Effect of 5-Methylcytosine on the Structure and Stability of DNA. Formation of Triple-Stranded Concatenamers by Overlapping Oligonucleotides

Abstract

A triple helix can be formed upon binding of a pyrimidine oligonucleotide to the major groove of a homopurine-homopyrimidine (R·Y) double-stranded DNA target site. Here, we report that this reaction can be influenced by base methylation. The pyrimidine strand 5′- TmCTmCTmCTmCTTmCT (mY₁₂), whose cytosine residues are methylated at C5, does not bind the duplex 5′-AGAGAGAGAAGA·3′-TCTCTCTCTTCT (R₁₂·Y₁₂) to yield a 12-triad triplex, as would be expected from these DNA sequences. Rather, a complex of overlapping oligonucleotides, which we define concatenamer, is formed. The concatenamer is clearly evidenced by Polyacrylamide gel electrophoresis (PAGE) since it migrates with a smeared band of very low mobility. The stoichiometry of the concatenamer, determined by both UV mixing curves and electrophoresis, is surprisingly found to be (R₁₂· 2mY₁₂)_n, thus showing that the unmethylated Y₁₂ strand is excluded from the complex. Denaturation experiments performed by ultraviolet absorbance (UV) and differential scanning calorimetry (DSC) show that the concatenamers melt with a single and highly cooperative transition whose Tm strongly depends on pH. Overall, the data point to the conclusion that the concatenamers are in triple helix, where the methylated mY₁₂ strand is engaged in both Watson-Crick and Hoogsteen base pairings, thus displacing the Y₁₂ strand from the R₁₂·Y₁₂ duplex. A possible mechanism of concatenamer formation is proposed. The results presented in this paper show that 5-methylcytosine brings about a strong stabilizing effect on both double and triple DNA helices, and that pyrimidine oligonucleotides containing 5-methylcytosine can displace from R·Y duplexes the analogous non-methylated strand. The advantage of using methylated oligonucleotides in antisense technology is discussed.