Search

Adenosine receptors play pivotal roles in physiological processes. Adenosine A 3 receptor (A 3 R), the most recently identified adenosine receptor, is expressed in various tissues, exhibiting important roles in neuron, heart, and immune cells, and is often overexpressed in tumors, highlighting the therapeutic potential of A 3 R-selective agents. Recently, we identified RNA-derived N 6 -methyladenosine (m 6 A) as an endogenous agonist for A 3 R, suggesting the relationship between RNA-derived modified adenosine and A 3 R. Despite extensive studies on the other adenosine receptors, the selectivity mechanism of A 3 R, especially for A 3 R-selective agonists such as m 6 A and namodenoson, remained elusive. Here, we identify tRNA-derived N 6 -isopentenyl adenosine (i 6 A) as an A 3 R-selective ligand via screening of modified nucleosides against the adenosine receptors. Like m 6 A, i 6 A is found in the human body and may be an endogenous A 3 R ligand. Our cryo-EM analyses elucidate the A 3 R-G i complexes bound to adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), m 6 A, i 6 A, and namodenoson at overall resolutions of 3.27 Å (adenosine), 2.86 Å (NECA), 3.19 Å (m 6 A), 3.28 Å (i 6 A), and 3.20 Å (namodenoson), suggesting the selectivity and activation mechanism of A 3 R. We further conduct structure-guided engineering of m 6 A-insensitive A 3 R, which may aid future research targeting m 6 A and A 3 R, providing a molecular basis for future drug discovery., (© 2024. The Author(s).)

The study's goals are to fabricate PLGA nanoparticles (PNPs) loaded with apigenin (AP) and adenosine (AD) using a microfluidic preparation method to a standard emulsification method and investigate the possible heart-protective effects of AP-AD PNPs made using the emulsification method. Compared to microfluidics, the emulsification method fabricated small-size nanoparticles, which are better at encapsulating drugs, retaining more drugs, and having a low viscosity for the myocardial infarction (MI) injection. TheMI model was developed using SD rats injected under the skin with 85 mg/kg doxorubicin (DOX) for 2 days. The metabolic results showed that our AP-AD PNPs accelerated the blood flow in rats with MI, which increased the amounts of AP and AD in the circulatory system. This led to significant improvements in the cardiac index and lower amounts of AST, LDH, and CK in the blood. A histopathological study using Hematoxylin&eosin, and TUNEL staining showed that cardiac function had improved and apoptosis had decreased. Moreover, tests that checked the amounts of IL-6, TNF-α, NO, GSH, MDA, and SOD showed that AP-AD PNPs may help treat MI by reducing oxidative stress and inflammation, making it a potentially useful therapeutic approach., (© 2024. The Author(s).)

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits-the potent influence of Ψ55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. Growing cells under nutrient depleted conditions also revealed a novel condition-specific increase in m1A58 modification on some tRNAs. A global and isoacceptor-specific classification strategy was developed to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Laser-induced graphene (LIG) possesses characteristics of easy handling, miniaturization, and unique electrical properties. We modified the surface of LIG by electropolymerizing β-cyclodextrin (β-CD), which was used to immobilize antibodies on the electrode surface for highly sensitive detection of targets. N 6-methyladenosine (m6A) is the most prevalent reversible modification in mammalian messenger RNA and noncoding RNA, influencing the development of various cancers. Here, β-CD was electropolymerized to immobilize the anti-m6A antibody, which subsequently recognized the target m6A. This was integrated into the catalytic hydrogen peroxide-hydroquinone (H 2 O 2 -HQ) redox system using phos-tag-biotin to generate electrochemical signals from streptavidin-modified horseradish peroxidase (SA-HRP). Under optimal conditions, the biosensor exhibited a linear range from 0.1 to 100 nM with a minimum detection limit of 96 pM. The method was successfully applied to the recovery analysis of m6A from HeLa cells through spiking experiments and aims to inspire strategies for point-of-care testing (POCT).

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.

N6-Methyladenosine (m 6 A) is a prevalent RNA post-transcriptional modification that plays crucial roles in RNA stability, structural dynamics, and interactions with proteins. The YT521-B (YTH) family of proteins, which are notable m 6 A readers, functions through its highly conserved YTH domain. Recent structural investigations and molecular dynamics (MD) simulations have shed light on the mechanism of recognition of m 6 A by the YTHDC1 protein. Despite advancements, using MD to predict the stabilization induced by m 6 A on the free energy of binding between RNA and YTH proteins remains challenging due to inaccuracy of the employed force field and limited sampling. For instance, simulations often fail to sufficiently capture the hydration dynamics of the binding pocket. This study addresses these challenges through an innovative methodology that integrates metadynamics, alchemical simulations, and force-field refinement. Importantly, our research identifies hydration of the binding pocket as giving only a minor contribution to the binding free energy and emphasizes the critical importance of precisely tuning force-field parameters to experimental data. By employing a fitting strategy built on alchemical calculations, we refine the m 6 A partial charge parameters, thereby enabling the simultaneous reproduction of N6 methylation on both the protein binding free energy and the thermodynamic stability of nine RNA duplexes. Our findings underscore the sensitivity of binding free energies to partial charges, highlighting the necessity for thorough parametrization and validation against experimental observations across a range of structural contexts.

