Nature Protocols最新文献

{"title":"Preparation of homogeneous ZnSeTeS quantum dots","authors":"Fan Cao, Sheng Wang, Zhixin Chen, Shizheng Zhang, Qianqian Wu, Jiaqi Zhang, Xuyong Yang","doi":"10.1038/s41596-026-01340-2","DOIUrl":"10.1038/s41596-026-01340-2","url":null,"abstract":"Quantum dots (QDs) have emerged as promising candidates for next-generation display owing to their exceptional optoelectronic properties. However, despite substantial advancements in QD synthesis, the blue-emitting QDs, especially heavy-metal-free blue ones, still underperform compared with their red and green counterparts. ZnSeTe QDs offer a viable ecofriendly alternative for blue emissions, but their performance is limited by spectrum broadening (linewidth >20 nm) and structural instability. These issues stem from compositional inhomogeneity, which is primarily induced by Te aggregation during synthesis. Recently, we realized the synthesis of homogeneous quaternary-alloyed ZnSeTeS QDs through a synergistic strategy of reactivity modulation and isoelectronic control. This Protocol enables precise bandgap tuning in the blue spectral region (450–475 nm) by controlling the Te ratio, while ensuring high color purity and stability of QDs. Furthermore, the as-prepared ZnSeTeS QDs exhibit outstanding electroluminescence performance, with a peak external quantum efficiency of 24.7% and half-life of 29,600 h at 100 cd cm−2, and demonstrate strong potential for applications such as solid-state lighting and bioimaging owing to their high stability and low toxicity. Here we detail a synthesis Protocol for ZnSe0.94Te0.03S0.03/ZnSe/ZnS core/shell/shell QDs via a hot-injection method using zinc carboxylate and anionic phosphine precursor, systematically outlining the design and preparation of precursors and QDs, post treatments (including purifications) and characterization methods, including time-resolved photoluminescence spectroscopy. The entire process typically requires 11–12 h for QD synthesis and 6 h for characterizations, demanding only accessible chemistry knowledge and routine colloidal synthesis techniques. Quantum dots are used in many display and imaging applications. Preparing blue quantum dots that are heavy-metal-free has been challenging. This Protocol describes the tunable synthesis of ZnSeTeS quantum dots with narrow emission bands in the violet–blue range.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 8","pages":"3719-3735"},"PeriodicalIF":18.4,"publicationDate":"2026-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147654744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The trRosettaRNA server for RNA structure prediction.","authors":"Wenkai Wang, Xiaocheng Liu, Zhenling Peng, Jianyi Yang","doi":"10.1038/s41596-026-01356-8","DOIUrl":"https://doi.org/10.1038/s41596-026-01356-8","url":null,"abstract":"<p><p>Similar to proteins, many RNAs fold into three-dimensional (3D) structures to perform biological functions. Here we present the trRosettaRNA server, a web-based platform for automated RNA 3D structure prediction using deep learning. The primary input is the nucleotide sequence of a target RNA, with the option to upload custom multiple sequence alignments and secondary structures. The server uses an end-to-end neural network for automated 3D structure prediction, followed by an energy optimization step to resolve structural violations. As an automated server, trRosettaRNA is distinguished by its state-of-the-art modeling accuracy, flexible input options and comprehensive visualization of prediction results. trRosettaRNA has been successfully applied in various contexts, including predicting structures for Rfam families lacking known 3D structures, where representative cases of high-confidence structure predictions were found to align well with subsequent experimental observations. Utilizing up to 5 central processing unit (CPU) cores in parallel on our computer cluster, the server takes a median time of about 1 h to predict structures for RNA sequences with about 200 nucleotides. The standalone package for trRosettaRNA offers distinct advantages such as enhanced data privacy for sensitive sequences, the ability to bypass server queues and integration into high-throughput automated pipelines. Importantly, the open-source nature of the package empowers researchers to directly modify the codebase for specialized research needs or to develop derivative tools by fine-tuning the underlying neural network. The web server and standalone package of trRosettaRNA are available at https://yanglab.qd.sdu.edu.cn/trRosettaRNA/ and https://github.com/YangLab-SDU/trRosettaRNA2 , respectively.