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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. 

Nature encodes the information required for life in two fundamental biopolymers: nucleic acids and proteins. Peptide nucleic acid (PNA), a synthetic analog comprised of nucleobases arrayed along a pseudopeptide backbone, has the ability to combine the power of nucleic acids to encode information with the versatility of amino acids to encode structure and function. Historically, PNA has been perceived as a simple nucleic acid mimic having desirable properties such as high biostability and strong affinity for complementary nucleic acids. In this feature article, we aim to adjust this perception by highlighting the ability of PNA to act as a peptide mimic and showing the largely untapped potential to encode information in the amino acid sequence. First, we provide an introduction to PNA and discuss the use of conjugation to impart tunable properties to the biopolymer. Next, we describe the integration of functional groups directly into the PNA backbone to impart specific physical properties. Lastly, we highlight the use of these integrated amino acid side chains to encode peptide-like sequences in the PNA backbone, imparting novel activity and function and demonstrating the ability of PNA to simultaneously mimic both a peptide and a nucleic acid.