs. The positive charge allows a high affinity for most cell membranes that are negatively charged under physiological conditions. Liposomes usually follow intracellular pathways of receptor-mediated endocytosis. One such example of a cationic lipid carrier is N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP), which is widely used for internalisation of AS molecules. A major drawback to this delivery method is that once the liposome enters the cell as an endosome, they must release AS in order for interaction with their target RNA. Additives to cationic liposomes, such as dioleylphosphatidylethanolamine (DOPE), have now been shown to destabilise the endosomal membrane leading to release of the AS ODNs (Lysik et al., 2003). When DOPE is combined with an amphipathic lipid, fusion of the liposome and endosome can only occur at low pH, resulting in AS escape into the intracellular environment (Fattal et al., 2004).

Porphyrin derivatives as delivery vehicles have been shown to display promising properties in respect to transporting AS across cellular membranes (Benimetskaya et al., 1998; Flynn et al., 1999; Dass, 2002; Kocisova et al., 2006). Porphyrin molecules are water-soluble, lipophilic and positively charged and therefore able to interact with the negatively charged DNA. A study by Kralova and colleagues (Kralova et al., 2003) established that porphyrin-ODN complexes were able to traverse cellular and endosomal membranes and dissociate, allowing accumulation of ODN within the nuclei of primary leukaemia cells. They have been prominently used as complexes but some investigators have successfully conjugated or covalently linked porphyrin moieties to AS ODNs (Le Doan et al., 1990).

Chapter 1: Introduction 47

Covalent conjugation of AS ODNs to macromolecules like dendrimers (Yoo et al., 2000) and cell-penetrating peptides (Jarver et al., 2004) have also been shown to allow AS ODNs to traverse the cellular membranes. The most commonly used cell- penetrating peptide is the PNA which has been shown to successfully and specifically down regulate target mRNAs in both in vitro and in vivo studies (reviewed in (Jarver et al., 2004; Kaihatsu et al., 2004).

1.7.6 Biodistribution, Pharmacokinetics and Toxicity Theoretically, the concept of antisense therapy seems ideal, however, the ODN must successfully enter the cell, avoid destruction in lysosomes and various intra and extra- cellular nucleases, and hybridise with the target mRNA with sufficient affinity and specificity in order to effect inactivation or degradation. In addition, in complex living organisms such as mammals, antisense molecules are subjected to complex distribution and clearance and come into contact with many different proteins, cells and tissue types. For example, AS ODNs bind strongly to plasma proteins such as albumin and α2- macroglobulin (Levin, 1999; Geary et al., 2002).

Independent of sequence, it is known that 50% of the carbon from AS ODNs is exhaled as carbon dioxide, 15-25% excreted in the urine and faeces, and 20-25% remain in tissues 10 days after a single dose (Vidal et al., 2005). Unmodified ODNs are degraded rapidly in the plasma with a half-life of 5 minutes (Agrawal et al., 1995b) and are also rapidly eliminated from the body (Ma et al., 2000). AS ODN clearance from tissue is much slower than from plasma, with tissue half-lives of 1-5 days (Vidal et al., 2005). Phosphorothioated ODNs and ribozymes, are delivered to a wide range of tissues with accumulation primarily in the kidneys, liver, spleen and bone marrow (Desjardins et al., 1996; Zhao et al., 1998).

In general, AS ODNs have very acceptable toxicity profiles at the doses required for antisense activity (Vidal et al., 2005). AS ODNs produce dose-dependant, transient and mild to moderate toxicities in rodents, primates and humans (Chan et al., 2006). Acute, reversible toxic effects tend to occur where the highest concentration of ODNs has accumulated (Henry et al., 1999). Dose-limiting toxicities reported in trials include thrombocytopaenia, hypotension, fever, enhanced liver enzymes and hyperglycaemia (Jason et al., 2004; Vidal et al., 2005).

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Toxic effects associated with AS ODNs, primarily activation of the complement cascade and inhibition of the clotting cascade, are based on the chemical nature of the backbone instead of sequence specific effects (Jason et al., 2004). Toxicity in the most commonly studied oligonucleotide, phosphorothioated AS ODNs, is a result of the sulfur backbone modification that non-specifically interacts with cellular proteins. Sequence specific effects include immune stimulation due to an un-methylated cytosine-phosphorous-guanine (CpG) motif in the sequence that can be recognised by Toll-like receptor-9 in immune cells. This recognition results in the release of cytokines, B-cell proliferation, T and natural killer cell activation and antibody production (Vollmer et al., 2004).

At present, the only antisense molecule approved by a regulatory agency is Vitravene™ (Formivirsen; ISIS Pharmaceuticals). Vitravene™ targets peripheral cytomegalovirus retinitis in AIDS patients by local intravitreous administration (Vitravene-Study-Group, 2002a). Side-effects and toxicities associated with Vitravene™ include inflammation and increased eye pressure in approximately 25% of patients (Vitravene-Study-Group, 2002b). These toxicities appear to be dose- and schedule-dependant but not clearly attributable to the antisense treatment (Vitravene-Study-Group, 2002a). There are several antisense molecules in advanced stages of clinical development (phase 3) including Alicaforsen (ISIS Pharmaceuticals) targeting Chrohn’s disease and Genasense (Aventis and Genta) targeting cancer.

1.7.7 Biological Activity of the 10-23 DNAzyme The potential of the 10-23 DNAzyme to suppress gene function in biological systems has been explored by a number of groups. The biological activity of DNAzymes is comparable to, and often surpasses, the activity of other antisense molecules such as antisense and ribozymes. Many in vitro gene suppression studies utilising DNAzymes have been based on viral diseases and some findings are summarised in Table 1.3. Other diseases that have been targeted in vitro include tuberculosis, Huntington’s disease, and various types of cancers (Table 1.3). Considerable reductions of target cellular RNA and protein, by up to 70-90%, has been achieved. However, the true potential of DNAzymes has been demonstrated in animal models.

Disease Target Modification Delivery Activity Reference Method HIV-1 gag none Lipofectin 95% viral inhibition (Sriram et al., 2000) HIV-1 CCR5 none Lipofectin Decreased cell membrane fusion (Goila et al., 1998) HIV-1 env none Lipofectamine 77-81% suppression of viral load; suppression of viral replication (Zhang et al., 1999) HIV-1 TAT/rev 10 G residues at 3' +/- Lipofectin Taken up by human macrophage cell line without Lipofectin; (Unwalla et al., 2001) inhibition of HIV-1 activity; protection when challenged with HIV-1 HIV-1 TAR none Lipofectin 10-12 fold reduction in mRNA; >80% reduction of viral antigen; (Chakraborti et al., significant inhibition of gene expression in primary and chronically 2003) infected cells Hepatitis B virus HBx none Lipofectamine Target mRNA and protein levels significantly reduced (Hou et al., 2006) Hepatitis B virus HBx none Lipofectin 4-6 fold reduction in intracellular target RNA and protein levels (Goila et al., 2001) compared to control cells Hepatitis C virus HCV Phosphorothioate Lipofectamine 32% and 48% reduction in viral target RNA in two human cell lines (Trepanier et al., 2-base cap 2006) Influenza virus PB2 N3'-P5' DOTAP >99% suppression of viral expression in cells (Takahashi et al., phosphoramidite 2004) bonds at 3’ and 5’ Respiratory RSV Phosphorothioate Added directly Inhibition of target protein expression; 7 log reduction of viral yield; (Xie et al., 2006) syncytial virus 2-base cap/3' to media suppression of wild human strains in clinic cholesterol Tuberculosis icl Phosphorothioate Added directly Decreased M. tuberculosis survival in macrophages (Li et al., 2005) 2-base cap to media Vascular disease c-myc 3'-3' inversion DOTAP Blocked target protein expression; 80% suppression of cell growth (Sun et al., 1999) Huntington’s huntingtin 3'-3' inversion Lipofectamine 85% protein suppression (Yen et al., 1999) disease Cancer (Chronic Bcr-abl 2' O-methyl cap Lipofectin Apoptotic morphology; 99% suppression of reporter expression (Warashina et al., myelogenous 1999) leukemia, CML) Cancer (CML) Bcr-abl Phosphorothioate Cytofectin 40% protein suppression; 50% cell growth inhibition; 53-80% growth (Wu et al., 1999) 2-base cap inhibition Cancer PKCα 3'-3' inversion DOTAP Cell inhibition in four of five cancer cell lines; up to 83% reduction in (Sioud et al., 2000) protein levels Pancreatic survivin phosphorothioate Oligofectamine Increase in apoptotic cells; inhibition of PANC-1 cell growth (Liang et al., 2005) carcinoma cap Table 1.3 Efficacy of DNAzymes targeting diseases in vitro.

Disease Target Modification Delivery Activity Reference Method Squamous cell c-Jun 3'-3' inversion FuGENE6 Inhibition of SCC proliferation and suppression of solid (Zhang et al., 2006) carcinoma SCC tumour growth in mice (SCC) Breast EGR-1 3'-3' inversion Intra-tumoral Protein suppression; inhibition of cell proliferation; (Mitchell et al., 2004) carcinoma injection inhibition of solid tumour growth in mice Tumour EGR-1 3'-3' inversion FuGENE6; Blocked angiogenesis in mice; inhibited breast carcinoma (Fahmy et al., 2003) angiogenesis direct in mice; repressed neovascularisation of rat cornea administration Inflammation c-Jun 3'-3' inversion FuGENE6; Reduction in vascular permeability and inflammation in (Fahmy et al., 2006) direct mice and rats administration Vascular c-Jun 3'-3' inversion Intravitreal Inhibition of retinal neovascularisation in mice (Khachigian et al., disease administration 2002) Cardiovascular EGR-1 3'-3' inversion Superfect; Reduced target protein; inhibition of cell proliferation and (Santiago et al., 1999; disease adventitial wound repair; inhibition of protein expression, cell Lowe et al., 2001; Lowe /endoluminal proliferation and neointima formation after mechanical et al., 2002; Bhindi et delivery injury to rat and pig carotid artery wall in vivo. Reduced al., 2006) myocardial infarct size in rats. Cardiovascular VDUP1 3'-3' inversion Superfect; 70% reduction in target cellular mRNA levels; decrease in (Xiang et al., 2005) disease direct apoptosis; 2 fold increase in cell survival. intracardiac Prolonged reduction in cardiomyocyte apoptosis as well injection as marked reduction in myocardial scar formation in rats. Vascular PKC- 3'-3' inversion Lipofectin; Reduced target protein levels specifically by >60% in (Nunamaker et al., disease epsilon electroporation mice 2003)

Table 1.4 Efficacy of DNAzymes targeting diseases in vivo.

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Specific down-regulation of target genes in vivo has been established in the areas of vascular, cardiovascular disease and cancer models (Table 1.4). Khachigian and co- workers in particular, have greatly contributed to knowledge in these areas. They have demonstrated the efficacy of DNAzymes targeting cardiovascular disease, tumour angiogenesis, squamous cell carcinoma and inflammatory disease in various animal models (Table 1.4). These ground-breaking studies have confirmed that DNAzymes are able to act in a specific manner with minimal side-effects in vivo, when delivered directly to the diseased tissue. Although there has been considerable success in these preclinical studies, there are as yet, no reported DNAzymes in phase I clinical trials.

1.8 Application of Antisense Therapy to Malaria

In the past, there have been several studies that have utilised AS ODNs to down- regulate target gene expression and in turn, hinder malarial parasite growth. The majority of the studies have been reliant on the findings that, once an erythrocyte is parasitised, transport pathways are altered and the parasite is able to take up macromolecules, including antisense RNA and DNA, from the surrounding medium via a 50-70nm 'parasitophorous duct' or NPP (Ginsburg et al., 1985; Rapaport et al., 1992; Taraschi, 1999).

The first published malarial antisense study involved knockdown of P. falciparum DHFR-TS in a cell-free system (Sartorius et al., 1991). The expression of DHFR-TS was reduced by up to 90% by 45μM of AS ODN targeting the translation initiation site. Furthermore, incubation of chloroquine-resistant and chloroquine-sensitive P. falciparum cultures with phosphorothioated AS ODNs directed against DHFR-TS, resulted in a significant inhibition of parasite growth and invasion (Rapaport et al., 1992). An antisense study on the hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA of P. falciparum also confirmed the spontaneous uptake of radioactive ODNs by parasitised RBCs and inhibition of parasite cultures by up to 50% with 40μM phosphoramidite ODNs (Dawson et al., 1993).

A subsequent study by Clark and co-workers, attributed the reduced parasite growth by unmodified and phosphorothioated AS ODNs, to a non-sequence specific effect, namely inhibition of RBC invasion (Clark et al., 1994). Ramasamy and co-workers also

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claimed non-specific inhibition was evident at the re-invasion stage. They attributed this to the polyanionic phosphorothioated AS ODNs binding to the parasite recognition molecule, sialic acid, on the surface of the RBC (Ramasamy et al., 1996). Barker and colleagues investigated further and concluded that at concentrations below 0.5μM, phosphorothioated AS ODNs targeting several malarial genes significantly inhibited parasite growth in a sequence-dependant mode (Barker et al., 1996). Further studies by Barker confirmed this, but also showed that by reducing phosphorotioate linkages and including other modifications, a more specific effect was seen at concentrations >0.5μM (Barker et al., 1998). By contrast, Ramasamy and colleagues demonstrated that specific antisense effects could not be demonstrated over three cycles of schizogony or when cationic liposomes were used and further showed other polyanions had the same effect as phosphorothioated AS ODNs on parasite growth (Kanagaratnam et al., 1998). A more comprehensive study illustrated sequence-specific down-regulation of malarial aldolase mRNA and protein, as well as parasite inhibition of 50% with as little as 11nM of phosphorothioated AS ODNs (Wanidworanun et al., 1999). However, non-specific effects were seen with 0.1μM and 0.3μM phosphorothioated AS ODNs.

There have been only two studies since that utilise AS ODNs against malaria cultures. In the first, malarial topoisomerase II was targeted with phosphorothioated AS ODNs in a sequence-specific manner, inhibiting parasite culture growth by up to 47% at 0.5μM after 48h (Noonpakdee et al., 2003). However, in the latest study, the same investigators have taken a different approach whereby phosphorothioated AS ODNs targeting malarial topoisomerase II were complexed with chitosan to form solid nanoparticles. These chitosan:ODN nanoparticles successfully reduced malarial growth in a 48h assay significantly more than free phosphorothioated AS ODNs and controls (Foger et al., 2006).

1.9 Experimental Background

It had been previously established that a specific ribozyme, CPSRz4, targeting the junction of PfCPSII insertion II region, effectively kills P. falciparum in laboratory cultures (Flores et al., 1997). Exogenous delivery of CPSRz4 to parasite cultures at 0.5μM was shown to reduce malarial viability by up to 55% (Flores et al., 1997). Inhibition bioassays were carried out with synchronised ring-stage parasites over 24h to

Chapter 1: Introduction 53

eliminate the possibility of non-specific inhibition of parasite invasion by polyanionic molecules. The mode of inhibition was assessed to be both sequence-specific and stage-specific, coupled with a marked decrease in total PfCPSII enzyme activity from cultures treated with CPSRz4.

Studies utilising fully phosphorothioated AS ODNs were also undertaken to examine alternative targets in the PfCPSII mRNA transcript both within and outside of the insert regions for a more suitable site of inhibition. Eleven PfCPSII phosphorothioated AS ODNs and two controls, scrambled and sense, were designed and tested for in vitro inhibition of P. falciparum growth. Comparable levels of inhibition of parasite growth, to that seen with CPSRz4, were detected for many of the phosphorothioated AS ODNs, reducing malarial viability by up to 45% at 0.5μM concentrations (Katrib et al., 1997). Although, inhibition with control ODNs was also high (25%), there was a statistically significant difference with non-control phosphorothioated AS ODNs. The CPSRz4 region remained one of the most accessible in terms of parasite inhibition.

New chimeric DNA-RNA ribozymes were the next constructs to be tested and consisted of DNA binding arms and an RNA catalytic core. The stability of the chimeric molecule was significantly increased compared to ribozymes. The all-RNA ribozymes were shown to be fully degraded in 5min in 0.1% human serum while 10% of the chimeric equivalent was still intact (Flores et al., 1999). However, the reduction in parasitaemia of the chimeric ribozymes was significantly lower (44%) at the 0.5μM concentration than seen with CPSRz4 (55%).

Subsequent catalytic nucleic acids tested were based on the 10-23 DNAzyme and were designed to target the same insertion II region of PfCPSII. These new constructs, M5L and M8 both suppressed parasite growth by up to 50% at 0.5μM concentrations. These molecules were significantly more effective than the catalytically inactive (antisense) control, which inhibited proliferation by 26% (Flores, unpublished data).

Chapter 1: Introduction 54

1.10 Aims

The next logical step of nucleic acid therapy against malarial CPSII was to test these DNAzymes in an in vivo malarial model. The rodent malarial parasite P. berghei was chosen as suitable for an in vivo animal trial due to the vast amount of literature on antimalarial drug efficacy testing in this system. As DNAzymes require binding to the target sequence by complimentary base-pairing, the nucleotide sequence of the CPSII gene from P. berghei first needed to be elucidated. Therefore, the first aim of this body of work was to determine whether the insertions existed in the CPSII gene from P. berghei and if so, to determine the nucleotide sequence of these insertion regions.

A second aim was to isolate the nucleotide and deduced amino acid sequence of the insertion regions and the conserved region between the insertions of CPSII, from P. chabaudi and P. vivax. As this project was commenced prior to the Plasmodium genome sequencing projects, this was to be achieved by a PCR based strategy involving design and use of degenerate primers from the highly conserved regions of the gene. The resulting information was expected to shed light on the inter-species sequence variation and predicted amino acid nature of the insertions. It would also serve a dual purpose of allowing future synergistic DNAzymes to be designed that target CPSII from these organisms.

The third aim of this thesis was to design new synthetic DNAzymes targeting insertion II of CPSII from P. berghei. Further characterisation of these DNAzymes was to be carried out by utilisation of in vitro cleavage assays and cleavage kinetics studies to assess their efficacy at cleaving the target mRNA. The final aim was thus to use a suitable candidate DNAzyme in an in vivo rodent malaria trial in order to test the ability of the DNAzyme to reduce parasite growth in a complex mammalian system.

CHAPTER 2

Materials & Methods

Chapter 2: Materials & Methods 56

2 MATERIALS AND METHODS

2.1 Materials

2.1.1 General Reagents Tris (hydroxy methyl) methane (tris-base), DL-dithiothreitol (DTT), N-2- hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), polyethylene glycol (PEG8000), N,N,N',N'-tetramethylethylenediamine (TEMED), D-sorbitol were purchased from Sigma Chemical Co., St. Louis, U.S.A. Agarose (DNA grade), ampicillin (sodium salt), ethidium bromide (EtBr), 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (Xgal), isopropyl-β-D-galactopyranoside (IPTG), were purchased from Progen Industries Ltd, Darra, Queensland, Australia. NickTM columns, deoxynucleotides and dideoxynucleotides were purchased from Pharmacia, North Ryde, N.S.W, Australia. Caesium chloride (CsCl) and glycogen (DNA grade) were purchased from Boehringer Mannheim, North Ryde, N.S.W, Australia. Recycled caesium chloride was prepared by Associate Professor T. Stewart using the method described previously (Farrell, 1981).

Yeast extract and tryptone were obtained from Oxoid, Carlton, NSW FMC Seakem® GTG® and SeaPlaque® GTG® agarose were purchased from Edwards Instrument Company, Narellen, NSW Amresco acrylamide (19:1 acrylamide:bis-acrylamide) was purchased from Astral Scientific, Gymea, N.S.W, Australia. X-ray film (Fuji RX) was purchased from Hanimex Australia, Brookvale, N.S.W, Australia. RPMI 1640 media and saponin were purchased from ICN Flow, Seven Hills, N.S.W, Australia. All other reagents were of analytical or molecular biology grade.

2.1.2 Radio-chemicals

Aqueous [γ-32P] ATP (3Ci/μmole), [α−32P] dATP (3 Ci/μmole), and [α-32P] UTP (3Ci/μmole) were obtained from Amersham, Buckinghamshire, U.K.

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2.1.3 Oligonucleotides The sequences of all oligonucleotides used in PCR are shown in Appendix 1. Oligonucleotides for use in PCR were purchased in lyophilised form from Gibco BRL - Life Technologies Pty. Ltd., Mulgrave, Victoria. Stocks of 100μM concentration were made by re-suspension in sterile water and stored at –80˚C. Dilutions to 20μM were then made prior to use in PCR and stored at –20˚C. All DNAzymes and synthetic RNA oligonucleotides were purchased in lyophilised, reverse phase HPLC purified form from Oligos Etc. Oregon, USA. Sequences of the DNAzymes are listed in Table 4.1. Sequences of the synthetic RNA substrates are listed in Appendix 2. MD14, 15, 25 and 26 were resynthesised with the 3'-inverted thymidine base at the 3' end of the molecule to offer protection against nucleases for in vivo studies. The DNAzymes were also resuspended in sterile water and stored at -80˚C. Gel-purified synthetic RNA oligonucleotides were purchased with a 2'-O-methyl group on the ribose moiety. They were resuspended in sterile DEPC-treated water, an aliquot taken as a working stock and the remainder re-lyophilised and stored at –80˚C. The sequence of DNAzyme M5L is shown in Appendix 2. MD14 and M5L, used in Plasmodium inhibition bioassays, were synthesised with a single 3' phosphorothioate linkage and a C9 amino linker at the 5' end for conjugation to methylpyrroporphyrin XXI ethyl ester. A separate conjugate was also synthesised with a FITC molecule at the 3' end to be used in uptake studies. Methylpyrroporphyrin DNAzymes were conjugated and HPLC-purified by Dr. T. Rede (St. Vincent’s Hospital, Sydney, Australia).

2.1.4 Enzymes and Reaction Kits T4 polynucleotide kinase, T4 DNA ligase, T4 DNA polymerase, shrimp alkaline phosphatase, Klenow fragment of E. coli DNA polymerase I, the Random prime DNA labelling kit and crude bovine pancreatic ribonuclease A (RNase A) were purchased from Boehringer Mannheim, North Ryde, N.S.W, Australia. Crude RNase A was made free of DNase using the method of Sambrook et al., 1989. Restriction endonucleases, Agar ACE™, RNA transcription kit, and the pGEM®-T Easy vector kit used for TA cloning and sequencing of PCR products, were purchased from Promega Corporation, Rozelle, NSW, Australia. A map of the pGEM®-T Easy vector is located in Appendix 3. A range of Taq DNA polymerases were used including Bioline Bio-X-act™ Taq DNA polymerase purchased from Fisher-Biotec, Subiaco, W.A, Australia;

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Platinum®Taq DNA polymerase and Platinum®Pfx DNA polymerase purchased from GibcoBRL® Life Technologies, Gaithersburg, M.D, USA. The Gentra Systems GENERATION™ Capture Column™ Kit was used for genomic DNA extractions from whole blood and was purchased from Diagnostic Technologies, N.S.W, Australia. The Stratagene RNA transcription kit was purchased from Integrated Sciences, Willoughby, NSW, Australia. The QIAquick™ gel extraction kit was used for extraction of DNA from agarose gels and purchased from QIAGEN, Clifton Hill, Victoria, Australia.

2.1.5 Parasite Species P. falciparum FCQ-27 from Papua New Guinea was provided by the Army Malaria Research Unit (Ingleburn, NSW, Australia). P. berghei QIMR and ANKA infected blood samples were generously donated by Professor Allan Saul, Queensland Institute of Medical Research (QIMR), Australia. P. berghei K173 strain and P. chabaudi ADAMI blood was kindly donated by Professor Nick Hunt, Dept. of Pathology, Sydney University, Australia. P. vivax genomic DNA used in PCR was extracted from a patient blood sample kindly supplied by John Walker, Westmead Hospital, NSW, Australia. The patient contracted P. vivax whilst in Vietnam.

2.1.6 Bacterial Strains and Helper Phage The following strain of E. coli was used for cloning and sequencing: q JM101: sup E, thi, D(lac-proAB), [F', lacI , ZΔM15, proAB, traD36] The following helper phage was used for infection of E. coli cells for production of single-stranded plasmid DNA : M13K07 helper phage (Vieira et al., 1987).

2.2 General Methods

2.2.1 Sterilisation and Containment Heat-stable solutions and plastic wares (microfuge tubes and pipettor tips) were sterilised by autoclaving at 120˚C, 125kPa for 15min. Following autoclaving, the plastic wares were dried at 80˚C overnight. Heat-labile solutions were filter-sterilised using disposable 0.2μm Millipore microfilters. Glassware was sterilised by dry heat at

Chapter 2: Materials & Methods 59

180°C overnight. All biologically contaminated material was autoclaved at 125kPa, 120˚C for 20min prior to disposal.

2.2.2 Disposal of Waste Clinical waste contractors disposed of all autoclaved biological waste. Corrosive, organic and toxic waste such as phenol, chloroform, ethidium bromide and acrylamide were separated and disposed of by chemical waste contractors. Syringe needles, contaminated glass pipettes and other sharp objects were disposed of in sharps bins.

2.2.3 Standard Stock Solutions The following is a list of stock solutions and buffers used for the research described in this thesis: Agarose Gel Loading Dye (10x): 60% (w/v) Sucrose, 0.1% (w/v) bromophenol blue and 40mM EDTA pH 8.0 dNTP mix: 0.125mM each nucleotide, pH 7.8 E buffer: 40mM Tris-acetate, 2mM EDTA pH 8.0

Kinase buffer: 70mM Tris-HCl, 10mM MgCl2 and 5mM dithiothreitol, pH 7.6

Klenow buffer: 50mM Tris-HCl, 10mM MgSO4, 0.1mM DTT, pH 7.2 Labelling mix: 333.5mM NaCl, 1.375μM dGTP, 1.375μM dCTP and 1.375μM dTTP

Ligase buffer (5x): 250mM Tris-HCl pH 7.6, 50mM MgCl2, 25% (w/v) polyethylene glycol 8000 and 5mM dithiothreitol Multi-Core™ Buffer (10x): 250mM Tris-acetate, pH 7.8, 1M potassium acetate, 100mM magnesium acetate and 10mM dithiothreitol One-Phor-All™ buffer (10x): 100mM Tris-acetate pH 7.5, 100mM magnesium acetate and 500mM potassium acetate Phosphate-buffered saline (PBS): 0.25g/l KH2PO4, 0.84g/l K2HPO4 and 9g/l NaCl, adjusted to pH 7.4 with 2M KOH

RBC lysis buffer: 1mM NH4HCO3 and 114mM NH4Cl RNase A: 10mg/ml RNase A in 10mM Tris-HCl buffer pH 7.5 - boiled (15 min) to remove traces of DNases Stop solution: 0.3% each of Bromophenol Blue and Xylene Cyanol FF, 10mM EDTA pH 7.5 and 97.5% deionized formamide

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Taq DNA polymerase buffer: 10mM Tris-HCl, 1.5mM MgCl2, 50mM KCl and 0.01% (w/v) gelatin, pH 8.3 TBE buffer (10x): Tris base 432g/l, boric acid 220g/l, EDTA 37.2g/l TE buffer: Tris-HCl 10mM and EDTA 0.1mM, pH 8.0

TM10 Buffer (10x): 0.5M Tris-HCl pH 7.5 and 0.1M MgCl2

2.2.4 Dialysis Tubing Preparation Dialysis tubing used for DNA purification was treated to remove contaminants by boiling (10min) in 2% (w/v) (NH4)2CO3, and 2mM EDTA for 10min. The tubing was then thoroughly rinsed in distilled water, autoclaved and stored at 4˚C in 50% (v/v) ethanol in a sealed container.

2.2.5 Autoradiography PEI-cellulose strips (labelled with 32P ), were wrapped with plastic and exposed to Fuji RX film under glass , in a perspex box, for 5-10min. The RX film was then placed in

Kodak developing solution (5min), rinsed in H20, and then placed in Kodak fixing solution.

2.2.6 Polyacrylamide Gel Electrophoresis Polyacrylamide gels for visualising DNAzyme cleavage and integrity of oligonucleotides were prepared using the Amresco (19:1, acrylamide:bisacrylamide) 40% acrylamide concentrate, containing 8M urea and electrophoresed in TBE buffer. Polyacrylamide gel polymerisation was catalysed by the addition of 10% (w/v) ammonium persulphate (5μl/ml gel solution) and of TEMED (0.5μl/ml gel solution). Gels were pre-electrophoresed for 15-30min before samples were loaded. After electrophoresis, gels were fixed by gentle agitation in acetic acid (10% v/v) and methanol (10% v/v) for 5min before drying and exposure to phosphorimaging screens.

2.2.7 Growth and Maintenance of E. coli Cultures 2.2.7.1 Media Recombinant E. coli JM101 cultures were grown in either luria broth (LB) or superbroth (SB). LB media contained 10g/l tryptone, 5g/l yeast extract and 10g/l NaCl.

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Ampicillin (100μg/ml) was added to LB to make LB-amp broth and LB-amp plates. SB media contained 12g/l tryptone, 14g/l yeast extract and 0.5% (v/v) glycerol. For every

9 vol of SB, 1 vol of a separately autoclaved sterile phosphate buffer (17mM KH2PO4,

72mM K2HPO4) and 100 μg/ml of ampicillin were added.

E. coli JM101 were maintained on minimal media plates containing (per litre): 4.5g

KH2PO4, 10.5g K2HPO4, 1.0g (NH4)2SO4, 0.5g tri-sodium citrate, 12g agar. After sterilisation of the solution, 20% MgSO4 (1ml), 1% Vitamin B1 (0.5ml) and 20% D- glucose (10ml) were added.

2.2.7.2 Glycerol Stocks Glycerol stocks of cell lines and transformed cell lines were prepared by mixing fresh over night culture (86% v/v) with sterile glycerol (24% v/v) and snap freezing in a dry ice/ethanol bath. The stocks were then stored at -80°C.

2.2.7.3 Inoculation Overnight E. coli cultures were inoculated from a single colony from a stock plate and grown at 37˚C overnight with agitation. E. coli JM101 cultures were grown for 5h at 37˚C.

2.2.8 Growth and Maintenance of P. falciparum Cultures 2.2.8.1 Media and Culture Maintenance P. falciparum FCQ-27 was grown in continuous culture in type O+ human blood using a modified version of the technique described by Trager and Jensen (Trager et al., 1976). Serum was pooled from a minimum of 10 donors, aliquoted and frozen for further use.

Cells were maintained in complete RPMI media. The RPMI 1640 basal media was prepared by dissolving 1 packet of RPMI 1640 (with glutamine) powdered medium in 900ml of water and adding glucose (2g) and HEPES (5.94g). This solution was adjusted to pH 7.2 with KOH, made up to 1000ml and filter sterilised. The media was completed by addition of human serum (10%v/v), sterile NaHCO3 (32mM) and gentamycin (50μg/l). Complete media was prepared fresh and stored at 4˚C for no

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longer than a week. Red blood cells (RBCs) from a single donor, obtained from the Sydney Red Cross Blood Bank, were aseptically transferred into 500 ml bottles and 25ml were further aliquoted into 50ml tubes. The blood was then centrifuged for 10min at 1000rpm and the cells were repeatedly washed in PBS (25ml) until the supernatant was clear. The washed RBCs were then made up to 50ml with complete RPMI media. Cultures were kept at 2% haematocrit in complete RPMI 1640 medium (10ml) in 25cm2 tissue culture flasks. Every day the infected RBC cultures were assessed for percentage parasitaemia by blood smears and fed with fresh complete RPMI 1640 medium.

Cultures were gassed (5% CO2, 5% O2 and 90% N2) for 1min before incubation at 37˚C. Prior to weekends, the cultures were maintained at low parasitaemias (<0.5%- 1%).

2.2.8.2 Analysis of Parasitaemia A blood smear was prepared by placing a single drop of infected RBCs onto one end of a microscope slide, then smearing the cells with the edge of a second slide. The blood smeared slide of the infected RBCs was dried and stained with Diff Quick (Lab Aids, N.S.W, Australia). At least 500 RBCs were counted under the microscope (100x objective oil immersion lens) and the percentage parasitaemia was determined.

2.2.8.3 Synchronisation of Ring-stage Parasites P. falciparum cultures were synchronised at the ring-stage as described previously (Lambros et al., 1979). The infected RBCs were pelleted by centrifugation at 1000rpm for 5min in a 5ml centrifuge tube. The supernatant was discarded and the pellet resuspended in 3 volumes of filter sterilised D-sorbitol (5%). The tube was incubated for 10-20min on its side at room temperature, complete media (10ml) was added, followed by gentle mixing and centrifugation to remove the supernatant. The washed pellet was then resuspended in complete media and cultured in the usual manner or used immediately for drug inhibition assays.

2.2.8.4 Liquid Nitrogen Storage Parasitised red blood cell cultures were thawed from liquid nitrogen using the modified method of Butcher (Butcher, 1981). Rapid thawing at 37˚C was followed with an addition of 1ml of 8% (w/v) NaCl and thoroughly mixed. Following a slow addition of

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3ml of 6% (w/v) NaCl and 10-15ml of PBS, the culture was centrifuged for 10min at 3000rpm and washed twice in complete RPMI 1640 media. The cell culture was then placed in 80ml of complete media and cultured as in Section 2.2.8.1. Stocks of parasitised red blood cells were prepared by taking >8% ring-stage cultures and centrifuging at 3000rpm for 10min. The packed red blood cells were resuspended in 2ml of glycerol-mannitol solution consisting of 33.4% (w/v) glycerol, 220mM mannitol and 160mM NaCl, mixed thoroughly and placed in a cryogenic vial and immersed in liquid nitrogen.

2.3 General DNA Procedures

2.3.1 Phenol:chloroform Extractions Phenol:chloroform extraction was routinely used to remove contaminating proteinaceous material from DNA samples. An equal volume of equilibrated phenol (pH 7.4) was added and vortexed for 1min, followed by centrifugation for 2min (14 000g) at room temperature. The aqueous phase was transferred to a fresh tube and extracted (vortexed and centrifuged as above) twice with equal volumes of chloroform to remove residual phenol. The DNA was then ethanol precipitated (Section 2.3.2). In some cases, a single extraction with equal volumes of phenol/chloroform (1:1) or phenol/chloroform/isoamyl alcohol (50:49:1) was performed.

2.3.2 Ethanol Precipitations DNA was precipitated from solution by adding 0.1 volume of 3M sodium acetate pH 5.0 and 2.5 volumes of absolute undenatured ethanol. The samples were mixed well and chilled at -70˚C (powdered dry ice) for 15-20min or at -20˚C overnight. The precipitated DNA was pelleted by centrifugation at 14 000g at 4˚C for 15-30min. The pellet was then washed with cold 70% ethanol and re-centrifuged at 4˚C for 2min, vacuum dried (1min) or air dried (5min), then resuspended in the appropriate amount of TE buffer.

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2.3.3 Restriction Enzyme Digests Restriction enzyme digests were carried out in the buffer supplied for that enzyme by the manufacturer, or alternatively in Multi-Core™ reaction buffer (Promega, Section 2.2.3) or One-Phor-All™ buffer (Boehringer, Section 2.2.3) at the appropriate restriction assay conditions. Digests contained 4 units of enzyme per μg of DNA and were incubated for 1-2h at 37˚C in a water bath. Complete digestion of large fragments was checked by agarose gel electrophoresis (Section 2.3.4) before being used in further manipulations.

