Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

4-Isocyanoindole-2'-deoxyribonucleoside (4ICIN): An Isomorphic Indole Nucleoside Suitable for Inverse Electron Demand Diels-Alder Reactions.

Isomorphic nucleosides are powerful tool compounds for interrogating a variety of biological processes involving nucleosides and nucleic acids. We previously reported a fluorescent isomorphic indole nucleoside called 4CIN. A distinguishing molecular feature of 4CIN is the presence of a 4-cyano moiety on the indole that functions as the nucleobase. Given the known chemical reactivity of isonitriles with tetrazines through [4+1]-cycloaddition chemistry, we investigated whether conversion of 4CIN to the corresponding isonitrile would confer a useful chemical probe. Here we report the synthesis of 4-isocyanoindole-2'-deoxyribonucleoside (4ICIN) and the propensity of 4ICIN to undergo inverse electron demand Diels-Alder cycloaddition with a model tetrazine.

Synthesis and photophysical characterization of fluorescent indole nucleoside analogues.

Fluorescent nucleosides are useful chemical tools for biochemical research and are frequently incorporated into nucleic acids for a variety of applications. The most widely utilized fluorescent nucleoside is 2-aminopurine-2'-deoxyribonucleoside (2APN). However, 2APN is limited by a moderate Stokes shift, molar extinction coefficient, and quantum yield. We recently reported 4-cyanoindole-2'-deoxyribonucleoside (4CIN), which offers superior photophysical characteristics in comparison to 2APN. To further improve upon 4CIN, a focused library of additional analogues combining the structural features of 2APN and 4CIN were synthesized and their photophysical properties were quantified. Nucleosides 2-6 were found to possess diverse photophysical properties with some features superior to 4CIN. In addition, the structure-function relationship data gained from 1-6 can inform the design of next-generation fluorescent indole nucleosides.

SARS-CoV-2 nsp14 Exoribonuclease Removes the Natural Antiviral 3'-Deoxy-3',4'-didehydro-cytidine Nucleotide from RNA.

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3'-to-5' exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3'-deoxy-3',4'-didehydro-cytidine 5'-monophosphate (ddhCMP), incorporated by RdRp at the 3' end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3'-deoxy-cytidine monophosphate (3'-dCMP), whereas RdRp is more susceptible to chain termination by 3'-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP.

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

A Chemical Strategy for Intracellular Arming of an Endogenous Broad-Spectrum Antiviral Nucleotide.

The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.

Synthesis of 4-Cyanoindole Nucleosides, 4-Cyanoindole-2'-Deoxyribonucleoside-5'-Triphosphate (4CIN-TP), and Enzymatic Incorporation of 4CIN-TP into DNA.

4-Cyanoindole-2'-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2'-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4-cyanoindole-ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase.

4-Cyanoindole-2'-deoxyribonucleoside (4CIN): A Universal Fluorescent Nucleoside Analogue.

The synthesis and characterization of a universal and fluorescent nucleoside, 4-cyanoindole-2'-deoxyribonucleoside (4CIN), and its incorporation into DNA is described. 4CIN is a highly efficient fluorophore with quantum yields >0.90 in water. When incorporated into duplex DNA, 4CIN pairs equivalently with native nucleobases and has uniquely high quantum yields ranging from 0.15 to 0.31 depending on sequence and hybridization contexts, surpassing that of 2-aminopurine, the prototypical nucleoside fluorophore. 4CIN constitutes a new isomorphic nucleoside for diverse applications.