Bemnifosbuvir (AT-527) and AT-752 are guanosine analogues currently in clinical trials against several RNA viruses. Here, we show that these drugs require a minimal set of 5 cellular enzymes for activation to their common 5\'-triphosphate AT-9010, with an obligate order of reactions. AT-9010 selectively inhibits essential viral enzymes, accounting for antiviral potency. Functional and structural data at atomic resolution decipher N6-purine deamination compatible with its metabolic activation. Crystal structures of human histidine triad nucleotide binding protein 1, adenosine deaminase-like protein 1, guanylate kinase 1, and nucleoside diphosphate kinase at 2.09, 2.44, 1.76, and 1.9 Å resolution, respectively, with cognate precursors of AT-9010 illuminate the activation pathway from the orally available bemnifosbuvir to AT-9010, pointing to key drug-protein contacts along the activation pathway. Our work provides a framework to integrate the design of antiviral nucleotide analogues, confronting requirements and constraints associated with activation enzymes along the 5\'-triphosphate assembly line.

The trimethylguanosine (TMG) cap is a motif present inter alia at the 5\' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2\'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3\'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.

A new emissive guanosine analog CF3thG, constructed by a single trifluoromethylation step from the previously reported thG, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3thG suggests that the electronic effects of the trifluoromethyl group dominate its behavior and demonstrates its susceptibility to microenvironmental polarity changes. In vitro transcription initiations using T7 RNA polymerase, initiated with CF3thG, result in highly emissive 5\'-labeled RNA transcripts, demonstrating the tolerance of the enzyme toward the analog. Viability assays with HEK293T cells displayed no detrimental effects at tested concentrations, indicating the safety of the analog for cellular applications. Live cell imaging of the free emissive guanosine analog using confocal microscopy was facilitated by its red-shifted absorption and emission and adequate brightness. Real-time live cell imaging demonstrated the release of the guanosine analog from HEK293T cells at concentration-gradient conditions, which was suppressed by the addition of guanosine.

7-Deaza-2\'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2\'-deoxyisocytidine and purine-purine base pairs with 2\'-deoxyguanosine or 5-aza-7-deaza-2\'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2\'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2\'-deoxyisoguanosine/5-aza-7-deaza-2\'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2\'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

BACKGROUND: N7-methylguanosine (m7G) modification is one of the most prevalent RNA modifications in humans. Dysregulated m7G modifications caused by aberrant expression of m7G writers contribute to cancer progression and result in worse patient survival in several human cancers. However, studies that systematically assess the frequency and clinical relevance of aberrant m7G writer expression in a pan-cancer cohort remain to be performed.
OBJECTIVE: This study aims to systematically investigate the molecular alteration and clinical relevance of m7G methyltransferase in human cancers.
METHODS: We analysed genome, transcriptome and clinical data from the Cancer Genome Atlas Research Network spanning 33 types of human cancers for aberrant changes in genes encoding m7G writers.
RESULTS: We demonstrate that m7G writers are dysregulated in human cancers and are associated predominantly with poorer survival. By dividing patients into those with high and low m7G scores, we show that a lower m7G score is generally associated with immune infiltration and better response to immunotherapy.
CONCLUSIONS: Our analyses indicate the genetic alterations, expression patterns and clinical relevance of m7G writers across various cancers. This study provides insights into the potential utility of m7G writer expression as a cancer biomarker and proposes the possibility of targeting m7G writers for cancer therapy.

N-7methylguanosine (m7G) modification plays a crucial role in various biological processes and is closely associated with the development and progression of many cancers. Accurate identification of m7G modification sites is essential for understanding their regulatory mechanisms and advancing cancer therapy. Previous studies often suffered from insufficient research data, underutilization of motif information, and lack of interpretability. In this work, we designed a novel motif-based interpretable method for m7G modification site prediction, called Moss-m7G. This approach enables the analysis of RNA sequences from a motif-centric perspective. Our proposed word-detection module and motif-embedding module within Moss-m7G extract motif information from sequences, transforming the raw sequences from base-level into motif-level and generating embeddings for these motif sequences. Compared with base sequences, motif sequences contain richer contextual information, which is further analyzed and integrated through the Transformer model. We constructed a comprehensive m7G data set to implement the training and testing process to address the data insufficiency noted in prior research. Our experimental results affirm the effectiveness and superiority of Moss-m7G in predicting m7G modification sites. Moreover, the introduction of the word-detection module enhances the interpretability of the model, providing insights into the predictive mechanisms.

