Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5'-GT-2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3' (R^2APCW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3' (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R^2APCW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19° C to 33° C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R^2APCW triplex and up to six YOYO molecules to CW hairpin.

Key words: R-triplex DNA; Stabilization; YOYO; Melting temperature.