2.3.4 Agarose Gel Electrophoresis 2.3.4.1 Quantitative Agarose Gel Electrophoresis Various DNA samples, such as genomic DNA; supercoiled plasmids; linear plasmids; digested DNA; and PCR products, were analysed by electrophoresis on 0.8% to 2.0% (w/v) agarose gels. Samples were run with agarose gel loading dye and electrophoresed in E buffer containing 0.5mg/ml EtBr. Mini gels (60 x 60 x 41 mm) were electrophoresed at a constant current of 110mA until the loading dye had run approximately 2/3 of the length of the gel. Bands were visualised with a UV transilluminator at 254nm. Gels were photographed using a Mitsubishi Video Copy- processor and Mitsubishi K65HM thermal paper.

2.3.4.2 Preparative Agarose Gel Electrophoresis To purify a particular DNA sample, 1% (w/v) Seakem™ GTG agarose or 1% SeaPlaque™ low melting agarose was used. Care was taken to ensure sterility of all equipment. Gel forming apparatus and the electrophoresis tanks were cleaned, rinsed with 70% ethanol and distilled water before allowing to air dry. Seakem™ agarose was weighed out using sterile techniques, and electrophoresis was carried out in sterile E buffer. Bands were visualised with a handheld UV lamp in order to minimise damage to the DNA sample. The DNA sample was then isolated from the low-melting agarose gel by AgarACE™ agar digesting enzyme (Promega) according to the manufacturers specification. Isolation of DNA fragments from Seakem™ agarose was achieved by a QIAquick® gel extraction kit (Qiagen) or Gene Clean II® (Bio101) according to the manufacturers instructions.

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2.3.5 Estimation of DNA Concentration and Size Linear DNA was electrophoresed on agarose gels (Section 2.3.4) along side markers (λDNA/HindIII or pUC/HinfI DNA fragments) of known size to allow estimation of size by graphical methods. Supercoiled DNA was electrophoresed alongside supercoiled plasmids of known size and concentration. The concentration of a sample of DNA was estimated by two methods:

(i) comparing the intensity of bands of the DNA sample on an agarose gel with those of bands containing a known amount of DNA. (ii) by measuring the absorbance of the DNA at 260nm by using a Pye- Unicam SPG- 450 UV/VIS spectrophotometer or a Beckman Du750 spectrophotometer. An absorbance of 1.0 at 260nm, corresponds to 50μg/ml of dsDNA; 40μg/ml of ssDNA and 33μg/ml of oligonucleotide. The purity of the DNA sample was determined by the comparison of absorbance at 260nm with the absorbance at 280nm. An A260/A280 ratio of greater than 1.8 indicated low or no protein contamination.

2.3.6 Small-scale Plasmid Preparations A variation on the method of Ish-Horowicz and Burke, the alkali lysis method, was used to prepare approximately 10 μg of plasmid DNA (from a 2ml culture) for initial recombinant insert analysis (Ish-Horowicz et al., 1981). RNA was removed from the "miniprep" by incubation with Ribonuclease A (1μl of 10mg/ml) at 37˚C for 30min. This treatment was followed by phenol/ chloroform extraction (Section 2.3.1) and ethanol precipitation (Section 2.3.2)

2.3.7 Large-scale Plasmid Preparations When recombinant clones were identified as carrying the specific insert, they were subjected to a large-scale plasmid preparation procedure in order to obtain sufficient amounts of the plasmid for further work. The colonies containing positive clones were inoculated into 80 ml of sterile SB and ampicillin (Section 2.2.7) and incubated overnight at 37˚C with vigorous shaking. The cells were then isolated by centrifugation at 14 000g for 10min. DNA was extracted from the cell by using a scaled-up version of the Ish-Horowicz and Burke method (1981). However, instead of the phenol extraction and ethanol precipitation, the nucleic acids were precipitated in an equal volume of

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isopropanol for 20min at room temperature. The plasmid DNA was purified from the crude nucleic acid preparation by CsCl density centrifugation (Section 2.3.8).

2.3.8 Caesium Chloride Equilibrium Density Centrifugation Large-scale plasmid preparation and genomic DNA were purified from contaminating proteins and RNA by using CsCl density centrifugation. Crude DNA extractions were dissolved in TE buffer, pH 8.0 (3ml), to which was added CsCl (4.5g for plasmids and 4.3g for genomic DNA) followed by addition of 200μl of EtBr (10mg/ml). The mixture was then transferred to a 5ml Beckman Optiseal™ polyallomer ultracentrifuge tube and topped up with TE buffer. The tubes were capped and balanced to < 0.06g difference and centrifuged at 55 000rpm overnight at 20˚C in a Sorvall NVT90 rotor and Sovall Discovery™ 100 ultracentrifuge. The DNA band was visualised with long wavelength

UV light, removed with a syringe and extracted three times with CsCl:H2O (3:1) saturated isopropanol solution or until the sample was clear. The DNA sample was dialysed against TE buffer for 4h, with a change of buffer after 2h. The yield of DNA was spectrophotometrically determined and analysed by agarose gel electrophoresis to assess the purity.

2.3.9 Double-stranded Plasmid DNA Preparations for Sequencing A variation on the method of Ish-Horowicz and Burke (1981) was used to prepare recombinant DNA for automated sequencing. An overnight SB culture (2ml) was pelleted and the plasmid extracted by the standard alkaline lysis method. The mini- preps were treated with RNase A (20μg/ml) at 37˚C for 20min. The samples were then chloroform extracted twice and precipitated with an equal volume of 100% isopropanol and immediately centrifuged for 10min at room temperature. The DNA pellet was washed with cold 70% (v/v) ethanol then vacuum dried for 3min. The pellet was dissolved in sterile water (32μl) and re-precipitated by addition of 4M NaCl (8μl) and 13% PEG8000 (40μl). The samples were incubated on ice for 20min, then the DNA was pelleted by centrifugation for 15min at 4˚C. The pellet was once again washed with 70% ethanol then vacuum dried for 3min and resuspended in 20μl of sterile water. An aliquot (10%) was visualised and quantified by agarose gel electrophoresis. The samples were stored at -20˚C.

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2.3.10 Single-stranded Plasmid DNA Preparations for Sequencing The method of Sanger was used for preparation of single-stranded plasmid DNA for sequencing (Sanger et al., 1977). Recombinant E. coli JM101 pGEM®-T Easy phagemids were grown overnight in SB-amp broth (2ml) in the presence of helper phage M13K07 (50μl, 107pfu/ml). The cells were pelleted by centrifugation at 13 000rpm for 15min and the supernatant carefully transferred to a fresh tube. The phage were aggregated in 20% PEG8000 and 2.4M NaCl (0.3ml) at 4˚C for 30min and pelleted by centrifugation at 4˚C for 30min. The pellet was resuspended in TE buffer, phenol extracted for 20min at 55˚C and ethanol precipitated. The DNA was then resuspended in TE buffer and 10% of the volume was analysed by agarose gel electrophoresis. The sample was stored at -20˚C.

2.4 Isolation and Characterisation of CPSII Genes from Plasmodium

2.4.1 Isolation of Plasmodium Genomic DNA 2.4.1.1 Isolation of P. falciparum Genomic DNA Genomic DNA was prepared from cultures with 15-20% parasitaemia. The parasitise culture was centrifuged at 1000rpm for 10min, the supernatant discarded and the packed cells lysed by re-suspension in RBC lysis buffer to a ratio of 25ml:1ml of packed RBCs. The suspension was incubated on ice for 30min and then centrifuged at 5000rpm for 15min at 4˚C. The pellet was washed several times in the lysis buffer, each time removing the contaminating hemolysate supernatant, until the supernatant appeared clear. Genomic DNA was extracted by dissolving the resulting parasite pellet in 100mM Tris-HCl pH 8.0, 100mM EDTA containing 4% (w/v) sarkosyl, 100μg/ml Proteinase K (2ml), preheated to 60˚C then added at a ratio of 3.5ml:1ml of packed parasite pellet. The crude extract was then incubated at 60˚C overnight and the genomic DNA purified by CsCl density centrifugation as described in Section 2.3.8.

2.4.1.2 Isolation of P. berghei and P. chabaudi Genomic DNA Infected blood (1ml) was centrifuged (13 000rpm, 3min, 4˚C) and the serum removed. The pellet was washed 3 times in ice cold PBS (1ml). Saponin (0.075%, 1ml) was added to lyse the erythrocytes and the mixture incubated on ice for 10min. Parasites were pelleted by centrifugation (13 000rpm, 3min, 4˚C) and resuspended in 975μl of

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Tris/EDTA/Sarkosyl (0.1M/0.1M/4%). To the suspension was added 25μl of a pre- incubated (60˚C, 1h) mixture consisting of Proteinase K (4mg) in 1ml of Tris/EDTA/Sarkosyl (0.1M/0.1M/4%). The resultant mixture was then incubated overnight at 60˚C. Contaminating proteins were removed by sequential extraction with equal volumes of the following: phenol equilibrated with TE buffer, pH 8, phenol/chloroform/iso-amyl alcohol (50:48:2, v/v) and chloroform/iso-amyl alcohol (24:1, v/v). In each case, the aqueous phase was recovered by centrifugation (13000rpm, 5min). Ethanol precipitation was then performed to recover the DNA (Section 2.3.2.). The DNA was analysed spectrophotometrically for purity before use in PCR (Section 2.3.5).

2.4.1.3 Isolation of P. vivax Genomic DNA A blood sample from a patient infected with P. vivax was used to purify genomic DNA. The sample obtained had been frozen and thawed several times and the majority of the cells had lysed. Genomic DNA was extracted with GENERATION™ Capture Column™ (Gentra Systems) as outlined in the manufacturer’s instructions. The lysed P. vivax sample (200μl) was added to the matrix column and was allowed to absorb for up to an hour. DNA Purification Solution (400μl) was added and the column incubated for 1min at room temperature. The column was centrifuged at 12 000g for 10s and the eluant discarded. This process was repeated twice. DNA Elution Solution was then added and the column immediately centrifuged at 12 000g for 10s. DNA Elution solution (200μl) was again added and the column incubated for 10min at 99˚C. Centrifugation at 12 000g for 20s released the purified DNA from the column. The DNA was analysed spectrophotometrically for purity (Section 2.3.5) and used in PCR (Section 2.4.2.1).

2.4.2 Isolation of CPSII Gene Fragment Clones 2.4.2.1 PCR with Degenerate Primers PCRs were carried out in an MJ Research Minicycler™ in 0.5ml PCR tubes initially in a 20μl volume. When the correct product was amplified, the reaction volume was scaled up to 100μl. A 20μl reaction typically contained: dNTPs (0.2mM), MgCl2 (1.5- 4.5mM), Bio-X-act™ Taq DNA polymerase (1U) (Fisher-Biotec) and 10x OptiBuffer™ buffer; primers (20-40pmol each) and either genomic DNA (50-100ng) or super coiled

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plasmid DNA (5ng). In the case of nested PCRs, 1μl of the initial PCR mixture was used as template. The following cycling temperatures and times were most commonly used: (i) denaturation; 95˚C; 3min (ii) denaturation; 95˚C; 1min (iii) annealing; Tm-5˚C; 1min (iv) extension; 60˚C; 1-5min (v) repeat (ii)-(iv) for 29 more cycles (vi) final extension; 60˚C ; 10min However, for amplification of P. vivax genomic DNA, the traditional PCR extension temperature of 72˚C was used.

2.4.2.2 PCR Product Preparation An aliquot (5μl) of the PCR mixture was electrophoresed on an agarose gel against pUC18/HinfI or λ/HindIII size standards to ensure that a PCR product of expected size was obtained. The remaining PCR mixture was ethanol precipitated (Section 2.3.2) and purified by agarose gel electrophoresis (Section 2.3.4.2) and the final concentration of DNA determined (Section 2.3.5).

2.4.2.3 PCR Product Ligations Typical ligations were performed using 25ng of vector pGEM®-T Easy (Appendix 3), and an appropriate amount of purified insert to give a vector to insert molar ratio of 1:5. Ligation reactions were set up according to the manufacturers instructions in a total volume of 10μl. Ligation reactions were incubated overnight at 16˚C.

2.4.2.4 Preparation of Competent Cells and Transformations Competent E. coli JM101 cells were prepared fresh on the same day of transformations as described by Messing (Messing, 1983). LB medium was inoculated with 1% of an overnight E. coli culture and incubated with shaking at 37˚C until A590= 0.5 (2-2.5h). After chilling on ice for 10min, the cells were harvested by centrifugation (4000rpm for 5min at 4°C). The supernatant was discarded and the cell pellet gently resuspended in ice-cold 50mM CaCl2 to half the culture volume. The cells were kept on ice for 20min, re-centrifuged (4000rpm, 5min, 4˚C) and the pellet resuspended in 50mM CaCl2 (10%

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of the original volume of the culture). The competent cells were stored on ice until use. Competent cells were also stored for long-term use at -80°C after the addition of sterile glycerol to a final concentration of 15% (v/v). An aliquot (5μl) of the ligation reaction or 1ng of the intact plasmid were mixed with competent cells (200-300μl) and maintained on ice for 30min. The cells were then heat-shocked at 42˚C for 2min and LB medium (1ml) was added before incubation for 1h, at 37˚C, with shaking (Crouse et al., 1983). After harvesting the cells by centrifugation (10 000g for 15s), the supernatant was decanted leaving approximately 100μl, and the pellet resuspended. IPTG (100mM, 10μl) and X-gal (2% (w/v) in dimethylformamide, 50μl) were added for "blue-white" selection when required, and the suspension plated onto LB-amp plates.

2.4.2.5 Detection of Recombinant Clones Small-scale plasmid preparations were made of the suspected recombinants i.e. white bacterial colonies (Section 2.3.4). The plasmids were digested with the appropriate restriction enzyme and the insert size determined by agarose gel electrophoresis. A recombinant clone containing the insert of appropriate size was then grown on a larger scale (Section 2.3.7) and purified using caesium chloride density gradient centrifugation (Section 2.3.8).

2.4.3 Plasmodium CPSII Sequence Analysis 2.4.3.1 Generation of Nested Deletions The Erase-a-Base System (Promega) was used to generate single-stranded phagemid sub-clones containing progressive uni-directional deletions of DNA fragments for sequencing. Cloned DNA (20μg) was linearised with the two restriction enzymes required to produce a 5' overhang or blunt end (for digestion with exonuclease III) and 3' overhang (for protection from exonuclease III). The digest was heat inactivated and a sample (500ng) electrophoresed to check for complete digestion. The remainder of the digest was ethanol precipitated and resuspended in exonuclease III buffer (120μl, 1x). The DNA template was pre-warmed to 25˚C and the reaction commenced upon addition of exonuclease III (650U). At one minute intervals, an aliquot (5μl) was removed from the reaction tube and added to separate tubes on ice containing 15μl of S1 mix (1x S1 Buffer, 6U S1 nuclease). After 20min, the S1 digestion tubes were allowed to incubate at room temperature for 30 min. S1 stop buffer (2μl 0.3M Tris base, 50mM EDTA)

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was then added and the tubes heated to 70˚C for 10min to activate the S1 nuclease. The S1 digestion time samples (22μl) were then purified by preparative agarose gel electrophoresis (Section 2.3.4).

Separate aliquots (8μl) of each of the purified digests were subsequently incubated with Klenow fragment (0.5U) and 1 x Klenow Buffer (1μl 20mM Tris-HCl, pH 8.0, 100mM

MgCl2) and allowed to incubate for 5min at 37˚C. After this, 1μl dNTPs (0.125mM) was added and the incubation continued at 37˚C for a further five minutes. The total reaction (10μl) was then ligated with T4 DNA ligase (0.1U), 1mM ATP and 1x ligase buffer (Section 2.2.3) by incubation at room temperature for a minimum of 2h. The ligations were then transformed into E. coli JM101 as described in Section 2.4.2.4 and plated onto LB plates containing ampicillin (100μg/ml). The recombinant colonies were then used to produce single-stranded DNA (Section 2.3.10) which were then subjected to sequencing (Section 2.4.3.2).

2.4.3.2 Automated Sequencing using Big Dye Chemistry Double or single-stranded DNA, as prepared in Section 2.3.9 and 2.3.10, was used for automated sequencing. A typical sequencing reaction contained double-stranded plasmid DNA (1μg), purified PCR product (200-500ng) or single-stranded DNA (500ng), Big Dye reaction premix (4μl) (ABI sequencing kit), primer (5pmol) and water in a total volume of 20μl. The reactions were carried out in a Perkin-Elmer thermocycler. The cycling temperatures were: denaturation 96˚C for 10s, annealing 50˚C for 5s and extension at 60˚C for 4min for 25 cycles. The reaction volume was increased to 100μl and extracted once with 50μl TE-saturated Phenol/Chloroform (1:1) and ethanol precipitated (Section 2.3.3). The pellet was vacuum dried (3min) and stored at -20˚C. The sequencing reaction was electrophoresed with an Applied Biosystems 377 DNA sequencer and resolved by the Automated DNA Sequencing Analysis Facility (UNSW).

2.4.3.3 Bioinformatic Analysis of Sequencing Data

2.4.3.3.1 Sequence Contigs and Alignments Automated sequencing files were inserted into Autoassembler (Applied Biosystems) and contigs were generated. Once Contigs were complete, sequence analysis such as

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identification of ORFs, deduced amino acid sequence and content, was carried out in the program DNA Strider v1.2 (Marck, 1988). Multiple alignments of nucleotide and predicted amino acid sequences were performed by ClustalW available at www.ebi.ac.uk. Two multiple alignments were produced. In the first alignment, sequences obtained of CPSII from Plasmodium species were aligned. A profile of the percentage of identity along the multiple alignment was derived: non-overlapping windows corresponding to 50 aligned positions were considered, and for each window the number of positions occupied by identical amino acid residues among all sequences was counted. In the second, the P. falciparum CPSII was compared with homologues from other species including: Homo sapiens; Rattus norvegicus; Opsanus beta; Trypanosoma cruzii; Leishmania mexicana and Toxoplasma gondii (Appendix 4).

2.4.3.3.2 Hydrophobicity Profiles Hydrophobicity profiles were constructed using the Kyte and Doolittle hydrophobicity scale and a sliding window 20 amino acids long shifted by one amino acid (Kyte et al., 1982). Recurrence plots were constructed applying the RQA (Recurrence Quantitative Analysis) software described by Webber and Zbilut (Webber et al., 1994). A sequence is compared with itself in a dot-plot matrix. In analysing the CPSII protein, the corresponding hydrophobicity value of the Kyte and Doolittle scale was assigned to each amino acid. It was decided to consider dipeptides and a cut-off value of 0.5. According to this choice, any pair of dipeptides is compared with all the others along the protein by calculating the euclidean distance between the corresponding hydrophobicity values. Each dot in the recurrence plot correspond to a pair of dipeptides for which the distance is lower than 0.5.

2.4.3.3.3 CPSII Structural Analysis The domains of the Plasmodium CPSII proteins were predicted using the Conserved Domain Database available at www.ncbi.nih.gov. 3D homology modelling using DeepView- Swiss-PdbViewer v 3.7 (http://www.expasy.org/spdbv/) was performed with the P. falciparum CPSII sequence without insertion sequences, modelled on the solved crystal structure for E. coli CPS (PDB code: 1JDB, (Thoden et al., 1999)).

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Formatted: Bullets and 2.5 Design and Selection of DNAzymes Targeting PbCPSII Numbering Insertion II

2.5.1 DNAzyme Site Selection 2.5.1.1 Transcription Template Preparation Two methods were used to prepare linear template DNA for the production of a P. berghei CPSII insertion II transcript:

2.5.1.1.1 Linearised Clone DNA Plasmid DNA (15μg) containing the PbCPSII insertion II was linearised with PstI, EcoRV or NdeI (60U) for 2h at 37˚C. A sample (500ng) of the digest was then electrophoresed in 1% agarose to confirm linearity. The remainder of the digest was either gel purified using QIAquick® gel extraction kit (Qiagen) or Gene Clean II® (Bio101) according to the manufacturers instructions (Section 2.3.4.2). The sample was then eluted, ethanol precipitated (Section 2.3.2). The sample was then resuspended in

DEPC-treated H20 to a final concentration of 1μg/ul.

2.5.1.1.2 PCR Product DNA Linear template DNA was prepared by PCR in a mixture containing plasmid DNA (300ng). Universal primer (40pmol), which binds to a region outside the T7 promoter of the vector pGem®-T Easy and primer PbCPS16 (40pmol, Appendix 1), which was designed to anneal to the 3' end of the PbCPSII insertion II gene, were used to amplify the product. The reaction was carried out with Bio-X-Act™ Taq DNA polymerase

(2.5U) (Fisher-Biotec), Bio-X-Act™ 10x Opti-buffer™, dNTPs (0.2mM), MgCl2 (1.5mM) to a final volume of 100μl. The cycling temperatures used were 94˚C for 30s, 47˚C for 1min, 60˚C for 3min for 30 cycles with a final extension at 60˚C for 10min. The product was then purified by electrophoresis on a sterile 1% agarose gel (Seakem™) and extracted with Gene Clean II (Bio101) and resuspended in DEPC- treated H20 (40μl). A small aliquot (1μl) was electrophoresed on a 1% agarose gel and the amount of DNA estimated.

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2.5.1.2 In Vitro Transcription Initially, a 32P-labelled transcript was synthesised from both types of template DNA (1μg) by transcription in the presence of [α-32P] UTP and T7 RNA polymerase at 37˚C for 1h-3h using different RNA transcription kits from Promega and Stratagene. The final method decided upon for production of RNA substrate was using the PCR template (Section 2.5.1.1.2) in a 3h transcription with the Stratagene RNA transcription kit. The amount of radio-labelled transcript was determined by an incorporation assay, which involved spotting 1μl of the reaction on two separate filter discs. One was washed with 5% trichloroacetic acid solution for 5 min at room temperature to remove unincorporated radioactivity. The amount of [α-32P] UTP on both discs was determined by scintillation counting and the percentage incorporation was calculated, which was then used to calculate the amount (μM) of radio-labelled transcript. The transcript was then purified by phenol:chloroform extraction (Section 2.3.1) and ethanol precipitated (Section 2.3.2). The transcripts were also electrophoresed on a 4% denaturing polyacrylamide gel and assessed for purity of full-length transcripts. For the production of unlabelled RNA, the concentration was estimated spectrophotometrically.

2.5.1.3 Multiplex DNAzyme Cleavage Reaction The multiplex DNAzyme cleavage reaction was carried out essentially as described previously (Cairns et al., 1999). DNAzymes were separated into three groups, each with a corresponding primer extension oligonucleotide. DNAzymes in each group were combined and diluted to a final concentration of 5nM and 50nM in DEPC-treated H20. DNAzymes (5nM, 50nM) and unlabelled RNA transcript (0.2μM) were pre-equilibrated separately for 10 min at 37˚C in equal volumes of TM10 buffer (Section 2.2.3) and 0.01% SDS. The reaction was initiated by combining both DNAzymes and substrate together at t=0. After 1h, the reaction was stopped by immersion in ice if it was to be used immediately, or kept at -20˚C for further experimentation.

2.5.1.3.1 Primer Extension Each primer extension oligonucleotide was 5' end-labelled with polynucleotide kinase (1U), polynucleotide kinase buffer, and 10μCi of [γ-32P] ATP at 37˚C for 30min. The enzyme was heat inactivated at 75˚C for 5min. Primer extension was performed on the DNAzyme cleaved transcript with SuperScriptII reverse transcriptase (Gibco BRL Life

Chapter 2: Materials & Methods 75

Technologies). In each reaction, 2pmol of radio-labelled primer was combined with 400nM of RNA and denatured at 90˚C for 5min. The primer was allowed to anneal slowly between 65˚C and 45˚C before adding the 1st strand buffer (1x), DTT (5mM), dNTPS (500μM) and enzyme (100U) to a final volume of 20μl. The mixture was then incubated at 45˚C for 1h, then immersed in ice.

2.5.1.3.2 Cycle Sequencing Sequencing fragments corresponding to the targeted regions of the message were also generated by primer extension on the PbCPSII insertion II clone, using the same labelled primer extension oligonucleotides as in the above primer extension experiment, in the presence of chain terminating dideoxynucleotides (ddNTPs). Each of the four sequencing reactions contained 1pmol of radio-labelled primer, 0.87U AmpliTaq DNA polymerase (Perkin Elmer), PCR buffer, 2.5μM dNTPs and 10μM ddGTP or 100μM ddATP or 200μM ddTTP or 100μM ddCTP. This reaction was performed as a linear amplification over 25 cycles at 95˚C for 30s, 50˚C for 60s and 72˚C for 90s. Four sequencing reactions were performed for each primer extension oligonucleotide namely, MD32, MD33, and MD34.

2.5.1.3.3 Product Visualisation After primer extension, samples were combined with equal volumes of stop solution (Section 2.2.3) and heated at 80˚C for 5min before loading onto a 6% denaturing polyacrylamide gel. The gel was dried for 30min using a gel dryer, covered with plastic cling wrap and exposed overnight to a pre-blanked phosphorimaging screen and analysed with a Phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

2.5.1.4 Individual Cleavage Assays DNAzymes were analysed separately for their ability to cleave radio-labelled transcript. Briefly, the radio-labelled transcript (1μM) and individual DNAzyme (10μM) were pre- equilibrated separately for 10 min at 37˚C in equal volumes of TM10 buffer (Section 2.2.3) and 0.01% SDS. The two were then combined and kept at 37˚C for one hour after which a 5% aliquot was added to stop solution (Section 2.2.3) on ice. The samples

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were heated at 80°C for 5min, then electrophoresed on a 4% denaturing polyacrylamide gel. The gel was processed and samples analysed as in Section 2.5.1.3.3.

2.5.1.5 Computer Predicted Site Selection To analyse accessible sites by computer prediction, the program mFold (Zuker, 1989) was used (http://www.bioinfo.rpi.edu/applications/mFold/). The DNA sequence from the PbCPSII insertion II clone used in transcript preparation was inserted into the program and the RNA molecule with the lowest energy of conformation was assessed for single-stranded, accessible regions.

2.5.2 DNAzyme Cleavage and Kinetics 2.5.2.1 Single Turnover Cleavage Single turnover cleavage was performed on both in vitro transcribed radio-labelled transcript, as well as a short synthetic RNA substrate. The radio-labelled transcript (1μM) and individual DNAzyme (10μM) were pre-equilibrated separately for 10 min at 37˚C in equal volumes of TM10 buffer and 0.01% SDS. The two were then combined and kept at 37˚C and 5% of the reaction was taken at 0, 5, 10, 20, 30, and 60min and added to stop solution (Section 2.2.3) on ice. The samples were then were heated at 80°C for 5min and electrophoresed on a 4% denaturing polyacrylamide gel. For single- turnover kinetics on the short substrate, the RNA oligonucleotide (1μM) was 5' end- labelled with polynucleotide kinase (2U) (New England Biolabs), polynucleotide kinase buffer, and 10μCi of [γ-32P] ATP at 37˚C for 30min. The labelled substrate (0.2μM) and DNAzyme (2μM) were treated as above, however, the products were electrophoresed on a 15% denaturing polyacrylamide gel. The gel was dried down and exposed overnight to pre-blanked phosphor storage screens and the relative intensity of the cleavage products and the substrate were quantified as in Section 2.5.1.3.3.

2.5.2.2 Analysis of Single Turnover Kinetics Data Percent product was calculated as Total Product/(Total substrate + Total Product) x 100. A line of best fit was generated for the data (least-squares) in the program MacCurve Fit (Raner Software, Mt. Waverly, Australia) using the equation %P = %Pμ - C.exp [-kt] where %P is % Product, %Pμ is the %P at t=μ, C is the difference in %P between t=μ and t=0, and k is the first order rate constant. The first order rate constant Kobserved (kobs)

Chapter 2: Materials & Methods 77

was used to compare the cleavage efficiencies of the chosen DNAzymes. All experiments were performed in quadruplicate and the standard deviation determined.

2.5.2.3 Multiple Turnover Cleavage Multiple-turnover cleavage reactions were set up with 1600nM, 800nM, 600nM, 400nM and 200nM of in vitro transcribed radio-labelled substrate where the concentration of DNAzyme was kept constant at 5nM. Short synthetic RNA substrate was 5' end-labelled as for single-turnover reactions, however the concentration range used was 32nM, 16nM, 8nM, 4nM and 2nM. In this case, the concentration of DNAzyme was kept constant at 0.2nM. Time point aliquots (5%) were taken at 0, 5, 10, 20, 30, and 60min for each substrate concentration reaction. The products were treated and visualised as above (Section2.5.2.1).

2.5.2.4 Analysis of Multiple Turnover Kinetics Data The % P was calculated as above (Section2.5.2.2). Cleaved substrate (pM) was calculated by multiplying % P x [Substrate nM]. The slope of a plot of cleaved substrate (pM) against time (min) is equivalent to the initial velocity (Vo). The Vo for each substrate concentration was divided by the enzyme concentration [E] to calculate the Kobs. Kobs was then plotted against Kobs/[S] in a modified Eadie-Hofstee plot using the program Enzyme Kinetics v1.1 (Trinity Software). The equation of the line of best- fit y=b-mx, was used by the program to determine Km and Kcat where m = Km and b =

Kcat. A minimum of three experiments was performed (for statistical purposes) for each of the DNAzymes that were tested. The constants for each experiment were determined and the standard deviation across the triplicates was calculated.

2.6 Plasmodium Inhibition Studies

2.6.1 Inhibition of P. berghei In Vivo The P. falciparum in vivo inhibition studiy was carried out in collaboration with Prof. Nick Hunt, Department of Pathology, Sydney University.

2.6.1.1 Animal Trial Experimental Design Parasite: P. berghei K173 (PbK).

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Mouse strain: CBA

Six to eight week old female CBA mice were obtained from Blackburn Animal House, University of Sydney. Animals were maintained under standard conditions on a 12h 6 light/dark cycle and were fed ad libitum. Female mice (n=28) were inoculated with 10 parasitised erythrocytes from a PbK-infected donor mouse (day 10 post-infection) and these were randomly split into two groups, C and D. Group D received the parasite and MD14 while Group C received parasite and the vehicle, PBS. Two additional control groups were used, group A (n=14) received only PBS while group B (n=14) received only MD14.

2.6.1.2 MD14 Preparation and Storage The MD14 compound (2 lots of 10mg) was resuspended in sterile PBS, combined and aliquoted into four sterile tubes for the four consecutive days of injection and stored at -80˚C.

2.6.1.3 MD14 Toxicity Test To evaluate drug toxicity, a preliminary experiment was performed. Five female CBA mice were injected intravenously with 200μl stat dose of MD14 (5mg/kg). Animals were observed over a period of 7 days.

2.6.1.4 MD14 Drug Administration Animals were give a daily intravenous dose of MD14 (5mg/kg) via the tail vein, from day 0 (relative to parasite inoculation) to day 4, inclusive. Prior to drug administration, animals were warmed on a heat pad. Injections were given on alternate sides of the tail to decrease potential trauma.

2.6.1.5 Analysis of Parasitaemia To record parasite progression, the percentage parasitaemia was determined microscopically on methanol fixed, tail vein blood smears, stained with Diff Quick (Lab Aids, N.S.W, Australia). Parasitaemias were initially taken on day 4 p.i. and every second day thereafter. Slides were read coded and randomised by an independent researcher from Prof. Hunt’s laboratory. 200 RBCs /slide were counted and the average

Chapter 2: Materials & Methods 79

was recorded. The standard error was calculated based on the mean and analysed by student’s T-test.

2.6.1.6 Analysis of Organs Brain, kidney, liver, heart and spleen samples were fixed in formalin in case further analysis was needed.

2.6.2 Inhibition of P. falciparum In Vitro 2.6.2.1 DNAzyme Stability Studies in Human Serum Oligonucleotides (6-7pmol) were 5'-end labelled by incubation with 10μCi of [γ−32P] ATP (3μCi/pmol) and 0.5U of T4 polynucleotide kinase at 37˚C in kinase buffer for 30min. Phosphorylation was checked using 1μl (from a total reaction volume of 20μl) of sample analysed by thin layer chromatography on a PEI cellulose strip run in 0.75M KH2PO4 (pH 4.3) followed by autoradiography (Section 2.2.5). Labelled oligonucleotides were incubated in 10% human serum in RPMI media for 0, 2, 6 and 24h then analysed on a 20% (w/v) polyacrylamide gel and visualised by phosphorimaging to ensure their integrity (Section 2.5.1.3.3).

2.6.2.2 Parasite Inhibition Bioassays The assay used to determine the antimalarial activity of DHFR105 (Barker et al., 1996) was adopted for malaria inhibition assays with DNAzymes M5L, FITC-M5L, MPP- M5L and MPP-MD14. The assay was modified by exposing 4% synchronous ring- stage parasites (200μl/well, 2% haematocrit) to a range of concentrations of DNAzymes in PBS for 24h. Control wells were treated with PBS alone. A sample of the parasitised RBCs (10μl) were taken from each triplicate well and pooled for microscopic visualisation (Section 2.6.2.3). The remainder of the triplicate wells were pooled, RBCs harvested by centrifugation at 3000g and processed for flow cytometry (Section 2.6.2.4). For 48h bioassays, duplicate wells were set up with the same conditions for 24h bioassays, but after 24h, the media was removed and fresh DNAzyme and media were added and allowed to incubate for a further 24h. Experiments were performed in triplicate on different days and blinded, with the code revealed after data analysis. Final parasitaemias were based on the mean of

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microscopic (Section 2.6.2.3) and flow cytometry counts (2.6.2.4), the standard deviation determined and the results analysed by students T-test.

2.6.2.3 Analysis by Microscopic Visualisation A sample of the parasitised RBCs (10μl) were taken from each triplicate well and pooled. The cells were pelleted by centrifugation (2000g, 5s). Harvested parasitised red blood cells (1μl packed in 2μl media) were used in slide preparation and parasite growth data was determined by microscopic examination of thin blood smears stained as in Section 2.2.8.2. A minimum of 1000 cells per slide were counted to determine the proportion of infected cells. All data presented is the average of a minimum of three experiments of pooled triplicate samples for each oligonucleotide dilution. Differences in growth reduction were estimated by comparison with untreated controls.

2.6.2.4 Analysis by Flow Cytometry The supernatant was carefully removed from the parasitised red blood cell pellet and the pellet gently resuspended in 500μl of PBS. The cells were then stained with the fluorophore dihydroethidium (2.5μl, 20μg/μl stock in DMF) (Molecular Probes) by incubation at 37˚C for 30min in the dark to allow incorporation of the fluorophore into the parasite DNA. The cells were kept in the dark, on ice, until analysed by the flow cytometer. Controls included stained and unstained, uninfected and infected RBCs.

Samples were analysed on the MoFlo Multi Laser Sorter (MLS) flow cytometer by Cytomation® Inc. (Colorado, USA) and the data collected and analysed on v2 CyCLOPS Summit operating software, CYCLONE and SortMaster. The MoFlo laser setting used was:

Laser 1:Innova-90-5, wavelength 488nm Filters used: Blocking filter 488RB, stops all wavelengths before 488nm; Dichroic >555nm, wavelengths>555nm pass to next filter; Orange 590LP, wavelengths 590- 610nm collected; Dichroic 610 wavelengths >610nm allowed to pass to next filter.

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Gain and voltage settings were: PMT Gain Voltage Fsc 4 - Linear Ssc 1 500 Linear Fl1 0.5 525 Log Fl2 0.5 680 Log

The MoFlo used a 70μM tip to produce steam for the analysis of the sample. The sheath pressure was 34psi and the sample pressure was 36psi to analyse cells with a speed of 12 500 events per second. A histogram of cells was produced using forward and side scatter parameters to measure size and granularity of the cells. A representative population of cells was gated using pulse width parameter to discriminate between cells and debris. A homogenous population of cells was then analysed using either one or two fluorescence channels. For P. falciparum bioassays, parasitised RBCs were distinguished from uninfected RBCs by staining with dihydroethidium and observing the red fluorescence emission. For uptake studies two fluorescence channels were examined. FITC-labelled DNAzymes were examined for green fluorescence and parasites for red fluorescence. In all cases, gates were established based on the negative controls and a minimum of 100 000 cells per sample were counted.

