Studies on the Extension of Sequence-independence and the Enhancement of DNA Triplex Formation

Abstract

A more elaborate sequence-independent triple-helix formation viability study was carried out and extended from a recombination-like triple-helical DNA motif of a previous study (J. Mol. Recognition 14, 122–139 (2001)). The intended triple-helix was formed by mixing one part of a DNA hairpin duplex and one part of a single (or third) strand identical to one of the duplex strands and complementary to the other strand. In contrast to the common purine and pyrimidine motifs in triple-stranded DNA, the strands of the recombination-like motif are not monotonously built from pyrimidine only, or purine only, in the sequence. The stability of the recombination-like motif triplexes with varying sequences was monitored by UV thermal melting curves. The results showed that the order of the stability of the R-form DNA base triads (J. Mol. Biol., 239, 181–200 (1994)) is G*(G ○ C) > C*(C ○ G) > A*(A ○ T) >T*(T ○ A) (the Watson-Crick base pair is denoted in the parentheses) in 200 mM NaCl, at pH 7. In an attempt to increase the stability of the triplex in the recombination-like motif, we replaced cytidine by 5-methylcytidine (mC) of the third strand. There is a general trend that mC modification stabilizes the complex (<2 °C per mC). The complex is furthermore stabilized by Mg2+ ion. The Tm increases from 7 to 2 °C from less stable to highly stable triplex by 20 mM Mg2+ ion in solution.