Bio-protocol最新文献

{"title":"Probing the Luminal Compartment of 3D Organoids via Particle Tracking Microrheology.","authors":"Katrina N Lyon, Barkan Sidar, Cameron Dudiak, Grace Jordan, Diane Bimczok","doi":"10.21769/BioProtoc.5830","DOIUrl":"10.21769/BioProtoc.5830","url":null,"abstract":"<p><p>The mucus layer lining the human stomach is a critical barrier that protects the underlying epithelium from gastric acid and harmful pathogens such as <i>Helicobacter pylori</i>. The efficacy of this barrier relies on the structural integrity of the mucus, which is determined by various biochemical and biophysical features. Human gastric organoids-3D cellular models that resemble the stomach-contain mucus and have been used to investigate gastric disease. The luminal compartment of three-dimensional epithelial organoids represents a physiologically relevant but experimentally inaccessible microenvironment. In gastric organoids, luminal accumulation of mucus creates a confined viscoelastic hydrogel that mimics native gastric mucus. However, the small volume and topological confinement of organoids preclude conventional bulk rheometry. Here, we describe a particle tracking microrheology (PTM) protocol to measure the viscoelastic properties of the mucus within intact organoid lumina following microinjection of fluorescent microspheres. High-speed fluorescence imaging and particle trajectory analysis enable the quantification of viscous and elastic properties of the mucus through calculation of mean squared displacement (MSD), diffusive scaling exponent (alpha), and frequency-dependent storage (G') and loss (G'') moduli. This method enables rheological measurements in nanoliter-scale compartments without disrupting organoid architecture. We further discuss the impact of mucus heterogeneity and microstructure on scale-dependent mechanical behavior. This protocol is broadly applicable to other organoid systems and can be adapted to Transwell or organ-on-chip platforms for in situ luminal measurements. Key features • Enables rheological measurements in small (nanoliter scale) volumes. • Compatible with intact, Matrigel-embedded 3D organoids. • Allows in situ measurement without mucus harvest or purification. • Resolves microscale heterogeneity inaccessible to bulk rheometry and is compatible with functional screening of mucus-modifying drugs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5830"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598651/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042453","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":"A NanoLuc-Based Protease Biosensor for Highly Sensitive Detection of Intracellular Protease Activity: Applications to Apoptosis and Coronavirus Infection.","authors":"Masashi Arakawa, Eiji Morita","doi":"10.21769/BioProtoc.5833","DOIUrl":"https://doi.org/10.21769/BioProtoc.5833","url":null,"abstract":"<p><p>FlipNanoLuc is a highly sensitive protease biosensor based on the β-strand-flipping principle of NanoLuc luciferase, which is derived from <i>Oplophorus gracilirostris</i>. In the inactive configuration, one β-strand of NanoLuc is repositioned, thereby suppressing luciferase activity. Upon cleavage of the embedded protease recognition sequence by a target protease, the flipped β-strand is released, and luciferase activity is reconstituted. Incorporation of the LgBiT fragment (NanoBiT technology) yields strong luminescent output once the reporter is reconstituted, whereas the CL1-PEST1 degradation tag lowers background luminescence by promoting the degradation of the uncleaved, inactive form; together, these two modifications widen the dynamic range. A firefly luciferase normalization cassette connected via a P2A self-cleaving peptide is encoded in the same reporter plasmid, thereby eliminating the need for separate co-transfection. Because the readout directly reports intracellular protease activity in living cells, the system is suitable for detecting protease activation during apoptosis or viral infection and, in principle, for evaluating protease inhibitors and antiviral compounds. This protocol describes the following: (1) generation of HEK293T cells stably expressing FlipNanoLuc by retroviral transduction; (2) validation of reporter activity by protease overexpression; (3) detection of apoptosis using staurosporine; (4) detection of human coronavirus OC43 infection; and (5) detection of SARS-CoV-2 infection in BHK cells stably expressing hACE2. All luminescence assays employ dual-luciferase normalization and are compatible with standard 384-well plate readers. Key features • Highly sensitive detection of intracellular protease activity using a β-strand-flipping NanoLuc biosensor with dual-luciferase normalization encoded in a single plasmid. • Detects protease activity in living cells, including caspase-3 during apoptosis and 3CLpro during authentic OC43 and SARS-CoV-2 infection. • Quantitative luminescence readout in a 384-well plate format with a standard plate reader. • Adaptable to other proteases and to inhibitor or antiviral screening by exchanging the embedded recognition sequence.