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147639378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Automated cGMP optical labeling of FDA-approved antibodies for human use","authors":"Kamal Jouad,&nbsp;Marisa Hom,&nbsp;Ashtyn McAdoo,&nbsp;J. Oliver McIntyre,&nbsp;Eben L. Rosenthal,&nbsp;Adam J. Rosenberg","doi":"10.1038/s41596-026-01344-y","DOIUrl":"10.1038/s41596-026-01344-y","url":null,"abstract":"Monoclonal antibodies are commonly used as targeted therapies for autoimmune, infectious and oncologic diseases. Here, we describe a simple protocol to optically label monoclonal antibodies for use as molecular imaging agents for clinical investigation. In preclinical settings, optical imaging has complemented the strengths of nuclear imaging while offering higher resolution and a safer method for antibody-ligand engagement. However, translation of optically labeled monoclonal antibodies to the clinic has been slow because antibodies require a manual process in current good manufacturing practices (cGMP)-compliant facilities; the cost barriers to establish the investigational new drug application by using the traditional contract research organization pathway (&gt;US$1 million) exceed the resources of academic institutions and early-phase pharmaceutical companies. To address these challenges, we repurposed an existing radiolabeling cGMP method for optical labeling that uses an automated, self-contained synthesis module. This method depends on commercially available, single-use, cassette-based production, which simplifies the workflow and does not require a clean room facility. This automated production method reduces both the cost and time required to produce a clinical dose of near-IR fluorescently labeled monoclonal antibody–IRDye800CW, decreasing the development costs for pilot and initial batches by almost 90%, as well as the production time by 40% to 4 h plus quality control (~10 h total). Our cGMP manufacturing method can optically label any compatible monoclonal antibodies at any dedicated radiochemistry facility. We provide the detailed protocol for production of panitumumab–IRDye800CW and nivolumab–IRDye800CW under cGMP regulations, achieving excellent yield, optimal degree of labeling and high purity. This Protocol describes an automated approach for optical labeling of therapeutic monoclonal antibodies by using disposable commercial components, yielding a cGMP-grade product at reduced cost and without the need for a dedicated clean room facility.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 8","pages":"3795-3813"},"PeriodicalIF":18.4,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147632765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sensing and perturbing mammalian cell states with reprogrammable ADAR sensors (RADARS)","authors":"Jeremy Koob,&nbsp;Kaiyi Jiang,&nbsp;Samantha R. Sgrizzi,&nbsp;Fei Chen,&nbsp;Omar O. Abudayyeh,&nbsp;Jonathan S. Gootenberg","doi":"10.1038/s41596-025-01305-x","DOIUrl":"10.1038/s41596-025-01305-x","url":null,"abstract":"Reprogrammable Adenosine Deaminase Acting on RNA (ADAR) Sensors (RADARS) control RNA translation in mammalian cells, allowing for noninvasive sensing or perturbation of specific cell types based on transcriptional signatures. Upon base-pairing between a target RNA and a sensor RNA, RADARS leverages ADAR to edit a premature stop codon upstream of a gene of interest, thereby releasing translation of the desired cargo. These design principles enable sequence programmability, allowing RADARS to adapt more easily to new contexts than existing tools for targeting cell types. We describe a detailed protocol for performing experiments with RADARS, including designing, cloning and validating RADARS constructs targeting a transcript of interest. RADARS guide sequences can be designed with an intuitive web interface and cloned into existing constructs for downstream applications including imaging, sorting and sequencing. We outline recommendations for cargo choice, sensor design and ADAR system selection, enabling users to choose the best workflow depending on the desired application. Beginning with sensor design, the selection of top-performing RADARS guides can be completed in ~2 weeks, followed by a desired use case. Convenient engineering and application of RADARS for various applications enable the design and execution of various cell-targeting experiments. This protocol uses Reprogrammable Adenosine Deaminase Acting on RNA (ADAR) Sensors (RADARS) to robustly sense RNA transcripts inside eukaryotic cells, enabling detection of changes in gene expression or targeting and perturbation of specific mammalian cell types and states.