A novel N4,N4-dimethylcytidine in the archaeal ribosome enhances hyperthermophily.

Ribosome structure and activity are challenged at high temperatures, often demanding modifications to ribosomal RNAs (rRNAs) to retain translation fidelity. LC-MS/MS, bisulfite-sequencing, and high-resolution cryo-EM structures of the archaeal ribosome identified an RNA modification, N4,N4-dimethylcytidine (m42C), at the universally conserved C918 in the 16S rRNA helix 31 loop. Here, we characterize and structurally resolve a class of RNA methyltransferase that generates m42C whose function is critical for hyperthermophilic growth. m42C is synthesized by the activity of a unique family of RNA methyltransferase containing a Rossman-fold that targets only intact ribosomes. The phylogenetic distribution of the newly identified m42C synthase family implies that m42C is biologically relevant in each domain. Resistance of m42C to bisulfite-driven deamination suggests that efforts to capture m5C profiles via bisulfite sequencing are also capturing m42C.

Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs.

The functional analysis of epitranscriptomic modifications in RNA is constrained by a lack of methods that accurately capture their locations and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base resolution through sodium borohydride reduction to tetrahydroacetylcytidine (tetrahydro-ac4C), followed by cDNA synthesis to misincorporate adenosine opposite reduced ac4C sites, culminating in C:T mismatches at acetylated cytidines (RedaC:T). However, this process is relatively inefficient, resulting in <20% C:T mismatches at a fully modified ac4C site in 18S rRNA. Considering that ac4C locations in other substrates including mRNA are unlikely to reach full penetrance, this method is not ideal for comprehensive mapping. Here, we introduce "RetraC:T" (reduction to tetrahydro-ac4C and reverse transcription with amino-dATP to induce C:T mismatches) as a method with enhanced ability to detect ac4C in cellular RNA. In brief, RNA is reduced through NaBH4 or the closely related reagent sodium cyanoborohydride (NaCNBH3) followed by cDNA synthesis in the presence of a modified DNA nucleotide, 2-amino-dATP, that preferentially binds to tetrahydro-ac4C. Incorporation of the modified dNTP substantially improved C:T mismatch rates, reaching stoichiometric detection of ac4C in 18S rRNA. Importantly, 2-amino-dATP did not result in truncated cDNA products nor increase mismatches at other locations. Thus, modified dNTPs are introduced as a new addition to the toolbox for detecting ac4C at base resolution.

Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides.

The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.

Substrate specificity of Mycobacterium tuberculosis tRNA terminal nucleotidyltransferase toxin MenT3.

Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3'-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3'-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.

Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

Crosslinking of base-modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing.

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of putative amine/amide donors, U-to-C editing by KP6 could not be observed in vitro. C-to-U editing was found not to be concomitant with U-to-C editing, discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were highly enriched in interphase fractions consistent with covalent crosslinks to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed secondary peaks with mass shifts of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

Assessing the inhibitory effects of some secondary amines, thioureas and 1,3-dimethyluracil conjugates of (-)-cytisine and thermopsine on the RNA-dependent RNA polymerase of SARS-CoV-1 and SARS-CoV-2.

SARS-CoV-2 causes COVID-19, with symptoms ranging from mild to severe, including pneumonia and death. This beta coronavirus has a 30-kilobase RNA genome and shares about 80 % of its nucleotide sequence with SARS-CoV-1. The replication/transcription complex, essential for viral RNA synthesis, includes RNA-dependent RNA polymerase (RdRp, nsp12) enhanced by nsp7 and nsp8. Antivirals like molnupiravir and remdesivir, which are RdRp inhibitors, treat severe COVID-19 but have limitations, highlighting the need for new therapies. This study assessed (-)-cytisine, methylcytisine, and thermopsine derivatives against SARS-CoV-1 and SARS-CoV-2 in vitro, focusing on their RdRp inhibition. Selected compounds from a previous study were evaluated using a SARS-CoV-2 RNA polymerase assay kit to investigate their structure-activity relationships. Compound 17 (1,3-dimethyluracil conjugate with (-)-cytisine and thermopsine) emerged as a potent inhibitor of SARS-CoV-1 and SARS-CoV-2 RdRp, with an IC50 value of 7.8 µM against SARS-CoV-2 RdRp. It showed a dose-dependent reduction in cytopathic effects in cells infected with SARS-CoV-1 and SARS-CoV-2 replicon-based single-round infectious particles (SRIPs) and significantly inhibited SARS-CoV N protein expression, with EC50 values of 0.12 µM for SARS-CoV-1 and 1.47 µM for SARS-CoV-2 SRIPs. Additionally, compound 17 reduced viral subgenomic RNA levels in a concentration-dependent manner in SRIP-infected cells. The structure-activity relationships of compound 17 with SARS-CoV-1 and SARS-CoV-2 RdRp were also investigated, highlighting it as a promising lead for developing antiviral agents against SARS and COVID-19.

Chemistry of installing epitranscriptomic 5-modified cytidines in RNA oligomers.

