Elucidation of the sequence-specific third-strand recognition of four Watson-Crick base pairs in a pyrimidine triple-helix motif: T.AT, C.GC, T.CG, and G.TA.

We report a specific pattern of recognition by third-strand bases for each of the four Watson-Crick base pairs within a pyrimidine triple-helix motif as determined by PAGE: T.AT, C.GC, T.CG, and G.TA. Our recognition scheme for base triplets is in agreement with previous studies. In addition, we identified another triplet, T.CG, under physiological conditions, in which formation of triple helix was observed at equimolar ratios of the third strand and duplex target. Although different nearest-neighbor effects are expected, this finding extends the base-recognition code to all 4 base pairs in double-stranded DNA under physiological conditions. Base-composition analysis of putative triplex species provided independent evidence for the formation of triplex and confirmed the base-recognition code determined by PAGE. Moreover, the formation of triplex, as detected by gel electrophoresis, was seen to be an all-or-none phenomenon, dependent upon a single-base mismatch among 21 nucleotides. This result suggests a high specificity for the recognition of double-stranded DNA by a third strand. In addition, we report the surprising finding that triplex stability depends on the length and sequence of the target duplex DNA.