The Asp-tRNA Asn /Glu-tRNA Gln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics., (© 2024. The Author(s).)

Collagen hydrogel has been shown promise as an inducer for chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), contributing to the repair of cartilage defects. However, the precise molecular mechanism underlying this phenomenon remains poorly elucidated. Here, we induced chondrogenic differentiation of BMSCs using collagen hydrogel and identified 4451 differentially expressed genes (DEGs) through transcriptomic sequencing. Our analysis revealed that DEGs were enriched in the focal adhesion pathway, with a notable decrease in expression levels in the collagen hydrogel group compared to the control group. Protein-protein interaction network analysis suggested that actinin alpha 1 (ACTN1) and actinin alpha 4 (ACTN4), two proteins also involved in cytoskeletal recombination, may be crucial in collagen hydrogel-induced chondrogenic differentiation of BMSCs. Additionally, we found that N6-methyladenosine RNA methylation (m6A) modification was involved in collagen hydrogel-mediated chondrogenic differentiation, with fat mass and obesity-associated protein (FTO) implicated in regulating the expression of ACTN1 and ACTN4. These findings suggest that collagen hydrogel might regulate focal adhesion and actin cytoskeletal signaling pathways through down-regulation of ACTN1 and ACTN4 mRNA via FTO-mediated m6A modification, ultimately driving chondrogenic differentiation of BMSCs. In conclusion, our study provides valuable insights into the molecular mechanisms of collagen hydrogel-induced chondrogenic differentiation of BMSCs, which may aid in developing more effective strategies for cartilage regeneration., (© 2024. The Author(s).)

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m 6 A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCN V ) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCN V heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m 6 A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCN V heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-l-homocysteine (SAH) into L-homocysteine. As a result, the SAM-to-SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts, but there has yet to be a study looking at the potential downstream impact of DZNep treatment on proteins other than histones. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small-molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize the protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in the SAM/SAH levels. This study systematically evaluates DZNep's impact on the transcriptome, the proteome, and the thermal stability of proteins.

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Photothermal therapy (PTT) is a promising cancer treatment method due to its ability to induce tumor-specific T cell responses and enhance therapeutic outcomes. However, incomplete PTT can leave residual tumors that often lead to new metastases and decreased patient survival in clinical scenarios. This is primarily due to the release of ATP, a damage-associated molecular pattern that quickly transforms into the immunosuppressive metabolite adenosine by CD39, prevalent in the tumor microenvironment, thus promoting tumor immune evasion. This study presents a photothermal nanomedicine fabricated by electrostatic adsorption among the Fe-doped polydiaminopyridine (Fe-PDAP), indocyanine green (ICG), and CD39 inhibitor sodium polyoxotungstate (POM-1). The constructed Fe-PDAP@ICG@POM-1 (FIP) can induce tumor PTT and immunogenic cell death when exposed to a near-infrared laser. Significantly, it can inhibit the ATP-adenosine pathway by dual-directional immunometabolic regulation, resulting in increased ATP levels and decreased adenosine synthesis, which ultimately reverses the immunosuppressive microenvironment and increases the susceptibility of immune checkpoint blockade (aPD-1) therapy. With the aid of aPD-1, the dual-directional immunometabolic regulation strategy mediated by FIP can effectively suppress/eradicate primary and distant tumors and evoke long-term solid immunological memory. This study presents an immunometabolic control strategy to offer a salvage option for treating residual tumors following incomplete PTT., (© 2024. The Author(s).)

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N 6 -methyladenosine (m 6 A), N 1 -methyladenosine (m 1 A), 3-methylthymine (m 3 T), and 3-methyluracil (m 3 U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m 6 A, m 1 A, or m 3 T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O 2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O 2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O 2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O 2 ., Competing Interests: Competing interests statement:The authors declare no competing interest.

Loss of the translational reading frame leads to misincorporation and premature termination, which can have lethal consequences. Based on structural evidence that A1503 of 16S rRNA intercalates between specific mRNA bases, we tested the possibility that it plays a role in maintenance of the reading frame by constructing ribosomes with an abasic nucleotide at position 1503. This was done by specific cleavage of 16S rRNA at position 1493 using the colicin E3 endonuclease and replacing the resulting 3'-terminal 49mer fragment with a synthetic oligonucleotide containing the abasic site using a novel splinted RNA ligation method. Ribosomes reconstituted from the abasic 1503 16S rRNA were highly active in protein synthesis but showed elevated levels of spontaneous frameshifting into the -1 reading frame. We then asked whether the residual frameshifting persisting in control ribosomes containing an intact A1503 is due to the absence of the N6-dimethyladenosine modifications at positions 1518 and 1519. Indeed, this frameshifting was rescued by site-specific methylation in vitro by the ksgA methylase. These findings thus implicate two different sites near the 3' end of 16S rRNA in maintenance of the translational reading frame, providing yet another example of a functional role for ribosomal RNA in protein synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

β-Thalassemia is an inherited genetic disorder associated with β-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of β-thalassemia. This study conducted a virtual screening of certain compounds similar to 5'-deoxy-5'methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of β-thalassemia.

Purpose: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated., Methods: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m 6 A RNA methylation in nanoparticles-treated cells., Results: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m 6 A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m 6 A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m 6 A level of Sox1 mRNA, enhancing its stability., Conclusion: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m 6 A RNA methylation modification of Sox1 ., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Bai et al.)