2.6.2.5 Examination of DNAzyme Uptake by Confocal Laser Scanning Microscopy FITC-DNAzymes (0.1μM, 1μM and 5μM) were incubated with 10% ring-stage parasitised RBCs (200μl/well, 2% haematocrit) for 4h and 24h. The cells were harvested at those times by centrifugation at 3000g for 20s. The media was carefully removed and the pellet resuspended in 500μl of wash buffer (PBS, 2% human serum and 5mM EDTA). The cells were centrifuged and resuspended twice with wash buffer (500μl) and finally resuspended in a volume of 50μl. The tubes were then placed on ice and, after noting the time, 200μl of cold glycine buffer (0.5M Glycine, 0.1M NaCl and 1% human serum) was added. At exactly 1min after addition, 1ml of wash buffer was added to neutralise the pH. The cells were once again washed with wash buffer. The cells were pelleted again and resuspended in 500μl of cold PBS containing dihydroethidium (2.5μl, 20μg/μl stock in DMF). The tubes were incubated at 37˚C for 30min in the dark. A drop (2μl) was removed, placed onto a clean slide and protected

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with a sealed cover slip for analysis by confocal microscopy. The remainder of the prepared sample was used for flow cytometry (Section 2.6.2.4). For each experiment the relevant controls included: unstained, uninfected and parasitised RBCs; stained, uninfected and parasitised RBCs; unstained, uninfected and parasitised RBCs with DNAzyme; and stained, uninfected and parasitised RBCs with DNAzyme. The confocal microscope used in this study was the Leica TCS SP. Microscope settings and sample processing was performed by Paul Halasz (The Histology & Microscopy Unit, School of Medical Sciences, University of New South Wales). Image slices of 1μm depth using the 63x objective were processed using the Leica software. The lasers used for FITC and dihydroethidium were the argon and helium lasers.

CHAPTER 3

Isolation of the CPSII Gene from Five Species of Plasmodium Chapter 3: CPSII Genes from Plasmodium Species 84

3 ISOLATION OF THE CPSII GENE FROM FIVE SPECIES OF PLASMODIUM

3.1 Introduction and Aims

The objective of this research was to isolate the insertion sequences and surrounding conserved sequences of CPSII from other species of malarial parasites in order to further validate the insertions as catalytic nucleic acid chemotherapeutic targets. Confirmation of the presence and the sequence of the two insertions in the CPSII gene from the rodent malarial species, P. berghei, P. yoelii and P. chabaudi, would allow further testing of DNAzymes targeting these regions in a malarial animal model. Although P. vivax is not often associated with mortality, it is the most prevalent of the four human species and has a significant impact on morbidity and economic losses. In addition, as 99% of human malarial infections are caused by P. falciparum, P. vivax or mixed infections of both, an efficient synergistic chemotherapeutic would be highly beneficial. Isolation of CPSII insertion regions from P. vivax would allow design of an antisense molecule from consensus sequence in order to target both P. falciparum and P. vivax simultaneously. As a secondary goal, investigation of the composition of the insertions and their conservation amongst the different species of Plasmodium, would allow us to gain some insight into the nature of these extremely large insertion sequences, throughout the evolution of different species of Plasmodium.

In summary, the aims of this chapter were to: (1) isolate the CPSII insertion nucleotide and deduced amino acid sequences from P. berghei, P. chabaudi, P. yoelii and P. vivax; (2) isolate the surrounding core regions of CPSII from the same species and (3) examine and compare the nature of these regions.

Initially, the approach taken involved use of PCR with degenerate primers based on conserved regions to amplify CPSII gene fragments containing the two insertions from P. berghei, P. chabaudi and P. vivax. In the latter stages of the study, completion of several Plasmodium species sequencing projects allowed identification of this gene from P. yoelii by database mining.

Chapter 3: CPSII Genes from Plasmodium Species 85

3.2 Results

3.2.1 Isolation of P. berghei and P. chabaudi CPSII Genes 3.2.1.1 Stage 1: Isolation of Insertion I CPSII insertion I from P. berghei (QIMR strain) and P. chabaudi (Adami strain) was isolated and sequenced in full by Neil Davies (Davies, 1998). Two specific primers PbCPS13F and PbCPS14R (Appendix 1) were designed from this sequence in order to isolate (by PCR and sequencing) insertion I from two additional strains of P. berghei (K173 and ANKA). These P. berghei strains are used routinely in animal trials. The 860bp sequences for both strains were amplified independently, cloned into pGem®- T Easy (Appendix 3) and sequenced as outlined in Section 2.4.3.2. The sequence was analysed as outlined in Section 2.4.3.3.1, and found to be identical to that of the QIMR strain.

Although contamination of DNA seemed unlikely, as insertion I from the QIMR strain was isolated independently to ANKA and K173, further investigation was carried out. The PCR and cloning of amplified products was repeated with new reagents in order to eliminate the chance of DNA contamination; the results indicated that the sequence was again identical across all three P. berghei strains examined. Extensive polymorphisms have been well documented in genes determining surface antigens of merozoites of P. falciparum and P. vivax, including the merozoite surface protein 1 (MSP-1) (Cheng et al., 1993) and the apical membrane protein 1 (AMA-1) (Thomas et al., 1990). Variability in the MSP-1 gene was used to confirm the authenticity of the three P. berghei strains used to isolate insertion I. Primers B4F and B4R (Appendix 1) were designed targeting block 4 of the MSP-1 gene and PCR was conducted. The products were sequenced for each of the three strains and the results confirmed that the QIMR strain was indeed different to ANKA and K173, as stated in the literature (Figure 3.1). Since the strains of P. berghei used in amplifying insertion I were confirmed, it was assumed the sequences were correct.

Existing data suggests that the degree of genetic diversity in laboratory lines of P. berghei is low compared to the substantial enzyme polymorphisms occurring in P. chabaudi, P. vinckei and P. yoelii (Saul et al., 1997). In a study examining

Chapter 3: CPSII Genes from Plasmodium Species 86

polymorphisms in the P. berghei MSP-1 and AMA-1, it was found that eight out of ten laboratory lines examined had identical repeat units (Saul et al., 1997). Saul and co- workers (Saul et al., 1997) showed that K173 and ANKA had the same repeat units in both genes, but different repeat units were revealed for the QIMR strain. The identical sequence of CPSII across the three strains of P. berghei can be explained by the lack of heterogeneity often reported in the literature. This heterogeneity is thought to be due to mishaps during laboratory passage or to the specialised natural environment of P. berghei (Saul et al., 1997).

K173 QITTSGQSSTEPASTGTPSSGEVSTGTSTGGASAGVTNTGAATTGT------+K173 QITTSGQSSTEPASTGTPSSGEVSTGTSTGGASAGVTNTGAATTGT------ANKA QITTSGQSSTEPASTGTPSSGEVSTGTSTGGASAGVTNTGAATTGT------+ANKA QITTSGQSSTEPASTGTPSSGEVSTGTSTGGASAGVTNTGAATTGT------QIMR ------PASTGTPSSGEVSTGTSTGGASAGVTNTGAATTGTSTGGASAG +QIMR ------PASTGTPSSGEVSTGTSTGGASAGVTNTGAATTGTSTGGASAG ***********************************

K173 -TGTGAATTGTTGAEAVTTGNTGAEAATTGNTNTEVTQVQTV +K173 -TGTGAATTGTTGAEAVTTGNTGAEAATTGNTNTEVTQVQTV ANKA -TGTGAATTGTTGAEAVTTGNTGAEAATTGNTNTEVTQVQTV +ANKA -TGTGAATTGTTGAEAVTTGNTGAEAATTGNTNTEVTQVQTV QIMR VTNTGAATTGTTGTGAATTGTTGAEAVTTGNTGAEVTQVQIV +QIMR VTNTGAATTGTTGTGAATTGTTGAEAVTTGNTGAEVTQVQIV *.**********: *.***.*****.*****.:****** *

Figure 3.1 Amino acid alignment of variable region of MSP-1 from P. berghei strains. The strains shown above are: K173; ANKA and QIMR. + signifies the sequence determined in our laboratory strains.

3.2.1.2 Stage 2: Isolation of Insertion II The strategy employed to isolate insertion II from P. berghei K173 and P. chabaudi Adami involved designing a single set of degenerate primers to be used with DNA from both species (designated by the prefix PbCPS in the Appendix 1). The primers were designed based on PfCPSII sequence with a bias towards A/T rich codons due to the similar A/T content of P. berghei, P. chabaudi and P. falciparum. The orientation and relative locations of primers are schematised in Figure 3.2. Although sense and antisense degenerate primers should optimally be 200-500bp apart for successful PCR (Lowe et al., 1990), some of the primers designed were up to 2400bp apart. These primers were designed on the possibility that insertion II was not present or perhaps was significantly smaller than predicted. Along with these primers, positive control primers PbDHFRF1 and PbDHFRR1, were designed to amplify a 440bp segment of the DHFR gene from P. berghei (Appendix 1).

Chapter 3: CPSII Genes from Plasmodium Species 87

5F 5A 6F 7F

5' Ins II 3'

6R 12R 9R 10R 11R 7R 8R

13F 2A 5F

5' Ins I Ins II 3'

14R 17R 7R

Figure 3.2 Overview of the relative primer sites in the CPSII gene from P. berghei and P. chabaudi. (A) Primer sites used to amplify insertion II (Ins II). (B) Primers used to amplify insertion I (Ins I) and core regions. Degenerate primers are shown in black font. Specific primers are shown in red font. For sizes of PCR products, see Table 3.1 All primers have the prefix PbCPS in Appendix 1.

Specificity in PCR is influenced by conditions such as the temperatures and times of annealing, extension and denaturation, as well as by changes in the concentration of DNA, Mg2+, dNTPs, and Taq DNA polymerase. Initial annealing temperatures used were calculated as Tm-5˚C (Bej et al., 1991), but when non-specific products were evident, the annealing temperature was increased by increments of 2˚C. The extension stage was also found to be crucial. Due to the extreme A/T richness of the DNA in most Plasmodium species, results can be improved by performing extension at 60˚C rather than 72˚C (Su et al., 1996). The extension temperature was lowered from 72˚C to 60˚C, resulting in longer products being synthesised.

Genomic DNA from P. berghei and P. chabaudi was isolated (Section 2.4.1.2) and the amount to be used in PCR was optimised by using the DHFR primers as positive controls. Initially, DHFR primers were used in a typical 20μl PCR reaction containing 100ng of genomic DNA (Section 2.4.2.1). However, these conditions failed to produce

Chapter 3: CPSII Genes from Plasmodium Species 88

a PCR product. The DNA was serially diluted from 1:2 to 1:50 in order to reduce the concentration of contaminating haem that would inhibit the reaction. A 1:2 dilution generated the DHFR control as a single amplification product with an optimal yield. This dilution of DNA was then used as a standard amount in subsequent PCR reactions.

MgCl2 concentrations usually ranging between 1.5mM and 4.5mM were used in PCR reactions in order to increase specificity and yield. Excess nucleotides can be inhibiting to Taq DNA polymerase (Innis et al., 1988), however, lowering the concentration of dNTPs used had little effect on specificity. Different types of commercially available Taq DNA polymerase and their corresponding buffers were tested (Section 2.1.4) and were found to have an effect on the specificity of PCR.

Single primer controls were performed alongside PCRs to determine any unspecific products resulting from single primer amplification. All primers except PbCPS6R, PbCPS7R and PbCPS8R, resulted in amplifying many non-specific products. In order to increase the chance of amplifying the correct sequence, primers targeting the larger PCR fragments were used to facilitate a second round of PCR with nested or internal primers. For example, primers 5A and 11R were often used in a first round of PCR. An aliquot (1%) of this initial reaction was then used as a template for a further round of PCR with primer 6F and either 9R or 10R. Frequently only a single internal primer was used along with one of the primers from the first round. Analysis revealed that smearing was a common occurrence with these nested PCRs, but this was reduced by decreasing the amount of template used from the first round of PCR. Many products were visualised of incorrect and predicted size, but after cloning and sequencing, most were unspecific amplicons. The results of PCR with various combinations of primer pairs is summarised in Table 3.1.

Chapter 3: CPSII Genes from Plasmodium Species 89

PRIMER EXPECTED GENERATED GENERATED PRODUCT (bp) PRODUCT/S PRODUCT/S P. berghei (bp) P. chabaudi (bp) 5F/6R 500 - - 5F/9R 660 - - 5F/10R 700 - - 5F/11R 1200 - - 5F/12R 550 - 1000 5F/7R 2400 2000 2000 5F/8R 2550 200-1000 100-1500 5A/6R 200 150*, 1000 170* 5A/9R 350 - - 5A/10R 370 50-500 450 5A/11R 830 - - 5A/12R 250 150*, 50-500 150*, 220* 5A/7R 2100 1800 1800 5A/8R 2230 400-3000 1500-4000 6F/9R 170 200*, 350* 200* 6F/10R 180 600-1200 * 1000, 350 6F/11R 690 700* - 6F/7R 1945 - - 6F/8R 2060 - - 7F/8R 114 200 200-1500

Table 3.1 CPSII PCR products from P. berghei and P. chabaudi. Expected and generated PCR product sizes using degenerate primers on P. berghei and P. chabaudi genomic DNA (approximate values). “*” signifies amplification of a product in a second round of PCR from template generated in an initial round of PCR. Bolded font denotes the correct PCR product verified by sequencing.

Chapter 3: CPSII Genes from Plasmodium Species 90

As indicated in Table 3.1, the correct P. berghei and P. chabaudi products of 150bp and 170bp respectively, were amplified in a second round of PCR with primers 5A and 6R, using a template produced from an initial round of PCR with primers 5F and 6R. Another correct P. chabaudi product of 220bp resulted from a second round of PCR with primers 5A and 12R, on templates produced from a first round with primers 5A and 12R. These products were cloned into pGem®-T Easy and sequenced, revealing extensive homology with PfCPSII. As the 220bp product involved primer 12R that was designed from within PfCPSII insertion II sequence, the presence of the insertion was confirmed in P. chabaudi CPSII (PcCPSII). However, the size and nature of the insertion remained elusive. In order to isolate the full insertion II sequence, high fidelity Taq DNA polymerase was used to obtain a 2000bp gene fragment from both P. berghei and P. chabaudi using the two furthermost primers 5F and 7R (Figure 3.3 and Figure 3.4). At the same time, an 1800bp product from P. chabaudi and P. berghei was amplified with primers 5A and 7R (Figure 3.3 and Figure 3.4). The identity of the 2000bp product from P. berghei was investigated further by use of a nested PCR. This gene fragment was agarose gel-purified (Section 2.3.4.2) and used as a template in a second round of PCR with primers 6F and 11F that produced a 700bp product of the expected size. The 2000bp and 1800bp products from both species were cloned into pGem®-T Easy and partially sequenced to confirm the homology with PfCPSII insertion II.

Phagemid sub-clones containing progressive uni-directional deletions were generated for complete sequencing of the 1800bp and 2000bp clones (Section 2.4.3.1). The clones were digested with SalI and SacI to produce a 5' overhang and a 3' overhang, respectively. Both enzyme sites were found only in the multiple cloning site of the vector, enabling linearisation of the clone. The 5' overhang acts as a substrate for exonuclease III digestion, whilst the 3' overhang is resistant to degradation. At 25˚C, exonuclease II digests at a rate of 110bp/min, allowing generation, over time, of progressively smaller clones that can be used for sequencing. An example of uni- directional deletions performed on the 1800bp P. berghei CPSII (PbCPSII) clone is shown in Figure 3.5. At least two clones of each PCR product were sequenced in both orientations to validate the correct bases and eliminate errors incorporated by Taq DNA polymerase.

Chapter 3: CPSII Genes from Plasmodium Species 91

Lane 12345678910

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517 396 214 75

Lane 1. Size standard pUC18/HinfI (1μg) Lane 2. No DNA negative control Lane 3. Primer 5F Lane 4. Primer 5A Lane 5. Primer 7R Lane 6. Primer 8F Lane 7. 5F/ 7R Lane 8. 5A/7R Lane 9. 5F/8R Lane 10. 5A/8R

Figure 3.3 Agarose gel electrophoresis of P. berghei CPSII insertion II PCR products. 1% agarose gel of PCR products obtained using combinations of degenerate primers on genomic P. berghei DNA. Positive PCR products encompassing insertion II are highlighted by the arrows in lane 7 and lane 8. PCR conditions used in this instance were: 95˚C/ 3min; denaturation 95˚C/1 min; annealing 42˚C/1 min; extension 60˚C/3 min; 30 cycles with a final extension 60˚C/10 min.

Chapter 3: CPSII Genes from Plasmodium Species 92

Lane 1 234567

bp 1419

517 396 214

Lane 1. Size standard pUC18/HinfI (500ng) Lane 2. No DNA negative control Lane 3. Primer 5F Lane 4. Primer 5A Lane 5. Primer 7R Lane 6. 5F/ 7R Lane 7. 5A/ 7R

Figure 3.4 Agarose gel electrophoresis of P. chabaudi CPSII insertion II PCR products. 1% agarose gel of PCR products obtained using combinations of degenerate primers on genomic P. chabaudi DNA. Positive PCR products encompassing insertion II are highlighted by the arrows in lane 6 and lane 7. PCR conditions used in this instance were: 95˚C/ 3min; denaturation 95˚C/1 min; annealing 44˚C/1 min; extension 60˚C/3 min; 30 cycles with a final extension 60˚C/10 min.

Chapter 3: CPSII Genes from Plasmodium Species 93

Lane 1 2 3 4 5 6 7 8 9 10 11 12

Lane 1. Undigested clone T=0 min Lane 2. clone digestion T=2 min Lane 3. clone digestion T=4 min Lane 4. clone digestion T=6 min Lane 5. clone digestion T=8 min Lane 6. clone digestion T=10 min Lane 7. clone digestion T=12 min Lane 8. clone digestion T=14 min Lane 9. clone digestion T=16 min Lane 10. clone digestion T=18 min Lane 11. clone digestion T=20 min Lane 12. pGem®-T Easy vector control

Figure 3.5 Uni-directional deletions of the P. berghei CPSII insertion II gene fragment. Unidirectional deletions of pGem®-T Easy clone of P. berghei 5A/7F PCR product encompassing insertion II. Products in lane 2-11 are exonuclease digestion time points where digestion at 25˚C occurs at 110bp/min from the 5' overhang at the SalI site in a SalI/SacI double digestion of the clone.

The contigs were aligned in Autoassembler and inspected visually. In circumstances where the nature of a base was ambiguous in the two different clones, a third clone was then sequenced in the region to identify it. The complete assembled sequence was then analysed in DNA Strider for open reading frames, translation into protein and amino acid composition (Section 2.4.3.3).

Chapter 3: CPSII Genes from Plasmodium Species 94

3.2.1.3 Stage 3: Isolation of Core CPSII Regions After cloning and sequencing of the insertion II products for each species, specific primers 2A and 17R were designed from P. berghei and P. chabaudi consensus sequences (Figure 3.2B). This was done in order to amplify the CPSII gene sequence between insertions I and II. PCR products of approximately 2.4kb from each species were amplified (Figure 3.6). These PCR products were cloned, fully sequenced by uni- directional deletions and contigs were assembled and analysed as described previously. In order to obtain the final sequences of PbCPSII and PcCPSII, the nucleotide sequence data for insertion I, insertion II, core regions between, and flanking the insertions, were compiled in Autoassembler.

Lane 1 2 3 4 5

bp 2322 2027 1419

517 396

Lane 1. Size standard λ / Hind III (1μg) Lane 2. Size standard pUC18 / HinfI (1μg) Lane 3. No DNA negative control Lane 4. 2A/17 on P. berghei DNA Lane 5. 2A/17 on P. chabaudi DNA

Figure 3.6 Agarose gel electrophoresis of P. chabaudi and P. berghei CPSII core region PCR products. 1% agarose gel electrophoresis of PCR products obtained using primers CPS2A/CPS17R on genomic P. berghei and P. chabaudi DNA. Positive PCR products encompassing the core region from insertion I to insertion II are highlighted by the arrows in lane 4 and lane 5. PCR conditions used in this instance were: 95˚C/ 3min; denaturation 95˚C/30 s; annealing 47˚C/1 min; extension 60˚C/4 min; 35 cycles with a final extension 60˚C/10 min.

Chapter 3: CPSII Genes from Plasmodium Species 95

3.2.2 Isolation of the P. vivax CPSII Gene 3.2.2.1 Stage 1: Isolation of the Core CPSII Region P. vivax genomic DNA was isolated from human whole blood samples as described in Section 2.4.1.3. The samples had gone through multiple rounds of freeze thawing at Westmead Hospital prior to being made available for this study. This made it impossible to isolate parasite DNA uncontaminated with human DNA. The amount of DNA to be used in subsequent PCR reactions was determined by performing a control PCR. Control primers PvDHFRF1 and PvDHFRR1 (Appendix 1) targeting the P. vivax DHFR gene (de Pecoulas et al., 1998), were used in PCR reactions on serial dilutions of the DNA template. An optimal dilution of 1:10, that produced the 770bp DHFR positive control product, was used in further PCR reactions. Due to the presence of contaminating human blood, a different strategy was used for isolation of insertion sequences from the P. vivax CPSII gene where degenerate primers were designed with minimal degeneracy from core regions of high conservation between insertion I and insertion II (Figure 3.7).

Df K2f

5 Qf Wf Kf 2

5' Ins I Ins II 3'

1 Wr Kr Nr Gr Yr

Figure 3.7 Overview of the relative primer sites of the CPSII gene from P. vivax. Degenerate primer sites used to amplify insertion I (Ins I), insertion II (Ins II) and core regions. Degenerate primers are shown in black font. Specific primers are shown in red font. All primers have the prefix PvCPS in Appendix 1.

Initial PCR reactions, with various primer pair combinations from the core region, were performed as outlined in Section 2.4.2.1. The initial annealing temperatures chosen for each primer pair were Tm-5˚C and were increased by increments of 2˚C to reduce unspecific products. The products for all combinations tested are listed in Table 3.2. Again, many unspecific, smeared products were visualized. By use of nested PCR, two

Chapter 3: CPSII Genes from Plasmodium Species 96

correct PCR products of approximately 450bp and 600bp resulted from primers Wf and Kr (on Qf/Nr template) and Kf and Nr (on Wf/Nr template), respectively (Figure 3.8). The sequences revealed extensive homology with PfCPSII core sequences and allowed progression to stage 2 of the strategy, whereby specific primers were designed from this sequence.

PRIMER EXPECTED GENERATED PRODUCT (bp) PRODUCT/S (bp)

Qf/Wr 585 400-520* Qf/Kr 1000 550 Qf/Nr 1600 -

Wf/Kr 430 450* Wf/Nr 1020 - Wf/Gr 2950 - Wf/Yr 3000 - Kf/Nr 590 600* Kf/Yr 1060 -

Df/1 2100 220 K2f/1 2000 350, 450, 1500 2/Gr 1900 - 2/Yr 2000 1700

5/1 2400 2200

Table 3.2 CPSII PCR products from P. vivax. Expected and generated PCR product sizes using degenerate primers on P. vivax genomic DNA (approximate values). “*” signifies amplification of a product in a second round of PCR from template generated in an initial round of PCR. Bolded font denotes the correct PCR product verified by sequencing.

Chapter 3: CPSII Genes from Plasmodium Species 97

Lane 1 2 3 4 5 678 9

1419

517 396 214 75

Lane 1. Size standard pUC18 / HinfI (1μg) Lane 2. No DNA negative control Lane 3. Qf/Wr nested on Qf/Kr Lane 4. Qf/Kr nested on Qf/Nr Lane 5. Qf/Wr nested on Qf/Nr Lane 6. Kf/Nr nested on Qf/Nr Lane 7. Wf/Kr nested on Qf/Nr Lane 8. Kf/Nr nested on Wf/Nr Lane 9. Wf/Kr nested on Wf/Nr

Figure 3.8 Agarose gel electrophoresis of the core CPSII PCR products from P. vivax. 1% agarose gel of PCR products obtained using combinations of degenerate primers on genomic P. vivax DNA. Positive PCR products encompassing CPSII core regions are highlighted by the arrows in lane 7 and lane 8. PCR conditions used in this instance were: 95˚C/ 3min; denaturation 95˚C/1 min; annealing 38˚C/1 min; extension 72˚C/2 min; 30 cycles with a final extension 72˚C/5 min.

Chapter 3: CPSII Genes from Plasmodium Species 98

3.2.2.2 Stage 2: Isolation of the Insertion II Region Specific primers PvCPS1 and PvCPS2 were designed to be used with degenerate primers from the 3' and 5’ ends of the gene (Figure 3.7, Appendix 1). A 1.7kb PCR product was obtained for primer PvCPS2 and Yr that was verified by sequencing as containing insertion II (Figure 3.9). The product was cloned into pGem®-T Easy and subjected to uni-directional deletions for complete sequencing as described previously.

Lane 1 2 3 4 5 67

bp 1419

517

396 214 75

Lane 1. Size standard pUC18/HinfI (1μg) Lane 2. No DNA negative control Lane 3. Yr primer control Lane 4. Gr primer control Lane 5. 2 primer control Lane 6. Yr/ 2 Lane 7. Gr/2

Figure 3.9 Agarose gel electrophoresis of the P. vivax CPSII insertion II PCR product. 1% Agarose gel of PCR products obtained using combinations of degenerate primers with specific primer PvCPS2 on genomic P. vivax DNA. A positive PCR product encompassing insertion II is highlighted by the arrow in lane 6. PCR conditions used in this instance were: 95˚C/ 3min; denaturation 95˚C/1 min; annealing 42˚C/1 min; extension 72˚C/2.5 min; 35 cycles with a final extension 72˚C/5 min.

Chapter 3: CPSII Genes from Plasmodium Species 99

3.2.2.3 Stage 3: Isolation of the Insertion I Region PCR with the degenerate primers Df and K2f located 5’ to insertion I (Figure 3.7), in combination with specific primer PvCPS1, was unsuccessful in amplifying the insertion I region. This was most likely due to the degeneracy of the primers and the presence of human DNA that resulted in amplification of many unspecific bands. At this stage in the investigation, a P. vivax Belem Genome Survey Sequence (GSS) from a mung-bean nuclease-digested genomic DNA library became available. Stage 3 of the isolation of PvCPSII gene fragment involved mining the database with PfCPSII amino acid sequences. It resulted in a small homologous region of 138 amino acids (414bp) upstream from insertion I being found (GenBank accession number: AZ567044). The specific primer PvCPS5 was designed from this region, to be used in PCR with PvCPS1 and allowed isolation of a 2175bp product that encompassed insertion I (Figure 3.10). Complete sequencing of this fragment again involved the lengthy process of cloning, uni-directional deletions, sequencing and analysis of the various sequenced fragments as described previously. Finally, all sequences obtained for PvCPSII were compiled in Autoassembler and translated into nucleotide and amino acid sequences (Section 2.4.3.3.1). Lane 123

1419 517 396

Lane 1. Size standard pUC18/HinfI (500ng) Lane 2. No DNA negative control Lane 3. 5/1 Figure 3.10 Agarose gel electrophoresis of the P. vivax CPSII insertion I PCR product. 1% agarose gel of PCR product obtained using specific primers PvCPS5/PvCPS1 on genomic P. vivax DNA. A positive PCR product encompassing insertion I is highlighted by the arrow in lane 3. PCR conditions used in this instance were: 95˚C/ 3min; denaturation 95˚C/1 min; annealing 50˚C/1 min; extension 72˚C/2 min; 35 cycles with a final extension 72˚C/5 min.

Chapter 3: CPSII Genes from Plasmodium Species 100

3.2.3 Identification of the P. yoelii CPSII Gene 3.2.3.1 Data-mining A search of the Plasmodium sequence database revealed two sequenced fragments of CPSII from whole genome shotgun sequence of a third rodent species, P. yoelii yoelii (GenBank accession numbers: EAA16690 and EAA18435). The two gene sequences were aligned with the CPSII gene from P. falciparum in ClustalW. The two sequences overlapped and together encompassed the entire CPSII gene (6.48kb).

3.2.4 Sequence Analysis Results 3.2.4.1 Nucleotide Conservation Investigation into the nucleotide and amino acid content was performed by comparison of CPSII sequences obtained for P. berghei (4.35kb), P. chabaudi (4.22kb), P. vivax (4.45kb) and P. yoelii (4.34kb), encompassing both insertions, with the equivalent sequence of the P. falciparum CPSII gene (4.98kb) (Section 2.4.3.3.1). Insertion locations used in all analyses were as originally identified (Flores et al., 1994). The CPSII genes from P. falciparum, P. yoelii, P. berghei and P. chabaudi were all found to be highly A/T rich at 76%, 75%, 74% and 74% respectively (Table 3.3). The values for P. falciparum and P. yoelii CPSII are identical to the published values for whole genome exons (Carlton et al., 2002; Gardner et al., 2002). The P. vivax CPSII gene sequence, as expected, was substantially lower in A/T codons at 58%. It appears that insertion I in all species except P. vivax, is 4-5% higher in A/T content than the corresponding core region. However, insertion II is only 1-2% higher than the corresponding core region in the case of P. falciparum and P. berghei. Insertion II in the remaining three species has equal or lower A/T content than the corresponding core regions.

Species Insertion I Insertion II Core General P. falciparum 79 77 75 76 P. vivax 57 54 60 58 P. yoelii 79 74 74 75 P. berghei 77 74 73 74 P. chabaudi 77 71 73 74 Table 3.3 A/T content of insertion I, insertion II and core CPSII gene sequences. The overall or general A/T content (%) of the CPSII gene is also listed.

Chapter 3: CPSII Genes from Plasmodium Species 101

Nucleotide sequence alignments for CPSII from the five species of Plasmodium were generated in the ClustalW program (Section 2.4.3.3.1). A summary of the nucleotide identity for insertion I, insertion II and core regions of the five species of Plasmodium investigated, is given in Table 3.4. Clearly, the core regions are generally well conserved across the species, with the lowest homology being 75% between P. vivax and the rodent species P. chabaudi, P. berghei and P. yoelii. P. vivax shows highest homology with the other human infecting species, P. falciparum. As expected, the rodent species are at least 95% identical to each other in core regions. The insertions on the other hand are significantly less conserved. They show relatively poor homology between rodent and human species of Plasmodium. In particular, homology of insertion II is as low as 6% between P. vivax and P. chabaudi. Nucleotide identity of insertion II between the two human species is also low at only 12% (compared to 76% in core regions).

3.2.4.2 Amino Acid Conservation Amino acid alignments of the CPSII gene from the five species of Plasmodium examined (Figure 3.11) were generated using clustalW program (Section 2.4.3.3.1). This figure illustrates the contrast between the highly conserved core or classical regions and the poorly conserved insertions of CPSII. Table 3.5 reinforces this point, where amino acid identity for the five species is calculated across the core regions, insertion I and II separately. As with the nucleotide conservation results, there is high amino acid conservation within the core sequences; in contrast to the poorly conserved insertions. Core regions are as high as 98% identical amongst P. yoelii, P. chabaudi and P. berghei. The lowest identity in this region, although still well conserved at 88%- 89%, is of the human malarial species to the rodent species. For the insertion regions, the rodent malarial sequences show moderately low homology (42-49%) in insertion I, and even lower homology, in insertion II (28%-34%), when compared to the human malarial sequences. There is also significantly less homology between the human malarial insertions of CPSII. P. vivax insertion amino acid sequences are slightly higher in homology to P. yoelii than P. falciparum, which is not the case for core sequences.

Chapter 3: CPSII Genes from Plasmodium Species 102

A. CPSII Insertion I P. berghei P. yoelii P. chabaudi P. falciparum P. vivax P. berghei 100 95 91 53 42 P. yoelii 100 90 66 46 P. chabaudi 100 61 35 P. falciparum 100 43 P. vivax 100

B. CPSII Insertion II P. berghei P. yoelii P. chabaudi P. falciparum P. vivax P. berghei 100 93 86 37 35 P. yoelii 100 86 36 38 P. chabaudi 100 20 36 P. falciparum 100 12 P. vivax 100

C. CPSII Core region P. berghei P. yoelii P. chabaudi P. falciparum P. vivax P. berghei 100 97 95 83 75 P. yoelii 100 96 85 75 P. chabaudi 100 84 75 P. falciparum 100 76 P. vivax 100

Table 3.4 Nucleotide identity across Plasmodium CPSII genes. Nucleotide identity (%) for all pairs of compared sequences, for: (A) insertion I; (B) insertion II; and (C) core regions calculated based on clustalW alignments.

