Current protocols最新文献

{"title":"Investigating Mitochondrial Bioenergetics in Intact Heart and Brain Tissue Slices","authors":"Xavier R. Chapa-Dubocq,&nbsp;Sukanya G. Gakare,&nbsp;Walia Zahra,&nbsp;Meagan Donovan,&nbsp;Keishla M. Rodriguez-Graciani,&nbsp;Tanner Hoole,&nbsp;Scott Edwards,&nbsp;Patricia E. Molina,&nbsp;Michael C. Salling,&nbsp;Janos Paloczi,&nbsp;Liz Simon","doi":"10.1002/cpz1.70451","DOIUrl":"10.1002/cpz1.70451","url":null,"abstract":"<p>Mitochondria are essential for maintaining the high energetic demands of the heart and brain, generating ATP required for contractile function, neuronal signaling, and ionic homeostasis. In both tissues, metabolic flexibility is critical for maintaining bioenergetic efficiency, redox balance, and cellular viability. Despite their importance, existing experimental approaches to assess mitochondrial bioenergetic function present notable limitations. Isolated mitochondria and permeabilized cell assays provide precise control over substrates and respiratory states but disrupt organelle integrity and remove native cellular and extracellular context. Conversely, measurements in isolated or cultured cells preserve intact mitochondria but introduce phenotypic and metabolic artifacts. These constraints highlight the need for an intermediate platform that preserves native tissue architecture and cellular diversity while enabling quantitative assessment of mitochondrial bioenergetics. Building on prior demonstrations of respiration measurements in intact cardiac and neural tissue, we describe a tissue punch-based approach that enables region-specific analysis of mitochondrial function in small ex vivo tissue slices of intact heart and brain. This method preserves cytoarchitecture, and intercellular interactions while remaining compatible with high-resolution Seahorse respirometry analysis. Tissue punches allow multiple technical replicates from individual organs, reduce variability associated with isolation procedures, and enable assessment of regional metabolic heterogeneity, such as atrial versus ventricular myocardium or discrete brain regions. Here we present detailed and reproducible workflow protocols for brain and cardiac tissue punch preparation and extracellular flux analysis, including guidance on sample acquisition, punch sizing, normalization strategies, and data interpretation, with considerations for adapting the protocols across multiple pre-clinical models. © 2026 Wiley Periodicals LLC.</p><p><b>Support Protocol</b>: Preparation, Reagent Setup, and Instrumentation</p><p><b>Basic Protocol 1</b>: Cardiac tissue preparation</p><p><b>Basic Protocol 2</b>: Brain tissue preparation: rodents</p><p><b>Basic Protocol 3</b>: Brain tissue preparation: nonhuman primates</p><p><b>Basic Protocol 4</b>: Placement of tissue punches on Seahorse organoid plate</p><p><b>Basic Protocol 5</b>: Mitochondrial respiration measurements</p><p><b>Basic Protocol 6</b>: Tissue disruption for total protein quantification</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148852067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effectiveness of Telerehabilitation in Individuals with Upper Crossed Syndrome Across Diverse Age Demographics: Study Protocol for a Randomized Controlled Trial","authors":"Khushi Saxena,&nbsp;Zubia Veqar,&nbsp;Iram Iram,&nbsp;Musawir Mohsin Parsa,&nbsp;Shabnam Khan","doi":"10.1002/cpz1.70450","DOIUrl":"10.1002/cpz1.70450","url":null,"abstract":"<p><b>Background</b>: Upper crossed syndrome (UCS), characterized by muscular imbalances and postural misalignments such as forward head posture and rounded shoulders, is increasingly prevalent among sedentary populations. Traditional rehabilitation often lacks adherence and supervision, whereas telerehabilitation enhances both through real-time remote supervision. However, limited evidence exists comparing the effectiveness of telerehabilitation and home exercise programs for managing UCS across age groups, particularly regarding age-specific adherence and clinical outcomes. This study aims to compare the effectiveness of telerehabilitation-supervised programs versus home exercise programs in improving posture, muscle strength, and adherence in individuals with UCS across age groups.</p><p><b>Methodology</b>: A randomized controlled trial will be conducted with 56 asymptomatic UCS participants stratified by age. Participants will be randomly assigned to either a telerehabilitation group or a home exercise program (HEP) group. Both groups will follow an identical 6-week corrective exercise protocol (3 sessions/week). The telerehabilitation group will receive live video supervision via the Zoom platform, while the control group will follow printed paper handouts. Primary outcomes, including craniovertebral angle and forward shoulder angle, will be assessed using photogrammetry. Secondary outcomes include scapular muscle strength, deep cervical flexor endurance, and exercise adherence.</p><p><b>Discussion</b>: The expected findings will highlight the comparative effectiveness of telerehabilitation and HEP, and guide age-appropriate physiotherapeutic interventions and integration of digital platforms into routine musculoskeletal care.