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N 6 -(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance ( 1 H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Combination of tumor immunotherapy with photothermal therapy (PTT) is a feasible tactic to overcome the drawback of immunotherapy such as poor immune response. Via triggering the immunogenic cells death (ICD), PTT can stimulate the activity of immune cells, but meanwhile, the level of adenosine is elevated via the CD73-induced decomposition of ATP which is overexpressed accompanying with the PTT process, resulting in negative feedback to impair the immune stimulation. Herein, we developed a novel biomimetic photothermal nanodrug to specifically block CD73 for inhibition of adenosine production and more efficient priming of the suppressive immune microenvironments. The nanodrug, named as AptEM@CBA, is constructed by encapsulation of photothermal agent black phosphorus quantum dots (BPQDs) and selective CD73 inhibitor α, β-Methyleneadenosine 5'-diphosphate (AMPCP) in chitosan nanogels, which are further covered with aptamer AS1411 modified erythrocyte membrane (EM) for biomimetic camouflage. With AS1411 induced active targeting and EM induced long blood circulation time, the enrichment of the nanodrug tumor sites is promoted. The photothermal treatment promotes the maturation of dendritic cells. Meanwhile, the release of AMPCP suppress the adenosine generation via CD73 blockade, alleviating the impairment of adenosine to dendritic cells and suppressing regulatory T cells, synergically stimulate the activity of T cells. The combination of CD73 blockade with PTT, not only suppresses the growth of primary implanted tumors, but also boosts strong antitumor immunity to inhibit the growth of distal tumors, providing good potential for tumor photoimmunotherapy., (© 2024. The Author(s).)

The ability to sense chemical gradients and respond with directional motility and chemical activity is a defining feature of complex living systems. There is a strong interest among scientists to design synthetic systems that emulate these properties. Here, we realize and control such behaviors in a synthetic system by tailoring multivalent interactions of adenosine nucleotides with catalytic microbeads. We first show that multivalent interactions of the bead with gradients of adenosine mono-, di- and trinucleotides (AM/D/TP) control both the phoretic motion and a proton-transfer catalytic reaction, and find that both effects are diminished greatly with increasing valence of phosphates. We exploit this behavior by using enzymatic hydrolysis of ATP to AMP, which downregulates multivalent interactivity in situ. This produces a sudden increase in transport of the catalytic microbeads (a phoretic jump), which is accompanied by increased catalytic activity. Finally, we show how this enzymatic activity can be systematically tuned, leading to simultaneous in situ spatial and temporal control of the location of the microbeads, as well as the products of the reaction that they catalyze. These findings open up new avenues for utilizing multivalent interaction-mediated programming of complex chemo-mechanical behaviors into active systems., (© 2024. The Author(s).)

Adenosine, as a water-soluble active substance, has various pharmacological effects. This study proposes a layer-by-layer assembly method of composite wall materials, using hydroxypropyl-β-cyclodextrin as the inner wall and whey protein isolate as the outer wall, to encapsulate adenosine within the core material, aiming to enhance adenosine microcapsules' stability through intermolecular interactions. By combining isothermal titration calorimetry with molecular modeling analysis, it was determined that the core material and the inner wall and the inner wall and the outer wall interact through intermolecular forces. Adenosine and hydroxypropyl-β-cyclodextrin form an optimal 1:1 complex through hydrophobic interactions, while hydroxypropyl-β-cyclodextrin and whey protein isolate interact through hydrogen bonds. The embedding rate of AD/Hp-β-CD/WPI microcapsules was 36.80%, and the 24 h retention rate under the release behavior test was 76.09%. The method of preparing adenosine microcapsules using composite wall materials is environmentally friendly and shows broad application prospects in storage and delivery systems with sustained release properties.

Glycerophosphodiester phosphodiesterase (GDPD) is a highly conserved enzyme in both prokaryotic and eukaryotic organisms. It catalyses the hydrolysis of various glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols, which serve as building blocks in several biosynthetic pathways. This enzyme is a well-known virulence factor in many pathogenic bacteria, including Staphylococcus aureus, and is thus considered a potential drug target. In this study, competent E. coli BL21(DE3)pLysS expression cells were used to express the GDPD enzyme from vancomycin-resistant Staphylococcus aureus (VRSA), which was then purified using size exclusion and anion exchange chromatography. The hydrolytic activity of GDPD was evaluated on the non-physiological substrate bis(p-nitrophenyl) phosphate (BpNPP), which indicated functional activity of the enzyme. 79 drugs were evaluated for their inhibitory potential against GDPD enzyme by the colorimetric assay. Out of 79 drugs, 13 drugs, including tenofovir (1), adenosine (2), clioquinol (11), bromazepam (12), lamotrigine (13), sulfadiazine (14), azathioprine (15), nicotine (16), sitagliptin PO 4 (17), doxofylline (18), clindamycin phosphate (19), gentamycin sulphate (20), and ceftriaxone sodium (21) revealed varying degrees of inhibitory potential with IC 50 values in the range of 400 ± 0.007-951 ± 0.016 µM. All drugs were also evaluated for their binding interactions with the target enzyme by saturation transfer difference (STD-NMR) spectroscopy. 10 drugs demonstrated STD interactions and hence, showed binding affinity with the enzyme. Exceptionally, tenofovir (1) was identified to be a better inhibitor with an IC 50 value of 400 ± 0.007 µM, as compared to the standard EDTA (ethylenediaminetetraacetic acid) (IC 50  = 470 ± 0.008 µM). Moreover, molecular docking studies have identified key interactions of the ligand (tenofovir) with the binding site residues of the enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Branching is a critical step in RNA splicing that is essential for 5' splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5' splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2'-OH of the adenosine for nucleophilic attack on the 5' scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