Chapter 3: CPSII Genes from Plasmodium Species 103

P.yoelii SMLGKIVVYKNRNNINNIYKEINLFDPGQIDVTKYVCNHYIRVFKLKNITFINNEERNYM P.berghei CMLGKIVVYKNRNNINNIYKEINLFDPGQIDVTKYVCNHYIRVFKLKNITFINNEGRNYM P.chabaudi SMLGKIVVYKNRNNINNIYKEINLFDPGQIDVTKYVCNHYVRVFKLKNIEFINSDERNNI P.falciparum SMLG KIVIYKNRQHINKLYKEINLFDPGNIDTLKYVCNHFIRVIKLNNITYNYKNKEEFN P.vivax SMLG KIVIYKNAKNVNKLYKEISLFDPGQIDTIKYVCNHYIRVIKLGRVNYYNNCKGKED .******:*** :::*::****.*****:**. ******::**:** .: : . :

P.yoelii NKNSNYLYKENNTECESYSN------LENVASSFEKIDYNKNSNYHSLLRGKMNLLES P.berghei NKNSNYLYKENNKECDSYSN------LENVASSFEKIDYNKNSNYHSLLRGKMNLLES P.chabaudi NKNSNCLYKDNYTECDSYSN------LENVASSFEKSYYNKNLNYHSLLRGKMNLLES P.falciparum YTNEMITNDSSMEDHDNEINGSISN--FNNCPSISSFDKSESKNVINHTLLRDKMNLITS P.vivax SKGANDVNDYSPEEEENGSSSLTNGNGYNYQSVSSFEKMDFNKNSNQHSLLRGRDEPFNL .. . . : :. . * . .** *:***.: : :

P.yoelii SD-ENIKHYSNYINFNDSNDENNSFRNLYGICEYDKYLIDIDENKEYHENNLDEYGYYNI P.berghei SE-ENIKHYSNYQNFNDNNDENNSFRNLYGMCEHDKYLIDIEENKEYHENNLDEYGYYNI P.chabaudi SE-ENIKDYSNYLNFNDNNAENNPFRNLYGMCEYDKYLIDIEENKDYHEDNLDEYGYYNI P.falciparum SE-EYLKDLHNCN-FSNSSDKNDSFFKLYGICEYDKYLIDLEEHASFHYNNVDEYGYYDV P.vivax IRGDLCGHCSGYQEYSTNGGKKDAFFNLRGACEYDKYLIDLEENSSFHDGNVDQYGHYDV : . . :. .. :::.* :* * **:******::*: .:* .*:* :**:*::

P.yoelii EG------TKKHGCEQKEI P.berghei EG------TKKHGCGQKEI P.chabaudi EG------TKKQCCEHKEI P.falciparum NKNTNILSNNKIEQNNNNENNKNNKNNNNNEVDYIKKDEDNNVNSKVFYSQYNNNAQNNE P.vivax ELSLQ------RGGLSEGGNETCTASISDEGSAPQM : : . :

P.yoelii GKEFNMNNDNSTYIKKTMNNQTFLDLVNKR-NNDKEKIIVIVDCGIKNSIIKNLMKHGKD P.berghei GKEFNMNNDNSTYIKKTVSNKTFLDLVNKR-NNDKEKIIVIVDCGIKNSIIKNLMKHGKD P.chabaudi GKEMNMNNDNSNYIQKTMDNQTFLDLVNKR-NHEKEKIIVIVDCGIKNSIIKNLMKHGND P.falciparum HTEFNLNNDYSTYIRKKMKNEEFLNLVNKRKVDHKEKIIVIVDCGIKNSIIKNLIRHGMD P.vivax EATFNLNNDYSTYVKKKMNKGEFLSLVNKR-KSDAEKIIVIVDCGIKNSIIKNLMKNGKD :*:*** *.*::*.:.: **.***** . *******************:::* *

P.yoelii LPITYIIVPYYYNFNYIDYDAVLLSNGPGNPEKCKLLINNLKTSLQQNKLIFGICLGNQL P.berghei LPLTYIIVPYYYDFNYIDYDAVLLSNGPGNPEKCKLLINNLKTSLQQNKLIFGICLGNQL P.chabaudi LPLTYIIVPYYYDFNYIDYDAVLLSNGPGNPEKCKLLIDNLKVSLQQNKLIFGICLGNQL P.falciparum LPLT YIIVPYYYNFNHIDYDAVLLSNGPGDPKKCDFLIKNLKDSLTKNKIIFGICLGNQL P.vivax LPLT YIIVPYYYDYNSIDYDAVLLSNGPGDPKKCDFLIETLKKSLQKNNLIFGICLGNQL **:*********::* *************:*:**.:**..** ** :*::**********

P.yoelii LGISLGCETYKMRYGNRGVNQPVIELLNNRCYITSQNHGYCLKKKCXLKRKDLAISYINA P.berghei LGISLGCETYKMRYGNRGVNQPVIELLNNRCYITSQNHGYCLKKKCILKRKDLAISYINA P.chabaudi LGISLGCETYKMRYGNRGVNQPVIELLNNRCYITSQNHGYCLKKKCILKRKDLAISYINA P.falciparum LGIS LGCDTYKMKYGNRGVNQPVIQLVDNICYITSQNHGYCLKKKSILKRKELAISYINA P.vivax LGIS LGCETYKMKYGNRGVNQPVIQLVDNKCYITSQNHGYCLKKKSILKRKELAISYVNA *******:****:***********:*::* ***************. ****:*****:**

P.yoelii NDKSIEGISHKNGRFYTVQFHPEGNNGPEDTGFLFRNFILDAFNKKREYRENLPQNIIHI P.berghei NDKSIEGISHKNGRFYTVQFHPEGNNGPEDTGFLFRNFILDAFHKKREYREHLPQNIIHI P.chabaudi NDKSIEGISHKNGRFYTVQFHPEGNNGPEDTGFLFRNFILDVFHKKREYRENLPQNIIHI P.falciparum NDKS IEGISHKNGRFYSVQFHPEGNNGPEDTSFLFKNFLLDIFNKKKQYREYLGYNIIYI P.vivax NDKS VEGITHKNGRFYSVQFHPEGNNGPEDTSFLFKNFLIDMFNKKKRIRDISGHNIIYI ****:***:*******:**************.***:**::* *:**:. *: ***:*

P.yoelii KKKVLLLGSGGLCIGQAGEFDYSGTQAIKSLKECGIYVILVNPNIATVQTSKGLADKVYF P.berghei KKKVLLLGSGGLCIGQAGEFDYSGTQAIKSLKECGIYVILVNPNIATVQTSKGLADKVYF P.chabaudi KKKVLLLGSGGLCIGQAGEFDYSGTQAIKSLKECGIYVILVNPNIATVQTSKGLADKVYF P.falciparum KKKV LLLGSGGLCIGQAGEFDYSGTQAIKSLKECGIYVILVNPNIATVQTSKGLADKVYF P.vivax KKKV LLLGSGGLCIGQAGEFDYSGTQAIKSLKECGIYVILVNPNIATVQTSKGLADKVYF ************************************************************

Chapter 3: CPSII Genes from Plasmodium Species 104

P.yoelii LPVNCEFVEKIIKKEKPDFILCTFGGQTALNCALMLEQKKVLKKNNCLALGTSLESILIT P.berghei LPVNCEFVEKIIKKEKPDFILCTFGGQTALNCALMLEQKKVLKKNNCLALGTSLESILIT P.chabaudi LPVNCEFVEKIIKKEKPDFILCTFGGQTALNCALMLEQKKVLKKNNCLALGTSLESILIT P.falciparum LPVN CEFVEKIIKKEKPDFILCTFGGQTALNCALMLDQKKVLKKNNCQCLGTSLESIRIT P.vivax LPVN CEFVEKIIKKEKPDFILCTFGGQTALNCALMLEQKKVLKKNNCLALGTSLESILIT ************************************:********** .******** **

P.yoelii ENRSMFAEKLKEINEIIAPYGSAKNVEEAIEVANKIGYPILVRTTFSLGGLNSSFINNEE P.berghei ENRSMFAEKLKEINEIIAPYGSAKNVEEAVEVANKIGYPILVRTTFSLGGLNSSFINNEE P.chabaudi ENRSTFAEKLKEINEIIAPYGSAKNVEEAIEVANNIGYPILVRTTFSLGGLNSSFINNEE P.falciparum ENRT LFAEKLKEINERIAPYGSAKNVNQAIDIANKIGYPILVRTTFSLGGLNSSFINNEE P.vivax ENRS MFAEKLREINEIIAPYGSARNVEQAIEVANKIGYPILVRTTFSLGGLNSSFINNEE ***: **** *:**** *******:**::*:::**:*************************

P.yoelii ELIEKCKEIFLQTDNEIFIDKSLKGWKEIEYELLRDNKNNCIAICNMENIDPLGIHTGDS P.berghei ELVEKCKEIFLQTDNEIFIDKSLKGWKEIEYELLRDNKNNCIAICNMENIDPLGIHTGDS P.chabaudi ELIEKCKEIFLQTDNEIFIDKSLKGWKEIEYELLRDNKNNCIAICNMENIDPLGIHTGDS P.falciparum ELIE KCNKIFLQTDNEIFIDKSLQGWKEIEYELLRDNKNNCIAICNMENIDPLGIHTGDS P.vivax ELIK KCKEIFLQTDNEIFIDKSLKGWKEIEYELLRDNKNNCIAICNMENIDPLGIHTGDS **::**::***************:************************************

P.yoelii IVVAPSQTLSNSEYYKFREIALKVITHLNIIGECNIQFGINPKTGEYCIIEVNARLSRSS P.berghei IVVAPSQTLSNYEYYKFREIALKVITHLNIIGECNIQFGINPKTGEYCIIEVNARLSRSS P.chabaudi IVVAPSQTLSNYEYYKFREIALKVITHLNIIGECNIQFGINPKTGEYCIIEVNARLSRSS P.falciparum IVVA PSQTLSNYEYYKFREIALKVITHLNIIGECNIQFGINPQTGEYCIIEVNARLSRSS P.vivax IVVA PSQTLSNYEYYKFREIALKVITHLNIIGECNIQFGINPKTGEYCIIEVNARLSRSS *********** ******************************:*****************

P.yoelii ALASKATGYPLAYISAKIALGYDLVSLKNSITRKTTACFEPSLDYITTKIPRWDLNKFEF P.berghei ALASKATGYPLAYISAKIALGYDLVSLKNSITRKTTACFEPSLDYITTKIPRWDLNKFEF P.chabaudi ALASKATGYPLAYISAKIALGYDLISLKNSITRKTTACFEPSLDYITTKIPRWDLNKFEF P.falciparum ALAS KATGYPLAYISAKIALGYDLISLKNSITKKTTACFEPSLDYITTKIPRWDLNKFEF P.vivax ALAS KATGYPLAYISAKIALGYDLISLKNSITKKTTACFEPSLDYITTKIPRWDLNKFEF ************************:*******:***************************

P.yoelii ASNTMNSSMKSVGEVMSIGRTFEESIQKSLRCIDDNYLGFSNTYCIDWNEEKIIDELKNP P.berghei ASNTMNSSMKSVGEVMSIGRTFEESIQKSLRCIDDNYLGFSNTYCIDWNEEKIIDELKNP P.chabaudi ASNTMNSSMKSVGEVMSIGRTFEESIQKSLRCIDDNYLGFSNTYCIDWNEEKIIDELKNP P.falciparum ASNT MNSSMKSVGEVMSIGRTFEESIQKSLRCIDDNYLGFSNTYCIDWDEKKIIEELKNP P.vivax ASKT MNSSMKSVGEVMSIGRTFEESIQKSLRCIDDNYLGFSNTYCIDWDEAKIVDELKNP **:*********************************************:* **::*****

P.yoelii SPKRIDAIHQAFHLNMSIDVIHELTNIDYWFLYKFYNIYNLENKLKSLTLEQLSFYDLKY P.berghei SPKRIDAIHQAFHLNMSIDVIHELTNIDYWFLYKFYNIYNLENKLKSLTLEQMSFYDLKY P.chabaudi SPKRIDAIHQAFHLNISIDVIHELTNIDYWFLYKFYNIYNLENKLKSLTLEQLSFYDLKY P.falciparum SPKR IDAIHQAFHLNMPMDKIHELTHIDYWFLHKFYNIYNLQNKLKTLKLEQLSFNDLKY P.vivax SPKR IDAIHQAFHLNIPMEKIHELTHIEYWFLHKFYNIFNLQNRLKTLSLEQLSFYDLKY ***************:.:: *****:*:****:*****:**:*:**:*.***:** ****

P.yoelii YKKHGFSDKQIAHYLSYNVKTK------ESDVMKYRENIGLHPHIKVIDTLSA P.berghei YKKHGFSDKQIAHYLSYNAKTK------ERDVMKYRENIGLHPHIKVIDTLSA P.chabaudi YKKHGFSDKQIAHYLSYNVKTK------ESDVMKYRENMGLHPHIKVIDTLSA P.falciparum FKKH GFSDKQIAHYLSFNTSDNNNNNNNISSCRVTENDVMKYREKLGLFPHIKVIDTLSA P.vivax FKKH GFSDKQIAHYLSFNNPKVT------EATVMRYRESLGLHPHIKVIDTLSA :***************:* * **:***.:**.***********

P.yoelii EFPALTNYLYLTYQGTEHDVLPLNMKVK------KRKDHRLISREYNEIRDGIPH P.berghei EFPALTNYLYLTYQGTEHDVLPLNMKVK------KRKDHKLMGRDHNEIRNSIPQ P.chabaudi EFPALTNYLYLTYQGTEHDVLPLNMKVK------KIKDYRLMGREHNEVRG---- P.falciparum EFPA LTNYLYLTYQGQEHDVLPLNMKRKKICTLNNKRNANKKKVHVKNHLYNEVVDDKDT P.vivax EFPA LTNYLYLTYQGVEHDVLPLNMKRK------KKLPAEGTGKKANRAGKLVHP *************** ********** * : . .: *.

Chapter 3: CPSII Genes from Plasmodium Species 105

P.yoelii HWHKNN---NKVIY----DEFS------F P.berghei HWHKNNENNNKVVY----DEFS------F P.chabaudi --DTAHEHNNQIVY----DEFS------F P.falciparum QLHKENNNNNNMNS----GNVENKCKLNKESYGYNNSSNCINTNNINIENNICHDISINK P.vivax HMATEMQEKEAVKQLQMG------. : :

P.yoelii NQETSQQNGLNCSRADINDAEEANKVTNNDGENGEMQNTEKNPDYTTIPSCKYMNNYGNG P.berghei NHEASQQNGLNCLKADINNSEEAIKVTNNDGGKDGMQNKEQNPDYTTIPSCKYMNNYGNG P.chabaudi NQEISQQDGVNDLGSDIN------NAGEEGKIQNKEKHSDYTTIPSCKYMHNYANG P.falciparum NIKVTINNSNNSISNNENVETNLNCVSERAGSHHIYGKEEKSIGSDDTNILSAQNSNNNF P.vivax ---CDQNGYSETLGNDVDVTVMSTKVEGAVGKQVQMVGSSPLGGSKEGPLFSAEGAQMNV :. : : : * . . . . *

P.yoelii RYMGHEQNDMFNLKNFLKNNKREEGFSDNNQQDTIGDDNKKRCPFSGNKMINQNDYHNFG P.berghei RYMGHGRNDMFNLKNFLKNNKREEDFSDNNQQNSIGDDNKKKCPFSGN----KNDYHNFG P.chabaudi RYMRHGHNDMFNLKNFLKNKREEGFGDN------YGNEKKCPFSGHKMIGKSDYHNFG P.falciparum SCNNENMNKANVDVNVLENDTKKREDINTTTVFMEGQNSVINNKNKENSSLLKGDEEDIV P.vivax HASPSNQ------MLSKDGKEEKSIGSDETNVFSVKNCTNNVTFPNGSHIAGGAMKNNG . :: .: . . . . .:

P.yoelii IHQGMKCPMKEKKEN------CILNNKSYPNNTKNEQKNENGNPNDGNILEEFKSNRSSH P.berghei MHQGMKRPMKEKKEN------CILNNKCYPNSTKNEQKNENGNPNDGNHLEEFKSNRSSH P.chabaudi MHQGMKCPMGEKQEN------CMSNSKCCPYSTKNE----DGNQNDGNNLEEFRSNRSSH P.falciparum MVNLKKENNYNSVINNVDCRKKDMDGKNINDECKTYKKNKYKDMGLNNNIVDELSNGTSH P.vivax MQSTTEAGQNSKEAK------ESSANNTSKEGCSKKVPNREVLMNHRMDDISNRSSH : . : .. : :.. . : * : ** :**

P.yoelii STNDQLYLENCNTSEDDENNANPDKSMRMDDNRGNIN-NIANNSYYIRDSVYNNEYKINK P.berghei STNDQLYLENCNTSEDEENNVNPDKSMRMDDNRGNIN-NIANNSYYIRDSVYNNEYKINK P.chabaudi STNDLLYLENCNTSEDDENNTNHDKSMRMDDNRGHIN-NIANNSYYFRDSVYNNEYEINK P.falciparum STNDHLYLDNFNTS-DEEIGNNKNMDMYLSKEKSISN-KNPGNSYYVVDSVYNNEYKINK P.vivax STNDQLYLENFNTSDEEMTNKNGDAHYLSKKKRNFSDSKGAGNLYYLVDSVYNNEYKMSK **** ***: * *** :: . * : ..::. : : ..* **. ********::.*

P.yoelii MRELINNDSKGEVIKS------P.berghei MRELINNDNKSEVIKS------P.chabaudi MREFMNKDNENEFIKS------P.falciparum MKELIDNENLNDEYNNNVNMNCSNYNNASAFVNGKDRNDNLENDCIEKNMDHTYKHYNRL P.vivax MKELINSENTDSNDSVQCEQRGFVVKSGQVG------*:*:::.:. .. .

P.yoelii ------DKNFG---NSKCEYINSNKEKAKKINYNE P.berghei ------SKNFANSTNSKCEYINSNKEKAKKINYND P.chabaudi ------GKNFANITNGKCEYINIDKEHTKNINYNE P.falciparum NNRRSTNERMMLMVNNEKESNHEKGHRRNGLNKKNKEKNMEKNKGKNKDKKNYHYVNHKR P.vivax ------RRFGATPAAEDSLPMHVPHYAGRKTNVGNA . . : :

P.yoelii ENTMVYSMNEQEENE-KSFSNNNKMASKNNNNLMNSYNDDNKSDCFSELSYIQNHKKNIN P.berghei ENTMVYNTNEQEENENKSLSNNNRMISKNNNNLINSYNDDNKSDCFSELSYIQNHKKNIN P.chabaudi DNECRSPNN------SNTRRMASKNNSSLMNSYNDD-KSDCFSELSYIQNHKK--- P.falciparum NNEYNSNNIESKFNNYVDDINKKEYYEDENDIYYFTHSSQGNNDDLSNDNYLSSEELNTD P.vivax ESGKGDSLPHHESERERSKGRSSSKKRETKLNSSGINFDDGNSDCFSELSYLRNCTKSSD :. .. . : .: :.* :*: .*: .

P.yoelii YIENDYGYEDLYSGSDNCYSSYSISMNDENNDSSVLDYEEDELNSELSSINSE------P.berghei DIENDYGYEDLYSGSDNCYSSYSISMNDDNNDNSVLDYEEDGLNSELSSINSE------P.chabaudi ---NDYEYEDLYSRSSDNCYSSHSITMKGDNDSSVLDYEEDEFNSELSSINSE------P.falciparum EYDDDYYYDEDEEDDYDDDNDDDDDDDDDGEDEEDNDYYNDDGYDSYNSLSSSRISDVSS P.vivax AENDDIDDDDAYYNGGDDVYTCSTDNGMFDDYAESHNTFSSKESEGSSVYSEN------:* :: . : .: . : ......

Chapter 3: CPSII Genes from Plasmodium Species 106

P.yoelii -----NDNIFHEKFNDIGFKIIDNKSEMEKEKKKCFIVLGCGCYRIGSS P.berghei -----NDNIFYEKFNDIGFKIIDNKSEMKKEKKKCFIVLGCGCYRIGSS P.chabaudi -----NDNIFHEKFNGIGFKIINNKREMEKDKKKCFIVLGCGCYRIGSS P.falciparum VIYSGNENIFNEKYNDIGFKIIDNRNEKEKEKKKCFIVLGCGCYRIGSS P.vivax ------ENIFNEKFNDIGFKIIHDRNEKEKEKKKCFIVLGCGCYRIGSS :*** **:*.******.:: * :*:******************

Figure 3.11 Amino acid alignment of the Plasmodium CPSII proteins. Insertion I is shown in red text. Insertion II is shown in blue text. Yellow boxed text highlights conserved regions within the insertion sequences. White boxed text highlights conservation at insertion borders.

A. CPSII Insertion I P. berghei P. yoelii P. chabaudi P. falciparum P. vivax P. berghei 100 94 85 48 46 P. yoelii 100 85 49 46 P. chabaudi 100 47 42 P. falciparum 100 45 P. vivax 100

B. CPSII Insertion II P. berghei P. yoelii P. chabaudi P. falciparum P. vivax P. berghei 100 87 69 30 32 P. yoelii 100 69 30 34 P. chabaudi 100 28 32 P. falciparum 100 33 P. vivax 100

C. CPSII Core region P. berghei P. yoelii P. chabaudi P. falciparum P. vivax P. berghei 100 98 97 88 89 P. yoelii 100 98 89 89 P. chabaudi 100 89 89 P. falciparum 100 91 P. vivax 100

Table 3.5 Amino acid identity across Plasmodium CPSII proteins. Amino acid identity (%) for all pairs of compared sequences, for: (A) insertion I, (B) insertion II and (C) core regions calculated based on clustalW alignments.

Chapter 3: CPSII Genes from Plasmodium Species 107

In spite of general low amino acid identity of the insertion sequences, there is a high degree of similarity indicating semi-conservative changes (Figure 3.11, Table 3.6). Additionally, the borders as well as blocks within the insertions are highly conserved among all examined species (Figure 3.11). An unusual finding is the presence of extra insertions within PfCPSII insertions I and II that are significantly high in asparagine content. There also appears to be an extra insertion of 12 amino acids residues immediately prior to PfCPSII insertion II, which is also high in asparagine content.

Species Region % % % % % Identity Similarity Hydrophilicity N N,K, D,E,S P. falciparum CPSII - - 53 9 35 Insert 1 - - 74 23 57 Insert II - - 76 22 61

P. vivax CPSII 91 7 46 8 34 Insert 1 45 29 64 12 45 Insert II 33 27 64 10 47

P. yoelii CPSII 89 8 52 9 35 Insert I 49 25 73 21 54 Insert II 30 30 71 20 57

P. berghei CPSII 88 9 52 8 34 Insert I 48 26 73 21 54 Insert II 30 31 73 21 58

P. chabaudi CPSII 89 8 52 9 35 Insert I 47 27 72 20 53 Insert II 28 28 71 16 53

Table 3.6 Amino acid content across the different Plasmodium CPSII proteins. Amino acid identity and similarity were calculated compared to PfCPSII.

Chapter 3: CPSII Genes from Plasmodium Species 108

3.2.4.3 Hydrophilicity In contrast to the low general conservation observed in the insertions, hydrophilicity is stringently maintained for all Plasmodium insertions (Table 3.6). Furthermore, the hydrophilic amino acid residues asparagine (N), lysine (K), aspartic acid (D), glutamic acid (E) and serine (S) contribute substantially to the amino acid usage within the insertions (Table 3.6).

Recurrence plots were constructed on the deduced protein sequences to further investigate correlation between hydrophilicity and recurrence (Section 2.4.3.3.2). In a recurrence plot, a sequence is compared with itself in a dot-plot matrix, a pair of symmetrical dots being marked whenever the properties of an oligonucleotide of fixed length, orderly recur at two positions along the sequence (Webber et al., 1994). In analysing the CPSII proteins, the corresponding hydrophobicity value of the Kyte and Doolittle scale (Kyte et al., 1982) was assigned to each amino acid.

A comparison of the hydrophobicity profiles with the recurrence plots obtained for the Plasmodium CPSII proteins is shown in Figure 3.12. Regions characterised by a low level of recurrence (white stripes in the dot-plot) usually correspond to poorly hydrophilic segments and to a high percentage of identity, as already highlighted in the case of other Plasmodium proteins (Pizzi et al., 2000). It is clear that insertions (blue bars) correlate to regions of high recurrence or high redundant usage of a few amino acids (dark stripes in the dot-plot), high hydrophilicity and low identity. This indicates that the evolutionarily conserved blocks are involved in the core structure of the enzyme, whilst the hydrophilic regions correlating to insertions are extruded from the protein core. The recurrence plots also highlight differences between the Plasmodium CPSII proteins. The P. falciparum protein is characterised by a high redundant usage of few amino acids within insertions. P. berghei, P. yoelii (not shown) and P. chabaudi show very similar recurrence structures, as expected on the base of the sequence similarity, and it seems that the level of recurrence within insertions are slightly lower with respect to P. falciparum. On the other hand, P. vivax shows a poor recurrent regime and a different amino acid usage of insertion sequences. The recurrence plots emphasise the finding shown in Figure 3.11, namely, the borders as well as some of the central portions within the insertions, are well conserved.

Chapter 3: CPSII Genes from Plasmodium Species 109

100 90 % ID 80 70 60 50 40 30 20 10 0 2 2.00 1 1.50 1.00 0 0.50

-1 0.00 -0.50 -2 -1.00 -1.50 -3 -2.00 -4 -2.50 -3.00

Plasmodium falciparum Plasmodium vivax

2.00 2.00 1.50 1.50 1.00 1.00 0.50 0.50 0.00 0.00 -0.50 -0.50 -1.00 -1.00 -1.50 -1.50 -2.00 -2.00 -2.50 -2.50 -3.00 -3.00

Plasmodium berghei Plasmodium chabaudi

Figure 3.12 Recurrence profiles of the Plasmodium CPSII proteins. Top: % similarity between Plasmodium CPSII amino acid sequences. Bars: indicate insertions I and II. Red line: Kyte and Doolittle hydrophobicity scale; region below the horizontal line indicating increasing hydrophilicity. Dot plot: recurrence plot along the length of the Plasmodium CPSII proteins; regions characterised by low level of recurrence correspond to white stripes and regions of high recurrence correspond to black stripes.

Chapter 3: CPSII Genes from Plasmodium Species 110

3.2.4.4 Compositional Analysis Insertions in P. falciparum CPSII follow the same amino acid usage pattern as in low- complexity sequences of P. falciparum proteins (Pizzi et al., 2001). Results of amino acidic composition analysis are illustrated in Figure 3.13. Insertions and core sequences were analysed separately. Not surprisingly, core sequences share a similar pattern of amino acid usage for all Plasmodium species. The most frequent amino acids are always isoleucine, leucine and lysine. Insertions I and II are characterised by a similar amino acid usage however, asparagine is by far the most frequent amino acid, more than double that in the core regions. In fact, asparagine, aspartate and glutamate make up a substantial portion of the insertions and compose up to 40% of P. falciparum CPSII insertion II. P. vivax CPSII is the exception, where the usage of asparagine is also higher in the insertions, but serine emerges as the most frequent amino acid.

3.2.4.5 Structural Analysis An amino acid alignment was constructed of CPSII from other organisms and compared to Plasmodium CPSII (Appendix 4). PfCPSII appear to share the same primary structural organisation as other homologous eukaryotic species, however, the two large insertions specific for the parasite protein, disrupt the two major domains in ‘hinge’ regions. In Figure 3.14A, the general organisation into functional domains is schematised. According to the Conserved Domain Database, different functional domains recognisable by the presence of specific amino acidic signatures for P. falciparum CPSII can be distinguished. The amino terminus of the protein corresponds to the prokaryotic carA small chain and is composed of two functional subdomains: PSD or the CPSase small chain (Pfam00988.8) and GLNase (Pfam00177.8). This region corresponds to the GAT domain. The remaining protein is organised into the two large CPS synthetase subunits A and B (COG0458.1) separated by an oligomerisation domain (Pfam02787.8) and a methylglyoxal synthase (MGS)-like domain that is situated at the carboxyl terminus and is proposed to play a regulatory role (Pfam02142.8) (Saadat et al., 1999). This region corresponds to the CPS domain.

Chapter 3: CPSII Genes from Plasmodium Species 111

Insertions Core 25 P. falciparum CPSII 20

A. 10 % Usage %

0 ACDEFGHIKLMNPQRSTVWY 25 P. vivax CPSII 20

15 B. 10

0 ACDEFGH IKLMNPQRSTVWY 25 P. berghei CPSII 20

15 C. 10

0 ACDEFGH IKLMNPQRS TVWY

25 P. chabaudi CPSII 20 D. 15 10

0 ACDEFGHIKLMNPQRSTVWY 25 P. yoelii CPSII 20 E. 15 10

0 ACDEFGHIKLMNPQRSTVWY Amino Acid

Figure 3.13 Amino acid composition of Plasmodium CPSII insertions compared to core regions. % amino acid composition of core regions (red columns) and combined insertion sequences (blue columns) for the CPSII protein from: (A) P. falciparum; (B) P. vivax; (C) P. berghei; (D) P. chabaudi; and (E) P. yoelii.

Chapter 3: CPSII Genes from Plasmodium Species 112

A. 179-232 aa 418-603 aa Ins I Ins II

150 aa

PSDCPSase small chain GAT domain domain GLNaseGATase domain

CPS.ACPSase A large chain

OligomerisationOligomerisation domain domain CPS domain CPS.BCPSase B large chain CPS domain MGS-likeMGS-like domain B . Insertion II

Insertion I

CPS domain GAT domain

Figure 3.14 Structural characterisation of Plasmodium CPSII. (A) Schematic representation of the organisation of the CPSII protein indicating the functional domains to scale. Also indicated is the size and position of the Plasmodium insertions. (B) The proposed structure of the P. falciparum CPSII protein modelled on the solved crystal structure for E. coli CPS. The location of the insertions are indicated. The conserved CPS subdomains are highlighted in the same colours as seen in (A).

Chapter 3: CPSII Genes from Plasmodium Species 113

In order to demonstrate the location of the two non-globular domains from a 3D structural point of view, one of the solved X-ray structures for E. coli CPS (Thoden et al., 1999) was considered as a template. There is approximately 35% homology between the two sequences when insertions are removed. This seems to be an acceptable level of identity to generate a reliable alignment as it lies between the 40% identity found to almost always result in the right alignment (Saqi et al., 1998) and 30%, below which the alignment becomes difficult (Rost, 1999). In Figure 3.14B, a ribbon representation of the modelled P. falciparum CPSII is shown, where each fold unit is coloured according to the code used in Figure 3.14A. The insertion sites for P. falciparum CPSII are indicated. It is significant to note that the insertions are proportional in size to each of the sub-domains they interrupt. In addition, they are located in a symmetrical manner at two opposite surfaces. The modelled structure of P. falciparum CPSII implies that the additional non-globular insertions are located on the exterior of the protein, far away from the catalytic sites.

3.3 Discussion

The five Plasmodium CPSII genes investigated in this study encode the largest CPS enzymes characterised to date. This is due solely to the presence of two unusually large insertion sequences of 179-232 and 418-603 amino acids not found in CPSII from any other organism. The predicted molecular weight of PfCPSII is 275kDa (Flores et al., 1994) when insertions are included, which is substantially larger than the average 180kDa predicted size of CPS from other organisms. Protein-splicing or processing of insertions to yield a correctly folded, mature protein is possible. However, no such mechanism for removal of non-globular domains has ever been reported in Plasmodium. In fact, co-workers have recently proven the existence of the insert of PfCTPase in the mature enzyme (Yuan et al., 2005). Studies using crude extracts from P. berghei revealed a high molecular weight protein containing CPS activity, which was at the time assumed to be associated with ATCase (Hill et al., 1981). However, separate CPSII and ATCase activities were subsequently detected in P. berghei (Krungkrai et al., 1990). During this study, a P. falciparum proteome database became available (Florens et. al., 2002). Subsequently, a search of this database revealed a single, short peptide sequence of 21 amino acids corresponding to a region of PfCPSII insertion II. It therefore seems highly likely that the insertions of PbCPSII are not

Chapter 3: CPSII Genes from Plasmodium Species 114

processed and exist as an integral part of the mature protein, accounting for the unusually high molecular weight of the native enzyme.

Genome studies indicate that the frequency of insertions and low-complexity regions is particularly high in Plasmodium (Gardner et al., 1998; Carlton et al., 2002; Gardner et al., 2002). A popular theory for the role of insertions involves use of the regions for immune evasion. However, this is unlikely because of the numerous examples of housekeeping enzymes with insertions. Insertions that are maintained in the Plasmodium enzymes, that have been studied thus far, seem to be crucial for enzyme function. There is evidence that suggests insertions are necessary for enzyme activity by improving protein folding, stability and substrate binding (Gilberger et al., 2000; Clarke et al., 2003). In the study by Clarke and co-workers (Clarke et al., 2003), deletion of an insertion sequence in the P. berghei glucose-6-phosphate dehydrogenase domain abolished activity. Some of this lost activity was restored upon insertion of the P. falciparum equivalent. Other deletion studies of parasite-specific insertions of glutathione reductase showed diminished enzyme folding, stability and substrate binding on elimination of insertions (Gilberger et al., 2000). More recently, mutational studies on parasite-specific insertions of a subtilisin-like protease-1 indicated that these insertions were necessary for enzyme maturation and, in turn, activity (Jean et al., 2005).

Insertions in Plasmodium genes often correspond to introns in corresponding plant genes and it has been postulated that these insertion sequences may have evolved from introns by loss of splice sites and/or stop codons (Clarke et al., 2003). In support of this theory is the similar selective pressure and higher A/T content of their nucleic acid (Pizzi et al., 2000). This does not seem to be the case for CPSII. The gene encoding alfalfa CPSII has been characterised and contains two ORFs separated by a 97bp non- coding intergenic sequence between the GAT domain and CPS domain (Zhou et al., 2000), which clearly does not correspond to the insertion regions of Plasmodium CPSII. Furthermore, the A/T content of the CPSII insertions is, in general, no higher than in the surrounding core regions.

These insertions have been maintained in all five species of Plasmodium investigated here, appearing in precisely the same position but diverging in sequence composition

Chapter 3: CPSII Genes from Plasmodium Species 115

and in length. Comparison of the Plasmodium CPSII genes indicate that the insertions are poorly conserved relative to the orthodox or core regions of the gene, which are highly conserved. The two human Plasmodium genes and their deduced amino acid sequences show high homology to each other, as do the rodent Plasmodium genes. The rate of evolution within insertions is higher than core regions and borders, suggesting a significant difference in functional constraint. This is similar to the finding in γ- glutamyl cysteine synthetase (γ-gcs) from P. falciparum and P. berghei, where central portions diverge more rapidly than the borders of the insertions (Pizzi et al., 2000).

Frontali and Pizzi have shown that low complexity regions (LCRs) of Plasmodium can be identified through use of recurrence plots (Pizzi et al., 1999; , 2001). The same approach was used here with the Plasmodium CPSII predicted protein sequences, where recurrence plots were compared with hydrophobicity profiles. The inserts clearly correlate with regions of high recurrence, low complexity and high hydrophilicity, thus indicating they are unstructured domains. However, the insertions of Plasmodium CPSII are only partially unstructured and contain several blocks of amino acids that are well conserved across the five species examined. Recent evidence suggests that these amino acid blocks also correspond to characteristic hydrophobic clusters that are usually interpreted as functional and/or structural elements in protein (Pizzi, personal communication).

The recurrence plots also highlight the differences between CPSII sequences. The sequence for P. falciparum CPSII and the rodent malarial sequences show similar recurrence structure. P. vivax, on the other hand showed a poor recurrent regime and different amino acid usage that is most likely due to the unusually high G/C content when compared to other Plasmodium species examined. This is in agreement with the finding that amino acid compositions of LCRs reflect the action of constraints acting on the genome rather than on the protein (Pizzi et al., 2001; Xue et al., 2003; DePristo et al., 2006). However, in a study by Pizzi and Frontali, it was revealed that the observed frequency of the hydrophilic amino acid asparagine was double that of random prediction in LCRs and was not a function of unconstrained use of A-rich codons, rather a positive selection pressure probably due to the flexible nature of the amido side-chain (Pizzi et al., 2001).

Chapter 3: CPSII Genes from Plasmodium Species 116

The insertions of Plasmodium are highly hydrophilic in nature with asparagine, aspartate, glutamate and serine residues dominating. Asparagine (N), together with aspartic (D) and glutamic acid (E) comprise up to 40%, and when lysine (K) and serine (S) are included, they represent up to 61%, of amino acid usage in the case of PfCPSII insertion II. This is not uncommon in insertions of Plasmodium or in low-complexity regions. The P. falciparum proteome is known to have a strong bias towards the amino acid residues N, K, I, L, D and E (Musto et. al., 1995). Low complexity regions in particular, are enriched in hydrophilic residues, particularly N and K (Pizzi and Frontali, 2001). A survey of the P. falciparum proteome revealed that the N content of PfCPSII insertions was almost double that of the average N content of the overall proteome (Chanda et. al., 2005). In addition, D and S were significantly higher than the average content found in the overall proteome. Interestingly, there was a marked decrease in the hydrophobic residues I, L and P in the insertions of PfCPSII compared to the overall proteome content. The gene encoding γ-gcs from P. falciparum has three large insertions that are also unusually high in N, D and S content (Luersen et al., 1999). These hydrophilic residues in LCRs have been postulated to form disordered or non- globular tertiary structures favouring these amino acid residues (Newfeld et al., 1994).

Glutamine/asparagine rich domains have been found in a large number of prokaryotic and eukaryotic proteins with a diverse range of functions (reviewed in(Michelitsch et al., 2000)). Analyses of these regions have led to the suggestion that they have been evolutionarily selected for as modular mediators of protein-protein interactions termed “polar-zippers” because of the flexible capacity of their side-chains to form hydrogen bond networks (Perutz, 1994). In support of this theory, there has been experimental evidence showing glutamine/asparagine rich domains mediating specific protein-protein interactions (David et al., 1997; Bailleul et al., 1999). Papenbrock and co-workers demonstrated physical interaction among the three subunits of Mg-chelatase of tobacco, encoded on a cDNA that contains a unique glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment (Papenbrock et al., 1997). The asparagine rich insertion regions of Plasmodium CPSII may well function in protein-protein interactions on the surface of the enzyme.

