The binding of a variety of deoxyribo and ribo homo- and copolynucleotide complementary duplexes to agarose-linked homopolynucleotide affinity columns has been studied. The results provide information concerning the specificity of recognition of complementary base pairs of nucleic acids through a mechanism that involves triple-helix formation under physiological conditions of ionic strength, pH, and temperature. The method employed made it possible, for the first time, to survey the full range of base triplets conceivable from the canonical nucleic acid bases and, in addition, hypoxanthine and thereby to differentiate between those triplets which can and cannot form. Certain previously observed features of the stereochemistry of double-helical targets for third-strand binding are confirmed, and some unrecognized features are elaborated. These include a general requirement for clusters of purine residues in one strand, protonation of third-strand C residues, the ability of natural third-strand residues to distinguish between A.T/U and G-C base pairs, and a capacity of third-strand (unnatural) I residues to recognize all base pairs within such clusters. Thus, the basis for a third-strand binding code is demonstrated.
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