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  1. 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.