This manuscript deals with cordycepin, an interesting secondary compound produced from entomopathogenic fungus, Cordyceps. It has attracted commercial interest due to its immense pharmacological importance beneficial to human health. In this study, the contents of cordycepin and its derivatives, like adenine and adenosine, were evaluated through solid-state fermentation using combinations of various grains as substrate. Treatment with grain combination numbers 2, 7, 8, and 9 exhibited higher cordycepin content (1.621, 1.929, 1.895, and 1.996 mg/g cordycepin, respectively) than control (rice). The grain combination number 7 exhibited significantly higher adenine content (700 mg/g) than the control and all other combinations. Treatments with grain combination numbers 2, 5, and 7 exhibited higher adenosine content (2.719, 2.938, and 3.392 mg/g, respectively); however, no significant increase in adenosine content was noted in any treatments. The biomass including fresh mycelium and fruit body was found higher in grain combination numbers 7 and 9, leading to enhanced cordycepin content. Overall, the increase in the fresh biomass significantly enhanced cordycepin accumulation. The level of cordycepin was recorded as higher than that of its derivatives, adenosine and adenine. The grain combination of rice, wheat, jowar, bajra, and sugarcane bagasse added to basal medium exhibited the highest cordycepin content and was found suitable for solid-state fermentation of Cordyceps militaris. To our understanding, the present study is the first to use combinations of cereals for the production of cordycepin from C. militaris., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N 6 -Methyladenosine (m 6 A) is a reversible chemical modification in eukaryotic messenger RNAs and long noncoding RNAs. The aberrant expression of RNA methyltransferase METTL3-METTL14 complex may change the m 6 A methylation level and cause multiple diseases including cancers. The conventional METTL3-METTL14 assays commonly suffer from time-consuming procedures and poor sensitivity. Herein, we develop a controllable amplification machinery based on MazF-activated terminal deoxynucleotidyl transferase (TdT)-assisted dendritic DNA structure assembly for ultrasensitive detection of METTL3-METTL14 complex activity in cancer cells and breast tissues. The presence of METTL3-METTL14 complex catalyzes the formation of m 6 A in detection probe, effectively preventing the cleavage of methylated detection probes by MazF. The methylated detection probes with 3'-OH termini can function as the primers for template-free polymerization catalyzed by TdT on magnetic beads (MBs), producing long chains of poly-thymidine (poly-T) sequences. Then poly-T sequences hybridize with signal probes that contain poly-adenine (poly-A) sequence, inducing TdT-mediated polymerization and the subsequent hybridization with more poly-A signal probes for generating dendritic DNA nanostructures assembled on MBs. After magnetic separation and elevated temperature treatment, the signal probes are disassembled from MBs to generate a high fluorescence signal. This method possesses excellent specificity and high sensitivity with a limit of detection (LOD) of 2.61 × 10 -15  M, and it can accurately quantify cellular METTL3-METTL14 complex at single-cell level. Furthermore, it can screen inhibitors, evaluate kinetic parameters, and discriminate breast cancer tissues from normal tissues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Development of resistance to platinum (Pt) in ovarian cancer remains a major clinical challenge. Here we focused on identifying epitranscriptomic modifications linked to Pt resistance. Fat mass and obesity-associated protein (FTO) is a N6-methyladenosine (m6A) RNA demethylase that we recently described as a tumor suppressor in ovarian cancer. We hypothesized that FTO-induced removal of m6A marks regulates the cellular response of ovarian cancer cells to Pt and is linked to the development of resistance. To study the involvement of FTO in the cellular response to Pt, we used ovarian cancer cells in which FTO was knocked down via short hairpin RNA or overexpressed and Pt-resistant (Pt-R) models derived through repeated cycles of exposure to Pt. We found that FTO was significantly downregulated in Pt-R versus sensitive ovarian cancer cells. Forced expression of FTO, but not of mutant FTO, increased sensitivity to Pt in vitro and in vivo (P < 0.05). Increased numbers of γ-H2AX foci, measuring DNA double-strand breaks, and increased apoptosis were observed after exposure to Pt in FTO-overexpressing versus control cells. Through integrated RNA sequencing and MeRIP sequencing, we identified and validated the enzyme nicotinamide N-methyltransferase (NNMT), as a new FTO target linked to Pt response. NNMT was upregulated and demethylated in FTO-overexpressing cells. Treatment with an NNMT inhibitor or NNMT knockdown restored sensitivity to Pt in FTO-overexpressing cells. Our results support a new function for FTO-dependent m6A RNA modifications in regulating the response to Pt through NNMT, a newly identified RNA methylated gene target., (©2023 American Association for Cancer Research.)

