Hybridization Kinetics Reveal Backbone-Dependent Binding of Peptide Nucleic Acid Analogs

Abstract

Antisense oligonucleotides (ASOs), short single-stranded nucleic acids, have emerged as an important class of RNA-targeting agents by binding through sequence-specific base pairing, resulting in alteration or blockage of RNA function. However, unmodified DNA-and RNA-based ASOs are prone to instability and enzymatic degradation. Peptide nucleic acids (PNAs) provide enhanced stability, affinity and sequence selectivity due to their N-(2aminoethyl)glycine backbone, but their limited aqueous solubility can restrict broader application. This limitation can be addressed by the incorporation of γ-miniPEG side chains improving the solubility while retaining favorable binding characteristics. As antisense efficacy strongly depends on target residence time, a quantitative understanding of hybridization kinetics is critical for candidate selection. Yet direct comparisons of γ-miniPEG-modified PNAs with RNA remain scarce. Here, we systematically compare the hybridization of RNA, PNA and γ-miniPEG-PNA toward complementary RNA strand using surface plasmon resonance (SPR) spectroscopy, which enables real-time analysis of association and dissociation kinetics beyond equilibrium duplex stability derived from melting temperature measurements. Our results shows that duplex dissociation slows with increasing strand length, whereas association behavior is largely governed by backbone chemistry. PNA exhibits particularly rapid target binding while maintaining strong affinity. Together, these findings elucidate how backbone structure governs hybridization dynamics and guide the rational design of PNA-based ASOs for RNA-targeting.