Effect pf 5-methylcytosine on the stability of triple-stranded DNA—a thermodynamic study

Abstract

We have previously shown that the pyrimidine oligonucleotide 5'CTTCCTCCTCT (Y11) recognizes the double-helical stem of hairpin 5'GAAGGAGGAGA-T4-TCTCCTCCTTC (h26) by triple-helix formation (1). In this paper, we report the effect on triplex formation of substituting the cytosine residues of Y11 with 5-methylcytosines (5meY11). In addition, we have studied the thermodynamics of the interaction between h26 and 5meY11. The results can be summarised as follows: (i) gel electrophoresis shows that at T = 5 degrees C and pH 5, both Y11 and 5meY11 form DNA triple helices with h26, whereas at pH 6.8 only the methylated strand binds to h26; (ii) pH-stability curves of the DNA triplexes formed from h26 + Y11 and h26 + 5meY11 show that Y11 and 5meY11 are semi-protonated at pH 5.7 and 6.7, respectively. Thus, it is concluded that cytosine methylation expands the pH range compatible with triplex formation by one pH unit; (iii) as the unmethylated triplex (h26:Y11), the methylated one (h26:5meY11) denatures in a biphasic manner, in which the low temperature transition results from the dissociation of 5meY11 from h26. The Tm of the triplex to h26 plus 5meY11 transition is strongly enhanced (about 10 degrees C) by cytosine methylation. A van 't Hoff analysis of denaturation curves is presented; (iv) DSC experiments show that triplex formation between 5meY11 and h26 is characterized by delta H = -237 +/- 25 kJ/mol and delta S = -758 +/- 75 J/Kmol, corresponding to an average delta H of -21 kJ/mol and delta S of -69 J/Kmol per Hoogsteen base pair; (v) the thermodynamic analysis indicates that the extra stability imparted to the triplex by methylcytosine is entropic in origin.

DNA triple helices: Biological consequences and therapeutic potential

“…These modifications have assisted in preventing the formation of unwanted intramolecular secondary structures within the TFOs. In the parallel pyrimidine triplex motif, the substitution of cytosine by 5-methylcytosine, N 6 -methyl-8-oxo-2-deoxyadenosine [52,53] or pseudoisocytodines [54] has been used to reduce the pH dependence of triplex formation. At neutral pH, the substitution of thymine by 5-propynyluracil stabilizes triplex formation [55].…”

Preorganization of DNA: Design Principles for Improving Nucleic Acid Recognition by Synthetic Oligonucleotides

“…Early studies with polymeric strands indicated an advantage in thermal stability with this substitution, 47,48 and more recent studies with short, well-defined sequences have confirmed a thermodynamic advantage as well. [49][50][51] One study found that the addition of each methyl group to uracil either in RNA or in DNA adds 0.1-0.5 kcal/mol of stability to double-and triple-helical structure. 51 It is thought that this effect is due to increased polarizability of methylated bases, which enhances van der Waals interactions with neighboring bases.…”

Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases

“…15,16 These limitations have been addressed by the incorporation of base and sugar modifications into pyrimidine TFOs. [17][18][19][20] On the contrary, TFOs form stable triplexes in the purine motif in a pH-independent manner. However, several groups have shown that binding of these guanine-rich (G-rich) TFOs to duplex DNA is significantly reduced at physiologic concentrations of monovalent cations, such as potassium (K + ).…”