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 7","pages":"3260-3285"},"PeriodicalIF":18.4,"publicationDate":"2026-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147609000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"APEX-seq maps transcriptome-wide subcellular RNA localization in living cells","authors":"Surbhi Sharma,&nbsp;Madeline E. Rasband,&nbsp;Xuemei Wang,&nbsp;Karen Tolentino,&nbsp;Sharon S. Huang,&nbsp;Furqan M. Fazal","doi":"10.1038/s41596-026-01342-0","DOIUrl":"10.1038/s41596-026-01342-0","url":null,"abstract":"Although we know a great deal about the subcellular locations of most proteins, our knowledge of where most RNAs localize within cells remains limited. As RNA subcellular localization determines the fate, function and regulation of both coding and noncoding RNAs, there has been substantial interest in developing new scalable approaches to study the location of RNAs in their native context. Furthermore, many locations, such as membrane-bound and membrane-less organelles, have traditionally been challenging to study owing to lack of suitable tools to interrogate their constituents. Here we describe a detailed protocol for APEX sequencing (APEX-seq) that yields transcriptome-wide information of the subcellular address of RNAs that can, in principle, be applied to any subcellular location, membrane or condensate. APEX-seq utilizes a genetically encoded engineered ascorbate peroxidase (APEX2) tagged to a specific protein that localizes it to a region of interest. In the presence of biotin-phenol and hydrogen peroxide, APEX2 catalyzes the biotinylation of RNAs in its vicinity, which can be purified using streptavidin beads and sequenced to reveal the RNA repertoire at that subcellular location. APEX-seq experiments can be carried out by laboratory personnel trained in molecular biology. The analysis of APEX-seq data requires familiarity with standard RNA sequencing workflows. With APEX2-expressing cell lines in hand, the entire procedure from labeling reaction to analysis can be completed in 1 week. We expect this proximity labeling approach to facilitate the unbiased discovery of RNAs localizing to different organelles and to generate hypotheses for the mechanisms and pathways involved in regulating these processes. This Protocol describes the transcriptome-wide labeling of RNAs in a particular subcellular compartment using proximity biotinylation by localized APEX2 enzyme and subsequent enrichment and sequencing of these transcripts.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 8","pages":"3736-3764"},"PeriodicalIF":18.4,"publicationDate":"2026-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147608939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Electrocatalytic reactions involving aqueous nitrate and nitrite.","authors":"Shunhan Jia, Ruhan Wang, Hanle Liu, Limin Wu, Libing Zhang, Qian Li, Xiaoyu Zhang, Junfeng Xiang, Zhijuan Zhao, Xiaofu Sun, Buxing Han","doi":"10.1038/s41596-026-01350-0","DOIUrl":"https://doi.org/10.1038/s41596-026-01350-0","url":null,"abstract":"<p><p>Electrocatalytic upgrading of aqueous nitrate ( <math> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>3</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </math> ) and nitrite ( <math> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </math> ) represents a sustainable and increasingly important approach for producing valuable nitrogenous chemicals from abundant and hazardous feedstocks. Recent advances have demonstrated the ability to selectively convert <math> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>3</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </math> / <math> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </math> into products such as inorganic nitrogenous species including ammonia (NH₃), hydroxylamine (NH₂OH), hydrazine (N₂H₄) and organonitrogen compounds (e.g., urea, oximes and amines). However, the absence of standardized protocols has hindered reproducibility and cross-laboratory comparisons. Herein, we present a comprehensive protocol for aqueous <math> <mrow> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>3</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> <mo>/</mo> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </mrow> </math> -involved electrocatalytic reactions. Our protocol includes electrode preparation, electrolyzer assembly, electrolysis, product quantification and purification, in situ characterization and technoeconomic analysis. The protocol includes essential safety guidelines for toxic intermediate handling. Moreover, it remains adaptable to different levels of experimental capability. This protocol is suitable for initial screenings and mechanistic investigations spanning small-scale reactors (<30 ml) to liter-level systems and takes ~2 weeks to complete. This work is designed for researchers in green and sustainable chemistry, electrocatalysis, nanotechnology and environmental science and aims to establish reproducible workflows that accelerate the development of electrochemical <math> <mrow> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>3</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> <mo>/</mo> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </mrow> </math> upgrading strategies. More importantly, we hope that the protocol can motivate researchers to design catalytic strategies to upgrade renewable chemical sources beyond <math> <mrow> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>3</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> <mo>/</mo> <msubsup><mrow><mi>NO</mi></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </mrow> </math> .