</p><p><b>Trial registration</b>: This trial was approved by the Institutional Ethics Committee of Jamia Millia Islamia (JMI), New Delhi, India (20/11/568/JMI/IEC/2025) and registered with CTRI as CTRI/2026/01/101387. © 2026 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148820627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analytical Workflow for Determining the Authenticity of Human Cell Lines and Tissue Samples by Short Tandem Repeat (STR) Genotype Analysis","authors":"Christopher T. Korch,&nbsp;Amanda Capes-Davis,&nbsp;Chao Shen","doi":"10.1002/cpz1.70439","DOIUrl":"10.1002/cpz1.70439","url":null,"abstract":"<p>This protocol is part of a two-article series [accompanying article: “A biomedical researcher's guide for analyzing short tandem repeat (STR) genotypes of human cell lines and in vitro tissue samples using three standard authentication algorithms”]. Here, we provide a stepwise procedure for using STR genotyping to determine the authenticity and relatedness of human cell lines and tissue samples used in biomedical research using two different cell line similarity tools (CLASTR of Cellosaurus and a provided Microsoft Excel spreadsheet computer file) together with three commonly used matching algorithms. Evaluation of the three algorithms is described in the accompanying article. This workflow protocol should be used in combination with the accompanying article. The applications of these three algorithms (Tanabe, Masters vs Query, and Masters vs Reference) to STR genotyping data are illustrated in the accompanying article. The different types of results are discussed along with suggestions for evaluating and resolving inconclusive match results. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Analytical workflow for determining the authenticity of human cell lines and tissue samples by short tandem repeat (STR) genotype analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148809636","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: CRISPR-Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella) patens","authors":"Shu-Zon Wu,&nbsp;Samantha E. Ryken,&nbsp;Magdalena Bezanilla","doi":"10.1002/cpz1.70456","DOIUrl":"10.1002/cpz1.70456","url":null,"abstract":"<p><i>Current Protocols</i> is issuing a correction for the following protocol article.</p><p>Wu, S.-Z., Ryken, S. E., &amp; Bezanilla, M. (2023). CRISPR-Cas9 genome editing in the moss <i>physcomitrium</i> (formerly <i>physcomitrella</i>) <i>patens</i>. <i>Current Protocols</i>, <i>3</i>, e725. doi: 10.1002/cpz1.725</p><p>In the above-referenced article:</p><p>In the “Reagents and Solutions” section, under “PRMB Medium”, the following sentences have been included:</p><p>Autoclave 500 mM CaCl₂ solution separately. Add 20 µL of CaCl₂ solution to every mL of PRMB medium before use.</p><p>The current version online now includes this correction and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13502213/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148809671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of Natural Killer Cell Serial Degranulation","authors":"Jens A. Niemann,&nbsp;Carsten Watzl","doi":"10.1002/cpz1.70452","DOIUrl":"10.1002/cpz1.70452","url":null,"abstract":"<p>Natural killer (NK) cells have been shown to be heterogeneous and to differ in their ability to kill target cells: although some NK cells show little cytotoxic activity, others sequentially kill multiple targets. The latter are called serial killers, and they perform this activity mainly via the release of cytotoxic granules. During this degranulation process, CD107a is exposed at the NK cell's surface. By staining this extracellular CD107a in a sequential manner, the ability of NK cells to serially degranulate while in co-culture with target cells can be analyzed. Here we describe a protocol for the detection and characterization of serial degranulating human NK cells. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Natural killer cell serial degranulation assay</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13502215/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148809614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human Stem Cell Transplantation, Immunosuppression, and Noninvasive In vivo Cell Tracking in the Mouse Brain","authors":"Kevin S. Chen,&nbsp;Kyle J. Loi,&nbsp;Lisa M. McGinley,&nbsp;Osama N. Kashlan,&nbsp;Diana M. Rigan,&nbsp;Shayna N. Mason,&nbsp;Jacquelin F. Kwentus,&nbsp;Andrew D. Carter,&nbsp;Masha G. Savelieff,&nbsp;Eva L. Feldman","doi":"10.1002/cpz1.70442","DOIUrl":"https://doi.org/10.1002/cpz1.70442","url":null,"abstract":"<p>Stem cell therapies have emerged as potential therapeutics of interest for many central nervous system diseases, including stroke, epilepsy, neurodegenerative diseases, and traumatic brain injury. Preclinical models are required to evaluate the migration of transplanted stem cells to various areas of the brain, monitor graft survival to improve immunosuppression regimens, and assess stem cell safety and their efficacy on disease outcomes. Understanding all these aspects of stem cells as therapeutics can help optimize parameters for translation to humans. To facilitate this overarching goal, we report a protocol for human stem cell transplantation into mouse fimbria fornix, the outflow white matter tract of the hippocampus, which facilitates stem cell dissemination to regions distant to the injection site. Although our protocol involves injecting human