Wright and Dyson have challenged the central dogma of structural biology that a folded

Chapter 3: CPSII Genes from Plasmodium Species 117

protein structure is necessary for function (Wright et al., 1999). They state that a large proportion of genes encode for low-complexity, non-globular proteins and that frequently, these proteins are involved in some of the most important regulatory functions in a cell, becoming structured when the protein binds to the target molecule. The functional advantages of such processes are numerous and include the ability to bind to several proteins (Wright et al., 1999). Important roles they play include the regulation of transcription and translation, cellular signal transduction, protein phosphorylation and the storage of small molecules. Furthermore, such ‘loopy’ or intrinsically unstructured regions have been postulated to be important for macromolecular assembly of large multi-protein complexes because of this ability to recognize many different biological targets (Liu et al., 2002).

The mammalian CAD complex and the bifunctional protein (CPSII/ATCase) of S. cerevisiae exhibit substrate channelling, a process which involves extensive interdomain interactions of the substrates and intermediates between the different enzyme domains (Serre et al., 1999). Additionally, the yeast complex has retained a 46kDa inactive DHOase domain that is thought to ensure the proper structural arrangement for channelling (Serre et al., 1998). In parasitic protozoa such as P. falciparum, T. gondii, B. bovis and T. bruzi, CPSII is a monofunctional enzyme (Nara et al., 2000). The hydrophilic insertions located on the exterior of Plasmodium CPSII, may become structured upon binding to target proteins. Their function may be to hold in close proximity either or both the ATCase and DHOase enzymes. X-ray crystallography of the E. coli CPS enzyme shows that the three active sites are connected via a molecular tunnel of 100Ǻ in length for substrate channelling (Thoden et al., 1997). It seems feasible that forming of a CAD-type complex by the insertions would be conducive to channelling of the substrate not only within the active sites of CPSII but sequentially to ATCase and DHOase, giving an evolutionary advantage by preventing the dilution of intermediate products. Further, the two sub-domains of the GAT domain of E. coli CPS are linked by a hinge-like loop that occurs in the same region as insertion I. Mutation studies on this region show that the hinge-loop plays a significant role in maintenance of the ammonia tunnel within the GATase subunit of CPS (Huang et al., 2000). Insertion I of Plasmodium CPSII occurs in this region and again supports the concept that the insertions playing a significant role in protein folding for possible tunnel formation to effect substrate channelling.

Chapter 3: CPSII Genes from Plasmodium Species 118

In support of this theory is the recently studied, hinge-linked bifunctional enzyme, S- adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) that contains parasite-specific insertions. It was shown that interference with these insertions reduces essential protein-protein interactions (Birkholtz et al., 2004). The first insertion was shown to be essential for both the decarboxylase activity as well as inter and intra-molecular protein-protein interactions. The second less structured insertion, was shown to be essential for enzyme activity. Furthermore, in a crystallography study of PfDHFR-TS, the first insert, located on the surface of the protein, stabilises the interdomain interactions between DHFR and TS domains. The second insert was shown to interact with the junction region between the two domains (Yuvaniyama et al., 2003).

The insertions within Plasmodial CPSII occur precisely at the sub-domain junctions of both the GAT domain and the CPS domain thereby not disrupting the classical conserved motifs. It appears that the genus Plasmodium has evolved with these two large insertions in a strategic location and maintained them throughout evolution for a specific purpose. In fact, for such large intervening sequences to be tolerated in a ‘house-keeping’ enzyme is surprising and eludes to a significant role. Furthermore, the fact that all species of Plasmodium investigated thus far have maintained the open reading frame of the genes, given the size variation across five species, also indicates that they play an important role.

CHAPTER 4

Selection and Cleavage Activities of DNAzymes Targeting PbCPSII Chapter 4: DNAzyme Selection & Cleavage Activities 120

4 SELECTION AND CLEAVAGE ACTIVITIES OF DNAZYMES TARGETING PbCPSII

4.1 Introduction

4.1.1 The 10-23 DNAzyme The 10-23 DNAzyme cleaves RNA in an enzymatic manner, by progression through multiple rounds of mRNA substrate binding, cleavage at purine-pyrimidine junctions and release of products (Stage-Zimmermann et al., 1998). Once a target sequence is determined, it is possible to design a wide range of these molecules spanning the length of the transcript. However, RNA secondary and tertiary structures can often disrupt the thermodynamics and kinetics of a cleavage reaction. Intramolecular hairpin structures, as well as intermolecular aggregates, are difficult to predict and some experimental techniques have been developed for detecting accessible sites in target RNAs (Section 1.7.2). In particular, in vitro synthesis of a full-length transcript can give a good indication of the natural conformation of the molecule even in the absence of cellular components. This chapter describes a thorough approach taken for the selection of DNAzymes targeting insertion II of CPSII from P. berghei.

4.1.2 The DNAzyme Kinetic Pathway A minimal kinetic pathway has been established for DNA-catalysed RNA cleavage and is represented in Figure 4.1 (Joyce, 2001). It is composed of four main species: enzyme (E) or DNAzyme; RNA substrate (S); DNAzyme/RNA substrate complex (E.S) and DNAzyme/RNA cleavage product complex (E.P1.P2).

The DNAzyme binding step is governed by K1 and dissociation from the RNA substrate is governed by K-1. In the presence of a divalent metal cation, the enzyme substrate (E.S) complex is cleaved to form E.P1.P2. The cleavage or reverse ligation step, is governed by K2 and K-2 respectively. The rate of dissociation of the product that is slowest to be released is governed by K3.

For efficient target hydrolysis to proceed, it is important that K1 be as close as possible 8 -1 -1 2+ to the rate of duplex formation (10 M min , 2mM Mg ) and that K2 be substantially

Chapter 4: DNAzyme Selection & Cleavage Activities 121

greater than K-1 for matched substrates only. In addition, K2 should substantially exceed K-2, and K3 should not be rate limiting for catalytic turnover (Joyce, 2001). The rate constants K2 and K-2 have been shown to be essentially the same for virtually all -1 10-23 DNAzymes. This general molecule has a catalytic rate (Kcat) of 0.1min under simulated physiological conditions in vitro (Santoro et al., 1998). Furthermore, the Km 8 10 for an ideal substrate is <1nM, providing a catalytic efficiency, Kcat/Km of 10 -10 M-1min-1 (Joyce, 2001). Therefore, it is generally accepted that the substrate binding and release steps, which are characterised by the well-understood RNA helix-coil transition, account for the difference in kinetic values for various DNAzymes in the literature.

108 M-1 min-1 0.1 min-1 10 min-1

K1 K2 K3 E + S E.S E.P1.P2 E + P1 + P2 K-1 K-2 K-3 10-3 min-1 10-3 min-1

Figure 4.1 Minimal kinetic pathway of the DNAzyme cleavage reaction. ‘E’ is the DNAzyme or enzyme, ‘S’ is the RNA substrate, ‘P1’ is the 5' RNA product, and ‘P2’ is the 3' RNA product. ‘K’ represents the elemental rate constant for each step of the pathway. Rate constants for an ideal 10-23 DNAzyme are shown (Joyce, 2001).

4.1.3 Single Turnover Kinetics Under single turnover conditions, an excess concentration of DNAzyme to substrate allows the cleavage step to be studied separately from the substrate-binding step. Ideally, every substrate molecule is bound to a DNAzyme, and a single cleavage event follows. In this case, the rate of formation of the enzyme-substrate complex is roughly equivalent to the rate of duplex formation and the limiting rate of the reaction is governed by the cleavage or chemical step. The observed rate of cleavage (Kobs) is equal to the sum of K2 and K-2. However, the latter ligation rate constant is usually 450 fold slower (Joyce, 2001), so that Kobs is assumed equivalent to K2 and is known as the cleavage rate constant.

Chapter 4: DNAzyme Selection & Cleavage Activities 122

4.1.4 Multiple Turnover Kinetics Conditions of substrate saturation, or multiple turnover kinetics, occur when the DNAzyme is limiting and many moles of substrate are cleaved for each mole of DNAzyme present, allowing investigation into the catalytic nature or turnover capacity of the DNAzyme. Under these conditions, the E.S complex is in rapid equilibrium with free E and S i.e. K-1 > K2. The reaction follows Michaelis-Menten behaviour and the rate constants Km and Kcat can be determined by the standard Michaelis-Menten graphical methods such as the Lineweaver-Burk plot or the Eadie-Hofstee plot. In general, the Eadie-Hofstee plot is considered more accurate and generally superior to the Lineweaver-Burk plot, whose main disadvantage lies in the compression of data points at high [S] and emphasises points at lower [S] (Eklund et al., 1976). The modified Eadie-Hofstee plot, where Kobs is plotted against Kobs/[S], is commonly used for estimating kinetic parameters of catalytic nucleic acids such as DNAzymes.

Km is a measure of the amount of DNAzyme bound to the RNA substrate in any form.

Kcat, in the case of RNA hydrolysis is equal to K2. Kcat is the number of times the

DNAzyme ‘turns over’ per unit time. The value of Kcat/Km, the second order rate constant, is indicative of catalytic efficiency. Therefore, the higher the catalytic efficiency, the lower the Km or binding of the DNAzyme to the RNA substrate. An effective catalytic nucleic acid requires a balance between binding strongly enough for specificity, yet being able to dissociate from the products rapidly in order to participate in the next reaction (Fedor et al., 1992).

4.2 Experimental Background and Aims

It has been demonstrated previously that DNAzymes M5L and M8 inhibited growth of P. falciparum cultures by up to 50%, as compared to control levels of 6%. The aims of this chapter were to: (1) design M5L and M8 PbCPSII equivalent DNAzymes; (2) design new DNAzymes targeting insertion II of PbCPSII; (3) investigate accessible regions of the target PbCPSII RNA transcript; (4) to further assess kinetic behaviour of accessible DNAzymes with the aim of finding a superior molecule for testing against rodent malaria infections.

Chapter 4: DNAzyme Selection & Cleavage Activities 123

4.3 Results

4.3.1 DNAzyme Selection 4.3.1.1 Design of New DNAzymes The nucleotide sequence of P. berghei CPSII insertion II region was examined for potential DNAzyme target sites by considering all possible purine-pyrimidine junctions. The first 1200nt of the P. berghei CPSII insertion II region resulted in 189 potential sites. Previous work by Cairns and co-workers confirmed that DNAzyme sequences with above average theoretical total free energies of hybridisation (∆G˚) lower than -20 k/cal/mol were generally active (Cairns et al., 1999). Hence, molecules that were predicted to bind with a ∆G˚ greater than -25 k/cal/mol, by the nearest neighbour method (Sugimoto et al., 1995), were excluded. Finally, 31 potential DNAzymes for this region were chosen and synthesized (Table 4.1). The DNAzymes designed were all 31-mers consisting of a central 10-23 consensus catalytic domain (15nt) and target- specific recognition arms (8nt) based around the central purine-pyrimidine pair (Figure 1.10) (Santoro et al., 1998)). Two of the DNAzymes corresponded to the original CPSRz4/M5L (MD5) and M8 (MD6) sites successfully tested against P. falciparum cultures.

4.3.1.2 DNAzyme Site Accessibility

4.3.1.2.1 Preparation of the Long RNA Substrate A mRNA substrate encompassing PbCPSII insertion II was transcribed in vitro with the 32 incorporation of P-UTP ribonucleotides to assess efficiency of transcription (section 2.5.1.2). Initially, it was difficult to obtain a full-length transcript in substantial quantities, most likely due to the size and A/T rich composition of the transcript. In order to optimise yields and the proportion of full-length molecules obtained, the following variables were addressed: origin of the transcription template; template purification methods; and various commercially available transcription kits.

Chapter 4: DNAzyme Selection & Cleavage Activities 124

DNAzyme Sequence 5'-3' Cleavage / Primer pair

MD32 G3073TT GGG ATG CTT CGT GAT TAA AGG A MD1 T2791CA TCA CAG GCT AGC TAC AAC GAC ACG TTC A/U MD2 G2812CC CTA TAG GCT AGC TAC AAC GAT TTC TCG A/U MD3 A2814AG CCC TAG GCT AGC TAC AAC GAA TTT TCT A/U MD4 T2824AT GAG GAG GCT AGC TAC AAC GAG AAG CCC A/U MD5 C2894CT TGA TAG GCT AGC TAC AAC GAG TAA AAT A/U MD6 T2896TC CTT GAG GCT AGC TAC AAC GAA TGT AAA A/U MD7 T2964CT GCC CAG GCT AGC TAC AAC GAT AGT TTA A/U MD8 C2974AT TAT GAG GCT AGC TAC AAC GAC TCT GCC A/U MD9 T3007AT GCC AAG GCT AGC TAC AAC GAG TTG TGG A/U

MD33 G3423CG TTT CAT ACC TTG ATG CAT TCC A MD10 T3073GT TGG GAG GCT AGC TAC AAC GAG CTT CGT A/U MD11 T3085AA GCC CAG GCT AGC TAC AAC GAT TTG TTG A/U MD12 C3148TC CAT CAG GCT AGC TAC AAC GAT ATT GGT A/U MD13 T3151TC CTC CAG GCT AGC TAC AAC GAC ATT ATT A/U MD14 G3163CA TTC CAG GCT AGC TAC AAC GAC CTT TCC A/U MD15 A3168TT TTG CAG GCT AGC TAC AAC GAT CCA TCC A/U MD16 T3209TG CAT GAG GCT AGC TAC AAC GAG GTA TTG A/U MD17 A3238CC TAC CAG GCT AGC TAC AAC GAT TCC ATA A/U MD18 T3241AT ACC TAG GCT AGC TAC AAC GAC ATT TCC G/U MD19 C3245CC ATA TAG GCT AGC TAC AAC GAC TAC CAT G/U MD20 G3247TC CCA TAG GCT AGC TAC AAC GAA CCT ACC A/U MD21 A3249TG TCC CAG GCT AGC TAC AAC GAA TAC CTA A/U MD22 T3256TC GCC CAG GCT AGC TAC AAC GAG TCC CAT A/U MD23 A3265CA TAT CAG GCT AGC TAC AAC GAT TCG CCC A/U MD24 G3328CT GGT TAG GCT AGC TAC AAC GAT ATC GCT A/U

MD34 G3632GG TTT ACA TTA TTT TCC TCA TCT T MD25 T3415AC CTT GAG GCT AGC TAC AAC GAG CAT TCC A/U MD26 G3423CG TTT CAG GCT AGC TAC AAC GAA CCT TGA A/U MD27 T3435TC TTT CAG GCT AGC TAC AAC GAT GGG CGT A/U MD28 G3514GT TGC CAG GCT AGC TAC AAC GAT TTC ATT A/U MD29 T3526CC CAT CAG GCT AGC TAC AAC GAT TGG GTT A/U MD30 G3529GT TCC CAG GCT AGC TAC AAC GAC ATT TGG A/U MD31 C3538TT CCA AAG GCT AGC TAC AAC GAG GTT CCC A/U

Table 4.1 DNAzymes designed against PbCPSII insertion II. Sequences in bold denote oligonucleotides for use in primer-extension performed in a multiplex assay. All 5' base numbers correspond to the corresponding binding base in the PbCPSII sequence (Genbank Accession number AF286897). Underlined sequences denote the conserved catalytic core sequence of the 10-23 DNAzyme.

Chapter 4: DNAzyme Selection & Cleavage Activities 125

Initially, the pGEM®-T Easy clone containing PbCPSII insertion II was linearised with the three different restriction enzymes (PstI, EcoRV and NdeI), the sites for which were located at the 3' end of the cloned insertion (Section 2.5.1.1.1). The PstI and EcoRV digested products produced a 1600nt transcript and the NdeI fragment, a 2000nt transcript. Digestion was assessed by agarose gel electrophoresis. However, this crude method of visualising DNA would not detect low levels of undigested plasmid, which, even in nanogram amounts, may interfere with transcript purity and yield. Therefore, the linearised clone was electrophoresed on a preparative agarose gel and purified from the gel by QIAquick® gel extraction kit (Section 2.3.4.2). Transcription was then performed using T7 RNA polymerase (Promega) on both gel extracted and non-purified linear plasmid. It was evident that transcription on non-purified templates generated RNA fragments of many sizes (Figure 4.2). In contrast, transcription products from the purified template resulted in a single RNA band, except for the NdeI template that resulted in both the full-length and a larger RNA. However, the yield from the purified template was markedly decreased when compared to transcription using the crude template (Figure 4.2). The EcoRV fragment generated a single RNA species that was slighter higher in yield when compared to the other transcripts and was chosen for all further in vitro transcription. The DNA template was also purified from gel slices using Gene Clean II® (Bio101), which was superior in producing a purer and higher yield transcript than the QIAquick® treated sample.

To improve the yield of the transcript, the transcription reaction time was increased from 1h to 3h, resulting in a five-fold increase in the yield of pure transcript (Figure 4.3). Linear template DNA was also prepared by PCR amplification on the recombinant plasmid using M13 forward and reverse primers to determine if the nature of the template would affect yield and purity (Section 2.5.1.1.2). At the same time, a Stratagene RNA transcription kit was tested for improvement in transcription. The corresponding 32P-labelled transcripts from the different templates and transcription kits are shown in Figure 4.4. The purity of the transcript was clearly improved by use of the Stratagene RNA transcription kit (Figure 4.4). Although there was a decrease in overall yield, the proportion of the full-length transcript appeared to be higher than that produced with the initial Promega T7 RNA polymerase kit.

Chapter 4: DNAzyme Selection & Cleavage Activities 126

Lane 123456 -

Full length RNA transcript +

Transcription products from: Lane 1. gel purified PstI template Lane 2. impure PstI template Lane 3. gel purified EcoRV template Lane 4. impure EcoRV template Lane 5. gel purified NdeI template Lane 6. impure NdeI template

Figure 4.2 Transcription products encompassing PbCPSII insertion II. Transcription products were generated by three different restriction enzyme treated plasmid templates. Templates were used in a transcription reaction either directly after digestion or after undergoing agarose gel purification.

Lane 132 -

Full length RNA transcript +

Lane 1. 1h transcription reaction Lane 2. 2h transcription reaction Lane 3. 3h transcription reaction

Figure 4.3 The 1600nt DNAzyme substrate. The final substrate was generated from gel purified EcoRV linearised template after 1h, 2h and 3h.

Chapter 4: DNAzyme Selection & Cleavage Activities 127

Lane 132 -

Full length RNA transcript

Transcript generated from: Lane 1. linearised plasmid/Promega transcription kit Lane 2. PCR template/Stratagene transcription kit Lane 3. linearised plasmid/Stratagene transcription kit

Figure 4.4 The radio-labelled 1600nt substrate. Transcripts were generated from 150μg of linearised plasmid or PCR generated template, using two different commercially available transcription kits.

The yield with the PCR product as template was significantly improved when compared with that using the linearised clone as template. Once transcription was optimised, an unlabelled transcript was produced for use in the multiplex cleavage assay using the PCR product as template.

4.3.1.2.2 The Multiplex Cleavage Assay A multiplex cleavage assay (Cairns et al., 1999) was used to simultaneously screen for the most accessible regions of the target RNA, as well as the most efficient molecules in terms of cleavage rates. The multiplex assay was performed as outlined in Section 2.5.1.3. Thirty-one unmodified DNAzymes (MD1-MD31) were split into three groups each with a corresponding primer extension oligonucleotide: MD32, MD33 and MD34 (Table 4.1, Figure 4.5).

Chapter 4: DNAzyme Selection & Cleavage Activities 128

Linear DNA template T7 transcription PbCPSII Insertion II start site Transcription

5' 3' 1600nt mRNA

Three separate cleavage and primer- extension reactions with radio-labelled primers MD32, MD33, MD34

5' 3' MD34 (7 DZs) 5' 3' MD33 (15 DZs) 5' 3' MD32 (9 DZs)

Products run on a sequencing gel with cycle sequencing of clone with corresponding extension primers

Figure 4.5 A schematic representation of the multiplex cleavage assay. The assay was used to screen for potential DNAzyme cleavage activity against PbCPSII insertion II.

DNAzymes of each particular group or multiplex reaction were then incubated for 1h at

37˚C with the mRNA substrate in the presence of 2.5mM MgCl2. In order to detect which DNAzymes in the group were cleaving, a primer extension reaction was carried out after the cleavage reaction, using the 5' 32P end-labelled extension oligonucleotide for that particular group. Hence, a radio-labelled fragment was synthesised from the oligonucleotide-binding site to the cleavage site of the mRNA substrate. To provide the exact size markers, cycle sequencing was carried out on the DNA template used to produce the transcript. The primer used for each sequencing reaction was the same oligonucleotide used for the extension reaction. Comparison of the primer-extension products with the cycle sequencing ladder allowed detection of the cleavage sites to the exact base, enabling active DNAzymes to be selected.

Chapter 4: DNAzyme Selection & Cleavage Activities 129

Two cleavage and primer-extension reactions were carried out with 50nM and 5nM concentrations of each DNAzyme in the particular group. The concentration of transcript or substrate used in the reaction was 0.2μM. As the transcript was in excess of the enzyme by at least four fold, and the enzyme was limiting, it allowed assessment of the more effective molecules. As a control, primer extension products on an intact transcript were compared with those obtained after the cleavage reactions. This was done in order to eliminate any unspecific products due to mis-priming, labile mRNA fragments, or products resulting from termination of the extension reaction due to secondary or tertiary structure of the RNA transcript. The labelled fragments were electrophoresed on a 6% polyacrylamide gel along with size markers and cycle sequencing products and visualised after exposure to a phosphorimaging screen (Figure 4.6). The site of cleavage for each DNAzyme was located by the deduced sequence. For the purpose of simplifying analysis, cleavage activities in the multiplex were assessed qualitatively and were designated an arbitrary scale. However, due to the poor resolution in the upper region of the gel and the close proximity of some DNAzyme sites to each other, particularly MD2/MD3 and MD26/MD27, it was difficult to determine exactly which of these DNAzymes were active.

4.3.1.2.3 Individual Cleavage For resolution of obscure sites and to exclude the possibility of interference or co- operation between the DNAzymes, the activity of each DNAzyme was also tested individually against a radio-labelled substrate and compared to those in the multiplex cleavage assay. Each DNAzyme was incubated with the radio-labelled 1600nt mRNA substrate in a cleavage reaction for 1h (Section 2.5.1.4). An aliquot of each reaction was electrophoresed on a 4% polyacrylamide gel and the proportion of products generated quantified by densitometry (Figure 4.7). The results are summarised in Table 4.2.

Chapter 4: DNAzyme Selection & Cleavage Activities 130

Non-specifc product

Lane 1. Cycle sequencing ‘G’ Lane 2. Cycle sequencing ‘A’ Lane 3. Cycle sequencing ‘T’ Lane 4. Cycle sequencing ‘C’ Lane 5. Primer extension on uncleaved transcript Lane 6. Primer extension with 50nM DNAzymes Lane 7. Primer extension with 5nM DNAzymes

Figure 4.6 The PbCPSII multiplex cleavage assay. DNAzyme multiplex samples electrophoresed on a 6% polyacrylamide gel. ‘MD32’, ‘MD33’ and ‘MD34’ lanes represent radio-labelled primer-extension products by these primers on the following: cycle sequencing with incorporation of dideoxy terminators; uncleaved transcript; transcript incubated with 50nM of group DNAzymes listed in Table 4.1; transcript incubated 5nM of the same DNAzymes. The most efficient cleavage products are indicted. An example of a non-specific product is also indicated.

Chapter 4: DNAzyme Selection & Cleavage Activities 131

A MD3 MD13 Uncleaved MD2 MD4 MD7 MD8 MD10 MD11 MD12 MD14 MD15 MD25 MD26 MD27 MD30 MD31

% Cleavage 2720 26 2887 70 45 38 32 20 18 bp Substrate 1100 900 bp 692 587 501 540 404 504 458

B MD20 Uncleaved MD14 MD9 MD16 MD17 MD18 MD19 MD21 MD22 MD23 MD24 MD28 MD29 MD1 MD5 MD6

% Cleavage 9090 26 4573516 4 532 33 7 35 16 Substrate bp 1100

900

692

bp 587

540 501 504 404 458

Figure 4.7 Individual cleavage reactions of the PbCPSII DNAzymes. Thirty-one DNAzymes were chosen for further individual cleavage analysis. Each DNAzyme was incubated separately with the radioactive RNA substrate under single turnover conditions. % cleavage of each DNAzyme after 1h is indicated in the corresponding lane. MD14 cleavage reaction was run on both gels as a positive control.

Chapter 4: DNAzyme Selection & Cleavage Activities 132

4.3.1.2.4 Computer Predicted Site Selection The experimental results were compared with accessible sites by the computer- predicted mRNA folding program mFold (Section 2.5.1.5) (Zuker, 2003). This program theoretically predicts RNA folding based on the lowest energy of conformation of the target molecule (Gibb's free energy, ΔG), hence allowing single-stranded regions to be determined. This includes scoring parameters for base-pair stacking, single dangling nucleotides, terminal mis-matches, and the lengths of secondary RNA structure features such as hairpin loops; internal loops; bulge loops; multi-branched loops; stems and pseudo-knots. Stems indicate regions of RNA base-pairing whereas different types of loops indicate varying regions of single-stranded RNA, which are more susceptible to nucleic acid pairing and cleavage (Figure 4.8). A summary of the findings is included with the experimental observations listed in Table 4.2.

B C

F D

Figure 4.8 Diagrammatic representation of RNA secondary structure. (A) A hairpin loop is where RNA folds back on itself. (B) An internal loop is where a short unpaired region exists between two stems. (C) If the internal loop is asymmetrical and only one 'strand' forms a loop, while the other continues directly from one stem to the other, it is referred to as a bulge. (D) In a multi-branched loop, several stems come together. (E) A stem is a double stranded (paired) region. (F) A pseudo-knot is a long- range interaction, where a loop pairs with another unpaired region.

Chapter 4: DNAzyme Selection & Cleavage Activities 133

DNAzyme Cleavage Multiplex Multiplex Cleavage Accessibility

Site (nt) Assay 50nM Assay 5nM Assay (%) (mFold)

MD1 404 + - 26 Internal loop MD2 425 ++ ++ 27 Stem MD3 427 ++ ++ 20 Internal loop MD4 437 - - - Hairpin loop MD5 (M5L) 507 - - - Internal loop MD6 (M8) 509 - - - Internal loop MD7 577 - - - Stem MD8 587 + - 26 Multi-branched MD9 620 - - 4 Hairpin loop MD10 689 - - - Multi-branched MD11 700 ++ +++ - Hairpin loop MD12 763 + - - Multi-branched MD13 766 + - 28 Bulge loop MD14 778 +++ ++ 90 Multi-branched MD15 783 + + 70 Multi-branched MD16 824 - - - Internal loop MD17 853 - - - Stem MD18 856 - - - Stem MD19 860 + - 5 Multi-branched MD20 862 + + 33 Multi-branched MD21 864 + - 7 Multi-branched MD22 871 + + 35 Multi-branched MD23 880 - - - Multi-branched MD24 943 + - 16 Hairpin loop MD25 1028 + + 45 Multi-branched MD26 1036 ++ +++ 38 Multi-branched MD27 1048 ++ +++ 32 Multi-branched MD28 1129 - - - Stem MD29 1141 - - - Hairpin loop MD30 1144 + - 20 Hairpin loop MD31 1153 + + 18 Stem

Table 4.2 Cleavage efficiencies of PbCPSII DNAzymes. The relative cleavage efficiency of PbCPSII insertion II DNAzymes as determined by multiplex assay or individual cleavage and compared to mFold predictions.

Chapter 4: DNAzyme Selection & Cleavage Activities 134

4.3.1.3 Summary of DNAzyme Selection Cleavage results from the multiplex reaction, individual cleavage reactions and mFold computer predictions are summarised in Table 4.2. Group 1 encompassed the first nine DNAzymes targeting the transcript. MD1, MD2 and MD3 DNAzyme sites were all predicted to lie within relatively inaccessible regions of the message when analysed by mFold. However, all three of these DNAzymes cleaved the mRNA substrate both in the multiplex cleavage experiment as well as the individual DNAzyme cleavage experiments, with low activity. MD4 was predicted to target a region of the message in a single-stranded accessible region. This molecule however, failed to cleave the target message under any of the conditions attempted. Interestingly, DNAzymes MD5 (M5L) and MD6 (M8), which had been successfully targeted against PfCPSII insertion II in the past, were predicted to be located in relatively inaccessible internal loops of the PbCPSII insertion II message. Both DNAzymes failed to produce any cleavage products under the conditions tested. As predicted, the MD7 region was inaccessible, and MD8, which was predicted to lie in a highly accessible region, successfully cleaved the message. MD1 and MD8 both showed detectable cleavage bands in the multiplex reaction at 50nM but none at the lower, more stringent conditions of 5nM. MD2 and MD3 showed detectable cleavage at 50nM as well as 5nM, indicating that they possess a higher turnover activity than other DNAzymes in this group. Overall, none of the DNAzymes in this portion of the message exhibited intermediate or high cleavage levels. It indicated that the mFold predictions for the secondary structure of the transcript were not reliable and this region is not ideal for nucleic acid therapy.

Group 2 contained 15 of the 31 DNAzyme sites, most of which were predicted to lie in open, single-stranded regions of the message. Of the twelve DNAzymes lying in open regions, MD10, MD16 and MD23 did not exhibit any form of cleavage activity. Interestingly, MD11 located 11 bases downstream from MD10, showed high levels of activity, cleaving at a much higher rate at the lower concentration of 5nM than 50nM. This unusual cleavage behaviour was exemplified by the complete absence of activity when tested alone in the 1h individual cleavage experiment, indicating DNAzyme co- operation in the multiplex. The most interesting feature of this group was DNAzymes MD12-MD15 whose cleavage sites were within 20 bases of sequence. The highest activities of all DNAzymes tested were clustered in this region, with MD14 cleaving 90% and MD15 cleaving 70% of the substrate in one hour, individually. These two

Chapter 4: DNAzyme Selection & Cleavage Activities 135

DNAzymes were the most outstanding in terms of cleavage efficiency and optimal accessibility in this region. Unusual behaviour was observed for MD21, which showed a very low activity, cleaving only 7% of the substrate after an hour. This cleavage site is two bases away from MD20 and eight bases away from MD22, both of which had intermediate cleavage activities of 33% and 35% respectively. MD24 behaved as expected, as the mFold algorithm predicted this DNAzyme site to lie in a stem loop region, which is only partially accessible. MD24 cleavage products were detected in the 50nM multiplex reaction but not in the 5nM reaction. When tested individually, only 16% of radio-labelled substrate was converted to its products as indicated by PAGE.

Group 3 spanned the final seven chosen DNAzyme sites. As predicted by mFold, MD25, MD26 and MD27 were active in both multiplex reactions and individual cleavage reactions and MD28 and MD29 showed no detectable cleavage. MD30 showed no activity in the multiplex assay, however, when subjected to separate cleavage, 20% of the substrate was cleaved after an hour. It suggested either interference between the DNAzymes in this region of the RNA substrate when the multiplex assay was performed or the inability to cleave under RNA saturating conditions. MD31 region was predicted to lie in a double-stranded stem region and showed low cleavage activity in the multiplex reaction and the single cleavage experiment.

4.3.2 DNAzyme Cleavage Kinetics Although conflicting cleavage activities were evident under different conditions, the four most efficient DNAzymes, MD14, MD15, MD25 and MD26, were chosen for further kinetic characterisation based on consistent cleavage activity under both multiplex and single turnover conditions. The following Section describes the kinetic analysis of the four chosen DNAzymes, performed on an in vitro transcribed mRNA substrate of 1600nt in length (long substrate). The transcription was performed with the uniform incorporation of 32P-labelled UTP as described previously (Section 2.5.1.2). Upon cleavage of this long substrate, two substrate products were detectable. Kinetic analysis was also performed on 32P end-labelled synthetic RNA substrates of 23nt-28nt

Chapter 4: DNAzyme Selection & Cleavage Activities 136

in length (short substrate). Cleavage of this end-labelled short substrate resulted in one product being detectable.

4.3.2.1 Cleavage of the Long Transcribed Substrate

4.3.2.1.1 Single Turnover Kinetics Under single turnover conditions, each DNAzyme was saturating (10μM) and was ten- fold in excess of the long substrate (1μM). Each DNAzyme was incubated with the substrate and aliquots were taken at 0, 5, 10, 20, 30 and 60 minutes and the reaction stopped (Section 2.5.2.1). The time point aliquots were electrophoresed on a 4% polyacrylamide gel and the radioactive bands detected by phosphorimaging and quantified by densitometry. The amount of cleaved substrate or product formed was calculated based on the ratio of pixel density as determined by densitometry. This was calculated for each time point and the percentage of cleaved substrate was then plotted against elapsed time, allowing the course of the cleavage reaction to be followed. The percentage of cleaved substrate and the Kobs were calculated as outlined in Section 2.5.2.2. Quadruplicate experiments were performed and the standard deviation determined.

A typical single turnover cleavage gel, showing radioactive products of expected size generated over time for each DNAzyme, is shown in Figure 4.9A. The course of the reaction is illustrated graphically in Figure 4.9B. The results clearly indicate that MD14 was the most effective DNAzyme, cleaving over 50% of the substrate in the first five -1 minutes of the reaction. The experimental cleavage constant or Kobs of 0.157 min also indicates that it is the most active of the four DNAzymes tested under these conditions (Table 4.3). Although MD15 behaved in a similar manner to MD14 with 70% of the -1 substrate being cleaved after 1h, it had a significantly lower Kobs of 0.057 min . MD25 -1 -1 and MD26, with Kobs of 0.027 min and 0.014 min respectively, both showed low levels of cleavage with less than 50% and 25% of the substrate being cleaved after 1h. These results were consistent across the four experiments performed, as indicated by the low standard deviations (Table 4.3).

Chapter 4: DNAzyme Selection & Cleavage Activities 137

4.3.2.1.2 Multiple Turnover Kinetics When multiple turnover experiments were performed on the long substrate, the substrate to enzyme ratio was varied from 1600nM:5nM or 320:1 to 200nM:5nM or 40:1 (Section 2.5.2.3). Under conditions of saturating substrate, MD14 was the only DNAzyme to show any detectable cleavage products. Figure 4.10A shows the increase in cleavage products over time under different saturating substrate concentrations for this DNAzyme. The amount of cleaved substrate was analysed by densitometry. When plotted against time, the slope of the line of best fit is equivalent the initial velocity of the reaction (Voreg) which allowed estimation of the Kobs as described in Section 2.5.2.4

(Figure 4.10B). The Kobs was then plotted graphically against Kobs/[S] to generate a modified Eadie-Hofstee plot using the program Enzyme Kinetics. The values for the rate constants Km (slope) and Kcat (y-intercept) were produced for each of the three individual experiments and averaged allowing the standard deviation to be calculated (Section 2.5.2.4). Table 4.3 summarises the findings of kinetic parameters calculated for MD14. Although the value for the Km is high at 6.02μM, the standard deviation indicates it was consistently reproduced over the three experiments.

Chapter 4: DNAzyme Selection & Cleavage Activities 138

Figure 4.9 Single turnover cleavage of the in vitro transcribed long substrate. (A) Radio-labelled products of a single turnover cleavage assay on the long substrate as visualised on a 4% polyacrylamide gel. ‘S’ indicates full length substrate; ‘P1’ and ‘P2’ denote the two cleavage products and ‘T’ indicates time in minutes (0, 5, 10, 20, 30, 60). (B) % cleavage of the substrate over the course of the reaction as measured by densitometry.

Chapter 4: DNAzyme Selection & Cleavage Activities 139

A. 320:1 160:1 120:1 80:1 40:1

T=0’ 60’

P1 P2

Figure 4.10 Multiple turnover cleavage of the in vitro transcribed long substrate. (A) Radio-labelled products of a multiple turnover cleavage assay with MD14 on a long substrate as visualised on a 4% PAGE. The substrate:DNAzyme ratio varied from 1600nM:5nM to 200nM:5nM or 320:1 to 40:1. ‘S’ indicates full-length substrate; ‘P1’ and ‘P2’ denote the two cleavage products and ‘T’ indicates time in minutes (0, 5, 10, 20, 30 and 60). (B) Amount of cleaved substrate over the course of the reaction as measured by densitometry for each saturating substrate concentration. The slope of the line allows an estimation of Voreg (initial velocity by regression), which in turn allows determination of Kobs.

