Nature Protocols最新文献

{"title":"Detection of intramolecular protein dynamics on nanosecond-to-microsecond timescales by nanoparticle-assisted NMR spin relaxation (NASR).","authors":"Xinyao Xiang, Alexandar L Hansen, Lei Bruschweiler-Li, Rafael Brüschweiler, Mouzhe Xie","doi":"10.1038/s41596-025-01177-1","DOIUrl":"https://doi.org/10.1038/s41596-025-01177-1","url":null,"abstract":"<p><p>Proteins under physiological conditions have an intrinsically dynamic nature; they sample a multitude of different conformational substates that allow them to perform their biological functions. Protein motions can take place on a wide range of timescales. Although there are many different NMR experiments with sensitivity to different time windows, it has proven difficult to measure intramolecular motions that happen in the nanosecond-to-microsecond regime. Nanoparticle-assisted NMR spin relaxation (NASR) has recently been introduced to overcome this long-standing challenge. When colloidal nanoparticles are added to proteins in solution, the effective global tumbling of the protein molecules slows down, whereas the internal motions remain essentially unperturbed. NASR extends the protein dynamics observation window from picoseconds all the way into the microsecond range. In this protocol, the NASR effect is realized by using commercially available silica nanoparticles, and NMR measurements are acquired by using a standard high-field solution NMR spectrometer. NASR data analysis is shown to be straightforward. We demonstrate NASR by detecting sub-microsecond dynamics in the Switch I and II regions of oncogenic human KRAS and in the Loop I region of bacterial colicin-immunity protein Im7, among other protein constructs. When an isotope-labeled protein sample is available, this protocol can be executed in 2-5 d, including sample preparation, NMR experiments and data processing and analysis, to uncover potentially functionally important intramolecular dynamics at atomic resolution on timescales that are several orders of magnitude slower than what conventional spin relaxation experiments can observe.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591824","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":"Three-dimensional robotic structures fabricated and powered entirely with proteins.","authors":"Haiyang Jia, Huan Sun, Johannes Flommersfeld, Wentao Shi, Frank Siedler, Petra Schwille","doi":"10.1038/s41596-025-01186-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01186-0","url":null,"abstract":"<p><p>Assembling and upscaling biomolecular activity to perform work in man-made devices is a challenge in synthetic biology. Here we report the step-by-step process to construct fully protein-based micro-three-dimensional (3D) printed robotic structures, which are coated with and actuated by a minimal actomyosin cortex. This approach can be used to program self-powered soft robots assembled from multiple biomolecular modules, devising biophysical assays to quantify active forces produced in 3D and engineering smart 3D microchips for synthetic cell assembly. The procedure covers the establishment of 3D printing microstructures from protein materials, the assembly of actomyosin-based active coatings and the robotic structure design and characterization. The detailed step-by-step instructions will guide scientists in replicating the preparation procedures, facilitating the adoption of biomolecular microrobots and the development of 3D protein-based robotic technology and their applications. The procedure is suited for users with expertise in biomaterials and requires 15 d to complete.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144554002","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":"Expansion and fluctuations-enhanced microscopy for nanoscale molecular profiling of cells and tissues.","authors":"Dominik Kylies, Hannah S Heil, Arturo G Vesga, Mario Del Rosario, Maria Schwerk, Malte Kuehl, Milagros N Wong, Victor G Puelles, Ricardo Henriques","doi":"10.1038/s41596-025-01178-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01178-0","url":null,"abstract":"<p><p>Advances in super-resolution microscopy enable the molecular profiling of cells and tissues at the nanoscale level, surpassing the diffraction limit of conventional light microscopy. However, super-resolution techniques typically require access to expensive specialized equipment and extensive training, limiting their broad applicability. Here we provide a detailed protocol for combining expansion microscopy with enhanced super-resolution radial fluctuations analysis to achieve nanoscale resolution using conventional microscopes. Expansion microscopy physically enlarges the sample, while enhanced super-resolution radial fluctuations computationally enhances the image resolution by analyzing fluorescence fluctuations over time. By combining both, we achieve images with a resolution of 25 nm in combination with diffraction-limited microscopes. Our step-by-step instructions include the expansion of cells and tissue samples, the optimization of multispectral microscopy parameters and the implementation of quality control metrics to minimize artifacts. We further cover the use of quantitative tools such as NanoJ-SQUIRREL, which enable the assessment of resolution improvements and image fidelity. We discuss key considerations for each stage, including sample preparation, image acquisition, computational processing and downstream analysis. Potential pitfalls and troubleshooting strategies are also addressed. This protocol can be used for imaging a variety of sample types with multiple fluorescent labels. With nanoscale spatial resolution and molecular specificity, expansion-enhanced super-resolution radial fluctuations microscopy provides a flexible, accessible approach for investigating cellular ultrastructure, protein localization and interaction networks, suitable for applications in cell biology, histopathology and biomedical research. The procedure requires 3-4 d to complete, involving ~7-9 h of total bench, imaging and processing time and only requires basic expertise in tissue handling, molecular and cell biology, and microscopy.