De novo design of nanopore-like architectures and applications of nanopores to investigate the SARS CoV-2 helicase

Abstract

Protein nanopores form nanometer-sized holes in a lipid bilayer and are widely used as analytical tools to study the flow of ions and other target molecules through them. Recent advancements in nanopore technologies have enabled use of nanopores for nucleic acid sequencing, peptide or protein analysis, and small-molecule identification and filtration. Custom designed nanopores are a promising area of research that may lead to increased accuracy and specificity of nanopore assays, or enable assays that are not possible or otherwise unknown with the existing library of protein nanopores. Designing protein structures de novo requires two steps: designing protein backbones that will result in the desired function, and identifying amino acid sequences that will fold into the desired backbones at their lowest energy states. In chapter 1, I introduce the geometrical and energetic considerations in building protein backbones, with a central channel and a defined sensing region, for the membrane. Using the Rosetta framework, I present de novo design strategies of oligomeric aqueous alpha-helical channels around an ion-channel filter and transmembrane beta-barrels. I show the results of biochemical validation of hexameric aqueous alpha-helical channels, and the ion-conductance properties of the first de novo designed 8-stranded transmembrane beta-barrel. This work is a step towards reducing our dependence on naturally occurring ion-channels that are challenging to modify, and towards creating entirely de novo nanopores with an un-naturally optimal fit for their targets. In chapter 2, I present the application of a naturally occuring nanopore to investigate the helicase found in Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonstructural protein 13 (nsp 13) helicase is involved in the replication of the SARS-CoV-2 genome and a potential drug target for antivirals. Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a new technique that uses MspA, a biological nanopore, to monitor the movement of single enzyme molecules on DNA at high spatiotemporal resolution. Using SPRNT, I present the first single-molecule assay to monitor the kinetics of single-nucleotide steps of the SARS-CoV-2 helicase, nsp13. This assay provides a single-molecule platform for understanding how antiviral compounds affect nsp13 function and glean insights for their development.