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Journal of Molecular Biology Sep 2022RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding...
RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding free energies of helices and loops. Although the full set of parameters has only been traditionally available for the four common bases and backbone, it is well known that covalent modifications of nucleotides are widespread in natural RNAs. Covalent modifications are also widely used in engineered sequences. We recently derived a full set of nearest neighbor terms for RNA that includes N-methyladenosine (mA). In this work, we test the model using 98 optical melting experiments, matching duplexes with or without N-methylation of A. Most experiments place RRACH, the consensus site of N-methylation, in a variety of contexts, including helices, bulge loops, internal loops, dangling ends, and terminal mismatches. For matched sets of experiments that include either A or mA in the same context, we find that the parameters for mA are as accurate as those for A. Across all experiments, the root mean squared deviation between estimated and experimental free energy changes is 0.67 kcal/mol. We used the new experimental data to refine the set of nearest neighbor parameter terms for mA. These parameters enable prediction of RNA secondary structures including mA, which can be used to model how N-methylation of A affects RNA structure.
Topics: Adenosine; Entropy; RNA; RNA Folding
PubMed: 35588868
DOI: 10.1016/j.jmb.2022.167632 -
Pain Jul 2020Extracellular nucleosides and nucleotides have widespread functions in responding to physiological stress. The "purinome" encompasses 4 G-protein-coupled receptors...
Extracellular nucleosides and nucleotides have widespread functions in responding to physiological stress. The "purinome" encompasses 4 G-protein-coupled receptors (GPCRs) for adenosine, 8 GPCRs activated by nucleotides, 7 adenosine 5'-triphosphate-gated P2X ion channels, as well as the associated enzymes and transporters that regulate native agonist levels. Purinergic signaling modulators, such as receptor agonists and antagonists, have potential for treating chronic pain. Adenosine and its analogues potently suppress nociception in preclinical models by activating A1 and/or A3 adenosine receptors (ARs), but safely harnessing this pathway to clinically treat pain has not been achieved. Both A2AAR agonists and antagonists are efficacious in pain models. Highly selective A3AR agonists offer a novel approach to treat chronic pain. We have explored the structure activity relationship of nucleoside derivatives at this subtype using a computational structure-based approach. Novel A3AR agonists for pain control containing a bicyclic ring system (bicyclo [3.1.0] hexane) in place of ribose were designed and screened using an in vivo phenotypic model, which reflected both pharmacokinetic and pharmacodynamic parameters. High specificity (>10,000-fold selective for A3AR) was achieved with the aid of receptor homology models based on related GPCR structures. These A3AR agonists are well tolerated in vivo and highly efficacious in models of chronic neuropathic pain. Furthermore, signaling molecules acting at P2X3, P2X4, P2X7, and P2Y12Rs play critical roles in maladaptive pain neuroplasticity, and their antagonists reduce chronic or inflammatory pain, and, therefore, purine receptor modulation is a promising approach for future pain therapeutics. Structurally novel antagonists for these nucleotide receptors were discovered recently.
Topics: Adenosine; Chronic Pain; Humans; Neuralgia; Receptors, Purinergic; Structure-Activity Relationship
PubMed: 32187120
DOI: 10.1097/j.pain.0000000000001857 -
International Journal of Molecular... Feb 2020Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and... (Review)
Review
Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and nucleotides (nitrogen bases plus ribose and phosphate). These compounds are very common in nature, especially in a phosphorylated form. There is increasing evidence that purines are involved in the development of different organs such as the heart, skeletal muscle and brain. When brain development is complete, some purinergic mechanisms may be silenced, but may be reactivated in the adult brain/muscle, suggesting a role for purines in regeneration and self-repair. Thus, it is possible that guanosine-5'-triphosphate (GTP) also acts as regulator during the adult phase. However, regarding GBP, no specific receptor has been cloned for GTP or its metabolites, although specific binding sites with distinct GTP affinity characteristics have been found in both muscle and neural cell lines. Finally, even if the cross regulation mechanisms between the two different purines (ABP and GBP) are still largely unknown, it is now possible to hypothesize the existence of specific signal paths for guanosine-based nucleotides that are capable of modulating the intensity and duration of the intracellular signal, particularly in excitable tissues such as brain and muscle.
