Research Tools and Investigations of Interactions of SARS-CoV-2 NSP3 SUD with G-Quadruplexes

Abstract

G-quadruplexes (G4s) are DNA and RNA secondary structures that are able to regulate cellular processes such as transcription and translation. Various proteins can bind to quadruplex structures in order to stabilize or destabilize them. One such SARS-CoV encoded protein, NSP3, was found to bind to RNA G4s through its SARS unique domain (SUD) and inhibit viral replication. With the emergence of SARS-CoV-2, we investigated the binding of SARS-CoV-2 NSP3 SUD to TRF2 DNA and RNA G4s. Through electrophoretic mobility shift assays (EMSAs), we determined the affinity of SUD binding to TRF2 and TRF2-m. TRF2 is a G-quadruplex forming sequence, while TRF2-m contains several nucleotide mutations in order to prevent G-quadruplex formation. SUD bound to TRF2 DNA with a Kd of 340 nM but did not bind to TRF2-m DNA. Furthermore, SUD bound to TRF2 RNA with a Kd of 480 nM, and bound to TRF2-m RNA with a Kd of 500 nM. The differential binding of SUD to DNA G4s with higher affinity than RNA implies its potential as a DNA G4 probe. Additionally, we designed a mammalian bicistronic dual luciferase expression system in order to investigate the downstream effects of SUD binding to DNA and RNA G4s. This system utilizes a SUD expression vector as well as a quadruplex-containing dual luciferase reporter vector. Though we were able to successfully insert SUD into pcDNA3.1 for mammalian expression, the protein was unable to express in HEK293T cells. Regardless, we believe that through basic subcloning, this system can be adapted to assess the downstream binding effects of any insertable protein to any insertable DNA/RNA region. Furthermore, we also investigated the structure of SUD and identified a binding groove compatible with nucleic acid binding. Lastly, we used site-directed mutagenesis (SDM) to replace various tyrosine residues with the amber stop codon in order to incorporate the photocrosslinkable p-azido-L-phenylalanine (AzF) into SUD. Three AzF mutants were designed and expressed in BL21 E. coli and purified via FPLC: Y195-AzF, Y214-AzF, and Y241-AzF. We further displayed the crosslinking capabilities of these SUD mutants to G4 DNA, with Y195 and Y241 being the most efficient. We think that SUD’s affinity for G4s combined with AzF’s photocrosslinking capabilities would allow for probing of novel G4s, protein complexes, and interacting nucleic acid. This will ultimately allow us to better understand how quadruplexes and other complexes regulate cellular and viral processes