Variability in the stability of DNA-peptide nucleic acid (PNA) single-base mismatched duplexes: real-time hybridization during affinity electrophoresis in PNA-containing gels.

Abstract

The stability of all single-base mismatched pairs between a peptide nucleic acid 11-mer and its complementary DNA has been quantified in terms of their melting temperature and compared with the limited amount of data published to date. The strength of the interaction was determined by an automated affinity-electrophoretic approach permitting the visualization, in real time, of hybridization between a physically immobilized peptide nucleic acid and a complementary DNA migrating in an electric field. The dissociation constants are in the range of 10(-7) M (for mismatches) to 10(-10) M (for fully complementary DNA), which are in excellent agreement with solution studies. These and other thermodynamic constants can be accurately, rapidly, and reproducibly measured in this system at concentrations approaching dissociation conditions by using fluorescently labeled DNA in conjunction with commercial DNA sequencers. The stability of single-base mismatched peptide nucleic acid-DNA duplexes depends both on the position as well as on the chemical nature of the mismatch. The stability is at a minimum when the mutation is positioned 4 bases from either terminus (a loss of 20 degreesC or more in the melting temperature) but regains substantial stability when the mismatch is at the center of the duplex. The most stable mismatched pairs are G:T and T:T whereas destabilization is maximal for A:A and G:G. These observations are of significance in the design of probes for detecting mutations by hybridization.