| dc.description.abstract | The thesis entitled “Synthesis and Structural Studies of Self Complementary Oligodeoxyribonucleotides” is aimed at understanding the role of sequence specific backbone conformation of DNA oligomers in protein–DNA interactions and/or in conformational interconversions of DNA itself. Toward this goal, studies were carried out on the following aspects:
1. Chemical synthesis of oligodeoxyribonucleotides using an optimal amount of monomer.
2. Development of convenient and simple HPLC purification methods for synthetic oligonucleotides.
3. Synthesis and solution conformation of certain CG oligomers containing restriction endonuclease (RE) sites.
4. Synthesis and solution conformation of AT tetramers, and correlation of their conformation with the lack of RE specificity in these tetranucleotides.
5. Role of 5 phosphate and sequence in the B Z conformational transition of alternating CG hexamers in solution.
6. Synthesis and conformation of d(ACGT) and d(AGCT) , with special reference to B Z interconversion.
7. Significance of flanking bases in sequence specific recognition and cleavage by restriction enzymes.
To present these aspects cohesively and comprehensibly, the thesis has been divided into ten chapters. Some results that are off shoots of the thesis work but do not fit perfectly within the main framework have been included in three appendices.
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Chapter I - Introduction
Synthetic oligodeoxyribonucleotides are essential tools in genetic engineering. Although the first report of their chemical synthesis appeared in the early 1950s, research in this area gained major momentum in the 1960s due to sustained efforts by several groups. Consequently, solution phase phosphodiester synthesis gave way to solid phase phosphotriester and phosphoramidite chemistry. This evolution in synthesis methods is reviewed in Chapter I, along with structural studies on various oligo and polynucleotides relevant to this thesis.
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Chapter II - Methodologies
Describes general methodologies used during this work, including:
• purification/drying of solvents,
• preparation of various buffers,
• operational and data handling techniques for spectroscopic methods (UV, CD, ³¹P NMR, ¹H NMR including 2D methods),
• chromatographic methods (column, TLC, HPLC), and
• polyacrylamide gel electrophoresis.
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Chapter III - Monomer Preparation
Details the preparation of suitably protected monomers required for the nucleotide chain building process via phosphotriester or phosphoramidite chemistry. Also includes derivatization of silica matrices for nucleotide anchoring.
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Chapter IV - Solid Phase Oligonucleotide Synthesis
Describes the synthesis of oligodeoxyribonucleotides through chain elongation on silica support, using both phosphotriester and phosphoramidite routes. A manual synthesizer was used (glass column with sintered disc). It is shown that:
• For large batches (>20 mol) of short oligomers, coupling reactions can be repeated once or twice to achieve 94–96% coupling yields using only 4–6 fold excess monomer,
• Conventional syntheses require 10–20 fold excess for similar yields.
The repeated coupling strategy works well for both phosphoramidite and phosphotriester syntheses.
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Chapter V - Purification Methods
A novel purification strategy for self complementary oligonucleotides on a reverse phase C 18 column using volatile triethylammonium bicarbonate (TEAB) at ambient temperature is described. By injecting heat denatured samples, the usual problems of multipeak formation due to duplexes/multimers are avoided.
A similar methodology was developed for anion exchange HPLC, replacing conventional non volatile buffers at elevated temperatures with volatile buffers at room temperature.
A dual column strategy combining RP HPLC and AE HPLC was also developed. Methods for characterization and sequence analysis of 5 end labelled oligomers via gel electrophoresis are included.
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Chapter VI - Conformation of CG Tetramers
The closely related CG tetramers d(CCGG) and d(GGCC), containing RE sites for MspI/HpaII and HaeIII respectively, were studied using:
• UV, CD, ³¹P NMR, 1D and 2D ¹H NMR,
• gel filtration HPLC.
Findings:
• d(CCGG) in low salt, low temperature solution adopts a unique conformation distinct from d(GGCC).
• Possible contributing factors (aggregation, incomplete deprotection, triple helical structures, Z like conformations) were examined.
• Larger oligomers d(CCGGCCGG) show similar conformations to d(CCGG).
• Under simulated crystallization conditions (presence of spermine), both tetramers adopt conformations similar to their known crystal structures.
• CD and ³¹P NMR show that spermine induces significant conformational changes.
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Chapter VII - AT Tetramers and Lack of RE Specificity
The AT tetramers d(AATT), d(TTAA), and d(TATA) were studied using UV, CD, ³¹P NMR, and ¹H NMR.
Findings:
• These tetramers do not form proper duplexes in low salt (<0.1 M NaCl) at low temperature (<5 °C).
• Their inability to adopt stable duplex conformations likely leads to loss of RE specificity.
• In solution, they probably adopt an average local conformation incompatible with restriction enzyme recognition.
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Chapter VIII - B Z Transition Studies
Explores the influence of sequence and 5 phosphate on B Z transitions in CG hexamers.
Findings:
• pd(CG) and pd( C G) exist as B DNA at low temperature and low salt.
• High salt CD and UV spectra show that a 5 phosphate inhibits the salt induced B Z transition.
• As d(GC) does not undergo B Z transition, inhibition by a 5 phosphate is relevant only for d(CG) .
Molecular modelling suggests possible inhibitory hydrogen bonding involving the 5 phosphate.
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Chapter IX - d(ACGT) and d(AGCT) : Conformational Studies
Despite containing alternating pyrimidine–purine steps, d(ACGT) and d(AGCT) do not undergo B Z transition in solution, even at high salt or in the presence of Co³ hexamine. Anti Z DNA antibody does not bind them.
Restriction digestion experiments with RsaI and AluI show that cleavage requires flanking base pairs; otherwise no cleavage occurs.
Results are discussed in detail.
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Chapter X - Conclusions
Summarizes achievements and outlines future directions.
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Appendices
• Appendix I: Identification and characterization of an oily impurity (p chlorophenyl di cyanethyl phosphate) interfering with phosphotriester synthesis.
• Appendix II: Simple method to convert unreacted phosphoramidite into stable phosphodiester.
Appendix III: Modified method for preparing oligonucleotides for RE digestion using volatile buffers. | |
