High Accuracy Nanopore Sequencing

This project is an expansion of the scope of our current funding which was awarded to improve nanoporesequencing. In the course of our research to improve nanopore DNA sequencing, we have discovered anddeveloped a new high-resolution single-molecule tool to observe the motion of helicases and polymerases. Thetool, which we called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), is able to reveal thesingle-nucleotide steps of helicases and polymerases at unprecedented detail. These enzyme steps are so small(~0.3 nm) and fast (~ 1 ms) that no other existing single-molecule technique can resolve such motion in real timeand at physiological conditions. The genome of the SARS-CoV-2 virus encodes for an RNA-dependent RNApolymerase, also known as non-structural protein 12 (nsp12), and a superfamily 1B helicase, known as nonstructural protein 13 (nsp13). Both of these enzymes are essential and specific to the in vivo replication of manyviruses, making this genomic replication machinery an ideal drug target. Several drug candidates to inhibit theseenzymes exist, however, none are as effective as they need to be to stem the tide of the current pandemic. Manyquestions remain about how exactly these drugs interfere with the function of nsp12 or nsp13. SPRNT providesan opportunity to answer these questions. We aim to use SPRNT to analyze the single-molecule motion of nsp12and nsp13 in physiological conditions and in the presence of drugs against them. The exact knowledge of themechanisms by which various drugs inhibit nsp12 or nsp13 will enable more rational and rapid design of effectiveantiviral drugs that have the potential to stop COVID-19.