STRONG, SPECIFIC, MONODENTATE G-C BASE-PAIR RECOGNITION BY N-7-INOSINE DERIVATIVES IN THE PYRIMIDINE-CENTER-DOT-PURINE-PYRIMIDINE TRIPLE-HELICAL BINDING MOTIF

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(O Me), 3) were prepared and incorporated into tripler forming oligodeoxy nucleotides, designed to bind to DNA in the parallel (pyrimidine . pur ine-pyrimidine) motif, By DNase I footprinting techniques and UV-melti ng curve analysis it was found that, at ph 7.0, the 15mer oligonucleot ides d((TTTTTCTXTCTCTCT)-C-Me-C-Me-C-Me-C-Me) (C-Me = 5-methyl-deoxycy tidine, X = beta(7)H, beta(7)H(OMe)) bind to a DNA target duplex formi ng a H . G-C base triple with equal to slightly increased (10-fold) st ability compared to a control oligodeoxynucleotide in which the hypoxa nthine 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 concentr ation, no stable tripler exhibiting H . A-T, H . T-A or H . C-G base a rrangements could be found (target duplex concentration similar to 0.1 nM), Multiply substituted sequences containing beta(7)H residues eith er in an isolated [d(TTTTT beta(7)HT beta(7)HT beta(7)HT beta(7)HT bet a(7)HT)] or in a contiguous [d(TTT beta(7)H beta(7)H beta(7)H beta(7)H TTTT beta(7)HTTT)] manner still form triplexes with their targets of c omparable stability as the control (C-Me-containing) sequences at ph 7 .0 and high salt or spermine containing buffers, General consideration s 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 hy poxanthine to N7 of guanine, This model is supported by a molecular dy namics simulation, A general comparison of the tripler forming propert ies 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 recogniti on in the latter two cases.