The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 10 6 -fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: N6-Methyladenosine (m6A), the most common and reversible mRNA modification, has attracted considerable attention recently, and accumulating evidence indicates it has an important role in the progression of ischemic stroke (IS)., Areas Covered: We first reviewed m6A methylation modification enzymes, including m6A methyltransferases (METTL3, METTL14, and WTAP), demethylases (FTO and ALKBH5), m6A-binding proteins (YTH domain containing 1/2 [YTHDC1/2], YTHDF1/2/3, and insulin like growth factor 2 mRNA binding protein 1/2/3 [IGF2BP1/2/3]), and their-related functions. An alteration in the m6A methylation profile of IS has been reported and m6A is differentially expressed in IS. Thus, we then focused on the underlying mechanism of m6A methylation in IS and the involvement of atherosclerosis (AS), cerebral ischemia/reperfusion (IR) injury, inflammation, oxidative stress, and apoptosis. Furthermore, we also elucidated the effect of m6A-associated single-nucleotide polymorphisms (SNPs) on stroke and uncovered new causal variants for IS. The clinical application of m6A targeting drugs is still in its infancy and will be available in the future., Expert Opinion: Collectively, the information in the present review is a summary of the latest developments in m6A modification and highlights the mechanisms underlying IS pathogenesis, which may provide novel insights into the mechanisms and therapeutic targets for IS.

The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N6-methyladenosine (m6A) modifications. How the m6A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m6A)UCG hairpin, which reveals that the m6A base extends 5' stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m6A)UCG tetraloop shows that the (m6A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m6A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m6A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m6A)UCG hairpin. Our data show that m6A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m6A modifications may regulate lncRNA function by modulating RNA structure., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Abnormal N6-methyladenosine (m6A) modification is closely associated with the occurrence, development, progression and prognosis of cancer, and aberrant m6A regulators have been identified as novel anticancer drug targets. Both traditional medicine-related approaches and modern drug discovery platforms have been used in an attempt to develop m6A-targeted drugs. Here, we provide an update of the latest findings on m6A modification and the critical roles of m6A modification in cancer progression, and we summarize rational sources for the discovery of m6A-targeted anticancer agents from traditional medicines and computer-based chemosynthetic compounds. This review highlights the potential agents targeting m6A modification for cancer treatment and proposes the advantage of artificial intelligence (AI) in the discovery of m6A-targeting anticancer drugs. Three stages of m6A-targeting anticancer drug discovery: traditional medicine-based natural products, modern chemical modification or synthesis, and artificial intelligence (AI)-assisted approaches for the future., (© 2022. The Author(s).)

N 6 -methyladenosine (m 6 A) RNA modification confers an essential layer of gene regulation in living organisms, including plants; yet, the underlying mechanisms of its deposition on specific target mRNAs involved in key plant developmental processes are so far unknown. Here, we show that a core component of the rice m 6 A methyltransferase complex, OsFIP37, is recruited by an RNA-binding protein, OsFIP37-associated protein 1 (OsFAP1), to mediate m 6 A RNA modification on an auxin biosynthesis gene, OsYUCCA3, during microsporogenesis. This stabilizes OsYUCCA3 mRNA and promotes local auxin biosynthesis in anthers during male meiosis, which is essential for meiotic division and subsequent pollen development in rice. Loss of function of OsFAP1 causes dissociation of OsFIP37 with OsYUCCA3 and the resulting abolished m 6 A deposition on OsYUCCA3. Our findings reveal that OsFAP1-dependent m 6 A deposition on OsYUCCA3 by OsFIP37 constitutes a hitherto unknown link between RNA modification and hormonal control of male meiosis in plant reproductive development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

(1 R ,5 S )-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one, available by an efficient catalytic pyrolysis of cellulose, has been applied as a chiral building block in the synthesis of seven new nucleoside analogues, with structural modifications on the nucleobase moiety and on the carboxyl- derived unit. The inverted configuration by Mitsunobu reaction used in their synthesis was verified by 2D-NOESY correlations, supported by the optimized structure employing the DFT methods. An in silico screening of these compounds as inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase has been carried out in comparison with both remdesivir, a mono-phosphoramidate prodrug recently approved for COVID-19 treatment, and its ribonucleoside metabolite GS-441524. Drug-likeness prediction and data by docking calculation indicated compound 6 [=(3 S ,5 S )-methyl 5-(hydroxymethyl)-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrofuran-3-carboxylate] as the best candidate. Furthermore, molecular dynamics simulation showed a stable interaction of structure 6 in RNA-dependent RNA polymerase (RdRp) complex and a lower average atomic fluctuation than GS-441524, suggesting a well accommodation in the RdRp binding pocket.

Here, we performed N6-methyladenosine (m6A) RNA sequencing to determine the circRNA m6A methylation changes in the placentas during the pathogenesis of preeclampsia (PE). We verified the expression of the circRNA circPAPPA2 using quantitative reverse transcription-PCR. An invasion assay was carried out to identify the role of circPAPPA2 in the development of PE. Mechanistically, we investigated the cause of the altered m6A modification of circPAPPA2 through overexpression and knockdown cell experiments, RNA immunoprecipitation, fluorescence in situ hybridization and RNA stability experiments. We found that increases in m6A-modified circRNAs are prevalent in PE placentas and that the main changes in methylation occur in the 3'UTR and near the start codon, implicating the involvement of these changes in PE development. We also found that the levels of circPAPPA2 are decreased but that m6A modification is augmented. Furthermore, we discovered that methyltransferase‑like 14 (METTL14) increases the level of circPAPPA2 m6A methylation and that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) maintains circPAPPA2 stability. Decreases in IGF2BP3 levels lead to declines in circPAPPA2 levels. In summary, we provide a new vision and strategy for the study of PE pathology and report that placental circRNA m6A modification appears to be an important regulatory mechanism., (© 2021. The Author(s).)