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5833"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598650/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149043005","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":"An In Vitro Model to Study Drugs That Affect Macrophage Adhesion to Murine Brain Endothelial Cells After Proinflammatory Insults of LPS and Pilocarpine.","authors":"Abriel J Rivera-Rivera, Paola N Gracia-Ayala, Arot L Velázquez Pulliza, Antonio H Martins, Yancy Ferrer-Acosta","doi":"10.21769/BioProtoc.5825","DOIUrl":"10.21769/BioProtoc.5825","url":null,"abstract":"<p><p>Neuroinflammation disrupts blood-brain barrier (BBB) integrity, promoting leukocyte recruitment into the central nervous system and contributing to the progression of neurological disorders. This protocol describes a reproducible macrophage adhesion assay to evaluate interactions between immune cells and brain endothelial cells and to screen compounds with potential anti-inflammatory activity. Murine brain endothelial cells (bEnd.3) were cultured to confluency and exposed to inflammatory stimuli, such as lipopolysaccharide (LPS) or pilocarpine, a cholinergic muscarinic receptor agonist reported to induce inflammatory responses through seizure-associated neuroinflammatory mechanisms, in the presence or absence of candidate therapeutic compounds. In these studies, the natural flavonoid quercetin and the synthetic alkyl-lysophospholipid edelfosine were tested for their effects on macrophage adhesion. After 48 h of treatment, fluorescently labeled murine macrophages (RAW 264.7) were added to the endothelial monolayer, and adherent cells were quantified by fluorescence microscopy. The assay was validated using dexamethasone as an anti-inflammatory control and inflammatory stimulation with LPS or pilocarpine. As expected, dexamethasone reduced macrophage adhesion, whereas both LPS and pilocarpine significantly increased adhesion, demonstrating the assay's sensitivity to changes in endothelial inflammatory status. Overall, this protocol provides a reliable and accessible platform for investigating endothelial-immune cell interactions under neuroinflammatory conditions and for evaluating therapeutic compounds that may preserve BBB function and reduce inflammatory cell recruitment in neurological disease models. Key features • This protocol creates an in vitro model that simulates the luminal interface of the blood-brain barrier (BBB). • The protocol accounts for the 48-h interval required for maximal physiological expression of adhesion molecules at the cell surface. • The protocol quantifies macrophage adhesion to brain endothelial cells following inflammatory stimulation with lipopolysaccharide (LPS) or pilocarpine. • It enables evaluation of therapeutic candidates for their ability to modulate macrophage-brain endothelial cell adhesion under neuroinflammatory conditions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5825"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598658/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042470","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":"ChromID: A Protocol for Mapping Protein Chromatin Interactions in Living Cells.","authors":"Richard Cardoso da Silva, Douwe Ten Bulte, Tuncay Baubec","doi":"10.21769/BioProtoc.5807","DOIUrl":"10.21769/BioProtoc.5807","url":null,"abstract":"<p><p>Chromatin modifications regulate genome function by recruiting proteins that control transcription, genome organization, and DNA repair. Identifying the proteins associated with specific chromatin modifications is therefore essential for understanding how these regulatory processes operate. Traditional approaches, including chromatin immunoprecipitation and affinity purification coupled to mass spectrometry, have uncovered many chromatin-associated proteins. However, they often rely on crosslinking and chromatin fragmentation, which can disrupt native chromatin architecture and limit the detection of transient interactions. Here, we describe a proximity-labeling protocol for identifying the chromatin-dependent protein interactome associated with specific chromatin marks, termed ChromID. ChromID uses engineered chromatin readers (eCRs) fused to a promiscuous biotin ligase, which labels proteins in the immediate vicinity of the targeted chromatin mark. The protocol includes in vivo biotin labeling, nuclear extract preparation, streptavidin-based enrichment, and tryptic digestion for downstream LC-MS/MS analysis. The protocol has been validated across multiple cell types and chromatin contexts and can be extended to other chromatin-associated proteins, providing a versatile approach to profile chromatin-associated proteomes within their native cellular environment. Key features • Maps proteins associated with different chromatin modifications in living cells using engineered chromatin readers fused to TurboID, BASU, or other promiscuous biotin ligases. • Preserves native chromatin organization and captures transient chromatin-associated interactions that are often lost during conventional affinity purification workflows. • Validated across multiple chromatin contexts, including histone modifications, DNA methylation, transcription factors, RNA polymerase II, and DNA damage-associated chromatin states. • Applicable to diverse cell types and organisms and adaptable to other chromatin-associated proteins, including transcription factors and chromatin regulators.