From a genome-wide screen of RNAi molecules against SARS-CoV-2 to a validated broad-spectrum and potent prophylaxis.

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen of over 16,000 RNAi triggers against the SARS-CoV-2 genome, using a massively parallel assay to identify hyper-potent siRNAs. We selected Ten candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity (IC50 < 20 pM) and strong blockade of infectivity in live-virus experiments. We further enhanced this activity by combinatorial pairing of the siRNA candidates and identified cocktails that were active against multiple types of variants of concern (VOC). We then examined over 2,000 possible mutations in the siRNA target sites by using saturation mutagenesis and confirmed broad protection of the leading cocktail against future variants. Finally, we demonstrated that intranasal administration of this siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the gold-standard Syrian hamster model. Our results pave the way for the development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA.

Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.