Chapter 4: DNAzyme Selection & Cleavage Activities 140

Single Turn Over Multiple Turn Over

DNAzyme Kobs S.D Km S.D Kcat S.D Kcat/Km S.D (min-1) (µM) (min-1) (M-1min-1)

MD14 0.157 0.004 6.02 0.75 2.12 0.32 3.52x105 0.01

MD15 0.057 0.003 ------

MD25 0.027 0.007 ------

MD26 0.014 0.004 ------

Table 4.3 Kinetic constants for the cleavage of in vitro transcribed long substrate. Summary of kinetic constants estimated for single and multiple turnover kinetics on the 1600nt in vitro transcribed substrate. ‘S.D’ indicates the standard deviation as calculated across four single turnover experiments for each DNAzyme, and a minimum of three experiments under multiple turnover conditions.

4.3.2.2 Cleavage of Short Synthetic Substrates In order to test how effectively these four DNAzymes behave in the absence of secondary structure, both single and multiple turnover studies were conducted on short, synthetic RNA substrates. The synthetic RNA substrates corresponding to the length of the two arms of each DNAzyme including the cleavage junction (23nt) were designed and purchased with a 2'-O-methyl group on the ribose moiety to confer stability. As MD14 and MD15 cleavage sites were five bases apart, a single synthetic RNA that was slightly longer (28nt), was used for the kinetic studies. The sequences of the synthetic RNA molecules are listed in the Appendix 2. In addition, all kinetic experiments on the short substrate were performed with DNAzymes synthesized with a 3' inverted thymidine for stability against serum nucleases to assess whether the necessary clinical modifications affect kinetic activity.

4.3.2.2.1 Single Turnover Kinetics The RNA substrate was labelled immediately before use by 5' end-labelling with 32P- ATP as outlined in Section 2.5.2.1. The DNAzyme concentration (2μM) was in excess of the substrate concentration (0.2μM) by ten-fold as in the long substrate experiments. The cleavage reaction was commenced by addition of the DNAzyme to the substrate

Chapter 4: DNAzyme Selection & Cleavage Activities 141

and aliquots were again taken at 0, 5, 10, 20, 30 and 60 minutes. After PAGE, the bands were analysed by densitometry, which allowed the Kobs to be determined by graphical means (Section 2.5.2.2).

The cleavage over time, of end-labelled short substrates into single detectable products of expected size, is illustrated in Figure 4.11A. Cleavage occurred in an exponential manner, reaching a plateau for each of the four DNAzymes tested (Figure 4.11B). Under single turnover conditions on the short substrate, MD26 was slightly more -1 -1 efficient with a Kobs of 0.167 min , compared to MD14 (Kobs of 0.15min ) (Table 4.4).

MD15 and MD25 performed poorly in comparison, with a Kobs of 0.078 and 0.076min-1, respectively. Furthermore, the cleavage activity of MD14 virtually remained the same whereas, the activity for the remaining DNAzymes were higher.

4.3.2.2.2 Multiple Turnover Kinetics To further characterise the enzymatic properties of the four DNAzymes, multiple turnover reactions were performed on short substrate. In this case, the substrate to DNAzyme ratio was varied from 32nM:0.2nM or 160:1 to 2nM:0.2nM or 10:1, and the course of cleavage followed as stated previously (Figure 4.12). Figure 4.13 illustrates the modified Eadie-Hofstee plot used to calculate the Km (slope of the line) and Kcat (y- intercept) for each DNAzyme. The results of triplicate experiments performed on each DNAzyme and its respective substrate are summarised in Table 4.4. Of the four

DNAzymes, MD25 had the lowest Km value observed. However, when the standard deviation is taken into account, MD14 had the most consistent Km value of 10.99nM

(+/-1.11) compared to the highly variable Km of 8.54nM (+/- 5.59) for MD25. The Kcat value of 0.41 min-1 for MD14 indicates that this DNAzyme had a higher turnover -1 capacity than MD25 (Kcat 0.187 min ). The highest overall catalytic efficiency

(Kcat/Km ) was also seen with MD14.

Chapter 4: DNAzyme Selection & Cleavage Activities 142

Figure 4.11 Single turnover cleavage of the short synthetic substrate (A) Radio-labelled products of a single turnover cleavage assay on the short synthetic substrate as visualised on a 16% polyacrylamide gel. ‘S’ indicates full-length substrate; ‘P’ indicates the single end-labelled cleavage product and ‘T’ indicates time in minutes (0, 5, 10, 20, 30 and 60). (B) % cleavage of the substrate over the course of the reaction as measured by densitometry.

Chapter 4: DNAzyme Selection & Cleavage Activities 143

Figure 4.12 Multiple turnover cleavage of the short synthetic substrate. Radio-labelled cleavage products formed from the short substrate by individual DNAzymes MD14, MD15, MD25 and MD26 under multiple turn-over conditions. The substrate:DNAzyme ratio in this case varied from 32nM:0.2nM to 2nM:0.2nM or 160:1 to 10:1. ‘S’ indicates full length substrate; ‘P’ indicates the single end-labelled cleavage product and ‘T’ indicates time in minutes (0, 5, 10, 20, 30 and 60).

Chapter 4: DNAzyme Selection & Cleavage Activities 144

MD14 MD15 MD25 MD26 0.30

Kobs 0.20

0.10

0.000005 0.000010 0.000015 0.000020 0.000025 0.000030

Kobs/[S]

Figure 4.13 A representative modified Eadie-Hofstee plot for cleavage of the short substrate. Modified Eadie-Hofstee plots were generated by EnzymeKinetics v1.1. The Kobs -1 (calculated by linear regression) is plotted against Kobs (min ) over substrate concentration (nM). The slope of the line is the Km value and the y-intercept is the Kcat value.

Single Turnover Multiple Turnover

DNAzyme Kobs S.D Km S.D Kcat S.D Kcat/Km (min-1) (nM) (min-1) (M-1min-1)

MD14 0.150 0.019 10.99 1.11 0.41 0.06 3.76 x 107

MD15 0.078 0.006 31.04 6.48 0.56 0.09 1.79 x 107

MD25 0.076 0.007 8.54 5.59 0.19 0.08 2.19 x 107

MD26 0.167 0.020 40.56 27.5 0.27 0.25 6.61 x 106

Table 4.4 Kinetic constants for the cleavage of short synthetic substrate. Summary of kinetic constants estimated for single and multiple turnover kinetics on the short substrate by MD14, MD15, MD25 and MD26. ‘S.D’ indicates the standard deviation as calculated across a minimum of three experiments for each DNAzyme.

Chapter 4: DNAzyme Selection & Cleavage Activities 145

4.4 Discussion

4.4.1 DNAzyme Selection This study described the use of a multiplex assay and single turnover cleavage of individual DNAzymes to find accessible sites within the PbCPSII insertion II target mRNA and compared them to predicted sites of accessibility based on the computer algorithm mFold.

Analysis of the multiplex assay results indicated that 19 of the 31 DNAzymes designed showed a detectable cleavage product at 50nM and 11 at 5nM against the long RNA substrate. Cairns and co-workers reported that 8 of the 80 DNAzymes designed against a 0.75kb HIV transcript (HPV16 E6/E7), demonstrated substantial cleavage activity against long substrate at 5nM and that in general, these DNAzymes had a ∆G˚< -20 kcal/mol (Cairns et al., 1999). This can be explained using data from Sugimoto and colleagues for the thermodynamic stability of DNA-RNA hetero- duplexes (Sugimoto et al., 1995). They showed that one should aim for a predicted

∆G˚37 of –8 to –10 kcal/mole for each substrate-binding domain. As the DNAzymes targeting PbCPSII insertion II were selected based on this criterion, it is no surprise that a substantial proportion of DNAzymes exhibited detectable cleavage ability at 50nM and 5nM.

Although the multiplex method enabled rapid screening of 31 DNAzymes in this case, analysis of the results posed many difficulties. The length of the message comprising each group posed difficulties in producing optimal resolution of cycle sequencing products and hence made detection of the active DNAzymes more difficult. There were many products visible in the intact transcript lane, which may have been degradation products at the ends of the transcript or premature termination of the primer extension products due to the complex folding of the message. An interesting feature of this method was the presence of intense cleavage products that did not correspond to any DNAzyme sites and were not present in the non-cleaved transcript control. This is clearly not due to premature termination of extension due to RNA secondary structure and is most likely attributed to co-operation of the DNAzymes in some form to allow cleavage at unusual sites of the message or simply non-specific cleavage (Cairns et al., 1999).

Chapter 4: DNAzyme Selection & Cleavage Activities 146

In general, the most accessible regions of the transcript were found to be in the regions including the MD14 and MD15 DNAzyme sites, as well as the MD25-27 region. Interesting results were seen around the MD19-MD22 sites, whereby MD20 and MD22 showed moderate cleavage activity and MD21 whose site lay within two nucleotides of MD20 and seven nucleotides from MD22, showed very little cleavage activity. This type of behaviour is unlikely to be due to the secondary structure of the message and most likely due to the nature of the DNAzyme sequence itself. Cairns and co-workers witnessed a similar phenomenon and attributed it to DNAzymes inhibiting their own cleaving efficiency by forming stable intra-molecular hairpin structures (Cairns et al., 1999). Alternatively, this could be attributed to competition or interference between the DNAzymes in these over-lapping regions.

The lack of detectable activity of the DNAzymes equivalent to M5L and M8 is notable. Previously, these DNAzymes have been shown to cleave target RNA in in vitro cleavage studies as well as successful inhibition of P. falciparum laboratory cultures (Flores, personal communication). This is most likely attributed to sequence differences between PfCPSII and PbCPSII insertion II and therefore secondary structure of the transcribed RNA.

In the past, computer-predicted mRNA folding algorithms such as mFold have proven inadequate in predicting the complex secondary and tertiary structure of RNA molecules. This claim was substantiated in this study as the mFold predictions showed poor correlation to target site accessibility determined by both the multiplex cleavage assay and the individual cleavage assays. Furthermore, the difficulty encountered with prediction of PbCPSII insertion II RNA secondary folding using the program mFold (Zuker, 2003) was due to the many different folds predicted with similar free energies making analysis of the target sequence difficult. It has been frequently observed frequently that a small proportion of antisense nucleic acids engineered based on computer algorithms have given rise to significant reductions of target RNA levels within cells (Pan et al., 2001). In general, the individual cleavage experiments proved to be the most rapid, easily quantifiable and reliable method used to select for active and accessible DNAzyme sites in the long substrate.

Chapter 4: DNAzyme Selection & Cleavage Activities 147

These methods rely on the folding of the RNA in solution under physiological conditions to give an estimation of accessible sites. In this study, the target RNA was only part of the CPSII full-length transcript (estimated 7000nt in length) due to difficulties of in vitro transcription of such a large molecule. This is not optimal, and may give inaccurate information as to the true folding of the message and accessible sites. However, it is a valuable indicator of possible folding in neighbouring regions, far better than using short substrates with minimal secondary structure. Furthermore, regardless of the length of the transcript, dynamic conformational changes are rapidly occurring in living cells and cellular components may also inhibit or interfere with target site accessibility.

Schubert and co-workers have demonstrated a method by which to overcome target RNA inaccessibility, eliminating the need for intense screening methods (Schubert et al., 2004). They demonstrated that by introduction of 2’-O-methyl RNA or locked nucleic acid (LNA) monomers into the substrate recognition arms, a DNAzyme was able to degrade a previously inaccessible target RNA, thus indicating that nucleotides with high target affinity were able to compete successfully with internal structures. However, in addition to target site accessibility, the thermodynamics and kinetics of enzyme-substrate interactions play a major role in the efficiency of hydrolysis of an RNA target. DNAzyme turnover is highly dependent upon the efficient release of the cleaved RNA target that is hindered by high target affinity.

4.4.2 DNAzyme Kinetics By studying the activity of DNAzymes against a short, synthetic RNA substrate devoid of secondary structure, an indication is given of the catalytic efficiency or the turnover ability of the enzyme. In addition, substrates of length equal to DNAzyme hybridising arms allow examination of how the nature of the hybridising arms affects catalytic efficiency i.e. the binding and release steps. This is because once the substrate is bound, it is held by base-pairing and the products dissociate at a rate determined by the number of base pairs, their GC content, and to a lesser extent, their detailed sequence (Joyce, 2001). An optimal DNAzyme must bind specifically, yet be able to dissociate rapidly enough to allow efficient turnover rates.

Chapter 4: DNAzyme Selection & Cleavage Activities 148

Kinetic experiments performed on the long substrate allow the binding, cleavage and product release steps to be studied in conjunction with accessibility of a more complex, folded mRNA by the DNAzyme in question. Secondary and tertiary structures within the RNA substrate reduce K1 by impeding access to Watson-Crick base-pairing.

DNAzyme cleavage of a long substrate (1600nt) under single turnover conditions showed that MD14 was superior in cleavage efficiency to MD15, MD25 and MD26 -1 with a Kobs of 0.157min . There are numerous examples in the literature of cleavage efficiency of DNAzymes against long substrates under single turnover conditions with similar Kobs values (Cairns et al., 1999; Sun et al., 1999; Lowe et al., 2001; Khachigian et al., 2002; Zaborowska et al., 2002; Chakraborti et al., 2003; Schubert et al., 2003). However, it must be noted that DNAzyme activities in the literature were evaluated under different reaction conditions, namely, substrate length and often differing in pH, divalent metal ion type and concentration.

Under multiple turnover conditions, MD14 was the only DNAzyme tested that showed an ability to cleave the long substrate under conditions of saturating substrate. This is not unusual as cleavage rates with long RNA molecules under multiple turnover conditions are reported to be difficult or even impossible (Thompson et al., 1996). To date, there are only a few published examples of multiple turnover kinetics performed on long substrates, which are significantly shorter in length than that tested here (Zhang et al., 1999; Goila et al., 2001; Schubert et al., 2003).

Zhang and colleagues performed multiple turnover kinetic analysis on a long substrate of 414 bases encoding the V3 loop of HIV-1 (Zhang et al., 1999). DNAzyme DzV3-9 -4 -1 -2 was shown to have a Kcat of 2.85x10 min , apparent Km of 9.41x10 μM and the 3 -1 -1 Kcat/Km was 3x10 M min against this transcript. However, cleavage conditions, which would have a proven effect on these values, were not outlined. Goila and co- workers investigated the kinetic parameters of three DNAzyme targeting a 465 base transcript under the same conditions outlined in this thesis. The Km of these -1 6 DNAzymes was found to be 0.1-0.3μM, the Kcat 1.6-3.7min and the Kcat/Km 5x10 - 3.7x107M-1min-1 (Goila et al., 2001). To date, the largest published in vitro transcribed substrate used in a multiple kinetic analysis was the 900 base rhinovirus substrate (Schubert et al., 2003). Single and multiple turnover experiments were performed

Chapter 4: DNAzyme Selection & Cleavage Activities 149

under the same conditions as outlined in this thesis (10mM MgCl2, 37˚C). The Kobs values determined under single turnover conditions for a number of DNAzymes ranged -1 -1 -1 from 0.009min to 0.9min , which were comparable to the Kobs of 0.157min seen for

MD14 (Table 4.3). However, no kinetic parameters such as Km and Kcat/Km were quantified due to technical impracticality and difficulty. Instead, their measurement of activity was based on the initial velocity. Considering the size of the PbCPSII transcript, the data compares favourable with the limited published values available from other groups.

Most published multiple turnover kinetic data has been of cleavage experiments performed on short, complimentary, synthetic RNA substrates. Kinetic experiments were performed on short substrates to further quantify the kinetic behaviour of the DNAzymes by eliminating the effect of RNA secondary structure on cleavage. Under -1 single turnover conditions, MD26 showed a slightly higher Kobs value (0.167min ) or cleavage efficiency than MD14 (0.150min-1). This is not surprising and indicates that the turnover or release of products is better for MD26 when secondary structure constraints are minimised signifying the MD26 region of the long substrate lies within a less accessible region when compared to the MD14 region.

What is also clear is the similarity in cleavage profiles seen with all DNAzymes under these conditions (Figure 4.11) when compared to the single turnover profiles on the long substrate (Figure 4.9). Apart from MD14, whose activity remained constant, all DNAzymes showed improved activity on the short substrate. The results are again most likely attributed to the lack of inhibitory secondary structure seen with the short substrate and indicate the relative importance of screening DNAzymes against a more complex RNA molecule. Nevertheless, screening DNAzymes against short substrates gives a good indication of the molecules that have optimal catalytic activity. Under multiple turnover conditions, MD25 displayed a lower Km value than MD14. However,

MD14 possessed a higher Kobs than MD25 under single turnover conditions, and a lower Km than MD26, as well the highest catalytic efficiency (Kcat/Km approaching the ideal 108M-1min-1) under multiple turnover conditions. It indicated MD14 possessed the most efficient balance between specific binding, cleavage efficiency and product release of the four DNAzymes examined. In addition, the kinetic values of MD14 appeared to be in good agreement with those found in the literature of Kcat (approx.

Chapter 4: DNAzyme Selection & Cleavage Activities 150

-1 6 8 -1 -1 1min ), Km (nano molar range) and Kcat/Km (10 -10 M min ) (Santoro et al., 1997; Sun et al., 1999; Liu et al., 2001; Lowe et al., 2001; Unwalla et al., 2001; Cieslak et al., 2002; Cairns et al., 2003; Schubert et al., 2004; Takahashi et al., 2004).

Finally, it is also noteworthy that the DNAzymes tested against the short substrate had the 3'-inverted thymidine modification that may or may not have affected catalytic efficiency. In a study by Sun and co-workers, a slight loss of cleavage activity to a four-fold increase in cleavage activity dependent on arm length, was reported with DNAzymes possessing the 3'-inverted thymidine modification (Sun et al., 1999). By contrast, Schubert and co-workers showed no significant influence of this type of modification on cleavage activity (Schubert et al., 2003). Nevertheless, modified and unmodified MD14 seemed to yield consistent and effective cleavage data on both short synthetic substrate as well as PbCPSII insertion II RNA and seemed like a promising candidate for further in vivo clinical studies.

CHAPTER 5

Plasmodium Inhibition Studies Chapter 5: Plasmodium Inhibition Studies 152

5 PLASMODIUM INHIBITION STUDIES

5.1 Experimental Background and Aims

As discussed previously, the 10-23 DNAzyme has been used to inhibit gene expression in cell culture and in animal models, by targeting the specific destruction of various cellular mRNAs. It has been shown that malaria-infected RBCs have the ability to spontaneously take up nucleic acids such as AS ODNs, ribozymes and DNAzymes from their external environment. These molecules appeared to be taken up exogenously and effectively and specifically reduce P. falciparum growth in vitro (Section 1.8). In particular, sub-micro molar amounts of these molecules targeted against PfCPSII have routinely reduced parasite cultures. The final aim of this thesis was to further assess a candidate DNAzyme in a rodent malarial in vivo system.

Rodent malaria models are widely used to screen antimalarial compounds and, although imperfect, there is consensus that use of this model is almost an obligatory step for in vivo studies of new antimalarial therapeutics (Landau et al., 1998). The P. berghei model was chosen for this study due to the vast literature available on antimalarial drug testing in this system.

After encouraging kinetic studies of the DNAzyme MD14 targeting PbCPSII insertion II (Chapter 4), a situation arose for industry partners to sponsor a preliminary in vivo assessment of MD14 against P. berghei infections in mice. It was appreciated that an in vitro based P. berghei culture system was available at the time, however, this was laborious and would involve high costs and time for establishment and optimisation. Therefore, it was decided by industry partners that the immediate priority or aim of this project was to conduct a brief in vivo trial in the prospect of obtaining rapid and rewarding results. It was noted that additional, appropriate controls should have been considered such as scrambled sequence and sense controls, however these were not considered an initial priority.

Results from this preliminary in vivo trial proved largely unfruitful. There appeared to be minimal inhibition of P. berghei infections in mice by MD14 that may have been attributed to a number of possible reasons. The rest of this study focused on problem

Chapter 5: Plasmodium Inhibition Studies 153

solving and clarifying some of these possibilities which included: the poor synthesis of MD14; sub-optimal dosage concentration and/or timing of dosage; unfavourable pharmacokinetics; the lack of endogenous uptake of MD14 into parasitised RBCs or simply the ineffectiveness of MD14. Throughout this work it was found that indeed the synthesis of MD14 was not optimal and that parasitised RBCs did not appear to spontaneously take up DNAzymes. Consequently, different DNAzyme delivery methods were also addressed.

5.2 Results

5.2.1 Inhibition of P. berghei In Vivo To test the efficacy of the DNAzyme MD14 in a P. berghei rodent malaria model, a large-scale amount of MD14 was synthesised with a 3'-inverted thymidine (two batches of 10mg each). The DNAzyme batches were resuspended in sterile PBS, combined and stored in daily dose aliquots at -20˚C. Initially, toxicity of MD14 was assessed by injecting five CBA mice with a dose of 5mg/kg and observing them over a seven-day period. No adverse effects were noted.

Four groups of mice (n=14 each) were tested in the trial: group A was given PBS; group B was given MD14 alone; group C was infected with P. berghei K173 and given the vehicle PBS (Pbk + PBS); group D was infected with P. berghei K173 and given MD14 (PbK + MD14). Animals were given a daily intravenous dose via the tail vein, from day 0 (relative to parasite inoculation) to day 4, inclusive. Percentage parasitaemia was determined microscopically on tail vein blood smears. Parasitaemias were initially determined on day 4 post infection (P.I) and every second day thereafter.

Figure 5.1 shows percentage parasitaemia counted for group C (Pbk + PBS) and group D (Pbk + MD14) over twelve days. Parasitaemia progression appeared normal for all PbK infected mice. Overall, only a slight reduction in parasitaemia was observed between the PbK + PBS control group and the PbK + MD14 treated group.

Chapter 5: Plasmodium Inhibition Studies 154

PBS 70 *** MD14

50 a

40 ***

*** % Parasitaemi 20

10 ***

*** 0 4681012 Day (P.I)

Figure 5.1 Inhibition of P. berghei in vivo by MD14. Mice were infected with PbK on day 0 and given either PBS or MD14 in PBS for four consecutive days. Tail vein smears were taken every second day from day 4 post- infection (P.I) to day 12 post-infection when mice were euthanased. Parasitaemia was assessed by slide visualisation for n=14 mice for each group. The standard error was calculated and the results were evaluated by the students T-test for each day analysed. ‘***’ denotes a highly statistically significant difference between the two groups of P<0.001.

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The most notable reduction was seen on day 10 post-infection (6.3% difference in % reduction in parasitaemia) as compared to the difference seen on other days (3-3.35%). Surprisingly, the slight reduction seen on the days assessed was highly statistically significant when compared to the control group as analysed by the students T-test. This is attributed to the high sample size and the small variation in % parasitaemia within each group.

5.2.2 Investigation into Low Levels of DNAzyme Inhibition 5.2.2.1 DNAzyme Integrity After the lack of positive results of the in vivo animal trial, the integrity of the commercially synthesised large-scale batches of MD14 was investigated. Prior to the trial, a sample of each batch was 5'-end labelled and assessed for integrity by PAGE (Figure 5.2). The two samples appeared to be intact and ran at the expected size compared to the original small-scale synthesis of the compound. After re-constitution, the batches were combined and aliquoted into daily doses for use in the animal trial.

After completion of the animal trial, the integrity of the inverted thymidine modification was assessed in order to eliminate this as the reason for the minimal effect seen in the rodent malaria system. A sample of the MD14 animal trial stock was again 5' end- labelled with γ-32P and incubated in 10% human serum in RPMI media at 37˚C (Section 2.6.2.1). A sample of the original, small-scale synthesis of MD14 with the same modification produced for the kinetic studies was used as a positive control. Aliquots were taken at 0, 2, 6, and 24 hours and were resolved on a polyacrylamide gel. The samples were visualised by exposure to a phosphorimaging system (Section 2.5.1.6).

The results indicate that as little as 6 hours after incubation in 10% human serum, there appeared to be notable differences in stability between large-scale and small-scale batches of MD14 (Figure 5.3). Furthermore, after 24 hours, the majority of the large- scale batch of MD14 had been degraded by nucleases whereas a comparatively higher amount of the small-scale modified MD14 remained intact.

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Lane 1 2 3 4

Lane 1. Unmodified MD14 Lane 2. MD14; large-scale batch 1 Lane 3. MD14; large-scale batch 2 Lane 4. MD14; small-scale

Figure 5.2 Integrity of MD14 DNAzymes from different batches. Various MD14 DNAzymes with the inverted-T modifications were 5' end-labelled with 32P, electrophoresed on a 20% polyacrylamide gel and exposed to a phosphorimaging screen. The modified DNAzymes were also compared to a 5' end-labelled unmodified MD14 DNAzyme. All DNAzymes appeared intact and ran at the expected sizes.

A B

T (h) 0 2 6 24 0 2 6 24

Figure 5.3 Stability of modified MD14 in 10% human serum. (A) Small-scale batch of MD14 with an inverted thymidine modification. (B) Large- scale batch of MD14 with an inverted thymidine modification. DNAzymes were 5' end-labelled and incubated in 10% human serum/RPMI media at 37˚C. Aliquots were taken at 0, 2, 6 and 24 hours, electrophoresed on a 20% polyacrylamide gel and exposed to a phosphorimaging screen.

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5.2.2.2 Selective Uptake of DNAzymes into Parasitised RBCs Past results based on ribozyme, DNAzyme and antisense inhibition of CPSII in P. falciparum were in support of the endogenous uptake of AS ODNs by parasitised RBCs. At the time of the study described herein, co-workers reported the apparent lack of reduction in parasitaemia by M5L, a DNAzyme that routinely inhibited growth of laboratory cultures of P. falciparum (data not shown). In order to investigate this, and to rule out the possibility that P. berghei infected RBCs were simply not taking up MD14, an uptake experiment was performed on in vitro cultures of P. falciparum.

The DNAzyme M5L was re-synthesised with a FITC molecule attached at the 5' end. The FITC-M5L DNAzyme (0.1μM, 1μM and 5μM) was incubated with 10% ring-stage P. falciparum infected RBCs for 4h or 24h (Section 2.6.2.5). The cells were washed and stained with dihydroethidium (DHE), a fluorophore that intercalates between the bases of DNA. As the human RBC is lacking DNA, infected and non-infected RBCs were able to be distinguished from each other. A sample of the DNAzyme-treated cells was analysed by confocal microscopy (Section 2.6.2.5) and the remainder by flow cytometry (Section 2.6.2.4). At all conditions investigated, there appeared no evidence of co-localisation and therefore no support of the endogenous uptake of FITC-M5L molecule into the parasite. Figure 5.4 is a representative confocal microscopy image of this general outcome.

Flow cytometry analysis was also in support of this finding, as there appeared to be no evidence of the green fluorescence emitted by FITC in parasitised RBCs (data not shown). Furthermore, incubation of FITC-M5L (0.1μM, 1μM and 5μM) and M5L (0.1μM, 1μM and 5μM) with parasitised RBCs (Section 2.6.2.2) yielded no significant reduction in parasitaemia when compared to untreated cultures (data not shown).

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10µm 10µm

10µm

Figure 5.4 Confocal microscopy of DNAzyme uptake into P. falciparum infected RBCs. A representative image showing: (A) Red channel indicating trophozoite parasite DNA stained with DHE, also visible is the fainter, auto-fluorescence of uninfected RBCs; (B) green channel indicating FITC-M5L; (C) overlay image of FITC-M5L and parasitised RBCs. The lack of yellow regions, or regions of overlap, indicate that no DNAzymes were taken up by the parasite.

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The possibility that the parasites had mutated in long-term culture due to extended use of gentamycin was also investigated. Frozen cell stocks, stored at the time the bioassays were showing effective inhibition of cultures, were revived and used in several inhibition bioassays. Analysis of parasitaemia revealed that there was no significant reduction of parasite growth when compared to an untreated control culture (data not shown).

At the time of this study, changes had been made to the P. falciparum tissue culture process within the laboratory. The human RBCs that were routinely used in tissue culture prior to this project, labelled ‘Malsep’, were from patients that had been to malaria endemic regions and were unable to be used by the Australian Blood Bank and hence, were always readily available for tissue culture. At the commencement of this project, this blood type was no longer accessible and instead, expired blood was used. To investigate whether this may have had an effect on uptake of M5L, a single batch of ‘Malsep’ blood was obtained and used in confocal microscopy experiments (Section 2.6.2.5) and P. falciparum inhibition bioassays (Section 2.6.2.2). After extensive uptake experiments, it was determined that there was no evidence of endogenous parasite uptake of the FITC-M5L. Furthermore, no inhibition of P. falciparum cultures by M5L (0.25μM, 0.5μM and 1μM) was observed (Figure 5.5A).

A new batch of serum (batch 2), pooled from twenty donors, was obtained and used in culturing the parasite. There appeared to be a DNAzyme dose-dependant reduction in parasitaemia with parasites grown in serum batch 2, however, this was low (5%) and was not significantly different to control cultures when analysed by students T-test (Figure 5.5B). Further, micro-titre plates (Nunc) were compared to the initial brand used (Greiner), in order to eliminate DNAzymes non-specifically binding to the plastic. There appeared to be no significant effect on the efficacy of M5L in vitro (parasites in ‘expired’ blood) with either plate examined (Figure 5.5C). Finally, parallel cultures were grown in both ‘malsep’ and ‘expired’ blood and used in 24h bioassays in the two different micro-titre plates. Again, no statistically significant effect on inhibition of parasite cultures by M5L was observed (Figure 5.5D).

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0.25µM M5L A 0.50µM M5L 1.00µM M5L

0 % Growth Reduction % Growth -5 U Ma Expi n trea lse red -10 ted p Blood

0.25µM M5L B 0.50µM M5L 1.00µM M5L

0 Un B Batch atch % Growth Reduction tr eate -5 1 2 d

-10 Serum

Figure 5.5 P. falciparum 24h bioassays with M5L. Ring-stage parasites were incubated with M5L or PBS (untreated) for 24h, when the cells were harvested and assessed for parasitaemia. Parasites were grown in: (A) ‘malsep’ or ‘expired’ RBCs; (B) two different batches of serum; (C) “Nunc’ and ‘Greiner’ microtitre plates; and (D) combinations of the two blood samples in different microtitre plates. Bioassays were performed in triplicate on different days and the mean and standard deviation calculated. Analysis by students T-test revealed no significant differences between all M5L treated and untreated cultures. All bioassays were performed blind and the code revealed after analysis of parasitaemia.

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C 0.5µM M5L 1.0µM M5L

-5 % Growth Reduction U Nunc Gr -10 nt e reated ine r -15 Microtitre Plate

D 0.5µM M5L 1.0µM M5L

-5

% Growth Reduction U Mals M Expired/ E n x treate alse pi re -10 ep/ p /G d d N N /G un re unc re c in in -15 e r er Blood/Microtitre plate

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5.2.3 Optimising DNAzyme Delivery As the uptake and efficacy of the fluorescent-labelled DNAzyme M5L could not be detected under the bioassay conditions previously reported (Flores et al., 1997), other methods of delivery were to be studied. Namely, exogenous delivery of DNAzymes to P. falciparum infected RBCs with the aid of carrier molecules was investigated.

5.2.3.1 Liposomal Delivery Methods The ability of nine commercially available cationic transfection reagents to deliver FITC-labelled M5L into P. falciparum infected RBCs was evaluated using both flow cytometry and confocal microscopy by co-workers in the laboratory (Hillier and Eiszele, personal communication). In summary, four transfection reagents (Dmrie-C, DOTAP, Geneporter and Cellfectin) successfully delivered the fluorescent DNAzyme into both infected and uninfected RBCs with various levels of efficiency (Table 5.1). Geneporter appeared to lyse the majority of the cells, even at the lowest concentrations used to facilitate uptake, but was found to give the highest percentage uptake in the remaining intact cells. Dmrie-C and Cellfectin were found to give the highest transfection efficiency whilst maintaining cellular integrity. In uptake studies utilising confocal microscopy, FITC-M5L was detected inside both uninfected and infected RBCs when either Dmrie-C or Cellfectin was the carrier (Table 5.1).

P. falciparum inhibition bioassays were performed to determine whether DNAzymes encapsulated by Dmrie-C or Cellfectin, after being taken up by infected RBCs, were able to inhibit parasite growth. Ring-stage parasites were dosed at T=0 and 24 h with 1μM M5L and samples were taken at 24h and 48h for analysis. There appeared to be no effect of M5L on parasite growth as evaluated by microscopic analysis and flow cytometry (Hillier and Eiszele, personal communication). Further transfection of the chloramphenicol acetyl transferase (CAT) reporter plasmid complexed with the above- mentioned liposomal reagents yielded no CAT activity, indicating that the DNA was not released from the complex.

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Transfection % Uptake by flow Uptake detection Effect on Reagent cytometry by confocal parasite microscopy growth DOTAP 10-15% Cellfectin 30-35% + - Dmrie-C 30-35% + - Lipofectin - Lipofectamine - Lipofectamine 2000 - Geneporter 98% Geneporter 2 2% Fugene 6 -

Table 5.1 Transfection reagent mediated delivery of M5L. Uptake of 1μM FITC-labelled M5L into infected RBCs after 4h incubation at as detected by flow cytometry and confocal microscopy.

5.2.3.2 Methylpyrroporphyrin Conjugates As co-workers were investigating the effectiveness of cationic transfection reagents in the delivery of DNAzymes to P. falciparum parasites, investigation into novel carrier molecules was addressed. Porphyrin derivatives have been shown to be effective delivery vehicles for antisense molecules (Benimetskaya et al., 1998; Flynn et al., 1999; Dass, 2002; Kocisova et al., 2006). A collaboration was established with T. Rede and D. Ma (St Vincents Hospital, Sydney) for conjugation of methylpyrroporphyrin (MPP) to DNAzymes M5L and MD14 (Figure 5.6A).

Three HPLC purified MPP-conjugates were synthesised by collaborators and included: MPP conjugated to M5L (MPP-M5L) for use against P. falciparum cultures; MPP conjugated to MD14 (MPP-MD14) as a negative control against P. falciparum cultures. Both molecules were purchased with a single phosphorothioate linkage at the 3' end for protection against nucleases (Figure 5.6B). In addition, an MPP-M5L DNAzyme with a FITC molecule at the 3' end (MPP-M5L-FITC) was also produced, to be used in uptake studies.

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Figure 5.6 Methylpyrroporphyrin conjugates. (A) Chemical structure of methylpyrroporphyrin conjugated to an ODN (Rede, 2004). Reproduced with permission. (B) Schematic representation of methylpyrroporphyrin conjugated DNAzymes synthesised for inhibition of Plasmodium parasites. MPP is depicted in red; FITC is depicted in green and a 3' phosphorothioate linkage is depicted in blue.

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5.2.3.2.1 Uptake of MPP DNAzymes into P. falciparum Infected RBCs Initially, control cells that included uninfected RBCs and parasitised RBCs, were used to establish optimal laser scanning confocal microscope settings by Mr Paul Hasalz (The Histology and Microscopy Unit, University of NSW). Both control groups were divided into two samples, one of which was stained with DHE. The unstained samples allowed autofluorescence background levels of RBCs and parasitised RBCs to be determined. In addition, the stained samples allowed any excess DHE or bleeding of red light emission into the green channel to be minimised.

