Real-Time Monitoring of DNA Polymerase Function and Stepwise Single-Nucleotide DNA Strand Translocation through a Protein Nanopore

摘要

Single-molecule DNA sequencing with nanopores is a promising concept for advancing the next generation of rapid and low-cost sequencing technologies. A proposed approach in this field involves moving a single-stranded DNA (ssDNA) through a narrow pore under the influence of an electric field and identifying the nucleobases as they pass by a reading-head located within the pore. A key challenge is to work out how to ratchet DNA strand through a nanopore at the appropriate speed and time intervals that are commensurate with real-time sequential nucleobase identification. Here we describe an approach that exploits the intrinsic motor function of DNA polymerases to effect stepwise DNA strand translocation through an alpha-hemolysin (aHL) protein pore. The system is based on a single-species supramolecular nanopore construct, whose configurational dynamic features provide the means for real-time monitoring of DNA strand translocation and DNA polymerase function.