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5807"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561692/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957536","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":"Massively Parallel In Vitro Functional Analysis of Evolution-Derived Transcriptional Riboswitch Sequences.","authors":"Laura M Hertz, Julius B Lucks","doi":"10.21769/BioProtoc.5804","DOIUrl":"10.21769/BioProtoc.5804","url":null,"abstract":"<p><p>Riboswitches are structured non-coding RNA elements that regulate gene expression in response to small molecules; they serve as valuable systems in both public health and biophysical research by elucidating principles around RNA-ligand interactions, structure, and cellular function. Traditional approaches to studying riboswitches have relied on low-throughput techniques such as reporter assays or gel electrophoresis analysis of transcriptional products, which are limited in scalability. In this study, we present a high-throughput protocol to characterize the transcriptional activity of nearly 2,000 natural variants of the fluoride riboswitch in in vitro transcription. Starting with bioinformatics, we compiled a comprehensive dataset of riboswitch variants and then employed massive parallel oligonucleotide synthesis to generate an oligo pool of the riboswitch library. This pool was transcribed in vitro, converted into an Illumina-compatible next-generation sequencing (NGS) library, and analyzed to identify transcriptionally active riboswitch candidates. The workflow integrates natural riboswitch bioinformatic acquisition into a quantitative readout in a single streamlined pipeline, enabling large-scale exploration of transcriptional riboswitch function. This protocol offers a scalable method for mapping genotype-to-function relationships across transcriptional riboswitch families, accelerating the identification of functional variants for desired applications. Key features • Bioinformatics to acquire full sequences (aptamer + downstream expression platform) of naturally occurring riboswitches. • <i>E. coli</i> RNA polymerase in vitro transcription to characterize nearly 2,000 fluoride riboswitches and analysis with NGS.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5804"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561695/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957567","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":"Endoscopic Collection and Analysis of Gastric Fluid DNA: A Liquid Biopsy Methodology for Tumor Biomarker Discovery.","authors":"Francine Carla Cadoná, Adriane G Pelosof, Thais F Bartelli, Haejin In, Lianlian Wu, Israel Tojal da Silva, Renata Pasqualini, Wadih Arap, Diana Noronha Nunes, Emmanuel Dias-Neto","doi":"10.21769/BioProtoc.5809","DOIUrl":"10.21769/BioProtoc.5809","url":null,"abstract":"<p><p>Gastric cancer remains a major global health challenge, and reliable prognostic biomarkers are urgently needed to guide treatment decisions. Here, we present a simple and efficient protocol for a novel liquid biopsy approach based on quantifying gastric fluid DNA (gfDNA) collected during routine esophagogastroduodenoscopy (EGD). We have previously shown that gfDNA carries gastric cancer-derived mutations; moreover, its concentration increases with tumor progression and varies according to cancer prognosis. This empirically observed increase in gfDNA may mechanistically stem from enhanced cellular turnover, tissue disorganization, dysbiosis of the local microbiota, and/or fluctuations in immune cell infiltrates. Surprisingly, however, in patients diagnosed with gastric cancer, elevated gfDNA levels were also associated with improved survival. This paradoxical finding may be reconciled by an increased anti-tumor immune cell response in treatment-responsive gastric cancers, as well as by the contribution of non-tumoral DNA from inflammatory processes within the microenvironment of the stomach. Here, we detail a standardized protocol for gastric fluid collection and processing, designed to support downstream gfDNA quantification among other potential molecular applications. Key features • A novel liquid biopsy approach for discovering potential biomarkers in human gastric cancer. • Translation of simple gfDNA concentration analysis for the diagnosis and prognosis of gastric cancer. • gfDNA