THE CHEMICAL SYNTHESIS AND PHYSICAL CHARACTERISTICS OF SEVERAL OLIGODEOXY RIBONUCLEOTIDES: D(CGAGTTTGACGP) FORMS AN UNUSUALLY STABLE HAIRPIN LOOP STRUCTURE (DNA)
Abstract
I. A method for accurately determining the molar absorptivity of an oligodeoxyribonucleotide has been applied to a number of related single-stranded molecules. The values obtained were used in the analysis of the absorptivity-temperature profiles and circular dichroism spectra of each oligomer. From the results, we conclude that the undecamer CGAGTTTGACGp exists in a hairpin conformation with a transition midpoint of 40(DEGREES)C in water containing 0.12 M Na+. The structure is unusually stable in comparison with hairpins formed by other oligomers, and at least some of this stability must arise from interactions other than those associated with Watson-Crick base-pairing and stacking because the molecule comprised of the complementary sequence (CGTCAAACTCGp) does not adopt the corresponding structure, within the observed temperature range of 0 to 85(DEGREES)C. Analysis of the undecamer's absorptivity-temperature profiles in terms of a two-state model indicates that the hairpin-to-coil transition occurs with an enthalpy change that is substantially larger than expected if only Watson-Crick base-paired stacking effects are considered. It appears that sequence-dependent interactions within the loop can play an important role in determining hairpin stability. II. A new protected 5' ribonucleotide, 2',3'-O-bis-(4,4'-dimethoxytrityl)uridine 5'-(4-chlorophenyl phosphate), forms the basis of a chemical procedure for phosphorylating the 5' ends of DNA fragments synthesized by the phosphotriester approach. Condensation of this mononucleotide with the free 5' hydroxyl of an otherwide fully protected oligomer results in high-yield formation of a 5'-5' triester linkage. Subsequently, the terminal ribonucleotide of the deprotected product rU5'-5'd(N-N(,n)-N) can be cleaved by periodate oxidation of its 2',3' cis-hydroxyl system followed by (beta)-elimination, leaving its phosphate attached to the 5' hydroxyl group of the oligodeoxyribonucleotide.
Degree
Ph.D.
Subject Area
Biophysics
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