A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA. We discovered that the length-variable poly-Arg tail marked distinctly the molecular processes associated with the nanopore-mediated duplexes capture, trapping and unzipping. This enabled the detection of ssDNA targets via the signatures of (poly-Arg)-PNA-cDNA blockade events, rendered most efficient from the β-barrel entrance of the nanopore, and scaled proportional in efficacy with a larger poly-Arg moiety. We illustrate the approach by sensing synthetic ssDNAs designed to emulate fragments from two regions of SARS-CoV-2 nucleocapsid phosphoprotein N-gene.