Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine·purine-pyrimidine triple-helical binding motif

Abstract

The nucleoside analogs 7-(2'-deoxy-α-D-ribofuranosyl)hypoxanthine (α7H, 1), 7-(2'-deoxy-β-D-ribofuranosyl)hypoxanthine (β7H, 2) and 7-(2'-O-methyl-β-D-ribofuranosyl)hypoxanthine (β7H(OMe), 3) were prepared and incorporated into triplex 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(TTTTT(Me)CTXT(Me)CT(Me)CT(Me)CT) ((Me)C = 5-methyldeoxycytidine, X = β7H, β7H(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 (Me)C. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 μM third strand concentration, no stable triplex exhibiting H·A-T, H·T-A or H·C-G base arrangements could be found (target duplex concentration ~ 0.1 nM). Multiply substituted sequences containing β7H residues either in an isolated [d(TTTTTβ7HTβ7HTβ7HTβ7HTβ7HT)] or in a contiguous [d(TTTβ7Hβ7Hβ7Hβ7HTTTTβ7HTTT)] manner still form triplexes with their targets of comparable stability as the control ((Me)C-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 G-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 triplex forming properties of oligonucleotides containing β7H with those containing (Me)C or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.