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The Synthesis and Study of Oligo(Deoxy)Ribonucleotides and Their Analogues

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The Synthesis and Study of Oligo(Deoxy)Ribonucleotides and Their Analogues The Synthesis and Study of Oligo(deoxy)ribonucleotides and their Analogues. by Susanne Ebel A Thesis Presented for the Degree of Doctor of Philosophy University of Edinburgh 1993 \ 4/ 11i1t I declare that this thesis is my own composition and that the work of which it is a record was carried out by myself unless otherwise acknowledged. No part of this thesis has been submitted in any previous application for a higher degree. Susanne Ebel. 1993 2 I wish to thank - my supervisor Prof.; Tom Brown for offering the opportunity to do this Ph.D. and giving advice and encouragement throughout, - Dr. Brian Sproat for the generous gift of his biotin-phosphoramidite, -the OSWEL DNA Service for the use of their facilities, - Dr. A. Lane of the NIMR who did all the excellent NMR work on G.A mismatches, - Dr. P. Hendry of the OSORO for his help with ribozyme-cleavage analysis, - David Will for being the most critical of all the colleagues in the group, - all the people in the Brown group for being fun to work with, - and Edinburgh University for use of their facilities. Proton, Carbon-13 spectra were recorded by John Millar and Heather Grant, FAB-Mass spectra were recorded by Alan Taylor. 3 AbbreviaUanz A260 absorbance unit at 260nm [ml-1 ] AMP adenosine mono-phosphate ATP adenosine tn-phosphate Bio single-addition biotin phosphoramidite as shown in chapter 2 Bz benzoyl Cbio biotin-phosphoramidite as shown in chapter 2 conc. concentrated CPG controlled pore glass Ctmp 1 -(2-chloro-4-tolyl)-4-methoxypipenidi n-4-yl dA 2Oxy adenosine dO 2'-deoxy cytidine dG 2'-deoxy guanosine dil. dilute dmf dimethylformamidine DMT DNA oligodeoxynibonucleic acid dT 2vdeoxy thymidine EDTA ethylenediaminetetraacetic acid eq. equivalents Fmoc fluoren-9-yl-methoxycarbonyl FOD fast oligo deprotection Fpmp 1 -(2-fluorophenyl)-4-methoxypipenidin-4-yl h hour(s) HEG hexaethylene glycol HPLC high performance liquid chromatography iBu isobutyryl iPr isopropyl mm. minute(s) MMT mono meth oxytnityl mRNA messenger RNA Mthp 4-methoxytetrahydropyran-4-yl MuOH multiple hydroxyl phosphoramidite as shown in chapter 2 NAP nucleic acid purification 5 NB 2-nitrobenzyl NH40Ac ammonium acetate p phosphate pac phenoxyacetyl PAGE polyacrylamide gel electrophoresis pre-mRNA precursor messenger RNA Pu purine px pixyl Py pyridine pyr pyrimidine rA adenosine rC cytidine rG guanosine RNA oligoribonucleic acid RNAse H ribonuclease H rRNA ribosomal RNA rU uridine sec second(s) lEG tetraethylene glycol TBDMS t-butyl-dimethylsilyl THF tetrahydrofuran Thp tetrahydropyranyl tRNA transfer RNA UV ultraviolet 6 Abstract Laboratory procedures for the synthesis, workup and purification of oligoribonucleotides using RNA phosphoramidites protected with a) Fpmp- and b) TBDMS- groups on the 2'-hydroxyl functions have been developed and employed in the following projects: Modified minimised DNA/RNA ribozymes were synthesised and purified, which contained a) a dT4-loop, b) a hexaethylene glycol- or, C) a tetraethylene glycol-loop closing the catalytic site. Their catalytic activity was confirmed. The cleavage acitvity of the hexaethylene