The Tautomeric State of N4-Hydroxycytidine within Base-Paired RNA.

Antiviral nucleoside analogues (e.g., Molnupiravir, Remdesivir) played key roles in the treatment of COVID-19 by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The nucleoside of Molnupiravir, N4-hydroxycytidine (NHC), exists in two tautomeric forms that pair either with G or A within the RdRp active site, causing an accumulation of viral RNA mutations during replication. Detailed insights into the tautomeric states within base pairs and the structural influence of NHC in RNA are still missing. In this study, we investigate the properties of NHC:G and NHC:A base pairs in a self-complementary RNA duplex by UV thermal melting and NMR spectroscopy using atom-specifically 15N-labeled versions of NHC that were incorporated into oligonucleotides by solid-phase synthesis. NMR analysis revealed that NHC forms a Watson-Crick base pair with G via its amino form, whereas two equally populated conformations were detected for the NHC:A base pair: a weakly hydrogen-bonded Watson-Crick base pair with NHC in the imino form and another conformation with A shifted toward the minor groove. Moreover, we found a variable influence of NHC:G and NHC:A base pairs on the neighboring duplex environment. This study provides conclusive experimental evidence for the existence of two tautomeric forms of NHC within RNA base pairs.

The RNA methyltransferase METTL8 installs m3C32 in mitochondrial tRNAsThr/Ser(UCN) to optimise tRNA structure and mitochondrial translation.

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.

Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis.

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

RNA-Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA.

Deoxyribozymes are emerging as modification-specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA-cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3-methylcytidine (m3 C), N4 -methylcytidine (m4 C), and 5-methylcytidine (m5 C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10- to 30-fold accelerated cleavage of their target m3 C-, m4 C- or m5 C-modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mX C-detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3 C and m5 C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.

N6 -Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes.

RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave post-transcriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. We report that the native tRNA modification N6 -isopentenyladenosine (i6 A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i6 A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i6 A RNA modification. Together with deoxyribozymes that are strongly inhibited by i6 A, these results highlight that post-transcriptional RNA modifications modulate the catalytic activity of DNA in various intricate ways.

Highly Selective Butyrylcholinesterase Inhibitors with Tunable Duration of Action by Chemical Modification of Transferable Carbamate Units Exhibit Pronounced Neuroprotective Effect in an Alzheimer's Disease Mouse Model.

In this study, the carbamate structure of pseudo-irreversible butyrylcholinesterase (BChE) inhibitors was optimized with regard to a longer binding to the enzyme. A set of compounds bearing different heterocycles (e.g., morpholine, tetrahydroisoquinoline, benzimidazole, piperidine) and alkylene spacers (2 to 10 methylene groups between carbamate and heterocycle) in the carbamate residue was synthesized and characterized in vitro for their binding affinity, binding kinetics, and carbamate hydrolysis. These novel BChE inhibitors are highly selective for hBChE over human acetycholinesterase (hAChE), yielding short-, medium-, and long-acting nanomolar hBChE inhibitors (with a half-life of the carbamoylated enzyme ranging from 1 to 28 h). The inhibitors show neuroprotective properties in a murine hippocampal cell line and a pharmacological mouse model of Alzheimer's disease (AD), suggesting a significant benefit of BChE inhibition for a disease-modifying treatment of AD.