Topics: Brain; Embryonic Development; Guanine; Guanine Nucleotides; Guanosine; Guanosine Triphosphate; Humans; Muscles; Nervous System; Nucleosides; Nucleotides; Purines; Receptors, Purinergic
PubMed: 32111063
DOI: 10.3390/ijms21051591 -
Accounts of Chemical Research Aug 2022RNA editing or "epitranscriptomic modification" refers to the processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid....
RNA editing or "epitranscriptomic modification" refers to the processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid. These chemical alterations can be found on either the ribose sugar or the nucleobase, and although many are "silent" and do not change the Watson-Crick-Franklin code of the RNA, others result in recoding events. More than 170 RNA modifications have been identified so far, each having a specific biological purpose. Additionally, dysregulated RNA editing has been linked to several types of diseases and disorders. As new modifications are discovered and our understanding of their functional impact grows, so does the need for selective methods of identifying and mapping editing sites in the transcriptome.The most common methods for studying RNA modifications rely on antibodies as affinity reagents; however, antibodies can be difficult to generate and often have undesirable off-target binding. More recently, selective chemical labeling has advanced the field by offering techniques that can be used for the detection, enrichment, and quantification of RNA modifications. In our method using acrylamide for inosine labeling, we demonstrated the versatility with which this approach enables pull-down or downstream functionalization with other tags or affinity handles. Although this method did enable the quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely because of the similar reactivity profiles of inosine and pseudouridine or other nucleobases.Seeking to overcome the inherent limitations of antibodies and chemical labeling methods, a more recent approach to studying the epitranscriptome is through the repurposing of proteins and enzymes that recognize modified RNA. Our laboratory has used Endonuclease V, a repair enzyme that cleaves inosine-containing RNAs, and reprogrammed it to instead bind inosine. We first harnessed EndoV to develop a preparative technique for RNA sequencing that we termed EndoVIPER-seq. This method uses EndoV to enrich inosine-edited RNAs, providing better coverage in RNA sequencing and leading to the discovery of previously undetected A-to-I editing sites. We also leveraged EndoV to create a plate-based immunoassay (EndoVLISA) to quantify inosine in cellular RNA. This approach can detect differential A-to-I editing levels across tissue types or disease states while being independent of RNA sequencing, making it cost-effective and high-throughput. By harnessing the molecular recognition capabilities of this enzyme, we show that EndoV can be repurposed as an "anti-inosine antibody" to develop new methods of detecting and enriching inosine from cellular RNA.Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications, and exploiting the affinity of these biomolecules offers a promising new direction for the field of epitranscriptomics.
Topics: Inosine; RNA; RNA Editing
PubMed: 35900335
DOI: 10.1021/acs.accounts.2c00287 -
International Journal of Molecular... Feb 2023Purine nucleotides and nucleosides are involved in various human physiological and pathological mechanisms. The pathological deregulation of purinergic signaling... (Review)
Review
Purine nucleotides and nucleosides are involved in various human physiological and pathological mechanisms. The pathological deregulation of purinergic signaling contributes to various chronic respiratory diseases. Among the adenosine receptors, A2B has the lowest affinity such that it was long considered to have little pathophysiological significance. Many studies suggest that A2BAR plays protective roles during the early stage of acute inflammation. However, increased adenosine levels during chronic epithelial injury and inflammation might activate A2BAR, resulting in cellular effects relevant to the progression of pulmonary fibrosis.
