Abbas Thesis
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Thesis for doctoral degree (Ph.D.) 2008 Thesis for doctoral degree (Ph.D.) 2008 Targeting Nucleic Acids in Bacteria with Synthetic Ligands Targeting nucleic acids in bacteria with nucleic synthetic ligands Targeting Abbas Nikravesh Abbas Nikravesh From the Programme for Genomics and Bioinformatics Department of Cell and Molecular Biology Karolinska Institutet, Stockholm, Sweden TARGETING NUCLEIC ACIDS IN BACTERIA WITH SYNTHETIC LIGANDS Abbas Nikravesh Stockholm 2008 All previously published papers were reproduced with permission from the publisher. Published by Kar olinska Institutet. Printed by [name of printer] © Abbas Nikravesh, 2008 ISBN 978-91-7357-489-1 In the Name of God, the Beneficent, the Merciful I would like to dedicate this thesis to my dear late parents Fatemeh and Hassan Questions are the cure for human ignorance. Let us ask what we do not know, and solve our entire unknown (Mohammad Taghi Jafari). ABSTRACT There is a need for new antibacterial agents, and one attractive strategy is to develop nucleic acid ligands that inhibit pathogen genes selectively. Also, such ligands can be used as molecular biology probes to study gene function and nucleic acid structures. In this thesis, bacterial genes were selectively inhibited with antisense peptide nucleic acid (PNA) and the higher order structures formed by (GAA)n repeats were probed with the intercalator benzoquinoquinoxaline (BQQ). A majority of bacterial genes belong to tight clusters and operons, and regulation within cotranscribed genes has been difficult to study. We examined the effects of antisense silencing of individual ORFs within a natural and synthetic operon in Escherichia coli. The results indicate that expression can be discoordinated within a synthetic operon but only partially discoordinated within a natural operon. Bacteria use natural antisense mechanisms to regulate gene expression and an established model for sense/antisense RNA pairing is the hok /sok toxin–antitoxin (TA) locus. We aimed to test this model in cells and also the idea that sequestration of Sok- RNA could provide a novel antimicrobial strategy using antisense agents. The results support the hok /sok sense/antisense interaction model and the idea that PNA can out- compete this interaction and provide potent killing activity. Certain antisense agents are effective in bacteria, yet it is unclear how these relatively large molecules overcome stringent bacterial barriers. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in E. coli and slow efflux. Consistent with cell accumulation and retention, the post- antibiotic effect (PAE) of a PNA that targets the growth essential fatty acid biosynthesis gene acpP was greater than seven hours. At the DNA level, polypyrimidine/polypurine rich sequences have the potential to form intramolecular triple helix structures (H-DNA). Triplet (GAA)n repeats are pathological in Friedreich’s ataxia (FA) disease, and may form H -DNA. Here we probed for triplex structures in (GAA)n sequences using the triple-helix specific stabilizing compound BQQ. The results showed that E. coli plasmids carrying a (GAA)n repeat sequence form H -DNA, suggesting that these structures play a role in the pathology of FA . LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals. I. Rikard Dryselius, Abbas Nikravesh, Agne Kulyte, Shan Goh and Liam Good Variable coordination of cotranscribed genes in Escherichia coli following antisense repression BM C Microbiology.2006;6(97) II. Omid R. Faridani, Abbas Nikravesh, Deo Prakash Pandey, Kenn Gerdes, and Liam Good Competitive inhibition of natural antisense Sok-RNA interactions activates Hok-mediated cell killing in Escherichia coli Nucleic Acids Res. 2006;34(20):5915-22 III. Abbas Nikravesh, Rikard Dryselius, Omid R Faridani, Shan Goh, Majid Sadeghizadeh, Mehrdad Behmanesh, Anita Ganyu, Erik Jan Klok, Rula Zain and Liam Good Antisense PNA Accumulates in Escherichia coli and Mediates a Long Post- antibiotic Effect Mol Ther. 2007 Aug;15(8):1537-42 IV. Abbas Nikravesh, Helen Bergquist, Raquel Domingo Fernandez, Chi-Hung Nguyen, Liam Good & Rula Zain Friedreich’s ataxia (GAA)n triplex DNA structures are stabilized by benzoquinoquinoxaline derivatives Submitted, 2007 CONTENTS 1 Introduction...................................................................................... 1 1.1 Bacteria and Health..................................................................... 1 1.1.1 Bacterial Infection............................................................. 1 1.1.2 Treatment of bacterial infection............................................. 1 1.1.2.1 Antibacterial mechanism of action ......................................... 