Unzipping Mechanism of Free and Polyarginine-Conjugated DNA-PNA Duplexes, Preconfined Inside the α‑Hemolysin Nanopore

In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore’s vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore’s β-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping. This study identifies key particularities of DNA-PNA duplex unzipping as it takes place inside the nanopore and being preceded by entrapment in the vestibule domain of the α-HL. Our results are a crucial step toward understanding the nucleic acids duplexes unzipping kinetics variability, in confined, variable geometries.