glycol minizyme was superior to that of the tetraethylene glycol minizyme, but cleaved less efficiently than the dT4-minizyme. Tetra-biotinylated antisense RNA was synthesised and purified using two methods. The method using 2 successive additions of a multiple hydroxyl phosphoramidite followed by 1 addition of a single-addition biotin phosphoramidite, generated biotinylated antisense oligori bonucleotides of superior specificity in biotin/streptavidin based pre-mRNA depletion experiments. The stability of tandem GA mismatches was analysed using thermal denaturation techniques. On DNA tandem GA mismatches of the type 5' pyr GA pu 3' adopted an amino-type basepairing and were very stable, tandem AG mismatches were imino-paired and significantly less stable. The amino- type base pair has a distinctly shifted phosphorus resonance in the 31 p- NMR spectrum. Tandem GA mismatches in RNA did pair in the amino-form when flanked by 5' pyr. .pu 3' but were highly unstable, as were tandem AG mismatches. Tandem GA mismatches in DNA/RNA hybrid sequences were also unstable. Multiple GA mismatches of the type d(5' pyr GA Pu 3') did not destabilise duplexes compared to T.A base pairs. d(GA) n sequences formed homo-duplexes with exclusively G.A basepairing. 7 CONTNT Chtr 1: Th CiriiiiicI SynthsiiL1ri11 PUFNICaWn W ysic!L1lrit@1 INTRODUCTION 12 1. Enzymatic RNA Synthesis 13 2. Solid Phase Synthesis of RNA 13 2.1 Protecting Groups for the Exocyclic Amino Functions 14 2.2 Protecting Groups for the 2'-Hydroxyl Group 17 AIMS 28 RESULTS AND DISCUSSION 29 1. TBDMS-Method 32 1.1 Automated Solid Phase Synthesis 32 1.2 Cleavage and Deprotection 33 1.3 Deprotection of the 2'-Hydroxyl Function 36 1.4 Purification 36 1.5 Very Base Labile Protecting Groups on the 39 Exocydllc Amino Functions 2. Fpmp-Method 40 2.1 Solid Phase Synthesis 40 2.2 Cleavage from the Solid Support and Base- and 41 Phosphate-Deprotection 2.3 Cleavage of the 0-0-Fpmp-Groups 41 2.4 Purification and workup 42 3. The Synthesis of Sequences > 20 Nucleotides 43 4. Large Scale Synthesis 47 5. The Synthesis of Mixed DNA-RNA Oligonucleotides 48 CONCLUSIONS 48 REFERENCES 50 Caer 2: HMA Spfldng and INTRODUCTION 52 1. mRNA Splicing 53 2. Catalytic RNA 54 2.1 The Tetrahymena !VS Ribozyme 55 2.2 Catalytic Acitivity of RNAse P 57 2.3 Self-Cleavage Activity in Plant Pathogens 57 8 24 Reversibility of Ribozyme Cleavage 69 2.5 Ribozymes as Therapeutic Agents 70 3. The Synthesis of Biotinylated Oligoribonucleotides 77 3.1 Enzymic Method 77 3.2 Reaction of Uridyl-oligonucleotide with Periodate Followed by Biotin-Hydrazide and Reduction with Sodium Borohydride 77 3.3 Coupling of an Activated Biotin-Ester to Free Amino- Group on 5'-End of an Oligonucleotide 79 3.4 Synthesis of Biotinylated Phosphoramidite Building Blocks Using Modified Nucleosides 81 3.5 Synthesis of Non-Nucleoside Based Biotin Phosphoramidites 82 3.6 Introduction of Biotin Labels in Conjunction with Multiple Hydroxyl Phosphoramidites 84 RESULTS AND DISCUSSION 86 1. The Synthesis and Analysis of Modified Ribozymes 86 1.1 The Large Scale Synthesis of the Minimised Sequence Ribozyme 87 1.2 Synthesis of the Trans-Acting Minizyme 91 1.2.1 The minizyme substrate 92 1.2.2 The dT4-minizyme 92 1.2.3 The tetraethylene (TEG) and hexaethylene (HEG) glycol - minizymes 93 1.2.4 Catalytic substrate cleavage 97 1.2.5 Kinetic parameters