2 1.1.2.2 Antibacterial Resistance...................................................... 3 1.1.2.2.1 The history of antibiotic resistance..................................... 3 1.1.2.2.2 Antibacterial resistance mechanisms .................................. 4 1.1.2.2.3 Possible solutions to the problem of resistance...................... 4 1.2 Bacteria as an experimental system .................................................. 5 1.3 Nucleic acids and gene expression regulation ..................................... 5 1.3.1 DNA structure and gene expression regulation .......................... 5 1.3.1.1 Non-B -DNA structures ...................................................... 6 1.3.1.1.1 Intramolecular triple helix DNA (H -DNA).......................... 6 1.3.2 RNA structure and gene expression regulation .......................... 7 1.3.2.1 RNA higher order structures ................................................. 8 1.4 Nucleic acid ligands .................................................................... 8 1.4.1 DNA ligands .................................................................... 8 1.4.1.1 Small molecule ligands ....................................................... 9 1.4.1.1.1 Small molecule ligands as anticancer agents....................... 10 1.4.1.1.2 Small molecule ligands as antimicrobials........................... 10 1.4.1.1.3 Natural and synthetic source of small molecule ligands ......... 11 1.4.1.2 DNA binding proteins....................................................... 11 1.4.1.3 Oligonucleotides ............................................................. 12 1.4.1.3.1 Antigene applications of triplex forming oligonucleotide ....... 12 1.5 Antisense oligonucleotides .......................................................... 14 1.5.1 Natural Antisense RNA..................................................... 14 1.5.2 Synthetic Antisense agents ................................................. 15 1.5.2.1 Nucleic acid analogues...................................................... 16 1.5.2.2 Nucleic acid mimics......................................................... 17 1.5.2.2.1 Chemical properties of PNA .......................................... 17 1.5.2.2.2 Activity of PNA in Biological systems.............................. 17 2 Aims............................................................................................. 19 3 results and discussion........................................................................ 20 3.1 Paper I ................................................................................... 20 3.2 Paper II .................................................................................. 20 3.3 Paper III................................................................................. 21 3.4 Paper IV................................................................................. 23 4 Conclusions .................................................................................... 25 5 Future Perspectives ........................................................................... 26 6 Acknow ledgements........................................................................... 27 7 References...................................................................................... 28 LIST OF ABBREVIATIONS ACP acyl carrier protein AO antisense oligonucleotide CFU colony forming unit dsRNA double-stranded RNA EMSA electrophoretic mobility shift assay FA Friedreich’s ataxia hok host killing LNA locked nucleic acid MIC minimal inhibitory concentration miRNA microRNA MP methylphosphonate mRNA messenger RNA MRSA methicillin-resistant Staphylococcus aureus ncRNA non-coding RNA ON oligonucleotide PBP penicillin binding protein PABA para-aminobenzoic acid PCR polymerase chain reaction PMO phosphorodiamidate morpholino oligomers PNA peptide nucleic acid PS phosphorothioate RISC RNA induced silencing complex RNAi RNA interference rRNA ribosomal RNA RT -PCR real-time reverse transcriptase-PCR SD Shine-Dalgarno snRNA small nuclear RNA sok suppression of killing TFO triplex forming oligonucleotide tRNA transfer RNA UTR untranslated region 1 INTRODUCTION 1.1 BACTERIA AND HEALTH Bacteria are involved in many aspects of ecology and health. It seems likely that all species both benefit and suffer from interactions with bacteria. For example, we use bacteria in making yogurt, cheese and other fermented foods. Also, large numbers of bacteria live on the skin and in the digestive tract. The human gut contains more than 1000 bacterial species, which are generally beneficial [1]. Gut bacteria synthesize vitamins such as folic acid, vitamin K and biotin, and they ferment complex indigestible carbohydrates. Other useful bacteria in gut flora