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144554001","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":"A graphic and command line protocol for quick and accurate comparisons of protein and nucleic acid structures with US-align.","authors":"Chengxin Zhang, Lydia Freddolino, Yang Zhang","doi":"10.1038/s41596-025-01189-x","DOIUrl":"https://doi.org/10.1038/s41596-025-01189-x","url":null,"abstract":"<p><p>With the success of structural biology and the advancements in deep-learning-based structure predictions, rapid and accurate structural comparisons among macromolecular structures have become increasingly important in structural bioinformatics. US-align is a highly efficient, versatile, open-source program for sequential and nonsequential structure comparisons of proteins, RNAs and DNAs in pairwise and multiple alignment forms and applicable to both monomeric and multimeric complex structures. The core algorithm of US-align is built on a highly optimized, iterative superimposition and dynamic programming alignment process, guided with a unified and sequence length-independent scoring function, TM-score. The unique design of US-align not only ensures its high accuracy and speed compared with other state-of-the-art methods designed for specific alignment tasks but also makes it the only protocol that can be applied to multiple alignment tasks and allow a structural comparison across different molecular types, the latter of which is critical for template-based heteromolecular structure prediction and function annotations. Here we describe how to install and effectively utilize US-align as a command line tool, as an online web server, and as a plugin to commonly used molecular graphic systems such as PyMOL. US-align installation takes a few minutes to setup, while the actual alignment implementation can be completed typically within 1 s.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144554000","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":"Author Correction: Observing isolated synaptic vesicle association and fusion ex vivo.","authors":"Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J Peters, Nisha Gopal, Richard A Pfuetzner, Austin L Wang, Axel T Brunger","doi":"10.1038/s41596-025-01232-x","DOIUrl":"10.1038/s41596-025-01232-x","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144528996","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":"Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes.","authors":"H M Arif Ullah, Qiju Huang, Simone Chiola, Yueqi Wang, Alex Shcheglovitov","doi":"10.1038/s41596-025-01197-x","DOIUrl":"10.1038/s41596-025-01197-x","url":null,"abstract":"<p><p>We have developed a method for generating human telencephalic organoids from stem cell-derived isolated single neural rosettes. The use of single neural rosettes for generating organoids offers several important advantages. First, it mimics the development of neural tissue from a singular neural tube in vivo. Second, single neural rosette-derived organoids exhibit a relatively consistent and reproducible composition of telencephalic neural cells. Finally, single neural rosette-derived organoids demonstrate predictable organization of the identified neural cells around a single neural rosette-derived lumen and contain a large proportion of functionally mature neurons that generate action potentials and receive both excitatory and inhibitory synaptic inputs. These unique features of our protocol enable the study of the specification and organization of different neural cells in the developing human telencephalon, as well as modeling of neurodevelopmental disorders associated with disrupted neural networks. Here, we describe our protocols for generating CRISPR-Cas9-engineered human stem cells with a hemizygous SHANK3 deletion, stem cell-derived single neural rosettes and telencephalic brain organoids. We also offer insights on how to conduct single-cell RNA sequencing, immunohistochemistry and slice patch-clamp electrophysiology on these organoids. Completion of the protocols takes 5-6 months and requires experience working with cultured cells. We expect this protocol will prove useful for studies of human brain development and disease, as well as for advancing the development of new organoid-based biocomputers.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144512218","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":"Sonoafterglow nanoprobes for deep-tissue imaging of peroxynitrite.","authors":"Cheng Xu, Jingsheng Huang, Kanyi Pu","doi":"10.1038/s41596-025-01202-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01202-3","url":null,"abstract":"<p><p>Optical imaging of tumor biomarkers provides key diagnostic information about tumor status. Light-induced afterglow (photoafterglow) imaging provides a higher signal to background ratio than typical fluorescence imaging; however, both modalities face challenges in detecting biomarkers in deep tissues owing to the limited penetration depth of light. Here we provide instructions for synthesizing ultrasound-induced afterglow (sonoafterglow) nanoprobes (SNAP) for the deep-tissue imaging of peroxynitrite (ONOO^-), a biomarker specific for M1 macrophages and a proinflammatory tumor microenvironment. SNAPs are coassembled from initiators, afterglow substrates and amphiphilic polymers