VIR-CRISPR: Visual in-one-tube ultrafast RT-PCR and CRISPR method for instant SARS-CoV-2 detection.

Until now, corona virus disease 2019 (COVID-19) remained to be an enormous threat for global health. As one viral illness induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), versatile, rapid and sensitive method for SARS-CoV-2 detection in early stage is urgently needed. Here, we reported an ultrasensitive and visual in-one-tube detection method which could be accomplished within half an hour from sampling-to-result. By integrating all reactions in one tube, liquid handling steps were omitted and amplicon contamination could be totally avoided. Magnetic beads were employed to achieve the fast extraction of viral nucleic acid and increase the sensitivity. Using portable thermocycler and blue light, the fluorescent results could be directly observed by naked eyes. The proposed method is of higher specificity and sensitivity, nearly at single molecule level. More important, results demonstrated 100% positive detection rate for 40 clinical samples, which was consistent with standard RT-PCR. Thus, our method is considerably simple, rapid, sensitive and accurate, holding great promise for the instant detecting of viruses including SARS-CoV-2 and the next generation of molecular diagnosis.

2'- and 3'-Ribose Modifications of Nucleotide Analogues Establish the Structural Basis to Inhibit the Viral Replication of SARS-CoV-2.

Inhibition of RNA-dependent RNA polymerase (RdRp) by nucleotide analogues with ribose modification provides a promising antiviral strategy for the treatment of SARS-CoV-2. Previous works have shown that remdesivir carrying 1'-substitution can act as a "delayed chain terminator", while nucleotide analogues with 2'-methyl group substitution could immediately terminate the chain extension. However, how the inhibition can be established by the 3'-ribose modification as well as other 2'-ribose modifications is not fully understood. Herein, we have evaluated the potential of several adenosine analogues with 2'- and/or 3'-modifications as obligate chain terminators by comprehensive structural analysis based on extensive molecular dynamics simulations. Our results suggest that 2'-modification couples with the protein environment to affect the structural stability, while 3'-hydrogen substitution inherently exerts "immediate termination" without compromising the structural stability in the active site. Our study provides an alternative promising modification scheme to orientate the further optimization of obligate terminators for SARS-CoV-2 RdRp.

1'-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication.

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.