for the minizymes 105 CONCLUSIONS 105 SUGGESTIONS FOR FUTURE WORK 106 2. The Synthesis of Biotinylated Antisense RNA Probes 107 2.1 The Nucleoside-based Biotin Phosphoramidite Approach 108 2.2 Attachment of Biotin-Molecules using the Multiple Hydroxyl Phosphoramidite 112 2.3 Antisense Detection of Specific pre-mRNA Sequences 118 CONCLUSIONS 122 9 REFERENCES 123 ChWev 3 OflganuWeaddes INTRODUCTION 128 Structural Properties of Oligonucleotides 128 1.1 Conformational Properties of the Phosphate Backbone, the Furanose Sugar and the Purinel Pyrimidine Bases 128 1.2 Nucleotide Structure and Base Pairing Arrangements 134 1.2.1 base pair mismatches 135 1.2.2 G.A mismatches 140 1.2.3 polypurine.polypyrimidine tracts, triplex formation 142 1.3 DNNRNA Hybrids 147 Thermal Denaturation of Oligonucleotides 153 RESULTS AND DISCUSSION 159 1. Synthesis and Analysis of the Hybrid Duplex d(GTGAACTT) .r(AAGUUCAC) 159 2. Self-Association of d(GTGAACTT) 159 2.1 pH Dependence 164 2.2 The Use of Inosine as Structural Probe 164 2.3 The Effect of the Overhanging Ends on Duplex Stability 164 2.4 The Solution Structure of[d(GTGAAC1T)J2 165 3. Stabilities of T.A, G.A, A.G and LA Tandem Base Pairs 167 3.1 Tandem G.A Mismatches in DNA -Duplexes 167 3.2 Tandem G.A Mismatches in RNA-Duplexes 170 3.3 Tandem G.A Mismatches in DNNRNA Hybrid Duplexes 174 4. Multiple G.A Mismatches in Oligonucleotides 175 5. Contiguous G.A Mismatches 177 CONCLUSOONS 184 REFERENCES 185 Ulf] 190 Materials and Methods 190 NMR 191 Mass Spectra 191 Chromatography 191 Capillary Gel Electrophoresis 191 1. DNA Synthesis and Purification 192 2. RNA Synthesis and Purification 197 2.1 TBDMS-Method 197 2.2 Fpmp-Method 201 3. Polyacrylamide Gel Electrophoresis 214 4. [y32P]ATP 5' End Labelling of Oligonucleotides 215 5. Ribozyme Cleavage Reactions 215 S. Enzyme Digestion of Oligodeoxynucleotides 216 7. Ultraviolet-Melting Studies 216 8. Chemical Synthesis 217 9. Determination of the Extinction Coefficient of 2'-Deoxy Inosine 219 References 220 1. Extinction Coefficients of Oligonucleotides 221 2. Melting Temperatures and Thermodynamic Data of DNA Hexamers and Octamers I 225 3. Melting Temperatures of DNA Decamers 227 4. Thermodynamic Parameters of RNA Decamers 229 5. G.A Mismatches in DNA/RNA Hybrids (Buffer A) 232 6. UV-Melting Data of Multiple Tandem G.A Mismatches in Oligonucleotides 233 7. Melting Temperatures of d(XA) n Sequences (X=dT, dG or dl) 235 8. Capillary Electrophoresis Elution Profile of Ribozyme-Substrate [R44]. 237 9. Capillary Electrophoresis Elution Profile of Hexaethyleneg lycol- Ribozyme [R54]. 238 11 ct1I CceD Synnemsend P mvM c&Uan W OOigoowdlle©®s INTRODUCTION RNA is much more widespread in its occurrence and more diverse in its biological functions than DNA. The roles of messenger RNA (mRNA), transfer RA (tRNA) and ribosomal RNA (rRNA) in the biochemistry of the cell are well understood, but the recent discovery of self-splicing RNA and the isolation of RNA-protein complexes involved in eukaryotic mRNA processing serve to illustrate that there is a great deal to learn about the participation of RNA in biological processes. The availability of pure synthetic oligoribonucleotides in reasonable quantities will almost certainly prove to be of great importance in future research in molecular biology.
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