</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Patient-derived ependymoma and medulloblastoma tumoroids: generation, biobanking and drug screening","authors":"Chiara Lago,&nbsp;Gloria Leva,&nbsp;Marcel Kool,&nbsp;Evelina Miele,&nbsp;Luca Tiberi","doi":"10.1038/s41596-026-01347-9","DOIUrl":"10.1038/s41596-026-01347-9","url":null,"abstract":"Ependymoma and medulloblastoma are among the most common malignant pediatric brain tumors and contribute significantly to morbidity and mortality in affected children. Robust models for investigating these tumors’ biology and heterogeneity, and exploring alternative therapeutic options, are currently limited. Here we present a detailed protocol for the generation and maintenance of pediatric patient-derived tumoroids (pPDTs) and pediatric patient-derived xenograft tumoroids (pPDXTs) directly from primary ependymoma and medulloblastoma tumor specimens. The protocol extension expands on our previous method for human induced pluripotent stem cell-derived medulloblastoma and high-grade glioma cancer organoids, with which it shares key reagents and methodological steps. This optimized workflow ensures efficient tumoroid establishment, amplification, biobanking, cryopreservation and recovery. In addition, we describe a scalable, low-throughput drug screening approach using calcein-based live-cell staining and automated image analysis, enabling rapid assessment of therapeutic responses. This protocol provides a robust and reproducible platform for modeling pediatric brain tumors in vitro and will enable broader adoption of patient-derived tumoroid systems for mechanistic studies and preclinical drug screening in pediatric neuro-oncology research. The protocol takes 28–35 days for the generation of tumoroids and from 1 to 4 weeks for amplification, biobanking and downstream applications. The protocol requires at least 3–6 months to become proficient in handling patient-derived samples and generating tumoroids. This is a protocol for the generation of pediatric patient-derived ependymoma and medulloblastoma tumoroids from surgically resected patient tumor tissues or xenografts, including their use in drug screening and in downstream analyses.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"3834-3862"},"PeriodicalIF":18.4,"publicationDate":"2026-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tracking-seq: a universal off-target detection approach for CRISPR–Cas genome editing","authors":"Runda Xu,&nbsp;Tingting Cong,&nbsp;Junsong Yuan,&nbsp;Xuancheng Chen,&nbsp;Yinqing Li,&nbsp;Xun Lan,&nbsp;Ming Zhu","doi":"10.1038/s41596-025-01331-9","DOIUrl":"10.1038/s41596-025-01331-9","url":null,"abstract":"Tracking-seq is a highly sensitive method for genome-wide detection of off-target effects in cells edited with diverse genome editing modalities, including Cas9, cytosine base editors, adenine base editors and prime editors. Since most genome editors induce DNA repair pathways and generate single-stranded DNA (ssDNA) intermediates, Tracking-seq leverages this process by tracking replication protein A—a key protein that binds and protects ssDNA—to identify on-target and off-target events. Here we provide a detailed protocol for Tracking-seq, covering genome editing of cells, extraction of replication protein A-bound ssDNA, sequencing library construction and data analysis using our custom computational tool Offtracker. Tracking-seq is applicable to various genome editing scenarios with low cell input, delivering high-performance results. The entire workflow, from genome editing to data analysis, can be completed within 1–2 weeks, making it a rapid solution for assessing genome-wide off-target activity. This step-by-step protocol describes a versatile approach for assessing genome-wide off-target activity of diverse genome editors by tracking replication protein A—a key protein that binds single-stranded DNA intermediates.