Synthesis, deprotection, and purification of diphosphorylated RNA oligonucleotides.

Fergione, Sarah J; Fedorova, Olga; Pyle, Anna Marie

doi:10.26434/chemrxiv-2023-rf0bz

Cited by 1 publication

(1 citation statement)

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“…Synthesis of 5'-triphosphorylated RNA oligonucleotides 5'-triphisphorylated RNA oligonucleotides were synthesized on the MerMade 12 RNA-DNA synthesizer (Bioautomation) as previously described (1). Base and 2'-hydroxyl group deprotection and subsequent purification was carried out essentially as described 51 . Briefly, for base and phosphate group deprotection and removal of the oligonucleotide from the support, the polymer support was transferred to a glass vial and incubated with 4 ml of the mixture of 28-30% aqueous ammonium hydroxide (JT Baker) and 40% aqueous methylamine (MiliporeSigma) (1:1) at 65 oC for 15 min.…”

Section: Native Mass Spectrometry (Nms) Analysismentioning

confidence: 99%

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain

Small,

Fedorova,

Olinares

et al. 2023

Preprint

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SUMMARYThe enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5’-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

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Section: Native Mass Spectrometry (Nms) Analysismentioning

confidence: 99%