ORIGINS OF HIGH SEQUENCE SELECTIVITY - A STOPPED-FLOW KINETICS STUDY OF DNA RNA HYBRIDIZATION BY DUPLEX-FORMING AND TRIPLEX-FORMING OLIGONUCLEOTIDES/

Stopped-flow UV kinetics and thermal denaturation experiments are used to examine the origins of high sequence selectivity and binding affin ity of circular triplex-forming oligonucleotides with single-stranded DNA/RNA targets. These 34-nt probes are hybridized to a series of 12-n t target sequences which are fully complementary or which contain a si ngle mismatch. Also studied for comparison are standard 12-nt Watson-C rick DNA or RNA complements. Several novel findings are described: (1) Circular triplex-forming oligomers bind targets with very high thermo dynamic selectivity (up to 8-10 kcal/mol against a single-nucleotide m ismatch), while linear strands show only 2-3 kcal/mol selectivity. (2) Rates for triplex formation by circular ligands are much greater than other reported tripler formation modes and are nearly the same as for Watson-Crick duplex formation. (3) DNA-DNA and RNA/RNA; hybridization rates are similar for both duplex and triplex formation. (4) For both modes of binding, hybridization rates do not vary when a mismatch is introduced into the target, and, therefore, binding selectivity is ref lected in large variations in dissociation, rather than association ra tes. Finally, (5) binding selectivity of circular ligands becomes sign ificantly greater as pH is lowered; results indicate that the high seq uence selectivity of the circular DNA ligand is due in large part to t he special stability of the protonated C+G-C trial relative to unproto nated mismatched triads. The results are useful in the understanding o f properties of nucleic acid complexes in general and give insight int o optimum design for synthetic DNA-binding ligands.