N6-methyladenosine (m 6 A) modification is an important regulatory factor affecting diseases, including multiple cancers and it is a developing direction for targeted disease therapy. Here, we present the M6ADD (m 6 A-diseases database) database, a public data resource containing manually curated data on potential m 6 A-disease associations for which some experimental evidence is available; the related high-throughput sequencing data are also provided and analysed by using different computational methods. To give researchers a tool to query the m6A modification data, the M6ADD was designed as a web-based comprehensive resource focusing on the collection, storage and online analysis of m6A modifications, aimed at exploring the associations between m6A modification and gene disorders and diseases. The M6ADD includes 222 experimentally confirmed m 6 A-disease associations, involving 59 diseases from a review of more than 2000 published papers. The M6ADD also includes 409,229 m 6 A-disease associations obtained by computational and statistical methods from 30 high-throughput sequencing datasets. In addition, we provide data on 5239 potential m 6 A regulatory proteins related to 24 cancers based on network analysis prediction methods. In addition, we have developed a tool to explore the function of m 6 A-modified genes through the protein-protein interaction networks. The M6ADD can be accessed at http://m6add.edbc.org/.

N 6 -methyladenosine (m 6 A) plays an important role in various biological processes. Identifying m 6 A site is a key step in exploring its biological functions. One of the biggest challenges in identifying m 6 A sites is how to extract features comprising rich categorical information to distinguish m 6 A and non-m 6 A sites. To address this challenge, we propose bidirectional dinucleotide and trinucleotide position-specific propensities, respectively, in this paper. Based on this, we propose two feature-encoding algorithms: Position-Specific Propensities and Pointwise Mutual Information (PSP-PMI) and Position-Specific Propensities and Pointwise Joint Mutual Information (PSP-PJMI). PSP-PMI is based on the bidirectional dinucleotide propensity and the pointwise mutual information, while PSP-PJMI is based on the bidirectional trinucleotide position-specific propensity and the proposed pointwise joint mutual information in this paper. We introduce parameters α and β in PSP-PMI and PSP-PJMI, respectively, to represent the distance from the nucleotide to its forward or backward adjacent nucleotide or dinucleotide, so as to extract features containing local and global classification information. Finally, we propose the M6A-BiNP predictor based on PSP-PMI or PSP-PJMI and SVM classifier. The 10-fold cross-validation experimental results on the benchmark datasets of non-single-base resolution and single-base resolution demonstrate that PSP-PMI and PSP-PJMI can extract features with strong capabilities to identify m 6 A and non-m 6 A sites. The M6A-BiNP predictor based on our proposed feature encoding algorithm PSP-PJMI is better than the state-of-the-art predictors, and it is so far the best model to identify m 6 A and non-m 6 A sites.

The novel coronavirus disease COVID-19, caused by the virus SARS CoV-2, has exerted a significant unprecedented economic and medical crisis, in addition to its impact on the daily life and health care systems all over the world. Regrettably, no vaccines or drugs are currently available for this new critical emerging human disease. Joining the global fight against COVID-19, in this study we aim at identifying a potential novel inhibitor for SARS COV-2 2'-O-methyltransferase (nsp16) which is one of the most attractive targets in the virus life cycle, responsible for the viral RNA protection via a cap formation process. Firstly, nsp16 enzyme bound to Sinefungin was retrieved from the protein data bank (PDB ID: 6WKQ), then, a 3D pharmacophore model was constructed to be applied to screen 48 Million drug-like compounds of the Zinc database. This resulted in only 24 compounds which were subsequently docked into the enzyme. The best four score-ordered hits from the docking outcome exhibited better scores compared to Sinefungin. Finally, three molecular dynamics (MD) simulation experiments for 150 ns were carried out as a refinement step for our proposed approach. The MD and MM-PBSA outputs revealed compound 11 as the best potential nsp16 inhibitor herein identified, as it displayed a better stability and average binding free energy for the ligand-enzyme complex compared to Sinefungin.