Once the settings for the confocal microscope were optimised, 10% ring-stage parasites (2% RBC hematocrit) were incubated with the MPP-M5L-FITC conjugate (0.5μM, 1μM and 5μM). As the conjugate was light sensitive, all work was done with minimal light exposure and micro-titre plates were wrapped in aluminium foil and incubated at 37°C. The cells were harvested at 4h (rings) or 24h (trophozoites), washed and stained with DHE as described in Section 2.6.2.5. Only under the conditions of 5μM incubation and trophozoite stage, did there appear to be evidence of localisation of FITC emission with parasite DNA. Figure 5.7 illustrates one such example of a field containing a trophozoite infected RBCs stained with DHE after incubation with MPP- M5L-FITC DNAzyme. Two mature trophozoites and a young trophozoite are indicated in the centre of the field (Figure 5.7A). Also evident is the autofluorescence of uninfected RBCs. In the green channel, three fluorescing spots indicate positions of FITC localisation (Figure 5.7B). When the images are superimposed, it is clear that two of these spots localise to the mature trophozoites as indicated by the yellow colour. The third spot appears to localise to the surface of the RBC membrane, suggesting that the MPP-M5L-FITC conjugate is only being taken up by mature, trophozoite-infected RBCs.

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Figure 5.7 Confocal microscopy of DNAzyme uptake into trophozoite-stage P. falciparum infected RBCs. A representative image showing: (A) red channel indicating trophozoite parasite DNA stained with DHE. Also visible is the fainter, auto-fluorescence of uninfected RBCs; (B) green channel indicating MPP-M5L-FITC DNAzymes; and (C) Overlay image of MPP-M5L-FITC and parasitised RBCs. White arrows indicate regions of DNAzyme uptake into parasites. The yellow arrow indicates an infected RBC that has not taken up the DNAzyme. The green arrow signifies DNAzyme/s on the surface of an uninfected RBC.

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Even though endogenous uptake in parasitised cells was conclusively demonstrated by confocal microscopy, analysis with flow cytometry was inconclusive. Parasites were incubated with MPP-M5L-FITC conjugates as stated previously (Section 2.6.2.4). Uninfected and infected RBCs were incubated with MPP-M5L-FITC and half the sample stained with DHE. Controls for establishing flow cytometry settings included stained and unstained samples of uninfected and infected RBCs. Over 100 000 cells per sample were analysed by examining two fluorescence channels; green fluorescence (FL1) and red fluorescence (FL2) (Section 2.6.2.4). Figure 5.8 shows representative histograms of untreated, DHE stained, ring-stage infected erythrocytes. Figure 5.9 shows representative histograms of DHE stained, ring-stage infected erythrocytes incubated with the MPP-M5L-FITC (1μM) once the flow cytometer settings had been established with control populations. No visible increase in green events was detected in the cell sample incubated with the MPP-M5L-FITC conjugate (1μM). Representative histograms of untreated, DHE stained, trophozoite-stage infected erythrocytes are shown in Figure 5.10. Representative histograms of DHE stained trophozoite-stage infected erythrocytes incubated with the MPP-M5L-FITC (1μM), after the flow cytometer settings had been established with control populations, is shown in Figure 5.11. In this case, a slight but not statistically significant increase in FITC events was detected in the cell sample incubated with the MPP-M5L-FITC conjugate.

It was difficult to establish whether any cells were taking up MPP-M5L-FITC on account of the negligible events detected in the FITC channel. There appeared to be a statistically significant increase (P<0.05) in highly fluorescing red events in the FL2 channel for both ring and trophozoite parasites after incubation with the DNAzyme conjugate. However, there was no specific quantification or conclusive evidence of MPP-M5L-FITC uptake into parasitised cells by flow cytometry.

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RBCs

RBCs pRBCs

Figure 5.8 Flow cytometry histograms of DHE stained P. falciparum rings. The top panel depicts the FL1 channel with increasing green fluorescence on the x-axis versus increasing number of cells on the y-axis. The bottom panel depicts the FL2 channel with increasing red fluorescence on the x-axis versus increasing number of cells on the y-axis. The main cell population in both channels are uninfected red blood cells (RBCs: R9, R6) as determined by negative control samples (not shown). The R8 population in the FL2 channel indicate parasitised cells (pRBCs) fluorescing red from the presence of DHE. In this sample, 13.32% of RBCs are showing infection with ring- stage parasites. The R10 population in the FL1 channel indicates background green fluorescence (1.51%).

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RBCs

RBCs pRBCs

Figure 5.9 Flow cytometry histograms of DHE stained P. falciparum rings incubated with MPP-M5L-FITC. The top panel depicts the FL1 channel with increasing green fluorescence on the x-axis versus increasing number of cells on the y-axis. The bottom panel depicts the FL2 channel with increasing red fluorescence on the x-axis versus increasing number of cells on the y-axis. The main cell population in both channels are uninfected red blood cells (RBCs: R9, R6) as determined by negative control samples (not shown). The R8 population in the FL2 channel indicate parasitised cells (pRBCs) fluorescing red from the presence of DHE. In this sample, 16.66% of RBCs are showing infection with ring- stage parasites. The R10 population in the FL1 channel indicates cells fluorescing green from the presence of FITC (1.31%).

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RBCs

RBCs pRBCs

Figure 5.10 Flow cytometry histograms of DHE stained P. falciparum trophozoites. The top panel depicts the FL1 channel with increasing green fluorescence on the x-axis versus increasing number of cells on the y-axis. The bottom panel depicts the FL2 channel with increasing red fluorescence on the x-axis versus increasing number of cells on the y-axis. The main cell population in both channels are uninfected red blood cells (RBCs: R9, R6) as determined by negative control samples (not shown). The R8 population in the FL2 channel indicate parasitised cells (pRBCs) fluorescing red from the presence of DHE. In this sample, 12.49% of RBCs are showing infection with trophozoite-stage parasites. The R7 population in the FL1 channel indicates background green fluorescence (0.36%).

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RBCs

RBCs pRBCs

Figure 5.11 Flow cytometry histograms of DHE stained P. falciparum trophozoites incubated with MPP-M5L-FITC. The top panel depicts the FL1 channel with increasing green fluorescence on the x-axis versus increasing number of cells on the y-axis. The bottom panel depicts the FL2 channel with increasing red fluorescence on the x-axis versus increasing number of cells on the y-axis. The main cell population in both channels are uninfected red blood cells (RBCs: R9, R6) as determined by negative control samples (not shown). The R8 population in the FL2 channel indicate parasitised cells (pRBCs) fluorescing red from the presence of DHE. In this sample, 15.44% of RBCs are showing infection with trophozoite-stage parasites. The R7 population in the FL1 channel indicates cells fluorescing green from the presence of FITC (0.66%).

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5.2.3.2.2 P. falciparum Inhibition Bioassays with MPP DNAzymes The efficacy of MPP-M5L at inhibiting P. falciparum cultures in vitro was quantified. MPP-MD14 targeting P. berghei CPSII was used as a negative control DNAzyme. Both DNAzyme conjugates (1nM, 10nM and 100nM) were incubated with 4% synchronous ring-stage infected RBCs for 24h in a microtitre plate (Section 2.6.2.2). After 24h, a sample was taken from each triplicate well and combined to prepare a fixed microscopic slide for visual cell counts (Section 2.6.2.3). The slides were blinded and randomised by a colleague and a minimum of 1000 cells per slide was counted, after which the code was revealed. The remainder of the parasites from triplicate wells were pooled, blinded and processed for flow cytometry (Section 2.6.2.4). The mean and standard deviation of the mean of percentage parasite growth reduction was calculated from the combined microscopic and flow cytometry counts. Statistical analysis was performed on the data by use of the students T-test. The findings are summarised in graphical format in Figure 5.12.

After 24h in the presence of the MPP DNAzymes, up to 15% reduction in parasitaemia was observed for MPP-M5L and 7% for MPP-MD14 at the lowest concentration of DNAzyme used (1nM) (Figure 12A). However, there was no statistically significant difference found between the effects of the two DNAzymes upon parasite growth. At 10nM of MPP-M5L, a reduction in parasitaemia of up to 25% was observed and although MD14 also inhibited P. falciparum cultures (13.5%), the effect of MPP-M5L was significantly higher (P<0.05). This difference in the action of the DNAzymes was most notable at 100nM (P<0.001) even though the reduction in parasitaemia was unusually lower than that observed at 10nM.

After 48h in the presence of 1nM MPP DNAzymes, parasite inhibition was slightly higher than that observed at 24h (Figure 12B). However, there appeared to be no significant difference between the two compounds indicating that MPP-MD14 was non- specifically inhibiting P. falciparum cultures. At 10nM DNAzyme concentration, parasite cultures were inhibited by 30% by MPP-M5L and 26% by MPP-MD14 after 48h, again indicating non-specific activity. Similar to the 24h bioassay, a lower amount of growth reduction was seen the at 100nM concentration of 23% by MPP-M5L and

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10% MPP-MD14. In this instance, the effect on cultures by MPP-M5L was statistically significantly different to that seen by MPP-MD14.

A M5L MD14 40

25 * 20 ** 15

% Reduction in parasitaemia 5

0 1nM 10nM 100nM [Dz]

B M5L MD14

30 * 25

% Reduction in parasitaemia 5

0 1nM 10nM 100nM [Dz]

Figure 5.12 Inhibition of P. falciparum in vitro cultures by MPP-DNAzymes. Ring-stage parasites were incubated in varying concentrations both MPP-M5L and MPP-MD14 for 24h (A) and 48h (B). Growth reduction (%) was calculated by assuming untreated control cultures to be 100%. Parasitaemia was assessed by flow cytometry and slide visualisation for three experiments and the mean and standard deviation determined. MPP-M5L and MPP-MD14 were compared by the students T- test where ‘*’ signifies significant difference where P<0.05 and ‘**’ where P<0.005.

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5.3 Discussion

5.3.1 DNAzyme Suppression of P. berghei In Vivo The MD14 efficacy trial was established in order to examine the suppression of P. berghei infections in a rodent system. The trial conducted was based on the 4-day suppressive test commonly used to assess antimalarial treatment in rodent models and was performed as initially described by Peters (Peters, 1965) with some modifications (Section 2.6.1). The preliminary design of the trial was rudimentary and involved four groups of fourteen mice, two of which included MD14-treated (5mg/kg/day) and saline- treated mice. The trial served to gauge whether MD14 had any effects at all on suppression of parasitaemia and was by no means a comprehensive in vivo study of the effect terms of the DNAzyme MD14.

Analysis of parasitaemia from the MD14 treated mice, compared to the untreated mice, indicated the administration of MD14 effected a highly statistically significant reduction of P. berghei parasitaemia. However, the absolute effect on parasitaemia was minimal, with an average of 3-4% reduction in parasitaemia seen. On Day 4, the reduction in parasitaemia was minimal at less than 1% of control mice. After Day 4, the reduction in parasitaemia was 3-4% lower than control mice indicating that the last day of administration of MD14 seemed to have a positive effect on reduction in parasitaemia. Therefore this minimal effect observed is most likely attributable to the early suppression of parasitaemia in the first four days post-infection. The initial aim was in fact to reduce the low level of parasitaemia in the first four days, enough to allow the immune system to clear the parasite. However, under the outlined conditions, this seemed inadequate for the long-term reduction of parasitaemia. These results are encouraging and although the reduction was minimal, it was a highly statistically significant reduction in parasite growth in vivo and further investigation seems warranted. Many avenues of the in vivo study warrant further research.

Research into the dosing schedule may generate improved efficacy of MD14. Treatment with MD14 could be delayed until there are detectable levels of parasitaemia and by maintaining the drug for a greater length of time, the effect seen may be greater. A study using AS ODNs complementary to exp-1 mRNA, demonstrated that by injecting P. berghei infected mice via the intraperitoneum at the first sign of

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parasitaemia (2-3 days post-infection) daily for six consecutive days, the life span of treated mice was extended by a factor of 2-4 (Tan et al., 1996). This approach seems particularly promising as the reduction in parasitaemia appeared greater after Day 4, the last day of drug administration. This type of approach may also be more effective in assessing the efficacy of DNAzymes targeting Plasmodium CPSII, as this appears to be the only scientific work published to date on the successful administration of nucleic acid therapy in Plasmodium infected mice.

An alternative dosing strategy may yield interesting results and could involve mixing the DNAzyme and parasite together prior to injection to optimise uptake into parasitised red blood cells and minimise clearance of the DNAzyme in the biological system. However, this does not realistically represent the potential administration of treatment of humans infected with malaria. Dosing over a longer period of time may also significantly lower the levels of parasitaemia observed in this study. Akhtar and colleagues claim that the biological effects of AS ODNs are usually short-lived and are likely to require repeated administration (Akhtar et al., 2000). Furthermore, it has been observed that repeated daily intravenous injections of a phosphorothioated AS ODN in rats, are required to maintain concentrations in the plasma at steady-state levels due to rapid clearance (Agrawal et al., 1995a; Agrawal et al., 1995b).

Examination of the dose of MD14 administration is necessary. There are many references concerning successful DNAzyme doses used in vivo (Table 1.4). However, the doses used are low (usually μg quantities/kg body weight) compared to that used in this study (5mg/kg), as they are administered locally to areas such as the arterial wall in cardiovascular disease

By reproducing successful ribozyme and antisense dosages injected directly into the blood stream, we may have more success in terms of a greater effect on the inhibition of parasites. At the time this in vivo study was conducted, the dosage of MD14 chosen was based on two successful antisense mouse trials in the literature. Monia and co- workers successfully inhibited lung tumour growth by intravenous administration of 6mg/kg of anti-c-raf kinase antisense (Monia et al., 1996). Further, Klasa and colleagues showed that by targeting BCL-2 with an intravenous antisense dose of 5mg/kg/alternate day, they were able to significantly increase the life span of SCID

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mice with non-Hodgkin’s lymphoma (Klasa et al., 2000). ISIS 3521 dosed at 2 or 3 mg/kg also had no effect (Marshall et al., 2004). Tan and co-workers showed that as little as 0.8mg/kg of an AS ODN targeting exp-1 mRNA in P. berghei showed a significant effect at prolonging the life span of mice (Tan et al., 1996). However, as stated above, the dosing regime was substantially different to that used in this study.

The lack of effect seen may be due to unfavourable pharmacokinetics. The pharmacokinetic properties of phosphodiester and phosphorothioate AS ODNs have been well documented and parameters appear to be determined by the chemistry of the molecules rather than the actual nucleotide sequence (Jason et al., 2004). At the time this study was carried out, there had been no investigation into the pharmacokinetic properties of 3'-inverted thymidine modified molecules. However, one would expect similar behaviour to the parent phosphodiester ODN due to the modification having the same size and charge as other nucleotides.

Intravenous injection of AS ODNs showed that the oligonucleotide is degraded rapidly in the plasma with a half-life of about 5 minutes (Agrawal et al., 1995b). In addition, AS ODNs are eliminated from the body rapidly (1h) following administration (Ma et al., 2000). Phosphorothioated AS ODNs, on which most pharmacokinetic studies have been performed, are also rapidly cleared from the plasma in a dose-dependant manner, with plasma half-life ranging from 30-60 minutes (Geary et al., 2002). Agrawal and co- workers (1998) reported minimal uptake of antisense molecules by peripheral blood cells and preferential clearance of the molecules predominantly to the kidneys and liver. Systemically administered AS ODNs are strongly bound to serum proteins such as albumin and α2-macroglobulin with binding affinities in the micro to milli molar range (Levin, 1999; Geary et al., 2002). In addition, Saijo and co-workers (Saijo et al., 1994) found that when oligonucleotide ISIS3466 was injected intravenously, it was quickly cleared from the circulation in normal mice but was readily absorbed into the systemic circulation when injected via the peritoneum. Intravenous injection was chosen as the method for delivery in this study as the site of action of the asexual stage P. falciparum malaria in the RBC. However, as this subjects the DNAzyme to rapid clearance, intra- peritoneal injection may prove more effective in the reduction of parasitaemia in Plasmodium infected mice. Alternatively, continuous intravenous infusion may

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circumvent the rapid plasma clearance.

Direct injection of antisense and DNAzyme molecules into the blood stream also subjects DNAzymes to an abundance of serum nucleases. Complete degradation of naked DNA occurs rapidly in these circumstances. In a study by Dass and co-workers (Dass et al., 2002), unmodified DNAzymes were found to have a half-life of approximately 70min in human serum and plasma compared to the 3' inverted thymidine analogue, whose half-life was approximately 22h. Investigation into the large-scale synthesis of MD14 purchased for the animal trial revealed a lower stability of the molecule in the presence of human serum compared to smaller scale production of the DNAzyme used in kinetic studies indicating conditions for the animal trial were sub-optimal. The only difference between modified and unmodified DNAzyme, in this case, is the 3' linkage of the terminal thymidine (Figure 1.11). Both DNAzymes have the same size and nucleotide composition and therefore would not be detectable by polyacrylamide gel electrophoresis. This single difference between modified and unmodified DNAzyme can go undetected by HPLC methods that rely on separation based on differences in hydrophobicity, particularly at a larger scale when quality control would be more difficult. The inevitable degradation of the poorly synthesised large-scale MD14 in the presence of serum nucleases would most definitely have contributed to the lack of efficacy of this compound.

Finally, the lack of effect seen may be due to the inability of the parasitised RBCs to take up the DNAzyme or simply that the DNAzyme was ineffective at reducing target CPSII mRNA.

5.3.2 DNAzyme Efficacy against P. falciparum In Vitro After the completion of the P. berghei in vivo trial, it became apparent that co-workers in the laboratory were unable to detect reduction of P. falciparum laboratory cultures by M5L. The current inability of M5L to have any effect on parasite growth led to the question of whether in fact the DNAzymes were getting into the parasite and how well they were taken up. Fluorescently labelled M5L molecules were employed in P. falciparum bioassays to detect uptake using flow cytometry and confocal microscopy. Uptake of fluorescent-labelled oligonucleotide could not be detected under

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the bioassay conditions previously reported (Flores et al., 1997). The possibility of reduced availability or the impaired uptake of the DNAzyme required further exploration.

As discussed in Section 1.8, there has been notable debate regarding the specificity of antisense molecules directed at Plasmodium. Some investigators claim antisense reduction of parasite proliferation is attributable to non-specific hindrance of the re- invasion process instead of sequence specific down-regulation. It is therefore, possible that AS ODNs may be adhering to the sialic acid on the surface of erythrocytes instead of being taken up. In contrast, there is significant experimental evidence of the ready uptake of nucleic acids by parasitised erythrocytes in the literature. Rapaport et. al., reported the entry of radio-labelled AS ODNs into parasitised RBCs but not uninfected RBCs (Rapaport et al., 1992). Dawson and co-workers also supported this by reporting that uptake of 32P-labelled AS ODNs by non-parasitised erythrocytes was 1% of that observed by parasitised RBCs (Dawson et al., 1993).

The possibility that the parasites had mutated in long-term culture due to extended use of gentamycin was considered. Frozen cell stocks dating to the time the original study was conducted were used in uptake experiments and inhibition bioassays. Furthermore, culture conditions had been slightly changed over time. Such changes may have altered the availability of DNAzymes or the parasite’s ability to take up these molecules. 'Malsep' blood (previously used) and expired blood (currently used) were concurrently utilised to culture P. falciparum parasites for uptake studies and bioassays with M5L. Different types of micro-titre plates were examined along with differing batches of human serum used in culturing in bioassays. All of these variables were tested individually (in triplicate) and none resulted in the percent reduction in parasitaemia previously reported.

Previous inhibitory effects observed with M5L were only based on microscopic examination of thin blood smears. Microscopic examination of thin smears has shortcomings in the limited numbers of cells that can be analysed and therefore inaccuracies in determining the parasitaemia. It is also difficult to distinguish between live and sickly or dead parasites and the different life stages of the parasite. Although a recent study demonstrated similar results between microscopic counts and flow

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cytometry counts of five established antimalarial drugs (Contreras et al., 2004), it was found that flow cytometry was advantageous in analysing the effect of these drugs on different life cycle stages. In this study, as opposed to previous work, a thorough approach was taken that involved microscopic evaluation and flow cytometry to determine the levels of parasitaemia.

The apparent lack of uptake and efficacy of M5L observed in this study could not be explained and may be due to an unknown factor in the tissue culture or bioassay process that may have changed conditions over time. So the direction of the work changed to alternative methods of delivery. Exogenous delivery of DNAzymes to P. falciparum infected RBCs with the aid of a carrier molecules, such as cationic lipids and MPP- conjugates, was analysed.

Co-workers screened the ability of nine commercially available transfection reagents to deliver a fluorescent-labelled DNAzyme into P. falciparum infected RBCs using both flow cytometry and confocal microscopy. The general finding was that two of the transfection reagents, Dmrie-C and Cellfectin, were found to give the highest transfection efficiency and highest mean fluorescence, while maintaining cell integrity (Hillier and Eiszele, unpublished). Both transfection reagents were able to non- specifically transport FITC-labelled M5L into both uninfected and infected red blood cells. However, further bioassays with the liposomal DNAzyme complex did not have a significant effect on parasite survival. The results demonstrated that the DNAzymes were either not released from cationic lipid complex or that the DNAzymes were simply ineffective in reduction of the target mRNA. It appears the former may be the case as further studies showed no CAT activity when the CAT reporter plasmid was complexed with these liposomes and transfected into P. falciparum infected RBCs (Hillier and Eiszele, unpublished).

In the past, porphyrin derivatives have been shown to display promising properties with respect to transporting AS ODNs across various cell membranes (Benimetskaya et al., 1998; Flynn et al., 1999; Dass, 2002; Kocisova et al., 2006) and have also been shown to have low toxicity, serum compatibility and nuclease protective ability (Kralova et al., 2003). The uptake studies performed with the MPP-M5L-FITC conjugated DNAzyme demonstrated co-localisation of green fluorescence associated with these molecules to

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regions of red fluorescence associated with parasite DNA, indicating specific parasite uptake of the conjugate DNAzymes. However, levels of uptake determined by confocal microscope visualisation were low. Furthermore, co-localisation of fluorescence was only visualised after 24h when parasites were at trophozoite-stage and at the highest concentration of DNAzyme used (5μM). At ring-stage, the parasites have minimal DNA content and hence detection of parasites would have been difficult due to the much lower sensitivity of confocal microscopy compared to flow cytometry. The high concentration required for visualisation is not uncommon in confocal microscopy of MPP conjugates. In a study assessing uptake of MPP-conjugated antisense into DHL4 lymphoma cells, it was found that incubation with 1μM of the conjugate was needed for detection of cell uptake by flow cytometry compared to 20μm by confocal microscopy (Naqvi, 2002). In addition, the number of cells able to be screened by confocal microscopy is lower and uptake of some cells may have been missed. A more likely explanation is that uptake was observed after 24h due to the appearance of the NPPs that appear within the parasitised RBCs 12-16 hours after invasion (Ginsburg et al., 1985; Rapaport et al., 1992; Taraschi, 1999).

Uptake as assessed by flow cytometry did not yield conclusive data. There appeared to be few or no events detected in the green fluorescence channel. In contrast, there was a statistically significant increase in the red channel of parasites incubated in the presence of the conjugate DNAzyme compared to control ring and trophozoite parasites. This may indicate the additional far-red fluorescence and in turn, presence of methylpyrroporphyrin. However, more complex studies need to be conducted where the parasite stain can be more readily distinguished from the red fluorescence of porphyrin. It was expected that a corresponding increase in the FITC channel would have been observed but this wasn't the case. There is the possibility that the porphyrin moiety or FITC moiety may have become dissociated or degraded from the conjugate DNAzyme in the cell, or by degradation caused by light or freeze-thawing. The lack of green fluorescence can also be explained by the work of Benimetskaya and colleagues who demonstrated a substantial quenching of fluorescein emission by porphyrin in porphyrin:ODN complexes (Benimetskaya et al., 1998). They specified that a molar ratio of 1:2 of porphyrin to oligonucleotide resulted in virtually 100% quenching being observed. They also observed, as was found in the course of this work, that regions of yellow indicating co-localisation in fluorescence microscopy were infrequent. They

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claim that this was probably attributed to quenching or dissociation of the porphyrin moiety.

Preliminary inhibition bioassays were performed with MPP-M5L, and the negative control DNAzyme MPP-MD14. In a 24h P. falciparum bioassay, ring-stage parasites were incubated in the presence of the conjugate DNAzymes and allowed to progress through to late-trophozoite stage when they were harvested. Concentrations of DNAzymes were kept at 100nM and below to minimise any non-sequence specific effects due to the phosphorothioate linkage, as was observed by Wanidworan and colleagues (Wanidworanun et al., 1999). After 24h, it was found that there was a significant reduction in parasitaemia in MPP-M5L treated cultures (10nM and 100nM) compared to the negative control DNAzyme MPP-MD14 designed against P. berghei. This finding cannot be attributed to the non-specific blocking of the re-invasion process postulated by Ramasamy and colleagues as the parasites were harvested before this stage (Ramasamy et al., 1996).

A 48h P. falciparum bioassay involved allowing ring-stage parasites to go through the full asexual cycle where merozoites are released and allowed to re-invade more RBCs. After 48h incubation, it was found that there was a substantial reduction in parasitaemia by both DNAzymes at 1nM and 10nM. There appeared to be no significant difference between the MPP-MD14 and MPP-M5L except at the highest concentration used (100nM), where the difference between the two DNAzymes was significant. In general, the highest concentration of MPP-M5L showed the most notable and significant difference compared to MPP-MD14. Overall levels of inhibition were lower at 100nM concentrations and may possibly be explained by the aggregation effect of high porphyrin concentrations noted in the literature. It has been observed when cationic porphyrins bind to the phosphodiester backbone of an ODN, their molecules form extended aggregates on the ODN surface or precipitate (Benimetskaya et al., 1998; Kralova et al., 2003). The comparison here is tentative, as it is difficult to compare data obtained in this study because there is a physical linkage between the DNAzyme and the porphyrin as opposed to the porphyrin:ODN complexes used in the noted studies. However, Rede noted a similar response with MPP-conjugated AS ODNs (Rede, 2004).

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The ability of MPP-MD14 to inhibit cultures almost as effectively as MPP-M5L was evident, particularly after the parasites had completed the asexual cycle. This reduction in parasitaemia observed can be attributed to a number of non-specific effects.

A blast search using MD14 sequence against the P. falciparum genome was conducted to investigate a sequence-specific effect of MD14. The blast search revealed no significant matches between MD14 and the P. falciparum genome and therefore DNAzyme binding to other regions of the genome or transciptome is unlikely. Other sequence specific non-antisense effects can be attributed to specific nucleotide motifs, such as CpG and G-quartets that engage in specific interactions with DNA or proteins. However, the MD14 sequence used in this study does not contain any nucleotide motif known to inhibit cell growth at this point in time. This does not eliminate the possibility that growth inhibition is due to an unknown nucleotide motif that causes sequence-specific non-antisense activity.

The high levels of parasite inhibition by MPP-MD14 may also be attributed to a non- sequence specific effect usually as a result of the chemical nature of the oligonucleotide. A possible explanation for this non-specificity is the phosphorothioate effect explained by Ramasamy and colleagues (Ramasamy et al., 1996). Although, in this case the single phosphorothioate linkage at the 3' end was minimal and others have reported little non-specific activity with chimeric phosphorothioated antisense, particularly at the low concentrations used in this study (Barker et al., 1996; Barker et al., 1998; Wanidworanun et al., 1999).

There is much documented literature on the non-specific effects of different backbones of AS ODNs but little on the effects of the MPP molecule. It has been demonstrated in one study that porphyrins are G-quadruplex interactive agents that cause telomerase inhibition by chromosomal destabilisation (Izbicka et al., 1999). This could also explain the pronounced inhibition of P. falciparum cultures by both MPP-DNAzymes after parasite replication.

Rede observed significant inhibition of leukaemia cell lines by MPP-conjugated antisense and was able to conclusively demonstrate no adverse effects of MPP alone on these cell lines at concentrations under 50μM (Rede, 2004). However, it is not feasible

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to compare two completely different cell systems. Studies have been conducted on the effects of ferriprotoporphyrin IX (ferric heme), an intermediate of the malarial haemoglobin degradation pathway. It was demonstrated that this molecule is able to lyse normal erythrocytes (Chou et al., 1981), P. berghei infected erythrocytes (Orjih et al., 1981) and P. falciparum infected erythrocytes (Fitch et al., 1982). The apparent toxicity is reported to be a common characteristic of biological membranes. However, the concentration of ferric heme used in the P. falciparum study was 1mM. Even if comparisons could be made between two different porphyrin molecules, the concentration used in this study was 4-6 orders of magnitude lower than that in the study by Fitch and colleagues. In addition, lysis of cells in the bioassays was not apparent by visible comparison of cell pellets harvested in untreated and treated samples.

In hindsight, investigation into the possible adverse effects of MPP on malaria-infected red blood cells should have been addressed in this preliminary study. Despite the inhibitory effect of the control DNAzyme conjugate, it was clear that the MPP-M5L showed superior ability to reduce parasite growth under certain conditions. Further examination of the MPP-conjugates as antimalarials is justified.

CHAPTER 6

Conclusions and Future Directions

Chapter 6: Conclusions & Future Directions 185

6 CONCLUSIONS AND FUTURE DIRECTIONS

The de novo pyrimidine biosynthetic pathway of Plasmodium falciparum, the causative agent of malaria, is ideal for chemotherapeutic attack as the parasite is unable to salvage pre-formed pyrimidines and must rely on de novo synthesis Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo pyrimidine biosynthetic pathway of Plasmodium falciparum. The gene encoding CPSII from P. falciparum has previously been isolated and characterised in our laboratory and has a unique feature when compared to the human host CPSII gene. P. falciparum CPSII is much larger than the human counterpart due to presence of two large insertion sequences encoding 232 amino acids (insertion I) and 603 amino acids (insertion II). These insertions are unique to Plasmodium CPSII and are primarily absent in CPSII genes from other organisms isolated to date. Previously, targeting of the larger insertion at the mRNA level by nucleic acid enzymes such as ribozymes and DNAzymes, has shown that this region of CPSII is essential to parasite survival. The current study was undertaken to allow further progression of testing of these molecules in a malarial rodent system with the long-term aim of finding a much-needed human antimalarial in the future.

The first aim of this research project, was the isolation of the nucleotide sequence of the CPSII insertion regions from other species of Plasmodium and this was achieved. Elucidation of the gene sequence of CPSII from rodent malarial species, P. berghei, P. yoelii and P. chabaudi, will now allow further design and testing of CPSII-directed antisense molecules in a range of malarial rodent models. Furthermore, characterisation of the insertion regions from P. vivax CPSII will also allow the design of a much needed synergistic antisense molecule that can simultaneously target the two most debilitating species of human malaria, P. falciparum and P. vivax.

The second aim was to examine the nature of Plasmodium CPSII insertions. Little is known about the real functional role of Plasmodium insertions and even less is known about their distribution and evolution across Plasmodium species. Inspection of the sequences of Plasmodium CPSII from five species revealed that the insertions occur precisely at the junction of the subdomains of the GAT domain and the CPS domain in all species examined. The Plasmodium CPSII insertions are highly hydrophilic and

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generally poorly conserved with respect to amino acid identity, but contain highly conserved elements. By examination of the composition of the insertions and their location within the highly conserved structure of this enzyme, it is possible to postulate some potential roles that may be examined in the future: (1) the significant location of the insertions and their non-globular, hydrophilic nature indicates they are located on the exterior of the conserved protein structure for interaction with other molecules; and (2) the low-complexity asparagine rich regions, may function as ‘polar zippers’ that become structured when they bind to their target molecule. It seems feasible that the target molecules in this case may be other pyrimidine biosynthetic enzymes, thereby controlling substrate channelling of the intermediates of the pathway as seen with the CAD complex in mammals and the bifunctional counterpart in yeast.

This could be accomplished by protein-protein interaction studies, by use of the native enzyme or a recombinant protein. Due to the unusually large size and nature of Plasmodium CPSII, it has never been isolated in its native form or expressed in a recombinant form, making it difficult to obtain evidence as to the precise role the insertions play. The GAT domain from P. falciparum has recently been (heterologously) expressed in Dictyostelium discoideum (Hillier, personal communication). Future research can now be initiated on finding the potential binding partners of the GAT domain and in particular, insertion I. In addition, antibodies to this region may now be prepared and used in localisation studies and western blots to identify the location of the insertion. Further, mutational studies of insertion I in the recombinant protein may shed some light on the role this insert plays.

The third aim was to design and analyse the kinetics of new DNAzymes targeting insertion II of the P. berghei CPSII gene. In summary, of the thirty-one DNAzymes designed and tested against PbCPSII insertion II, seventeen showed detectable cleavage products by individual cleavage analysis. Of these, four showed significant cleavage activity and were then further characterised for kinetics of cleavage. Single and multiple turnover data on both the long substrate as well as the short synthetic substrate, demonstrated that the use of the long substrate was advantageous in predicting active DNAzymes both in terms of site accessibility and turn-over ability under more realistic conditions. From the comprehensive kinetic study conducted, it was evident that the activity of each of the DNAzymes MD14, MD15, MD25 and MD26, were sequence-

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specific, time- and dose-dependent. Further, MD14 displayed superior over-all kinetics with the best balance between binding strongly to the RNA substrate and rapid release of products. On this basis, MD14 was chosen as a suitable candidate for use in an in vivo rodent malaria trial.

The final aim was a preliminary in vivo P. berghei trial with a candidate PbCPSII DNAzyme. Intravenous injection of MD14 into P. berghei infected mice yielded a significant lowering of parasitaemia, although the levels of reduction achieved were low. The conditions applied were less than optimal and included the use of a DNAzyme, supplied commercially, with a poorly synthesised 3' modification for protection from serum nucleases. Future studies should involve achieving further reduction in the parasitaemia by changing the dose, dosing schedule, and method of administration in order to improve the pharmacokinetic distribution of the DNAzyme to the target site of the infected red blood cell. It is possible that MD14 was merely not effective at reducing the target mRNA to adequate levels for effective parasite inhibition. Moreover, the low levels of reduction seen in this study may also be attributed to lack of spontaneous uptake of DNAzymes by parasitised erythrocytes.

Inhibition of P. falciparum cultures by the DNAzyme M5L, as observed previously, could not be repeated. Furthermore, exogenous uptake of FITC-M5L could not be established by either confocal microscopy or flow cytometry. These results or the lack thereof, resulted in the question of whether or not parasitised erythrocytes are capable of endogenous uptake of short nucleic acids. M5L bioassays should be performed by an independent laboratory with different P. falciparum cultures, to clarify whether the inability to repeat previously published results is due to a culturing artefact or parasite mutation, or simply the inability of the parasite to take up antisense molecules from the surrounding medium.

The research in the remainder of the project was by no means comprehensive but instead represented a preliminary foray into delivery of nucleic acids into P. falciparum infected erythrocytes. Attempts at DNAzyme delivery to parasitised erythrocytes by cationic liposomal transfection reagents were successful, however, parasite inhibition was not observed and is most likely attributed to the lack of release of DNAzymes from the liposomal complex. Uptake of porphyrin-conjugated DNAzymes (MPP-M5L) into

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parasites was only tentatively established by confocal microscopy and flow cytometry due to problems with the detection of fluorescein. However, when parasites were incubated with MPP-M5L for 24h, a significant reduction in parasitaemia was observed compared to the negative control DNAzyme. Longer incubation resulted in an increase of non-specific inhibition by this control DNAzyme.

The results of the porphyrin delivery studies are encouraging and warrant further investigation. However, more controls need to be included in further experiments to minimise or account for the non-specific effects seen in the P. falciparum bioassays. These include the use of the inverted thymidine modification in substitution of the 3' phosphorothioate linkage; porphyrin toxicity studies; scrambled arm and sense sequence controls. In addition, a fluorophore more compatible with porphyrin should be substituted in place of FITC for future uptake studies. An essential component, not addressed in this initial study, would include the use of a CPSII enzyme assay and RT- PCR or northern blot analysis to confirm specific reduction in the CPSII mRNA target.

