Significance: Efficient cost-effective single-molecule sequencing platforms will facilitate deciphering complete genome sequences, determining haplotypes, and identifying alternatively spliced mRNAs. We demonstrate a single-molecule nanopore-based sequencing by synthesis approach that accurately distinguishes four DNA bases by electronically detecting and differentiating four different polymer tags attached to the terminal phosphate of the nucleotides during their incorporation into a growing DNA strand in the polymerase reaction. With nanopore detection, the distinct polymer tags are much easier to differentiate than natural nucleotides. After tag release, growing DNA chains consist of natural nucleotides allowing long reads. Sequencing is realized on an electronic chip containing an array of independently addressable electrodes, each with a single polymerase–nanopore complex, potentially offering the high throughput required for precision medicine.

SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.

SARS‐CoV‐2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA‐approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS‐CoV‐2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low‐fidelity polymerases and SARS‐CoV RNA‐dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host‐like high‐fidelity DNA polymerase. Using the same molecular insight, we selected 3’‐fluoro‐3’‐deoxythymidine triphosphate and 3’‐azido‐3’‐deoxythymidine triphosphate, which are the active forms of two other anti‐viral agents, Alovudine and AZT (an FDA‐approved HIV/AIDS drug) for evaluation as inhibitors of SARS‐CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS‐CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS‐CoV and SARS‐CoV‐2 RdRps, we expect these nucleotide analogues would also inhibit the SARS‐CoV‐2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad‐spectrum anti‐coronavirus agents.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.