Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics

Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.