Revised Manuscript Received February 24, 1992 abstract: A 32-base DNA oligonucleotide has been studied by one-and two-dimensional NMR spectroscopy and is shown to form a stable, pyr-pur-pyr, intramolecular triple helical structure, with a four C loop and a TATA loop connecting the Watson-Crick-and Hoogsteen-paired strands, respectively. This triplex contains fiveT-AT base triplets, two C+-GC base triplets, and an unusual GTA base triplet which disrupts the pyr-pur-pyr motif. The GTA triplet consists of a Watson-Crick TA base pair, with the T situated in the “purine strand” and the A situated in the “pyrimidine strand” and a G situated in the Hoogsteen-base-paired “pyrimidine strand” hydrogen bonded to the T. The base-pairing structure of the GTA triplet has been investigated and has been found to involve a single hydrogen bond from the guanine amino group to the 04 carbonyl of the thymine, leaving the guanine imino proton free. The specific amino proton involved in the hydrogen bond is the H2 (2) proton. This orients the guanine such that its sugar is near the thymine methyl group. The guanine sugar adopts an N-type (C3'-endo) sugar pucker in this triplet. The stability of the GTA triplet within pyr-pur-pyr triplexes is discussed.

Triple-stranded DNA has been postulated to play a role in several important biological functions, such as transcriptional regulation and recombination [Weinreb et al., 1990; Hsieh et al., 1990; Htun & Dahlberg, 1989; Mirkin et al., 1987; re-viewed in Wells et al.(1988)] andhas been promoted as a potentially powerful biological tool, such as a chemical nuclease to map chromosomes (Frangois et al., 1989a; Strobel et al., 1988; Strobel & Dervan, 1991; Perrouault et al., 1990) or as a highly sequence-specific repressor to block protein recognition of DNA (Cooney et al., 1988; Maher et al., 1989; Hanvey et