cutoff metrics that discriminate between groups and enable tumor prognostic classification. • gfDNA as a source for downstream profiling of genetic mutations and microbiome-related studies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5809"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561674/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148946379","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":"Determining the Age of Every Cell Within Each Budding Yeast Microcolony Combining Single-Cell Microencapsulation With Confocal Microscopy.","authors":"Carmen Ruger-Herreros, Irene Delgado-Román, María José García-Marcelo, Sebastián Chávez, Mari-Cruz Muñoz-Centeno","doi":"10.21769/BioProtoc.5813","DOIUrl":"10.21769/BioProtoc.5813","url":null,"abstract":"<p><p>Isogenic populations of <i>Saccharomyces cerevisiae</i> exhibit significant proliferative heterogeneity, with individual cells within a clonal culture displaying divergent growth rates and metabolic states. Investigating the origins of this variation requires a method to reconstruct the individual histories of cells within the population. This protocol describes a method for single-cell microencapsulation in alginate microspheres to create a physically stable, traceable, three-dimensional genealogical environment. By utilizing the alginate matrix to prevent daughter cell migration, the replicative history of a founder cell can be mathematically reconstructed. This is achieved by correlating the total cell count (N) within a developed microcolony with the total number of accumulated bud scars (n) visualized via confocal microscopy. Key features • Enables non-destructive 3D reconstruction of yeast genealogies, preserving spatial architecture and mapping all division events from a single founder cell. • Implements a robust mathematical formula to determine the founder's replicative age, linking past mitotic history to current microcolony growth dynamics. • Combines Flow Focusing^® microencapsulation with precise confocal Z-stack analysis to review lineage-specific phenotypic heterogeneity in isogenic populations. • Ideal for studying transgenerational inheritance and growth-rate diversification, providing a traceable, three-dimensional genealogical ecosystem for single-cell research.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5813"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561675/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148946292","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":"Cryo-EM Pipeline for Actin Filament End Structures.","authors":"Nicholas J Palmer, Roberto Dominguez","doi":"10.21769/BioProtoc.5794","DOIUrl":"10.21769/BioProtoc.5794","url":null,"abstract":"<p><p>Actin filaments undergo dynamic growth and disassembly at their ends, regulated by many actin-binding proteins. However, structural analysis of filament end dynamics has been challenging due to the low abundance of filament ends in cryo-electron microscopy (cryo-EM) micrographs, their intrinsic polymorphisms, and the diversity and flexibility of end-binding proteins. Here, we describe a standardized cryo-EM protocol for determining actin filament end structures. First, short actin filaments are generated either biochemically using capping or severing proteins or mechanically through shearing. Filaments are then vitrified under conditions optimized for each specific end-binding protein. We describe data collection parameters using a 300 kV Titan Krios G3i microscope, including optimized grid preparation and imaging settings. Finally, we present a data processing pipeline for filament end structure determination based on machine learning-based particle picking, masking, and sorting strategies. This protocol has enabled the determination of multiple high-resolution structures of free, capped, elongating, and depolymerizing actin filament ends, and we further discuss considerations for extending this approach to other end-binding proteins. Key features • Generation of short actin filaments for cryo-EM using capping proteins, severing proteins, or mechanical shearing. • Integrated data collection and image processing workflow for filament end structure determination. • Common challenges, solutions, and experimental considerations for diverse actin filament end-binding complexes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5794"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13592462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149019097","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":"Engineering MRI-Based Programmable Genetic Sensors Using the MAPPER Platform.","authors":"Asish N Chacko, Yuxin He, Raymond E Borg, Thomas Tang, Arnab Mukherjee","doi":"10.21769/BioProtoc.5795","DOIUrl":"10.21769/BioProtoc.5795","url":null,"abstract":"<p><p>Genetically encodable reporters that produce signals detectable in deep tissues offer a powerful tool for noninvasive monitoring of molecular events in vivo. Although magnetic