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4169-4191"},"PeriodicalIF":18.4,"publicationDate":"2026-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147513594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preparation of an activatable benzothiadiazole-based nanoprobe for multispectral optoacoustic and NIR-II fluorescence dual-mode imaging of liver injury","authors":"Yinglong Wu,&nbsp;Chaobang Zhang,&nbsp;Junjie Chen,&nbsp;Fang Zeng,&nbsp;Shuizhu Wu,&nbsp;Yanli Zhao","doi":"10.1038/s41596-026-01338-w","DOIUrl":"10.1038/s41596-026-01338-w","url":null,"abstract":"Light-excited dual-mode imaging that integrates multispectral optoacoustic tomography (MSOT) and near-infrared region II fluorescence (NIR-II FL) imaging allows complementary deep-tissue visualization with high anatomical resolution and molecular sensitivity, thereby enhancing the accuracy of biomedical diagnostics. Activatable probes further advance this approach by producing signals upon recognition of disease-related biomarkers, which reduces background interference and improves imaging specificity. BTPE-NO2@F127, a benzothiadiazole-based nanoprobe, exhibits selective activation of both optoacoustic and NIR-II FL signals in response to hydrogen peroxide (H2O2), a crucial early stage biomarker of liver injury, thus permitting cross-validated detection of hepatic damage in vivo with excellent signal-to-background ratio contrast. Here we present detailed procedures for preparation of the BTPE-NO2@F127 nanoprobe and its applications in MSOT/NIR-II FL dual-mode imaging of trazodone- or ischemia–reperfusion-induced liver injury in mice. Compared to conventional liver injury diagnostic methods, such as invasive tissue biopsy, ex vivo blood analysis and previously reported fluorescent/optoacoustic probes, BTPE-NO2@F127 offers real-time, in situ monitoring with high sensitivity and signal-to-background ratio, as well as mutually corroborating signals for increased reliability. The fabrication of BTPE-NO2@F127, including the chemical synthesis and characterization, requires ~17 d, while the in vitro validation of its H2O2 responsiveness takes ~5 d. Notably, the complete workflow of data acquisition and analysis for MSOT/NIR-II FL dual-mode imaging of liver injury in mice using BTPE-NO2@F127 can be accomplished within 10 h. The protocol is easy to follow and suitable for clinicians and researchers with a basic understanding of chemistry and bioimaging techniques. This is a protocol for the preparation and characterization of the activatable BTPE-NO2@F127 nanoprobe and its applications in multispectral optoacoustic and near-infrared region II fluorescence dual-mode imaging of liver injury models in mice.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4347-4376"},"PeriodicalIF":18.4,"publicationDate":"2026-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147504397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Live-cell imaging of RNA dynamics using bright and stable fluorescent RNAs","authors":"Fangting Zuo,&nbsp;Ni Su,&nbsp;Xin Xie,&nbsp;Li Jiang,&nbsp;Mengyue Fang,&nbsp;Yuzheng Zhao,&nbsp;Linyong Zhu,&nbsp;Xianjun Chen,&nbsp;Yi Yang","doi":"10.1038/s41596-026-01343-z","DOIUrl":"10.1038/s41596-026-01343-z","url":null,"abstract":"RNAs exhibit complex dynamics in cells, including expression, splicing, localization, translation and degradation, and these processes are highly coordinated and tightly regulated both spatially and temporally. To better understand the biological function of diverse RNAs, approaches that allow monitoring of RNA with high spatiotemporal resolution are essential. Fluorescent RNAs (FRs), fluorescent protein–like entities consisting of RNA aptamers and their cognate fluorogenic dyes, have emerged as a promising approach for imaging RNA dynamics in live cells. We recently reported the development of several high-performance FRs, named Pepper, Clivia and Okra, that show advantageous properties, including high cellular brightness and photostability, low ion dependence and/or large Stokes shifts, and have been used to image diverse RNA species in live cells. In this protocol, we provide easy, efficient and generalizable strategies for using FRs to visualize different RNA species in bacteria and mammalian cells by expressing the RNA of interest tagged with one or more copies of the aptamer. We also provide a detailed procedure for multiplexed RNA imaging using orthogonal FRs and the steps to perform super-resolution live imaging of RNAs. The protocol typically takes 5–7 d, including cloning, transfection of mammalian cells or transformation of bacteria, live imaging and results analysis. This protocol is applicable to the real-time monitoring of the localization and dynamics of RNAs of interest in live cells. This protocol provides guidelines for using fluorescent RNAs, entities consisting of an RNA aptamer bound to its cognate fluorogenic dye, for live imaging of the localization and dynamics of different RNA species in bacteria and mammalian cells.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 8","pages":"3765-3794"},"PeriodicalIF":18.4,"publicationDate":"2026-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147491415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}