Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation

There is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3’ antisense sequence for target recognition and a 5’-phosphorylated 2’-5’ poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or ΔH69/ΔV70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.

A model for the emergence of RNA from a prebiotically plausible mixture of ribonucleotides, arabinonucleotides and 2’-deoxynucleotides

The abiotic synthesis of ribonucleotides is thought to have been an essential step towards the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2′-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2′-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2′-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabino-nucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2′-deoxy- or arabino-nucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Solid-Phase Synthesis of Phosphorothioate/Phosphonothioate and Phosphoramidate/Phosphonamidate Oligonucleotides

We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)-phosphinate internucleotide linkage introduced into the oligonucleotide chain by H-phosphonate chemistry using nucleoside-O-methyl-(H)-phosphinates as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and phosphoramidite chemistries to synthesize diversely modified oligonucleotide strands.

Synthesis of PS/PO-chimeric oligonucleotides using mixed oxathiaphospholane and phosphoramidite chemistry

Chimeric oligonucleotides containing stereoregular phosphorothioate and natural phosphodiester linkages have been obtained on the solid phase support using nucleoside oxathiaphospholanes and commercially available nucleoside phosphoramidites.