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

ABSTRACT

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 PNADNA 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 DNADNA duplexes, PNADNA 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 PNADNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNADNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNADNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNADNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.