Nicotinamide mononucleotide (NMN) or Nicotinamide-1-ium-1-β-D-ribofuranoside 5'-phosphate is a nucleotide that can be converted into nicotinamide adenine dinucleotide (NAD) in human cells. NMN has recently attracted great attention because of its potential as an anti-aging drug, leading to great efforts for its effective manufacture. The chemical synthesis of NMN is a challenging task since it is an isomeric compound with a complicated structure. The majority of biological synthetic routes for NMN is through the intermediate phosphoribosyl diphosphate (PRPP), which is further converted to NMN by nicotinamide phosphoribosyltransferase (Nampt). There are various routes for the synthesis of PRPP from simple starting materials such as ribose, adenosine, and xylose, but all of these require the expensive phosphate donor adenosine triphosphate (ATP). Thus, an ATP regeneration system can be included, leading to diminished ATP consumption during the catalytic process. The regulations of enzymes that are not directly involved in the synthesis of NMN are also critical for the production of NMN. The aim of this review is to present an overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Nascent messenger RNA is endowed with a poly(A) tail that is subject to gradual deadenylation and subsequent degradation in the cytoplasm. Deadenylation and degradation rates are typically correlated, rendering it difficult to dissect the determinants governing each of these processes and the mechanistic basis of their coupling. Here we developed an approach that allows systematic, robust and multiplexed quantification of poly(A) tails in Saccharomyces cerevisiae. Our results suggest that mRNA deadenylation and degradation rates are decoupled during meiosis, and that transcript length is a major determinant of deadenylation rates and a key contributor to reshaping of poly(A) tail lengths. Meiosis-specific decoupling also leads to unique positive associations between poly(A) tail length and gene expression. The decoupling is associated with a focal localization pattern of the RNA degradation factor Xrn1, and can be phenocopied by Xrn1 deletion under nonmeiotic conditions. Importantly, the association of transcript length with deadenylation rates is conserved across eukaryotes. Our study uncovers a factor that shapes deadenylation rate and reveals a unique context in which degradation is decoupled from deadenylation., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

N6-Isopentenyladenosine ( i6A ) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives' (compounds 2a-m ) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2 , 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

Angustmycin A has anti-mycobacterial and cytokinin activities, and contains an intriguing structure in which an unusual sugar with C5'-C6' dehydration is linked to adenine via an N-glycosidic bond. However, the logic underlying the biosynthesis of this molecule has long remained obscure. Here, we address angustmycin A biosynthesis by the full deciphering of its pathway. We demonstrate that AgmD, C, A, E, and B function as D-allulose 6-phosphate 3-epimerase, D-allulose 6-phosphate pyrophosphokinase, adenine phosphoallulosyltransferase, phosphoribohydrolase, and phosphatase, respectively, and that these collaboratively catalyze the relay reactions to biosynthesize angustmycin C. Additionally, we provide evidence that AgmF is a noncanonical dehydratase for the final step to angustmycin A via a self-sufficient strategy for cofactor recycling. Finally, we have reconstituted the entire six-enzyme pathway in vitro and in E. coli leading to angustmycin A production. These results expand the enzymatic repertoire regarding natural product biosynthesis, and also open the way for rational and rapid discovery of other angustmycin related antibiotics., (© 2021. The Author(s).)

N 6 -methyladenosine (m 6 A) RNA modification is a fundamental determinant of mRNA metabolism, but its role in innate immunity-driven non-alcoholic fatty liver disease (NAFLD) and obesity is not known. Here, we show that myeloid lineage-restricted deletion of the m 6 A "writer" protein Methyltransferase Like 3 (METTL3) prevents age-related and diet-induced development of NAFLD and obesity in mice with improved inflammatory and metabolic phenotypes. Mechanistically, loss of METTL3 results in the differential expression of multiple mRNA transcripts marked with m 6 A, with a notable increase of DNA Damage Inducible Transcript 4 (DDIT4) mRNA level. In METTL3-deficient macrophages, there is a significant downregulation of mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathway activity in response to cellular stress and cytokine stimulation, which can be restored by knockdown of DDIT4. Taken together, our findings identify the contribution of METTL3-mediated m 6 A modification of Ddit4 mRNA to macrophage metabolic reprogramming in NAFLD and obesity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers and is characterized by high recurrence and heterogeneity, yet its mechanism is not well understood. Here we show that N 1 -methyladenosine methylation (m 1 A) in tRNA is remarkably elevated in hepatocellular carcinoma (HCC) patient tumour tissues. Moreover, m 1 A methylation signals are increased in liver cancer stem cells (CSCs) and are negatively correlated with HCC patient survival. TRMT6 and TRMT61A, forming m 1 A methyltransferase complex, are highly expressed in advanced HCC tumours and are negatively correlated with HCC survival. TRMT6/TRMT61A-mediated m 1 A methylation is required for liver tumourigenesis. Mechanistically, TRMT6/TRMT61A elevates the m 1 A methylation in a subset of tRNA to increase PPARδ translation, which in turn triggers cholesterol synthesis to activate Hedgehog signaling, eventually driving self-renewal of liver CSCs and tumourigenesis. Finally, we identify a potent inhibitor against TRMT6/TRMT61A complex that exerts effective therapeutic effect on liver cancer., (© 2021. The Author(s).)

