An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA

We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they ape known to form P-loops, which consist of a [PNA](2)-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding; rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. Ln contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed. A theoretical analysis of the data suggests that for (C + T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J + T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J + T)-containing PNA constructions may be advantageous for use in vivo. (C) 1999 Academic Press.