resonance imaging (MRI) is a standard technique for noninvasive clinical imaging, its wider application in detecting molecular activities has been constrained by the lack of programmable sensors. This limitation is in stark contrast to the widespread use of fluorescent reporter-derived sensors in cultured cells and in transparent specimens. To overcome this limitation, we recently developed the modular aquaporin-based protease-activatable probe for enhanced reporting (MAPPER) platform. This sensor engineering framework integrates a metal-free MRI reporter derived from human aquaporin-1 (hAqp1) with synthetic protease-based circuits. This integration facilitates the modular and scalable creation of a wide range of sensors by regulating protease activity through precise molecular events, such as protein-protein interactions, pharmacological inhibition, and second messenger signaling. In this paper, we present a detailed protocol for constructing and deploying sensors using the MAPPER paradigm. The protocol encompasses genetic design, lentiviral production, stable cell line generation, biochemical and microscopic validation of sensor function, diffusion-weighted MRI, and MR image analysis to quantify sensor signals in terms of the apparent diffusion coefficient. We describe two distinct MAPPER architectures: DD-MAPPER, which leverages protease-controlled protein degradation, and ER-MAPPER, which utilizes protease-controlled, subcellular trafficking. The MAPPER framework allows adaptation to various molecular targets without the need to redesign the core MRI reporter mechanism, making MAPPER a versatile platform for noninvasive biosensing in living cells and tissues. Key features • MAPPER enables programmable, protease-controlled switching of aquaporin-1-based diffusion weighted-MRI signals in genetically modified mammalian cells. • The protocol covers two complementary biosensor architectures (DD-MAPPER and ER-MAPPER) that exploit different post-translational regulatory mechanisms to modulate MRI signals. • Stable MAPPER cell lines are generated via lentiviral transduction, allowing long-term, selection-free biosensor expression across multiple mammalian cell types. • The sensor is fully modular; proteases and protease-based logic circuits can be substituted without altering the hAqp1 reporter, enabling rapid adaptation to new molecular targets.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5795"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561953/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957559","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":"R-Loop Modification and Quantification by Dot Blot.","authors":"Taehwan Yang, Yi-Ru Li, Blerta Xhemalçe","doi":"10.21769/BioProtoc.5806","DOIUrl":"10.21769/BioProtoc.5806","url":null,"abstract":"<p><p>RNA modifications and their \"writer,\" \"eraser,\" and \"reader\" proteins are emerging as key regulators of gene expression and DNA repair through dynamically regulating RNA:DNA hybrids, or R-loops, during transcription. Therefore, it is paramount to develop rigorous techniques for accurate analysis of R-loop modifications. A convenient method for analyzing RNA modifications within total RNA is by dot blot with specific RNA modification antibodies; however, analysis of the modification of the RNA moiety within R-loops presents specific challenges. Here, we provide a detailed protocol for the production or purification of DNA containing R-loops in vitro and from cells, and the analysis of the RNA moiety modifications by dot blot. The DNA containing R-loops is treated with either mock or RNase H, which specifically degrades the RNA within RNA:DNA hybrids, to control for the specificity of the signal as originating from R-loops. Known quantities of the mock or RNase H-treated DNA are then spotted on three membranes, each blotted with antibodies that recognize double-stranded DNA, RNA:DNA hybrids, or the specific RNA modification antibodies of interest, such as m⁶A or ac⁴C. Thus, this protocol is useful to both biochemists and cell biologists with scientific interests at the intersection of R-loops and epitranscriptomics. Key features • R-loops produced via in vitro transcription of R-loop-forming DNA sequences can serve as substrates for biochemical assays, quantification standards, or antibody specificity controls. • R-loop-containing DNA can be purified from cells after gene editing or various treatments to analyze how global R-loop levels and modifications are regulated. • This protocol was initially applied to the study of the role of NAT10 and ac⁴C modification of the RNA moiety within R-loops in human cells. • Requires 4-7 days to complete, depending on the origin of R-loops (in vitro transcription vs. from cells).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5806"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561690/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957514","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}