Simultaneous identification of viruses and SARS-CoV-2 variants with programmable DNA nanobait

Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid read-out via nanopore sensing. Here, we show this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants, as we demonstrated for several key SARS-CoV-2 variants with single-nucleotide resolution. Lastly, we show that nanobait discriminates between samples extracted from oropharyngeal swabs from negative and positive SARS-CoV-2 patients without pre-amplification. Our system allows for multiplexed identification of native RNA molecules, providing a new scalable approach for diagnostics of multiple respiratory viruses in a single assay.

Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease

SARS-CoV-2 is the causative agent of COVID-19. The highly conserved viral NSP15 endoribonuclease, NendoU, is a key enzyme involved in viral immune evasion, and a promising target for the development of new classes of antivirals. Yet, the broad variety of recognition sequences, complex assembly and kinetics, and lack of structural complexes hampers the development of new competitive or allosteric inhibitors for this target. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index of 2. By using cryo-EM at distinct pHs combined with X-ray crystallography and structural analysis, we demonstrate the potential for NendoU to shift between open and closed states, and assembly in larger supramolecular entities, which might serve as a mechanism of self-regulation. Further, we report results from a large fragment screening campaign against NendoU, revealing multiple new allosteric sites for the development of inhibitors.

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.