via the film rehydration method, a generic and facile approach that enables their rapid nanoconstruction (in 10 min), with high reproducibility, while also providing control over the nanoprobe concentration, which overcomes limitations of traditional nanoconstruction methods including solvent injection and emulsion-solvent evaporation. Following ultrasound stimulation, SNAPs emit sonoafterglow with bright near-infrared emission (peaking at 780 nm), with a long half-life (~2 min), and can be detected through biological tissues twice deeper than photoafterglow. We further develop SNAP into SNAP-M, which can be switched on only in the presence of ONOO^-, allowing the real-time in vivo imaging of a proinflammatory tumor microenvironment at an unprecedented tissue depth for optical imaging. This Protocol can be implemented by users with expertise in material science in 1 week for nanoprobe construction and characterization, 1-2 week for cell assays and 3-4 weeks for animal experiments.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144506862","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":"Consensus guidelines for the use of concurrent TMS-fMRI in cognitive and clinical neuroscience.","authors":"Alexandra Woolgar, Eva Feredoes, Moataz Assem, Yasmine Bassil, Til O Bergmann, Lysianne Beynel, Michael Burke, Robin F H Cash, Roch M Comeau, Marta M Correia, Erhan Genc, Gesa Hartwigsen, Jade B Jackson, Matthias Kienle, Patrik Kunz, Olga Leticevscaia, Bruce Luber, Maximilian Lueckel, Claus Mathiesen, Elizabeth Michael, Ole Numssen, Desmond J Oathes, Allyson C Rosen, Teresa Schuhmann, Anna-Lisa Schuler, Catriona L Scrivener, Axel Thielscher, Martin Tik, Yordan Todorov, Maria Vasileiadi, Christian Windischberger, Molly S Hermiller, Alexander T Sack","doi":"10.1038/s41596-025-01182-4","DOIUrl":"https://doi.org/10.1038/s41596-025-01182-4","url":null,"abstract":"<p><p>Concurrent transcranial magnetic stimulation (TMS) and functional magnetic resonance imaging (TMS-fMRI) provides a step-change in the toolkit of neuroscience research. TMS enables the noninvasive perturbation of ongoing human brain activity, and when coupled to fMRI for the simultaneous read-out of its effects across the brain, concurrent TMS-fMRI enables studies aimed at determining the causal inference of human brain-behavior relationships, with implications for both fundamental research and clinical application. Many of the technical barriers to TMS-fMRI implementation, such as hardware design and setups, have now been overcome, and the research community in the field is rapidly growing. Here, we present the guidelines set by an international consensus, from researchers at all levels and across the fields of cognitive and applied human neuroscience, for the experimental design and practical considerations of concurrent TMS-fMRI via 12 detailed use cases. These guidelines may facilitate the uptake of this approach and simplify the experimental design and planning stages.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485156","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":"Author Correction: Generation of proximal tubule-enhanced kidney organoids from human pluripotent stem cells.","authors":"Jessica M Vanslambrouck, Ker Sin Tan, Sophia Mah, Melissa H Little","doi":"10.1038/s41596-025-01233-w","DOIUrl":"10.1038/s41596-025-01233-w","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485155","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 and biomedical applications of single-metal atom catalysts.","authors":"Yang Liu, Rui Niu, Yinghui Wang, Hongjie Zhang, Yanli Zhao","doi":"10.1038/s41596-025-01199-9","DOIUrl":"10.1038/s41596-025-01199-9","url":null,"abstract":"<p><p>Nanocatalysts, including nanozymes, photocatalysts and sonocatalysts, have been investigated to trigger catalytic reactions in vivo to regulate biological microenvironments and stimulate therapeutic effects. Compared with lower metal atom utilization rate and catalytic activity of conventional nanocatalysts, single-metal atom catalysts (SACs) usually possess higher catalytic activity and selectivity owing to their well-defined structures and maximized atom utilization. Their properties are, however, strongly dependent on their composition and the preparation procedure. Here we describe the design, preparation and functionalization of SACs with single-metal atoms positioned within nitrogen-doped carbon supports. The SACs are prepared by pyrolysis of zeolitic imidazolate framework-8 (ZIF-8) or polydopamine-derived materials. Their properties depend on, for example, the metal chosen and atoms available for coordination; four example procedures are described: Cu-N₄ from Cu-ZIF-8, Ir-N₅ from Ir@ZIF-8 plus melamine, Co-PN₃ from triphenylphosphine@Co-ZIF-8 and Cu-SN₃ from ZnS@Cu-polydopamine. These SACs need to be functionalized to, for example, reduce aggregation and in vivo corona formation before they can be used in biological applications. In this Protocol, functionalization with the proteins (that is, cholesterol oxidase and pyruvate oxidase) is used as an example. The Protocol provides advice regarding physicochemical and functional characterization, as well as for performing experiments in tumor-bearing mice. The functional experiments were designed with the aim of identifying nanocatalysts with peroxidase-like activity that generate reactive oxygen species within areas of the tumor microenvironment that have increased levels of hydrogen peroxide. SAC synthesis takes 3-4 days, functional modification requires one extra day and the most basic and essential in vitro and in vivo assays require 2-3 months.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144336754","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}