The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha(7)H, 1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta(7)H, 2) and 7-(2'-O-methyl-beta-D-ribofuranosyl)hypoxanthine (beta(7)H(OMe), 3) were prepared and incorporated into tripler forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine . purine-pyrimidine) motif, By DNase I footprinting techniques and UV-melting curve analysis it was found that, at ph 7.0, the 15mer oligonucleotides d((TTTTTCTXTCTCTCT)-C-Me-C-Me-C-Me-C-Me) (C-Me = 5-methyl-deoxycytidine, X = beta(7)H, beta(7)H(OMe)) bind to a DNA target duplex forming a H . G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by C-Me. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 mu M third strand concentration, no stable tripler exhibiting H . A-T, H . T-A or H . C-G base arrangements could be found (target duplex concentration similar to 0.1 nM), Multiply substituted sequences containing beta(7)H residues either in an isolated [d(TTTTT beta(7)HT beta(7)HT beta(7)HT beta(7)HT beta(7)HT)] or in a contiguous [d(TTT beta(7)H beta(7)H beta(7)H beta(7)HTTTT beta(7)HTTT)] manner still form triplexes with their targets of comparable stability as the control (C-Me-containing) sequences at ph 7.0 and high salt or spermine containing buffers, General considerations lead to a structural model in which the recognition of the Gi-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine, This model is supported by a molecular dynamics simulation, A general comparison of the tripler forming properties of oligonucleotides containing beta(7)H with those containing C-Me or N-7-2'-deoxyguanosine (N(7)G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.
