Cooperative Binding of Oligonucleotides to Adjacent Sites of Single-Stranded DNA: Sequence Composition Dependence at the Junction

Abstract

The quantitative parameters of cooperative binding of deoxyribooligonucleotides to adjacent sites by double helix formation have been determined as a function of sequence composition at the junction. The base stacks 5′-Py/p-Py-3′, 5′-Pu/p-Py-3′ and 5′-Pu/p-Pu-3′ (p is phosphate group, Py and Pu are pyrimidine and purine nucleoside, respectively) including mismatches on the 3′-side of the junction were studied using complementary addressed modification titration (CAMT) at 25°C and pH 7.5, 0.16M NaCl, 0.02M Na₂HPO₄, 0.1 mM EDTA. The equilibrium binding constants of alkylating derivatives of 8-mer oligonucleotides (reagents) with 22-mer oligonucleotides (targets) were determined using the dependence of the target limit modification extents on the concentrations of the reagents. The parameters of cooperati vity were calculated as the ratio of binding constants of reagents in the presence and the absence of a second 8-mer oligonucleotides (effectors) occupying the adjacent site on the 22-mer targets. For the stacks 5′-Py/p-Py-3′ the parameters of coopera- tivity were around unity both for matched and mismatched nucleotides at the junction indicating the absence of cooperativity. The parameters of cooperativity for the stacks 5′-Pu/p- Pu-3′ were higher than for the stacks 5′-Pu/p-Py-3′ in perfect and non-perfect duplexes. Discrimination of mismatches was higher in nicked than in normal duplexes.