Topics: Humans; Adenosine; Idiopathic Pulmonary Fibrosis; Inflammation; Receptors, Purinergic P1; Disease Progression
PubMed: 36901855
DOI: 10.3390/ijms24054428 -
Bioscience Reports Mar 2015Adenosine-to-inosine (A-to-I) RNA editing, the most prevalent mode of transcript modification in higher eukaryotes, is catalysed by the adenosine deaminases acting on... (Review)
Review
Adenosine-to-inosine (A-to-I) RNA editing, the most prevalent mode of transcript modification in higher eukaryotes, is catalysed by the adenosine deaminases acting on RNA (ADARs). A-to-I editing imposes an additional layer of gene regulation as it dictates various aspects of RNA metabolism, including RNA folding, processing, localization and degradation. Furthermore, editing events in exonic regions contribute to proteome diversity as translational machinery decodes inosine as guanosine. Although it has been demonstrated that dysregulated A-to-I editing contributes to various diseases, the precise regulatory mechanisms governing this critical cellular process have yet to be fully elucidated. However, integration of previous studies revealed that regulation of A-to-I editing is multifaceted, weaving an intricate network of auto- and transregulations, including the involvement of virus-originated factors like adenovirus-associated RNA. Taken together, it is apparent that tipping of any regulatory components will have profound effects on A-to-I editing, which in turn contributes to both normal and aberrant physiological conditions. A complete understanding of this intricate regulatory network may ultimately be translated into new therapeutic strategies against diseases driven by perturbed RNA editing events. Herein, we review the current state of knowledge on the regulatory mechanisms governing A-to-I editing and propose the role of other co-factors that may be involved in this complex regulatory process.
Topics: Adenosine; Adenosine Deaminase; Animals; Humans; Inosine; RNA Editing; RNA-Binding Proteins
PubMed: 25662729
DOI: 10.1042/BSR20140190 -
DNA Repair Jan 20225',8-cyclo-2-deoxy nucleosides (cdPus) are the smallest tandem purine lesions including 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG). They...
5',8-cyclo-2-deoxy nucleosides (cdPus) are the smallest tandem purine lesions including 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG). They can inhibit DNA and RNA polymerases causing mutations, DNA strand breaks, and termination of DNA replication and gene transcription. cdPus can be removed by nucleotide excision repair with low efficiency allowing them to accumulate in the genome. Recent studies suggest that cdPus can be induced in damaged nucleotide pools and incorporated into the genome by DNA polymerases. However, it remains unknown if and how DNA polymerases can incorporate cdPus. In this study, we examined the incorporation of cdAs by human DNA repair polymerases, DNA polymerases β (pol β), and pol η during base excision repair. We then determined the efficiency of cdA incorporation by the polymerases using steady-state kinetics. We found that pol β and pol η incorporated cdAs opposite dT and dC with low efficiency, and incorporated cdAs were readily extended and ligated into duplex DNA. Using molecular docking analysis, we found that the 5',8-covalent bond in cdA disrupted its hydrogen bonding with a template base suggesting that the phosphodiester bond between the 3'-terminus nucleotide and the α-phosphate of cdATP were generated in the absence of hydrogen bonding. The enzyme kinetics analysis further suggests that pol β and pol η increased their substrate binding to facilitate the enzyme catalysis for cdA incorporation. Our study reveals unique mechanisms underlying the accumulation of cdPu lesions in the genome resulting from nucleotide incorporation by repair DNA polymerases.
Topics: DNA; DNA Polymerase beta; DNA Repair; DNA-Directed DNA Polymerase; Deoxyadenosines; Humans; Kinetics; Molecular Docking Simulation
PubMed: 34871863
DOI: 10.1016/j.dnarep.2021.103258 -
Biomolecules Mar 2022The immunosuppressive effect of adenosine in the microenvironment of a tumor is well established. Presently, researchers are developing approaches in immune therapy that... (Review)
Review
The immunosuppressive effect of adenosine in the microenvironment of a tumor is well established. Presently, researchers are developing approaches in immune therapy that target inhibition of adenosine or its signaling such as CD39 or CD73 inhibiting antibodies or adenosine A2A receptor antagonists. However, numerous enzymatic pathways that control ATP-adenosine balance, as well as understudied intracellular adenosine regulation, can prevent successful immunotherapy. This review contains the latest data on two adenosine-lowering enzymes: adenosine kinase (ADK) and adenosine deaminase (ADA). ADK deletes adenosine by its phosphorylation into 5'-adenosine monophosphate. Recent studies have revealed an association between a long nuclear ADK isoform and an increase in global DNA methylation, which explains epigenetic receptor-independent role of adenosine. ADA regulates the level of adenosine by converting it to inosine. The changes in the activity of ADA are detected in patients with various cancer types. The article focuses on the biological significance of these enzymes and their roles in the development of cancer. Perspectives of future studies on these enzymes in therapy for cancer are discussed.
Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Adenosine Monophosphate; Humans; Inosine; Neoplasms; Tumor Microenvironment
PubMed: 35327609
DOI: 10.3390/biom12030418 -
Medicina Oral, Patologia Oral Y Cirugia... Nov 2023The development and establishment of oral squamous cell carcinoma are confined to carcinogenesis, which involves oxidative stress via oxygen-free radical production as a...
BACKGROUND
The development and establishment of oral squamous cell carcinoma are confined to carcinogenesis, which involves oxidative stress via oxygen-free radical production as a hydroxyl radical (HO•), considered the most important cause of oxidative damage to basic biomolecules since it targets DNA strands. 8-Hydroxy-2´-deoxyguanosine (8-OHdG) is considered a free radical with a promutagenic capacity due to its ability to pair with adenosine instead of cytosine during replication.
MATERIAL AND METHODS
We collected 30 paraffin-embedded tissue samples of OSCC from patients treated between 2013 and 2018. We recorded risk habits, disease stage, disease free survival and death with at least 3 years of follow-up. 8-Hydroxyguanosine was evaluated by immunohistochemistry and subsequently classified as weak-moderate or strong positive expression. Additionally, we noted whether it was expressed in the cytoplasm and/or nucleus.
RESULTS
Most of the cases expressed 8-OHdG with a strong intensity (80%). All neoplastic cells were preferentially stained in only the cytoplasm (70.0%), but nuclear positivity was found in 30%, independent of the intensity. Based on the location in the cytoplasm and/or nucleus, tumors >4 cm showed a high frequency (95.5%) of 8-OHdG expression in only the cytoplasm, with a significant difference (p value 0.001). Additionally, overall survival was affected when immunoexpression was present in the cytoplasm and nucleus because all deaths were in this group were statistically significant (p value = 0.001).
CONCLUSIONS
All tumors showed DNA oxidative damage, and 8-OHdG was preferentially expressed in the cytoplasm. This finding was associated with tumor size and, when present in the nucleus, might also be related to death.
Topics: Humans; 8-Hydroxy-2'-Deoxyguanosine; Mouth Neoplasms; Deoxyguanosine; Carcinoma, Squamous Cell; DNA Damage; Oxidative Stress; Free Radicals
PubMed: 37471300
DOI: 10.4317/medoral.25924 -
Molecules (Basel, Switzerland) Mar 2021The de novo synthesis of piperidine nucleosides from our homologating agent 5,6-dihydro-1,4-dithiin is herein reported. The structure and conformation of nucleosides...
The de novo synthesis of piperidine nucleosides from our homologating agent 5,6-dihydro-1,4-dithiin is herein reported. The structure and conformation of nucleosides were conceived to faithfully resemble the well-known nucleoside drugs Immucillins H and A in their bioactive conformation. NMR analysis of the synthesized compounds confirmed that they adopt an iminosugar conformation bearing the nucleobases and the hydroxyl groups in the appropriate orientation.
Topics: Adenine; Adenosine; Magnetic Resonance Spectroscopy; Molecular Conformation; Nucleosides; Piperidines; Purine Nucleosides; Pyrimidinones; Pyrrolidines; Structure-Activity Relationship
PubMed: 33809603
DOI: 10.3390/molecules26061652