Background: N-6 methylation (m6A) pushes forward an immense influence on the occurrence and development of lung adenocarcinoma (LUAD). However, the methylation on non-coding RNA in LUAD, especially long non-coding RNA (lncRNA), has not been received sufficient attention., Methods: Spearman correlation analysis was used to screen lncRNA correlated with m6A regulators expression from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) repositories, respectively. Then, the least absolute shrinkage and selection operator (LASSO) was applied to build a risk signature consisting m6A-related lncRNA. Univariate and multivariate independent prognostic analysis were applied to evaluate the performance of signature in predicting patients' survival. Next, we applied Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) to conduct pathway enrichment analysis of 3344 different expression genes (DEGs). Finally, we set up a competing endogenous RNAs (ceRNA) network to this lncRNA., Results: A total of 85 common lncRNAs were selected to acquire the components related to prognosis. The final risk signature established by LASSO regression contained 11 lncRNAs: ARHGEF26-AS1, COLCA1, CRNDE, DLGAP1-AS2, FENDRR, LINC00968, TMPO-AS1, TRG-AS1, MGC32805, RPARP-AS1, and TBX5-AS1. M6A-related lncRNA risk score could predict the prognostic of LUAD and was significantly associated with clinical pathological. And in the evaluation of lung adenocarcinoma tumor microenvironment (TME) by using ESTIMATE algorithm, we found a statistically significant correlation between risk score and stromal/immune cells., Conclusion: M6A-related lncRNA was a potential prognostic and therapy target for lung adenocarcinoma., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13 C labeling approach, called 13 C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m 6 A) versus 7-methylguanosine (m 7 G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m 6 2 A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m 6 2 A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13 C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies have shown that RNA methylation modification can affect RNA transcription, metabolism, splicing and stability. In addition, RNA methylation modification has been associated with cancer, obesity and other diseases. Based on information about human genome and machine learning, this paper discusses the effect of the fusion sequence and gene-level feature extraction on the accuracy of methylation site recognition. The significant limitation of existing computing tools was exposed by discovered of new features. (1) Most prediction models are based solely on sequence features and use SVM or random forest as classification methods. (2) Limited by the number of samples, the model may not achieve good performance. In order to establish a better prediction model for methylation sites, we must set specific weighting strategies for training samples and find more powerful and informative feature matrices to establish a comprehensive model. In this paper, we present HSM6AP, a high-precision predictor for the Homo sapiens N6-methyladenosine ( m 6 A ) based on multiple weights and feature stitching. Compared with existing methods, HSM6AP samples were creatively weighted during training, and a wide range of features were explored. Max-Relevance-Max-Distance (MRMD) is employed for feature selection, and the feature matrix is generated by fusing a single feature. The extreme gradient boosting (XGBoost), an integrated machine learning algorithm based on decision tree, is used for model training and improves model performance through parameter adjustment. Two rigorous independent data sets demonstrated the superiority of HSM6AP in identifying methylation sites. HSM6AP is an advanced predictor that can be directly employed by users (especially non-professional users) to predict methylation sites. Users can access our related tools and data sets at the following website: http://lab.malab.cn/~lijing/HSM6AP.html The codes of our tool can be publicly accessible at https://github.com/lijingtju/HSm6AP.git .

Alzheimer's disease, the most common form of dementia in the elderly, is a kind of neurodegenerative disease. However, its pathogenesis and diagnosis remain unclear. M6A is related to nervous system development and neurodegenerative diseases. Here in this study, using multiple RNA-seq datasets of Alzheimer's brain tissues, along with bioinformatic analysis, we innovatively found that m6A reader protein IGF2BP2 was abnormally highly expressed in Alzheimer's patients. After compared between Alzheimer's and normal brain samples, and between IGF2BP2- high and IGF2BP2- low subgroups of Alzheimer's patients, we took the shared differentially expressed genes as the relevant gene sets of IGF2PB2 affecting Alzheimer's disease occurrence for subsequent analysis. Then, weight gene correlation analysis was conducted and 17 functional modules were identified. The module that most positively correlated with Alzheimer's disease and IGF2PB2-high subgroups were mainly participated in ECM receptor interaction, focal adhesion, cytokine-cytokine receptor interaction, and TGF-beta signaling pathway. Afterwards, a hub gene-based model including 20 genes was constructed by LASSO regression and validated by ROC curve for Alzheimer diagnosis. Finally, we preliminarily elucidated that IGF2BP2 could bind with mRNAs in a m6A-dependent manner. This study first elucidates the pathogenic role of IGF2BP2 in Alzheimer's disease. IGF2BP2 and its relevant m6A modifications are potential to be new diagnostic and therapeutic targets for Alzheimer's patients.

Pseudogenes may play important roles in cancer. Here, we explore the mechanism and function of a pseudogene WTAPP1 in the progress of pancreatic ductal adenocarcinoma (PDAC). WTAPP1 RNA was significantly elevated in PDAC and was associated with poor prognosis in patients. Overexpression of WTAPP1 RNA promoted PDAC proliferation and invasiveness in vitro and in vivo . Mechanistically, N 6 -methyladenosine (m 6 A) modification stabilized WTAPP1 RNA via CCHC-type zinc finger nucleic-acid binding protein (CNBP), resulting in increased levels of WTAPP1 RNA in PDAC cells. Excessive WTAPP1 RNA bound its protein-coding counterpart WT1-associated protein (WTAP) mRNA and recruited more EIF3 translation initiation complex to promote WTAP translation. Increased WTAP protein enhanced the activation of Wnt signaling and provoked the malignant phenotypes of PDAC. Decreasing WTAPP1 RNA significantly suppressed the in vivo growth and metastasis of PDAC cell lines and patient-derived xenografts. These results indicate that m 6 A-mediated increases in WTAPP1 expression promote PDAC progression and thus may serve as a therapeutic target. SIGNIFICANCE: This study reveals how aberrant m 6 A modification of the WTAPP1 pseudogene results in increased translation of its protein-coding counterpart to promote Wnt signaling, which contributes to pancreatic cancer progression., (©2021 The Authors; Published by the American Association for Cancer Research.)