Studies of 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C) and 5-carboxycytidine (ca5C) modifications as products of the 5-methylcytidine (m5C) oxidative demethylation pathway in cellular mRNAs constitute an important element of the new epitranscriptomic field of research. The dynamic process of m5C conversion and final turnover to the parent cytidine is considered a post-transcriptional layer of gene-expression regulation. However, the regulatory mechanism associated with epitranscriptomic cytidine modifications remains largely unknown. Therefore, oligonucleotides containing m5C oxidation products are of great value for the next generation of biochemical, biophysical, and structural studies on their function, metabolism, and contribution to human diseases. Herein, we summarize the synthetic strategies developed for the incorporation of hm5C, f5C and ca5C into RNA oligomers by phosphoramidite chemistry, including post-synthetic C5-cytidine functionalization and enzymatic methods.

NBCR-ac4C: A Deep Learning Framework Based on Multivariate BERT for Human mRNA N4-Acetylcytidine Sites Prediction.

N4-acetylcytidine (ac4C) plays a crucial role in regulating cellular biological processes, particularly in gene expression regulation and disease development. However, experiments to identify ac4C in a wet lab are time-consuming and costly, and the learning-based methods struggle to capture the underlying semantic knowledge and relations within sequences. To address this, we propose a deep learning approach called NBCR-ac4C based on pretrained models. Specifically, we employ Nucleotide Transformer and DNABERT2 to construct contextual embedding of nucleotide sequences, which effectively mine and express context relations between different features in the sequence. Convolutional neural network (CNN) and ResNet18 are then applied to further extract shallow and deep knowledge from context embedding. Depending on extensive experiments for the prediction of ac4C sites in nucleotide sequences, we observe that NBCR-ac4C outperforms general learning-based models. It achieves the highest accuracy (ACC) of 83.51% and an Area Under the Receiver Operating Characteristic Curve (AUROC) of 89.58% on an independent test set. Moreover, the proposed model, compared to the current state-of-the-art (SOTA) model LSA-ac4C, demonstrates higher ACC and AUROC by 0.81-3.7% and 0.05-1.58%, respectively. The data set and code are available on https://github.com/2103374200/NBCR to facilitate further discussion on NBCR-ac4C.

A path from synthesis to emergency use authorization of molnupiravir as a COVID-19 therapy.

Coronaviruses are a group of enveloped viruses with non-segmented, single-stranded, and positive-sense RNA genomes. It belongs to the 'Coronaviridae family', responsible for various diseases, including the common cold, SARS, and MERS. The COVID-19 pandemic, which began in March 2020, has affected 209 countries, infected over a million people, and claimed over 50,000 lives. Significant efforts have been made by repurposing several approved drugs including antiviral, to combat the COVID-19 pandemic. Molnupiravir is found to be the first orally acting efficacious drug to treat COVID-19 cases. It was approved for medical use in the UK in November 2021 and other countries, including USFDA, which granted approval an emergency use authorization (EUA) for treating adults with mild to moderate COVID-19 patients. Considering the importance of molnupiravir, the present review deals with its various synthetic strategies, pharmacokinetics, bio-efficacy, toxicity, and safety profiles. The comprehensive information along with critical analysis will be very handy for a wide range of audience including medicinal chemists in the arena of antiviral drug discovery especially anti-viral drugs against any variant of COVID-19.

Discovery of A G-Quadruplex Unwinder That Unleashes the Translation of G-Quadruplex-Containing mRNA without Inducing DNA Damage.

To explore the mechanisms and therapeutic strategies for G-quadruplex (G4) mediated diseases, it is crucial to manipulate and intervene in intracellular G4 structures using small molecular tools. While hundreds of G4 stabilizers have been developed, there is a significant gap in the availability of G4 unwinding agents. Here, we propose a strategy to disrupt G-quadruplexes by forming G-C hydrogen bonds with chemically modified cytidine trimers. We validated a good G4 unwinder, the 2'-F cytidine trimer (2'-F C3). 2'-F C3 does not inhibit cell growth nor cause severe DNA damage at a concentration below 10 µM. Moreover, 2'-F C3 does not affect gene transcription nor RNA splicing, while it significantly enhances the translation of G4-containing mRNA and upregulates RNA splicing, RNA processing and cell cycle pathways. The discovery of this G4 unwinder provides a functional tool for the chemical modulation of G4s in living cells.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Study of protonated dimers of cytosine, cytidine, and deoxycytidine using survival yield method and quantum mechanics calculations.

