Single-Molecule, Real-Time Dissecting of Peptide Nucleic Acid–DNA Duplexes with a Protein Nanopore Tweezer

Abstract

Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.

The applications of nanopores in studies of proteins

“…The blockade current and dwell time of different conjugates were distinguishable. In 2018, Ciuca et al [58] designed and prepared peptide nucleic acids (PNAs) to facilitate detection of peptides and DNA. In their experiment, the opposite charged peptides and DNA would separate to thread through the nanopore under the influence of an electric field.…”

The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences

“…Since their discovery as structural DNA analogs containing an uncharged N-(2aminoethyl) glycine-based pseudopeptide backbone and mimicking DNA by forming Watson-Crick complementary duplexes with normal DNA [34,35], charge-neutral peptide nucleic acids (PNAs) have demonstrated tremendous potential for antigene and antisense therapy, functional genomics, or as a probe, useful in the toolbox for diagnosis and detection [36][37][38][39][40][41][42][43][44][45], and constitute an excellent model substrate for devising innovative approaches directed at nucleic acids' primary structure reading.…”

The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide-Nucleic Acid Sequences

“…Since their discovery as structural DNA analogues containing an uncharged N-(2aminoethyl)-glycine-based pseudopeptide backbone and mimicking DNA in forming Watson-Crick complementary duplexes with normal DNA [34,35], charge neutral peptide nucleic acids (PNAs) demonstrated tremendous potential to the growing field of nucleic acids nanotechnology, as well as medicinal chemistry and molecular biology [36][37][38][39][40][41][42][43][44][45], and constitute an excellent model substrate for devising innovative approaches directed at nucleic acids primary structure reading.…”