Triple helix formation: binding avidity of acridine-conjugated AG motif third strands containing natural, modified and surrogate bases opposed to pyrimidine interruptions in a polypurine target

A critical issue for the general application of triple-helix-forming oligon ucleotides (TFOs) as modulators of gene expression is the dramatically redu ced binding of short TFOs to targets that contain one or two pyrimidines wi thin an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix forma tion. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in oppositio n to pyrimidines and purines substituted into selected positions within a p aradigm 15-base polypurine target sequence. We found that substitutions clo ser to the intercalator end of the TFO (positions 4-6) had a more deleterio us effect on the dissociation constant (K-d) than those farther away (posit ion 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful tripler formation (K(d )s of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base s elective than cytosine, binding to T greater than or equal to 4 times bette r than to A, G or C. None of the TFOs tested showed avid binding when C res idues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K-d of 200 nM, significantly better than the other designs. Molecul ar modeling showed that the 3-nitropyrrole T:A triad is isomorphous with th e A A:T triad, and suggests novel parameters for evaluating new base triad designs.