In conclusion, the insertions of CPSII have been maintained in several species of Plasmodium and therefore it is likely that they have a specific, important role in enzyme function. However, due to their poorly conserved nucleotide identity, indicating that they are rapidly evolving, they seem less ideal as targets for nucleic acid therapy but instead more amenable to traditional chemotherapy. It is also evident from this study, and the fact that very little has been published in this field in recent years, that uptake or delivery of nucleic acid molecules to Plasmodium infected red blood cells needs to be addressed in more detail if it is to become a feasible antimalarial strategy.

Appendix

Appendix 190

APPENDIX

Appendix 1

Primers targeting the CPSII gene from P. berghei, P. chabaudi and P. vivax. ‘A, T, G and C represent the four bases: adenine; thymine; cytosine and guanine. The degenerate alphabet is as follows: Y=C or T; R = A or G; K = G or T; M = A or C; W = A or T; S = G or C; D = A, G or T; and N = A, T, C, or G.

Species Primer Sequence Name 5' to 3' P. berghei B4F ACC GAA GGA CAA ATA ACT ACA P. berghei B4R TTT ATA GTT TTT GCA ATT TCT TT P. berghei PbDHFR1F AGA TGA TTT ATT TCC TAT TTT A P. berghei PbDHFR1R CCT CAT TTG GAT TTA TTT CTT TTG P. berghei / P. chabaudi PbCPS5F WSW GTW GGW GAA GTW ATG P. berghei / P. chabaudi PbCPS5A GGW TTW TCW GAT AAA CA P. berghei / P. chabaudi PbCPS6F ACW AAT TAT YTW TAT YTW ACW TA P. berghei / P. chabaudi PbCPS6R TAA GTY AAR TAY ARR TAR TT P. berghei / P. chabaudi PbCPS7F TCT GTA GAR TTY GAY TGG P. berghei / P. chabaudi PbCPS7R GCW SWC CAA TCA AAT TCW AC P. berghei / P. chabaudi PbCPS8R TCW TCR AAR TAH ARY YTR TC P. berghei / P. chabaudi PbCPS9R TAA TTG WGT RTC YTT RTC RTC P. berghei / P. chabaudi PbCPS10R CAY TTR TTY TCW ACR TTW CC P. berghei / P. chabaudi PbCPS11R CAT CAC WAT RTC YTC YTC RTC P. berghei / P. chabaudi PbCPS12R CT TTT CAT RTT YAA WGG VA P. berghei / P. chabaudi PbCPS2A TTT ATA TGG AAT GTG TG P. berghei / P. chabaudi PbCPS17 ACA TCA CGT TCT TTT GTT TTG P. berghei / P. chabaudi PbCPS13F GTA TGC TGG GAA AGA TTG TAG P. berghei / P. chabaudi PbCPS14R TAA TTA ACA ATT TAC ATT TTT CTG G P. berghei PbCPS16 GAT AAT TCT GAA AAA CAG TCA CTT P. vivax PvDHFRF1 GGG GTC TGG GCA ATA AGG GG P. vivax PvDHFRR1 GCA CGG GCA CGC GGC GGG G P. vivax PvCPSDf GAY ACX AGR GCX CTX ACX AA P. vivax PvCPSK2f AAR GAR ATH AAY CTI TTY GAY CCH GG P. vivax PvCPSQf ATY GGR CAR GCM GGR GAR TTY GAY TA P. vivax PvCPSWf TGG AAR GAR ATY GAR TAY GA P. vivax PvCPSWr TCR TAY TCR ATY TCY TTC CA P. vivax PvCPSKf AAR ATY CCM MGR TGG GA P. vivax PvCPSKr TCC CAY CTK GGM ATY TT P. vivax PvCPSNr TAN GTY ARR TAY ARR TAR TTN GT P. vivax PvCPSGr CCR ATY CTR TAR GAX CCR GAX CC P. vivax PvCPSYr ATR AAR CTR CTY TAD TGK TGK CT P. vivax PvCPS1 GAA GGA GCG ACG ACG ATG CTG P. vivax PvCPS2 GAG GTA CCG AGA GAG TTT AGG P. vivax PvCPS5 GGC GAT GGT GGG GTA CCC GG

Appendix 191

Appendix 2

The sequence of synthetic RNA substrates complementary to the four chosen DNAzymes. ‘A, U, G and C represent the four bases: adenine; uracil; cytosine and guanine.

DNAzyme substrate Sequence 5' to 3' MD14/15 UGG AGG AAA GGA UGG AAU GCA AAA UAA A MD25 UUU UGG AAU GCA UCA AGG UAU GA MD26 UGC AUC AAG GUA UGA AAC GCC CA

The sequence of DNAzyme M5L. Underlined bases represent the core catalytic motif of the 10-23 DNAzyme.

5' TGT TCT TGA GGC TAG CTA CAA CGA CTT GAT AAG 3'

Appendix 192

Appendix 3

Restriction map of the pGEM®-T Easy Vector courtesy of Promega Corporation.

Appendix 4

Amino acid alignment of CPSII from other organisms compared with P. falciparum illustrating the position of the insertions. CPSII domains are highlighted in the same colours as shown Figure 3.14.

______H.sapiens ------MTRILTAFKVVRTLKTGFGFTNVTAHQKWKFSR-----PGIRLLSVKAQTAHIVLEDGTKMKGYS-GHPSS------VAGEVVFNTGLGGYPEAITDP R.norvegicus ------MTRILTACKVVKTLKSGFGLANVTSKRQWDFSR-----PGIRLLSVKAQTAHIVLEDGTKMKGYSFGHPSS------VAGEVVFNTGLGGYSEALTDP O.beta ------MTKLLLASSAVKFLRDGCVVSRRVGNR-ATWLA-----QQTKLFSVKAQTAHLVLEDGTRMKGYS-FHNAS------VSGELVFNTGLVGYPENLTDP T.gondii MPHSGGRRAVAPIYP-LDLAGRLRPAMLVLADGTEFLGYSFGYPGSVG------GEVVFNTGMVGYPESLTDP L.mexicana ------MEHYAKAELVLHGGERFEGYSFG------YEES------VAGEVVFATGMVGYPESLSDP T.cruzii ------MFGEKVKASLVLHGGECFEGYSFG------YEES------VAGEVVFATGMVGYPEAMTDP P.falciparum ------MTSEFWPDLDFKTVGR---LILEDGNEFVGYSVGYEGCKGNNSISCHKEYRNIINNDNSKNSNNSFCNNEENNLKDDLLYKNSRLENEDFIVTGEVIFNTAMVGYPEALTDP

PSD subdomain ______H.sapiens AYKGQILTMANPIIGNGGAPDTTALDELGLSKYE---SNGIKVSGLLVLDYS--KDYNHWLATKSLGQWLQEEKVPAIYGVDTRMLTKIIRDKGTML-KIEFEG------184 R.norvegicus AYKGQILTMANPIIGNGGAPDTTARDELGLNKYME--SDGIKVAGLLVLNYS--HDYNHWLATKSLGQWLQEEKVPAIYGVDTRMLTKIIRDKGTMLGKIEFEG------187 O.beta SYRGQILTLTYPIVGNYGVPNTEEVDELGLRRYV---SDRIQVSGLLVQTYC--HEYSHWNSVKSLGQWLQDEQVPALYGIDTRMLTKIIRDQGTVL-GIEFDG------183 T.gondii SYEGQILVLTYPLIGNYGVPSSEKDE-HGLPKYFE--GDRIYVRALVVADYDNAAVTAHFRAENSLSAWMNTHKVPAIAGVDTRALTKHLREVGCMLGKIVVLS------167 L.mexicana SYHGQILVLTSPMVGNYGVPR-VEEDLFGVTKYFESTDGRIHVSPVVVQEYC--DQPDHWEMYETLGAWLRKNKVPGMMMVDTRSIVLKLRDMGTALGKVLVAG------149 T.cruzii SYQGQILVLTSPMIGNYGIPP-IETDHFGLTKYFESMGGEIHVSAVVVSEYC--DEPAHWQMWETLGQWLRRNNIPGIMMVDTRHIVLKLREMGTALGKVVVND------150 P.falciparum SYFGQILVLTFPSIGNYGIEKVKHDETFGLVQNFE--SNKIQVQGLVICEYS--KQSYHYNSYITLSEWLKIYKIPCIGGIDTRALTKLLREKGSMLGKIVIYKNRQHINKLYKEINLFD 225

Insertion I

H.sapiens ------R.norvegicus ------O.beta ------T.gondii ------EEEERRSGLSLSALAALPSATAAEQRG------194 L.mexicana ------T.cruzii ------P.falciparum PGNIDTLKYVCNHFIRVIKLNNITYNYKNKEEFNYTNEMITNDSSMEDHDNEINGSISNFNNCPSISSFDKSESKNVINHTLLRDKMNLITSSEEYLKDLHN-CNFSNSSDKNDSFFKLYG 345

H.sapiens ------QPVDFVDPNKQNIAEVSTKDVKVYGKGN------213 R.norvegicus ------QSVDFVDPNKQNIAEVSTKDVKVFGKGN------216 O.beta ------QPVEITDPNKNNVAEVSTKETKVFGKGN------212 T.gondii ------ENDATVTPDKAEAR--LRVERRQAALTMWEEAIRNKAKNLPWEDPNKDNVALVSRKEVRVYKSTVVDPNLR------264 L.mexicana ------NDVPFMDPNTRNVAEVSTKTRVTHGHGT------178 T.cruzii ------KDVPFFDPNVRHVAEVSTKTRSTYGHGT------179 P.falciparum ICEYDKYLIDLEENASFHYNNVDEYGYYDVNKNTNILSNNKIEQNNNNENNKNNKNNNNNVDYIKKDEDNNVNSKVFYSQYNNNAPNNEHTEFNLNNDYSTYIRKKMKNEEFLNLVNKR 465

GLNase subdomain ______H.sapiens -----PTKVVAVDCGIKNNVIRLLVKRG------AVHLVPWNHDFTKMEYDGILIAGGPNPALAEPLIQNVRKILESDRKEPLFGISTGNLIT-LAAGAKTYKMSMANRGQNQPVLNI 320 R.norvegicus -----PTKVVAVDCGIKNNVIRLLVKRG------AEVHLVPWNHDFTQMDYDGLLIAGGPNPALAQPLIQNVKKILESDRKEPLFGISTGNIITGLAAGAKSYKMSMANRGQNQPVLNI 325 O.beta -----PIKVVAVDCGVKHNIIRLLVKRG------VEHLVPWNQDLMSLDYDGLFISNGPDPALAQTLINNVRKVMESDRTQPVFGICMGNQIT-AAAGAQSYKLPMGNRGQNQPVVNL 319 T.gondii -----DVLILCVDCGMKYNIYRQLLHSKFEHCNI-ILKVVPWDFDFGNDEFDGLFISNGPDPERCEKTVANIRRVME--RKIPIFGICLGNQLLALAAGARTYKMKYGNRGMNQPVIDL 376 L.mexicana ------LRILVIDMGVKLNQLRCLLKHD------VTLIVVPHDWDITTELYDGLFITNGPNPQMCTSTIRSVRWALQ--QDKPIFGICMGNQMLCPPAGGTTYKMKYGHRGQNQPCKCN 284 T.cruzii ------LVILVIDMGVKLNSLRCLLKYD------VTLIVVPHDWDITKETYDGLFISNGPNPQMCTKTIEHVRWAIT--QDKPIFGICMGNQILALAAGGSTYKMKYGHRGQNQPSTSR 285 P.falciparum KVDHKEKIIVIVDCGIKNSIIKNLIRHGMDLP-L-TYIIVPYYYNFNHIDYDAVLLSNGPDPKKCDFLIKNLKDSLT--KNKIIFGICLGNQLLGISLGCDTYKMKYGNRGVNQPVIQL 581

______H.sapiens TNKQAFITAQNHGYALDN-TLPAG--WKPLFVNVNDQT-EGIMHESKPFFAVQFHPEVTP-PIDTEYLFDSFFSLIKKGKATTITSVLPKPALVASRV-VSKVLILGSGGLSIGQAGEFD 435 R.norvegicus TNRQAFITAQNHGYALDN-TLPAG--WKPLFVNVNDQTNEGIMHESKPFFAVQFHPEVSP-PTDTEYLFDSFFSLIKKGKGTTITSVLPKPALVASRVEVSKVLILGSGGLSIGQAGEFD 442 O.beta MTDQAFITAQNHGYGINSDSLPEG--WSPLFINANDGT-EGIMHKTKPVFTAQFHPEANG-PTDTEFLFDAFISLIKSGKNANIVSVMPKMPEAPSRL-VSKVLVLGSGGLSIGQAGEFD 435 T.gondii RTSRCYITPQNHGFAVDESTLPR--DFLPLFVNANDRSNEGIIHRTLPFFSAQFHPEASG-PTDTFYLFGDFIASIMKAQ----TLKQVHTTPFSFPQKFQKVLLLGSGGLSIGQAGEFD 490 L.mexicana IDDRVVITTQKPGFAVDFKTLPSD-EWEEYFTNSNDGSNEGLWHKTKPFCSVQFHPEGRC-PQDTEYLFSEYVCRVKGSK------VKEVAKFKPRKVLVLGAGGIVIAQAGEFD 392 T.cruzii SDGHVFITTQNHGFAVDFKSVSQD-EWEECFYNPNDDSNEGLRHRTKPFFSVQFHPEGRC-PQDTEYLFGGVIAHVKESK------VKEASKYKPRKVLVLGAGGIVIAQAGEFD 393 P.falciparum VDNICYITSQNHGYCLKKKSILKRKELAISYINANDKSIEGISHKNGRFYSVQFHPEGNN-PEDTSFLFKNFLLDIFNKK----KQYREYLG-YNIIYIKKKVLLLGSGGLCIGQAGEFD 696

______H.sapiens YSGSQAVKAMKEENVKTVLMNPNIASVQTNEVGLKQAD-VYFLPITPQFVTEVIKAEQPDGLILGMGGQTALNCGVELFKRGVLKEYGVKVLGTSVES-MATEDRQLFSDKLNEINEKIA 553 R.norvegicus YSGSQAVKAMKEENVKTVLMNPNIASVQTNEVGLKQADAVYFLPITPQFVTEVIKAERPDGLILGMGGQTALNCGVELFKRGVLKEYGVKVLGTSVESIMATEDRQLFSDKLNEINEKIA 562 O.beta YSGSQAVKAMKEENLQTVLMNPNIASVQTNEVGTKQAD-VYFLPVTPEFVTEVIKIERPDGILLSMGGQTALNCGVELFRRGILKKYGVQVLGTSVES-MATEDRQLFSDKLVEINEKIA 553 T.gondii YSGSQAIKALKEQNIFVVVVNPNIATVQTSQH---MADRVYFLPVTDEFVTKVIEKEMPDGILCTFGGQTALNCAVKLHEQGVLAKFGCKILGSPIEAIIATEDRKVFAAKLEEIGEKVA 607 L.mexicana YSGSQCLKSLREEGMETVLINPNIATVQTDDE---MADHIYFVPLTVEAVERVIEKERPDGILLGWGGQTALNCGVKLDELGVLKKYNVQVLGTPVSVIAVTEDRELFRDTLLQINEQVA 509 T.cruzii YSGSQCLKALSEEGIETVLVNPNIATVQTDDE---MADQIYFVPITAEAVERVIEKERPDGIMLAWGGQTALNCGLEMDRLGILKKYNVQVLGTPISTITVTEDRDLFRNALLQINEHVA 510 P.falciparum YSGTQAIKSLKECGIYVILVNPNIATVQTSKG---LADKVYFLPVNCEFVEKIIKKEKPDFILCTFGGQTALNCALMLDQKKVLKKNNCQCLGTSLESIRITENRTLFAEKLKEINERIA 813

CPS.A subdomain ______H.sapiens PSFAVESIEDALKAADTIGYPVMIRSAYALGGLGSGIC-NRETLMDLSTKAFAMTN-QILE-KSVTGWKEIEYEVVRDADDNCVTVCNMENVDAMGVHT-DSVVVAPAQTLSNAEFQMLR 670 R.norvegicus PSFAVESMEDALKAADTIGYPVMIRSAYALGGLGSGICPNKETLMDLGTKAFAMTN-QILE-RSVTGWKEIEYEVVRDADDNCVTVCNMENVDAMGVHTGDSVVVAPAQTLSNAEFQMLR 681 O.beta PSIAVETVPDALKAAEQIGYPVMIRSAYALGGLGSGLC-TKEKLEDMAQKALAMSS-QILE-KSLLGWKEVEYEVVRDVADNCVTVCNMENFDPLGIHT-DSIVVAPSQTLSNEEYHKLR 670 T.gondii ESAAATNTEEAVQAAKAIGYPVLIRAAFALGGLGSGFAEDEETVRRICKEAFSHSS-QVFD-KSLKGWKEVEYEVVRDCKNNCITVCNMENLDPLGIHTGDSIVVAPSQTLSNEDYYRLR 726 L.mexicana KSAAVTSVEEAVVASKDIGFPMMVRAAYCLGGQGSGIVENMAELRHKVEVALAASP-QVLE-ESVAGWKEIEYEVVRDIYDNCITVCNMENFDPMGVHTGESIVVAPSQTLSNDEFHHLR 628 T.cruzii KSLAVTSIEEAVGASKVIGFPLMLRAAYCLGGQGSGIVYNEEELRHKVGVALAVSP-QVLE-ESVAGWKEVEYEVVRDIYDNCITVCNMENFDPMGTHTGESIVVAPLQTLTSDEYHMLR 629 P.falciparum PYGSAKNVNQAIDIANKIGYPILVRTTFSLGGLNSSFINNEEELIEKCNKIFLQTDNEIFD-KSLQGWKEIEYELLRDNKNNCIAICNMENIDPLGIHTGDSIVVAPSQTLSNYEYYKFR 933

______H.sapiens RTSINVVRHLGIVGECNIQFALHPTSMEYCIIEVNARLS-SSALASKATGYPLAFIAAKIALGIPLPEIKNVVSGKTSACFEPSLDYMVTKIPRWDLDR-HGTSSRIGSSMKSVGEVMAI 788 R.norvegicus RTSINVVRHLGIVGECNIQFALHPTSMEYCIIEVNARLSRSSALASKATGYPLAFIAAKIALGIPLPEIKNVVSGKTSACFEPSLDYMVTKIPRWDLDRFHGTSSRIGSSMKSVGEVMAI 801 O.beta ETAIKVVRHLGIVGECNIQYALHPTSLEYCIIEVNARLS-SSALASKATGYPLAFVAAKLALGIPLPEIKNAVSQKTTACFEPSLDYIVTKIPRWDLDR-QGMSREIGSSMKSVGEVMAI 788 T.gondii DTALKVIRHFGIVGECNIQYALDPNSEKYYIVEVNARLSRSSALASKATGYPLAYIAAKLALGSTLVELSNSVTKETTACFEPSLDYVVTKVPRWDLRKFESCDPLMGSAMKSVGEVMAI 846 L.mexicana SASIKIIRHLGIVGECNIQYGLDPFSHRYVVIEVNARLSRSSALASKATGYPLAHVATKIALGKGLFEITNGVTKTTMACFEPSMDYIAVKMPRWDLHKFNMVSQEIGSMMKSVGEVMSI 748 T.cruzii SASIKIIRHLGIVGECNIQYGLDPTSHRYVVIEVNARLSRSSALASKATGYPLALVAAKIALGKGLFEIANGVTKTTMACFEPSLDYIVVKVPRWDLSKFNMVSQNIGSMMKSVGEVMAI 749 P.falciparum EIALKVITHLNIIGECNIQFGINPQTGEYCIIEVNARLSRSSALASKATGYPLAYISAKIALGYDLISLKNSITKKTTACFEPSLDYITTKIPRWDLNKFEFASNTMNSSMKSVGEVMSI 1053

______H.sapiens GRTFEESFQKALRMCHPSIEGFTPRLPMNKEWPSN------LDLR-ELSEPSSTRIYAIAKAIDDNMSLDEIEKLTYIDKWFLYKMRDILNM 873 R.norvegicus GRTFEESFQKALRMCHPSVDGFTPRLPMNKEWPAN------LDLRKELSEPSSTRIYAIAKALENNMSLDEIVKLTSIDKWFLYKMRDILNM 887 O.beta GRTFEESIQKALRMCHPSIDGFMPRLPLKKDWADS------HDLQ-DLAVPSSTRIFSLAKAFHKGMSVDLIHQLTFIDKWFLYKLQRITQM 873 T.gondii GRTFEESLQKALRMVDEKAGGFDESVCHFFSTDEDCAPSLPGSDFKTSSSGECMRGGCGRTDSGAERQAALLEAELRRPSPNRIWALALAFQLGWTVDALHEKTKIDKWFLSKLQNINDI 966 L.mexicana GRTFEEAMQKAIRMVDPSYTGFSIPDRFAG-ADFDY------MEHIRHPTPYRLFAICRALLDGHSAEELYQMTKITRVFLYKLEKLVRL 831 T.cruzii GRTFEEALQKALRMVDPSHTGFDVPPRLEAKKNWDH------MQDLKVPTPDRIFAICRALHEGVSVETIHEMTRINLFFLNKLHKLILL 833 P.falciparum GRTFEESIQKSLRCIDDNYLGFSNTYCIDWDEKK------IIEELKNPSPKRIDAIHQAFHLNMPMDKIHELTHIDYWFLHKFYNIYNL 1136

Oligomerisation domain ______H.sapiens EKTLKGLNS---ESMT-ETLKRAKEIGFSDKQISKCLG------LTEAQRELRLKKNIHPWVKQIDTLAAEYPSVTN-YLYV-YNGQEHDVN------954 R.norvegicus DKTLKGLNS---ESVTEETLRQAKEIGFSDKQISKCLG------LTEAQRELRLKKNIHPWVKQIDTLAAEYPSVTN-YLYVTYNGQEHDIK------970 O.beta HQQLADYDS---DTVT-DLLLMAKQDGFSDRQVGEILG------SNEKARDLRHSHSIKPWVKQIDTLAAEYPAMTN-YLYC-YHGEEHDLD------954 T.gondii KRQLTQLTL---DDLTRADFFYIKKYGFSDRQIAQYLMNSPS------AAALSQFDRRRRLHLGVRPSVKQIDTLAAEFPAHTN-YLYLTYQGIDDDVSPLAATPSVSAVFAGAR 1072 L.mexicana SMATSTLYANRLTEMPRENLLSMKAHGFSDRQLAQLLN------TTAADRARRVELNVMPLIKQIDTVAGEYPAAQCCYLYSTYNAQRDDVP------918 T.cruzii QNHMLGQYKGKMNTMPRDCLLKMKANGFSDAQIAKYFL------CTADDRESRMELKITPKVKQIDTVAGEIPASQCGFLYTSYNAYHDDVE------920 P.falciparum QNKLKTLKL---EQLSFNDLKYFKKHGFSDKQIAHYLSFNTSDNNNNNNNISSCRVTENDMKYREKLGLFPHIKVIDTLSAEFPALTN-YLYLTYQGQEHDVLPLNMKRKKICTLNNKR 1252

Insertion II H.sapiens ------R.norvegicus ------O.beta ------T.gondii ------L.mexicana ------T.cruzii ------P.falciparum NANKKKVHVKNHLYNELVDDKDTQLHKENNNNNNMNSGNVENKCKLNKESYGYNNSSNCINTNNINIENNICHDISINKNIKVTINNSNNSISNNENVETNLNCVSERAGSHHIYGKEEK

H.sapiens ------R.norvegicus ------O.beta ------T.gondii ------AEKRE------EENAETCRDDEDESLLRRLSKS------1099 L.mexicana ------T.cruzii ------P.falciparum SIGSDDTNILSAQNSNNNFSCNNENMNKANVDVNVLENDTKKREDINTTTVFMEGQNSVINNKNKENSSLLKGDEEDIVMVNLKKENNYNSVINNVDCRKKDMDGKNINDECKTYKKNKY1492

H.sapiens ------R.norvegicus ------O.beta ------T.gondii ------L.mexicana ------T.cruzii ------P.falciparum KDMGLNNNIVDELSNGTSHSTNDHLYLDNFNTSDEEIGNNKNMDMYLSKQKSISNKNPGNSYYVVDSVYNNEYKINKMKELIDNENLNDEYNNNVNMNCSNYNNASAFVNGKDRNDNLEN1612

H.sapiens ------R.norvegicus ------O.beta ------T.gondii ------L.mexicana ------T.cruzii ------P.falciparum DCIEKNMDHTYKHYNRLNNRRSTNERMMLMVNNEKESNHEKGHRRNGLNKKNKEKNMEKNKGKNKDKKNYHYVNHKRNNEYNSNNIESKFNNYVDDINKKEYYEDENDIYYFTHSSQGNN1732

______H.sapiens ------FDDHGMMVLGCGPYHI970 R.norvegicus ------FDEHGIMVLGCGPYHI986 O.beta ------FKDNGTMVLGCGPYHI970 T.gondii ------SSARLRTGEGD------APGKQCFVVLGCGCYRI1127 L.mexicana ------FTERMYAVLGCGVYRI934 T.cruzii ------FTERMYAVFGCGVYRI936 P.falciparum DDLSNDNYLSSEELNTDEYDDDYYYDEDEEDDYDDDNDDDDDDGEDEEDNDYYNDDGYDSYNSLSSSRISDVSSVIYSGNENIFNEKYNDIGFKIIDNRNEKEKEKKKCFIVLGCGCYRI1852

______H.sapiens GSSVEFDWCAVSSIRTLRQLGKKTVVVNCNPETV-TDFDECDKLYFEELSLERILDIYHQEACGGCIISVGGQIPNNLAVPLYKNGVKIMGTSP-QIDRAEDRSIFSAVLDELKVAQAPW1088 R.norvegicus GSSVEFDWCAVSSIRTLRQLGKKTVVVNCNPETVSTDFDECDKLYFEELSLERILDIYHQEACNGCIISVGGQIPNNLAVPLYKNGVKIMGTSPLQIDRAEDRSIFSAVLDELKVAQAPW1106 O.beta GSSVEFDWCAVSSIRALRQMGKKTVVVNHNPETV-TDFDECDRLYFEELTLERILDISQQEGCVGTIVSVGGQIPNNLAVPLHKNGVKILGTSP-QIDRAEERSTFSKILDDLGVAQAPW1088 T.gondii GSSVEFDWSAVSCVRTLRSLGHHAIVVNCNPETVSTDYDVSDRLYFEDLSLETVLNIWDIEAPAGVIISVGGQTPNTLCSALEKQGVRIVGTSVAAIDCCEDRHKFSRLCDELNIDQPRW1247 L.mexicana GNSVEFDYGGVLVARELRRLGNKVILINYNPETVSTDYDECDRLYFDEVSEETVLDILTKERVRGVVISLGGQIVQNMVLSLKKSGLPILGTDPANIDMAEDRNKFSKMCDNLGVPQPEW1054 T.cruzii GNSVEFDYGGVLVARELRRLGKKVILINYNPETVSTDYDECDRLYFEEVSEETVLDILLKERIQGVVISLGGQIVQNMALRLKQHGLPILGTDPVNVDKAENRHKFSKMCDELGVLQPEW1056 P.falciparum GSSVEFDWSAIHCVKTIRKLNHKAILINCNPETVSTDYDESDRLYFDEITTEVIKFIYNFENSNGVIIAFGGQTSNNLVFSLYKNNVNILGSSAQSVDCCENRNKFSHLCDSLKIDQPKW1972

CPS.B subdomain ______H.sapiens KAVNTLNEALEFAKSVDYPCLLRPSYVLSGSAMN-VFSEDEMKKFLEEATRVSQEHPVVLTKFVEGAREVEMDAVGKDGRVISHAISEHVEDAG-HSGDATLMLPTQTISQGAIEKVKDA1206 R.norvegicus KAVNTLNEALEFANSVGYPCLLRPSYVLSGSAMNVVFSEDEMKRFLEEATRVSQEHPVVLTKFIEGAREVEMDAVGKEGRVISHAISEHVEDAGVHSGDATLMLPTQTISQGAIEKVKDA1226 O.beta SAVNSLDDAFTFANRVGYPCLLRPSYVLSGSAMN-AYGEEEMKRFLEEAAQVSQEHPVVITKFICGAREVEMDAVAKNGKVLCHAITEHVEDAG-HSGDATLMLPTQTISQGALEKVKIA1206 T.gondii KEFTDLRTAKAFCQEVGYPVLVRPSYVLSGAAMRVVTDDEQLDAFLKIAAVVSGESPVVISKFVENAKEVEFDSVACRGEIVNFAISEHVENAGTHSGDATLILPGQKLYVETIRRVKKI1367 L.mexicana ISATSVEQVHEFCDRVGYPALVRPSYVLSGSAMAVIANKEDVTRYLKEASFVSGEHPVVVSKYYEDATEYDVDIVAHHGRVLCYGICEHVENAGVHSGDATMFLPPQNTDKDTMKRIYDS1174 T.cruzii ILSTIVEQVHEFCKQVGFPTLVRPSYVLSGSAMAVIASAADINRYLEEAALVSGEHPVVVSKYYEGAMEYDVDIVAHHGRVLCYAICEHVENAGVHSGDATMFLPPQNTEKEVMKRIYNT1176 P.falciparum NKFTKLSKAIQFANEVKFPVLVRPSYVLSGAAMRVVNCFEELKNFLMKAAIVSKDNPVVISKFIENAKEIEIDCVSKNGKIINYAISEHVENAGVHSGDATLILPAQNIYVETHRKIKKI2092

______H.sapiens TRKIAKAFAISGPFNVQFLVKGN-DVLVIECNLRA-RSFPFVSKTLGVDFIDVATKVMIGNVDEKHLPTLDHPIIPADYVAIKAPMFSWPRLRD-DPILRCEMASTGEVACFGEGIHTA 1323 R.norvegicus TRKIAKAFAISGPFNVQFLVKGN-DVLVIECNLRASRSFPFVSKTLGVDFIDVATKVMIGSVDEKHLPTLEQPIIPSDYVAIKAPMFSWPRLRDADPILRCEMASTGEVACFGEGIHTA 1345 O.beta TRKIAHALEISGPFNTQFLVKGN-DVMVIECNLRA-RSFPFVSKTIGVDFIKVATKVMTGPLDESSLPSLENPIIPVDYVGIKAPMFSWPRLRE-DPVLRCEMASTGEVACFGPNIYSA 1323 T.gondii SQKLARALQVSGPFNIQFICKQN-DVKVIECNLRASRTFPFISKAFNVNLIDLATKVMIGPVTP-----LPIHLMDLSFVCVKVPVFSFARLRGCDPVLGVEMRSTGEVACFGASKHEA 1481 L.mexicana VNRIAEKLDVVGPMNVQFLLTAEGHLRVIEANVRKFRSVPFVSKTLGISFPSVMVSAFLAKDQN----LVPIKRAKMTHIGCKASMFSFIPLAGADPILGVEMASTGEIGVFGRDKHEV 1290 T.cruzii TALIAEELDVVGPMNIQFLFTKDKQLRVIEANIRSSRSVPFVSKTLGISFPAVMVSAFLSHDSN----LVPIKRARMTHIGCKASVFSFNRLAGADPILGVEMASTGEIGVFGRDKKEV 1292 P.falciparum SEKISKSLNISGPFNIQFICHQN-EIKIIECNLRASRTFPFISKALNLNFIDLATRILMGDVKP-----INISLIDLEYTAVKAPIFSFNRLHGSDCILGVEMKSTGEVACFGLNKYEA 2206

MGS-like domain ______H.sapiens FLKAMLSTGFKIP--QKGILIGIQQS-FRPRFLGVAEQ-HNEGFKLFATEATSDWLN------ANNVPATPVAWPSQ------EGQNP1395 R.norvegicus FLKAMLSTGFKIP--QKGILIGIQQS-FRPRFLGVAEQLHNEGFKLFATEATSDWLN------ANNVPATPVAWPSQ------EGQNP1418 O.beta FLKAMLSTGFKLP--QKGILIGIQHS-FRPHFLATAQQ-KDEGFKLYATEATSAWLC------ANDVPSTPVAWPS------DNADS1394 T.gondii FLKALISAGVPLPLEKRTILISAGPLWSKMELEPYFKILLDLGFTIYATEGTYRFLMNSVVRGQGTHLPGNASPASDSGLRTPTTAESDADACIRAKYASRIIRVRKPIVGSNESHNGGH1601 L.mexicana FLKAMLCQNFRIP--KKGVFFSIDVDSQTEALCPYIQHLVGRGLKVYGTANTAAVLHEYG------IECEVLLQRSELPSG-----DACESNR1370 T.cruzii FLKAMLCQNFRYP--QRGVFISCDVDAMAEDLCPTLS--ASDRFPVFTSKQTSRVLADYG------IPHTVLTQRHEDSE------1362 P.falciparum LLKSLIATGMKLP--KKSILISIKNLNNKLAFEEPFQLLFLMGFTIYATEGTYDFYSKFLES------FNVNKGSKFHQRLIKVHN------KNAEN2289

H.sapiens SLSSIRKLIRDGSIDLVINLPNN------TKFVHDN-YVIRRTAVDSGIPLLTNFQVKLFAEAVQK---SRKVDSKSLFHYRQYSAGKAA------1476 R.norvegicus SLSSIRKLIRDGSIDLVINLPNNN------TKFVHDN-YVIRRTAVDSGIALLTNFQVKLFAEAVQK---ARTVDSKSLFHYRQYSAGKAA------1500 O.beta NLPSIKRLISEGHIDLVVNLPNN------TRQVKDN-FLIRRMAIDYGVPLITNDQVKLFAEAIHS---ARSLDTTSLFHYRQKEGTQPKTQQG------1479 T.gondii QSPHALSLIESGKVEMVINVPDSM------NHRAATNGYLMRRTATDCGVPLLTNVKVSMFVEALNKKE-AKEAQGRSFWDIRSWDEYWPQK------1687 L.mexicana PAVYDEEVAKKEKFDLVIQLRDKRRDFVLRRCTRETAPPDYWVRRLAVDYNIPLLTEPSLKMFCEFMDLPASSIEVEPFRHYVPKIYHKVENNNCAMLRCHKVGLMITNNNDSKVLALR 1490 T.cruzii -PTFDTAVAVKEKFDLVIQLRDKRQDFMLRRCTQENATADYWIRRLAVDYNHSLLTEPNVRMFCETLDVDVKEIEIEPFRLYVPRVYNKMENDNYTMLHRHKVGLCITSTNDSKVLAIS 1481 P.falciparum ISPNTTDLIMNHKVEMVINITDTL------KTKVSSNGYKIRRLASDFQVPLITNMKLSLFIDSLYRKF-SRQKERKSFYTIKSYDEYISLV------2375

H.sapiens ------R.norvegicus ------O.beta ------T.gondii ------L.mexicana LSQEGLNITCFHAYLGGSDIDHFEQAFQSLNVPVEVVDLRSEIANSAFDLIMCQSADEHHNWHLSKLSWYIFGKYLIPVMRHVRMSVVAQTSKQNKKEAGFEKLRGTATARRWLSTTLGA1610 T.cruzii LREEKIALTCFHACLGG------IKNNSEEIAEQFRSIGSTSRAHRPPH--1524 P.falciparum ------

H.sapiens --- R.norvegicus --- O.beta --- T.gondii --- falciparum --- L.mexicana TRG 1613 T.cruzii ---

References References 200

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