Synthesis and characterization of oligodeoxynucleotides containing 4-O-methylthymine

Abstract

The carcinogenicity of N-nitroso compounds is believed to result from the alkylation of DNA, particularly on O-6 of the guanine and O-4 of the thymine residues. In order to study the base-pairing properties of 4-O-methylthymidine (T*) residues and the structural changes produced in DNA by the presence of this alkylated nucleoside, the oligodeoxyribonucleotides T*GCG, CGCAAGCTT*GCG, CGCGAGCTT*GCG, and CGCAAGCTTGCG were synthesized by the phosphotriester approach in solution. The 4-O-methylthymidine required for oligonucleotide synthesis was prepared by treating the 4-(3-nitro-1,2,4-triazolo) derivative of 3',5'-bis-O-(methoxyacetyl)thymidine with 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in methanol solution. The susceptibility of the 4-O-methyl group of T* toward nucleophiles enables this group of 4-O-methylthymidine-containing oligomers to be labeled by a direct exchange reaction with [13C]- or [14C]methanol in the presence of DBU. Although it has been previously suggested that 4-O-methylthymine forms stable base pairs with guanine, the thermal melting profiles of the double helices formed by these dodecamers suggest that the presence of 4-O-methylthymine paired to either adenine or guanine destablizes the helix. The melting curve of the sequence containing a 4-O-methylthymine residue base paired to guanine was biphasic and similar to that of an analogous sequence containing 6-O-methylguanine paired to thymine.