Zheng, Qinguo; (1995) Chemical synthesis of DNA containing modified bases by post-synthetic substitution and application of modified DNA to the study of protein-DNA interaction. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Because of the limitations of most methods for modified oligonucleotide synthesis, an alternative strategy (post-synthetic substitution strategy) was developed for synthesis of DNA with modification on the 4-position of thymine and the 6-position of guanine. This was achieved by incorporation of versatile nucleotides into DNA which contain a leaving group which is sufficiently stable to withstand the conditions of DNA synthesis but can be substituted by nucleophiles, after synthesis, to produce a series of oligomers each containing a different modified base. 4-Triazolothymidine phosphoramidite was prepared and inserted into the dodecanucleotide AGCGAAXTCGCT (X standing for 4-triazolothymine). By treating the oligomer with different nucleophiles the parent oligomer containing thymine (T) and other five oligomers were prepared each containing a different modified base: O4-methylthymine (TOMe), O4-ethylthymine (TOEt), 5-methylcytosine (TNH2), N4-dimethylamino-5-methylcytosine (TDH), or 4-thiothymine (TS). As a further development of the above method, a fully deprotected and purified versatile oligonucleotide containing 4-phenylthiothymine was prepared with the advantage of making oligonucleotides containing very labile modified thymines. Oligomers containing 5-methyl-N4,N4-ethanocytosine or 4-azidothymine were prepared by treating the versatile oligomer with ethyleneimine or sodium azide (NaN3), respectively. A post-synthetic approach was also developed for the preparation of oligonucleotides containing guanine modified at the 6-position. The versatile guanine monomer N2-phenylacetyl-6-(2,4-dinitrophenyl)thio-2'-deoxyguanosine-3'-phosphoramidite was prepared and incorporated into an oligomer. The oligomer containing this versatile guanine (GSø) was converted into oligomers containing 6-thioguanine (GS), 2,6-diaminopurine (GNH2), 2-amino-6-methylaminopurine (GNMe), O6-methylguanine (GOMe), or guanine (G) by treatment with appropriate reagents after synthesis. The interactions between λ-phage Cro repressor and its operator DNA were investigated by a photochemical cross-linking approach using 21 mer oligonucleotides containing the sequence of the OR3 operator of λ-phage in which one of guanine or thymine was substituted with 6-thioguanine or 4-thiothymine. Upon irradiation with long-wave UV light (~360 nm) the oligonucleotides containing these thiobases cross-linked to the Cro protein with different efficiency, depending on the position of the substitution. The cross-linking results are consistent with the Cro-DNA interaction model and also support the suggestion that cross-linking can occur only on those amino acid residues at the interface of the DNA-protein complexes. However an attempt to identify the amino acid residues covalently linked to DNA was unsuccessful, mainly due to the instability of cross-linked Cro-DNA complexes.

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