RATIONALE: Cytosine and its conjugates are prone to form protonated, triply-bonded dimers. Therefore, the nucleic-acid cytosine-rich sequence forms the four-stranded noncanonical secondary structure known as the intercalated motif (i-motif). This process has resulted in studies on cytosine protonated dimers. This communication focuses on the protonated dimers of cytosine and its nucleoside using the survival yield (SY) method and quantum mechanics calculations. METHODS: To obtain the precursor ion fragmentation curve, the plot of SY against Ecomδ , the product ion spectra of the protonated dimers were obtained using a Waters/Micromass Q-TOF Premier mass spectrometer. Quantum mechanics calculations were performed using GAUSSIAN 16, and full geometry optimizations and energy calculations were performed within the density functional theory framework at B3LYP/6-31G(d,p). RESULTS: The precursor ion fragmentation curve allowed the rating of the gas-phase stabilities of the analyzed protonated dimers. Substitution of sugar moiety at N1 cytosine atom decreased the gas-phase stabilities of the protonated dimers. The deoxycytidine dimer was found to be more stable than the cytidine dimer and cytidine-deoxycytidine dimer. Quantum chemical calculations indicated that cytosine aminohydroxy tautomer may be involved in the formation of protonated cytosine-cytosine nucleoside dimers but not in the formation of cytosine dimers. CONCLUSIONS: The results obtained for nucleoside dimers indicated that the SY method may reflect the i-motif stabilities observed under physiological conditions. Therefore, the analysis of other protonated dimers of variously substituted cytosine-cytosine nucleoside using the SY method may be important to study the effect of cytosine substitution on the i-motif stabilities. Cytosine tautomer containing C2-OH… N(2H)-C4 moiety may be involved in the formation of protonated cytosine-cytosine nucleoside dimers.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A.

Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.

Protonation-Dependent Sequencing of 5-Formylcytidine in RNA.

Chemical modification of cytidine in noncoding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remain unknown due to a lack of sensitive site-specific sequencing technologies. Here, we report a protonation-dependent sequencing reaction for the detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human transfer RNA (tRNA). Finally, we demonstrate the utility of our method to characterize a patient-derived model of 5fC deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA and more broadly provide a molecular foundation for using optimized sequencing reactions to better understand the role of oxidized RNA cytidine residues in diseases.

Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA.

The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

Computational Analysis of Molnupiravir.

In this work, we report in-depth computational studies of three plausible tautomeric forms, generated through the migration of two acidic protons of the N4-hydroxylcytosine fragment, of molnupiravir, which is emerging as an efficient drug to treat COVID-19. The DFT calculations were performed to verify the structure of these tautomers, as well as their electronic and optical properties. Molecular docking was applied to examine the influence of the structures of the keto-oxime, keto-hydroxylamine and hydroxyl-oxime tautomers on a series of the SARS-CoV-2 proteins. These tautomers exhibited the best affinity behavior (-9.90, -7.90, and -9.30 kcal/mol, respectively) towards RdRp-RTR and Nonstructural protein 3 (nsp3_range 207-379-MES).

Potential COVID-19 Drug Candidates Based on Diazinyl-Thiazol-Imine Moieties: Synthesis and Greener Pastures Biological Study.

A novel series of 1-aryl-N-[4-phenyl-5-(arylazo)thiazol-2-yl)methanimines has been synthesized via the condensation of 2-amino-4-phenyl-5-arylazothiazole with various aromatic aldehydes. The synthesized imines were characterized by spectroscopic techniques, namely 1H and 13C-NMR, FTIR, MS, and Elemental Analysis. A molecular comparative docking study for 3a-f was calculated, with reference to two approved drugs, Molnupiravir and Remdesivir, using 7BQY (Mpro; PDB code 7BQY; resolution: 1.7 A°) under identical conditions. The binding scores against 7BQY were in the range of -7.7 to -8.7 kcal/mol for 3a-f. The high scores of the compounds indicated an enhanced binding affinity of the molecules to the receptor. This is due to the hydrophobic interactions and multi-hydrogen bonds between 3a-f ligands and the receptor's active amino acid residues. The main aim of using in silco molecular docking was to rank 3a-f with respect to the approved drugs, Molnupiravir and Remdesivir, using free energy methods as greener pastures. A further interesting comparison presented the laydown of the ligands before and after molecular docking. These results and other supporting statistical analyses suggested that ligands 3a-f deserve further investigation in the context of potential therapeutic agents for COVID-19. Free-cost, PASS, SwissADME, and Way2drug were used in this research paper to determine the possible biological activities and cytotoxicity of 3a-f.

Isoxazole Nucleosides as Building Blocks for a Plausible Proto-RNA.

The question of how RNA, as the principal carrier of genetic information evolved is fundamentally important for our understanding of the origin of life. The RNA molecule is far too complex to have formed in one evolutionary step, suggesting that ancestral proto-RNAs (first ancestor of RNA) may have existed, which evolved over time into the RNA of today. Here we show that isoxazole nucleosides, which are quickly formed from hydroxylamine, cyanoacetylene, urea and ribose, are plausible precursors for RNA. The isoxazole nucleoside can rearrange within an RNA-strand to give cytidine, which leads to an increase of pairing stability. If the proto-RNA contains a canonical seed-nucleoside with defined stereochemistry, the seed-nucleoside can control the configuration of the anomeric center that forms during the in-RNA transformation. The results demonstrate that RNA could have emerged from evolutionarily primitive precursor isoxazole ribosides after strand formation.