Neurodegenerative diseases and brain tumours represent important health challenges due to their severe nature and debilitating consequences that require substantial medical care. Interestingly, these conditions share common physiological characteristics, namely increased glutamate, and adenosine transmission, which are often associated with cellular dysregulation and damage. Guanosine, an endogenous nucleoside, is safe and exerts neuroprotective effects in preclinical models of excitotoxicity, along with cytotoxic effects on tumour cells. However, the lack of well-defined mechanisms of action for guanosine hinders a comprehensive understanding of its physiological effects. In fact, the absence of specific receptors for guanosine impedes the development of structure-activity research programs to develop guanosine derivatives for therapeutic purposes. Alternatively, given its apparent interaction with the adenosinergic system, it is plausible that guanosine exerts its neuroprotective and anti-tumorigenic effects by modulating adenosine transmission through undisclosed mechanisms involving adenosine receptors, transporters, and purinergic metabolism. Here, several potential molecular mechanisms behind the protective actions of guanosine will be discussed. First, we explore its potential interaction with adenosine receptors (A1R and A2AR), including the A1R-A2AR heteromer. In addition, we consider the impact of guanosine on extracellular adenosine levels and the role of guanine-based purine-converting enzymes. Collectively, the diverse cellular functions of guanosine as neuroprotective and antiproliferative agent suggest a multimodal and complementary mechanism of action.

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients\' A>I(G) RNA-editing profiles.

BACKGROUND: Uveal melanoma (UVM) stands as the predominant type of primary intraocular malignancy among adults. The clinical significance of N7-methylguanosine (m7G), a prevalent RNA modifications, in UVM remains unclear.
METHODS: Primary information from 80 UVM patients were analyzed as the training set, incorporating clinical information, mutation annotations and mRNA expression obtained from The Cancer Genome Atlas (TCGA) website. The validation set was carried out using Gene Expression Omnibus (GEO) database GSE22138 and GSE84976. Kaplan-Meier and Cox regression of univariate analyses were subjected to identify m7G-related regulators as prognostic genes.
RESULTS: A prognostic risk model comprising EIF4E2, NUDT16, SNUPN and WDR4 was established through Cox regression of LASSO. Evaluation of the model\'s predictability for UVM patients\' prognosis by Receiver Operating Characteristic (ROC) curves in the training set, demonstrated excellent performance Area Under the Curve (AUC) > 0.75. The high-risk prognosis within the TCGA cohort exhibit a notable worse outcome. Additionally, an independent correlation between the risk score and overall survival (OS) among UVM patients were identified. External validation of this model was carried out using the validation sets (GSE22138 and GSE84976). Immune-related analysis revealed that patients with high score of m7G-related risk model exhibited elevated level of immune infiltration and immune checkpoint gene expression.
CONCLUSIONS: We have developed a risk prediction model based on four m7G-related regulators, facilitating effective estimate UVM patients\' survival by clinicians. Our findings shed novel light on essential role of m7G-related regulators in UVM and suggest potential novel targets for the diagnosis, prognosis and therapy of UVM.

Cellular senescence is characterized by a decrease in protein synthesis, although the underlying processes are mostly unclear. Chemical modifications to transfer RNAs (tRNAs) frequently influence tRNA activity, which is crucial for translation. We describe how tRNA N7-methylguanosine (m7G46) methylation, catalyzed by METTL1-WDR4, regulates translation and influences senescence phenotypes. Mettl1/Wdr4 and m7G gradually diminish with senescence and aging. A decrease in METTL1 causes a reduction in tRNAs, especially those with the m7G modification, via the rapid tRNA degradation (RTD) pathway. The decreases cause ribosomes to stall at certain codons, impeding the translation of mRNA that is essential in pathways such as Wnt signaling and ribosome biogenesis. Furthermore, chronic ribosome stalling stimulates the ribotoxic and integrative stress responses, which induce senescence-associated secretory phenotype. Moreover, restoring eEF1A protein mitigates senescence phenotypes caused by METTL1 deficiency by reducing RTD. Our findings demonstrate that tRNA m7G modification is essential for preventing premature senescence and aging by enabling efficient mRNA translation.