neural stem cells expressing insulin-like growth factor 1 into an Alzheimer's disease mouse model, it can be altered for application to diverse stem cells and cellular therapeutics, secreting various trophic factors, at multiple dosing schemes, and in different central nervous system targets and disorder models. Our approach is robust and highly reproducible, with demonstrated success in improving memory performance in an Alzheimer's disease mouse model and achieving long-term survival of transplanted cells (up to 32 weeks) in immunocompetent mice, facilitating stem cell studies in the context of neuroimmune crosstalk. Our primary protocol involves administering an anti-CD4/anti-CD40L monoclonal antibody cocktail to prevent graft rejection, with bioluminescence imaging to track transplanted stem cells <i>in vivo</i>. We also present an alternate immunosuppression protocol that relies on tacrolimus and mycophenolate mofetil, which is less costly, albeit less effective (especially in the longer term), than the CD4-CD40L antibody regimen. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Human stem cell transplantation into mouse hippocampus, antibody-based immunosuppression, and <i>in vivo</i> tracking by bioluminescence imaging</p><p><b>Alternate Protocol</b>: Immunosuppression by tacrolimus and mycophenolate mofetil</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70442","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148784596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systematic Necropsy and Tissue Sampling in the Neotenic Axolotl (Ambystoma mexicanum)","authors":"Andy Cano-Botero,&nbsp;Juliana Zapata-Cardona,&nbsp;Belfran Carbonell-Medina,&nbsp;Berardo de Jesús Rodríguez","doi":"10.1002/cpz1.70448","DOIUrl":"https://doi.org/10.1002/cpz1.70448","url":null,"abstract":"<p>Axolotls (<i>Ambystoma mexicanum</i>) are neotenic salamanders widely used as experimental models in regenerative biology, developmental research, toxicology, and pharmacology. Despite their extensive use in laboratory research and conservation programs, standardized necropsy procedures for this species remain limited and poorly documented. The absence of systematic protocols limits the consistency of postmortem examinations and complicates the interpretation of pathological findings in both experimental and diagnostic contexts. Here, we present a detailed and reproducible necropsy protocol for the neotenic axolotl based on 17 systematic necropsies performed under controlled laboratory conditions. The procedure describes step-by-step methods for external examination, access to the cranial cavity, exposure of the spinal cord, and systematic exploration of the coelomic cavity. Particular emphasis is placed on anatomical orientation, preservation of organ relationships, and strategies to minimize tissue damage during dissection. The protocol also includes guidelines for tissue sampling, fixation, and transport for downstream laboratory analyses such as histopathology and microbiology. Protocol refinement revealed practical considerations not previously described for this species, including variability in hepatic size and the absence of coelomic fat bodies in examined specimens, which simplified visceral exposure and influenced dissection strategy. These findings highlight the need to adapt necropsy techniques to species-specific anatomical characteristics. This protocol provides a practical reference for veterinarians, pathologists, and researchers working with <i>A. mexicanum</i>, facilitating standardized postmortem examinations and improving the reliability and reproducibility of diagnostic and research findings in this important amphibian model. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: External examination</p><p><b>Basic Protocol 2</b>: Organs of the head and spinal cord examination</p><p><b>Basic Protocol 3</b>: Organs of the coelomic cavity examination</p><p><b>Support Protocol</b>: Collection, preservation and transport of specimens for laboratory analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148784857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Protein Editing Method to Modify Proteins in Live Mammalian Cells","authors":"Jenna N. Beyer,&nbsp;Kaitlyn Toy,&nbsp;George M. Burslem","doi":"10.1002/cpz1.70443","DOIUrl":"https://doi.org/10.1002/cpz1.70443","url":null,"abstract":"<p>Protein editing, as mediated by protein trans-splicing, offers the ability to manipulate, label, and modify proteins in vitro and in live mammalian cells. This ability to observe and control proteins with this high resolution is a burgeoning area of interest within the chemical biology field. In this protocol, we outline the general approach for setting up a cellular protein editing experiment, emphasizing the planning process for a successful protein editing experiment. Additionally, we include methods to install and subsequently label a click chemistry handle <i>p</i>-azido-phenylalanine, a non-canonical amino acid (ncAA) within the intein donor, such that this ncAA and any label can be edited into a protein in live mammalian cells. Overall, we anticipate that this protocol should serve as a generalizable roadmap for planning and carrying out a protein editing experiment. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Expression of intein donor containing pAzF</p><p><b>Basic Protocol 2</b>: Purification and labeling of recombinant intein donor</p><p><b>Basic Protocol 3</b>: Preparation of mammalian cells for editing experiments</p><p><b>Basic Protocol 4</b>: Protein editing via electroporation</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70443","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148784846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Techniques for Estimation of Characteristics and Shelf-Life of Dried Pineapple (Ananas comosus) Slices","authors":"Anas Ejaz Yasmeen Shaikh,&nbsp;Payel Dasgupta,&nbsp;Hari Niwas Mishra","doi":"10.1002/cpz1.70437","DOIUrl":"https://doi.org/10.1002/cpz1.70437","url":null,"abstract":"<p>Although a drying technology majorly aims at producing dried pineapple slices for enhancing their shelf-life, it also simultaneously affects other characteristics of pineapple slices, e.g., nutrients, bioactive compounds, quality attributes, microbiological and enzymatic activities, physical properties, and sensory attributes, such as taste, texture, appearance, and flavor. The optimization of drying technologies requires consideration of overall attributes of the slices. Thus, it is important for pineapple drying industries to have knowledge of the techniques that can be used for characterization of dried pineapple slices. In this overview article, detailed procedures have been compiled for characterization of dried pineapple slices. Articles were extracted from Scopus (search phrase: “pineapple” AND “slices” AND “drying” OR “dehydration”) and techniques were compiled for raw material characteristics, sample preparation, and the characteristics of dried pineapple slices namely as nutritional (moisture, proximate, vitamins C and B-complex, pectin, minerals), physicochemical (water activity, total soluble solids, pH, and water activity), functional (phenolic compounds, flavonoids, carotenoids, antioxidant and enzymatic activity), physical (mechanical properties, microstructure, shrinkage and porosity, shape), quality (color, texture, volatiles, rehydration, sensory), and shelf-life (microbiological, sorption, packaging, and storage) characteristics. © 2026 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148784458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrative in silico and in vitro screening of small molecules targeting RNA","authors":"Sabrina Toews,&nbsp;Megan Ken,&nbsp;Harald Schwalbe","doi":"10.1002/cpz1.70431","DOIUrl":"https://doi.org/10.1002/cpz1.70431","url":null,"abstract":"<p>Targeting structured RNA elements with small molecules has emerged as a promising yet technically challenging strategy for antiviral drug discovery. Here, we present a comprehensive and experimentally validated workflow for the integrative <i>in silico</i> and <i>in vitro</i> screening of RNA-binding small molecules. The approach is exemplified using conserved RNA elements from the SARS-CoV-2 genome, including the 5’-terminal stem-loop 1 and the programmed –1 ribosomal frameshift pseudoknot, but is broadly applicable to other structured RNAs. The workflow integrates high-resolution RNA structural ensemble generation with virtual screening (VS) and nuclear magnetic resonance (NMR)-based experimental validation. Conformational ensembles generated by fragment-assembly approaches serve as targets for docking chemically diverse fragments and lead-like libraries. Top-ranked compounds are prioritized through consensus scoring and evaluated using ligand- and RNA-observed NMR experiments to confirm binding, characterize interaction modes, and assess specificity. Such ranking allows for NMR-guided fragment optimization, which enables systematic improvement of solubility, affinity, and selectivity through iterative medicinal chemistry in the pharmaceutical pipeline. Detailed procedures are provided for library preparation, ensemble-based VS, hit validation, data interpretation, and progression toward functional assays, together with practical considerations, quality-control parameters, and troubleshooting guidance to ensure reproducibility. By combining computational and experimental strategies that select for high-specificity ligands within a unified framework, this set of protocols accelerates the discovery and optimization of RNA-targeting small molecules and provides a scalable platform for RNA-focused drug discovery. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Target and library preparation for virtual screening</p><p><b>Basic Protocol 2</b>: Ensemble-based virtual screening of low-molecular-weight compounds against RNA targets</p><p><b>Basic Protocol 3</b>: Comparative hit prioritization and selectivity filtering for RNA-binding small molecules</p><p><b>Basic Protocol 4</b>: Preparation of RNA samples for <i>in vitro</i> validation of small-molecule binding</p><p><b>Basic Protocol 5</b>: NMR-based <i>in vitro</i> screening of RNA–small molecule interactions</p><p><b>Support Protocol</b>: Preparation of ligand stocks and NMR-based quality control</p><p><b>Basic Protocol 6</b>: Characterization and prioritization of validated RNA-binding hits</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 8","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70431","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148784456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}