Sequence limitations of triple helix formation by alternate-strand recognition

Until recently, oligonucleotide-directed triplex formation has been limited to oligopurine tracts of target DNA. Triplex formation by alternate-strand recognition relaxes this limitation by allowing triplexes to form at 5'-(Pu)m(Py)n-3' and 5'-(Py)m(Pu)n-3' sequences, with the third strand pairing first with purines on one strand and then switching to pair with purines on the other strand. In this study, the interaction of several oligonucleotides with the potential to form triplexes by alternate-strand recognition at the sequence 5'-A8C8A8-3' was studied by chemical probing and affinity cleaving. The results show that triplex formation can be readily accomplished at the 5'-A8C8-3' part of the sequence; however, base triplet formation is disrupted on either side of the strand switch and the Watson-Crick helix is distorted in such a way as to expose the N7 positions of purines adjoining the strand switch. Triplex formation is weak or nonexistent at the 3'-most A8 block, despite the opportunity for recruiting a spacer sequence for the second (C8-A8) strand switch by "slippage". This finding indicates that the C8-A8 strand switch is energetically unfavorable, although pairing at other 5'-(Py)n(Pu)n-3' sequences has been observed, with or without a spacer [Beal, P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478; Jayasena, S. D., & Johnston, B. H. (1992) Nucleic Acids Res. 20, 5279-5288]. Thus, alternate-strand recognition may not be feasible for certain sequences of 5'-(Py)m(Pu)n-3', at least under the conditions examined.