Nature Protocols最新文献

{"title":"Exploring proteins and protein–ligand complexes through residue interaction networks","authors":"Sol C. Begue, Emanuela Leonardi, Giovanni Minervini, Silvio C. E. Tosatto","doi":"10.1038/s41596-026-01334-0","DOIUrl":"10.1038/s41596-026-01334-0","url":null,"abstract":"Protein structures provide a wealth of information regarding biological functions and underlying mechanisms. The growing availability of high-quality structure predictions and extended molecular simulations has further expanded the potential to leverage these data in a myriad of different ways. Yet, an abundance of data can obscure important information, making it difficult to focus on biologically relevant features. Residue interaction networks (RINs) address this challenge by condensing structural data into subsets of well-defined noncovalent molecular interactions. In this Protocol, we explore how the RIN generator (RING) software can be used to gain biological insights by constructing detailed RINs for proteins and protein–ligand complexes. We provide a step-by-step guide to performing both single- and multi-state protein analyses using the RING web server and a stand-alone software package. In addition, we include a dedicated procedure for sequential multi-file analysis, which can be performed exclusively through the command-line interface. All potential inputs and outputs are explained in detail, along with strategies for downstream data processing. Designed for researchers in biology and related fields with minimal or no programming experience, the entire workflow can be completed in <45 min. Residue interaction networks (RINs) describe noncovalent molecular interactions within and between proteins. This Protocol uses RING software to generate these networks from protein structures toward understanding protein structure and function.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4250-4277"},"PeriodicalIF":18.4,"publicationDate":"2026-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147486329","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":"Establishing and analyzing the Simplified Human Intestinal Microbiota (SIHUMI) as a versatile in vitro gut microbiome model with qPCR-based strain-level tracking","authors":"Natalia S. Ríos Colombo, Mariana Perez-Ibarreche, Pranav Lanka, R. Paul Ross, Colin Hill","doi":"10.1038/s41596-026-01336-y","DOIUrl":"10.1038/s41596-026-01336-y","url":null,"abstract":"A major challenge in microbiome research is the inherent complexity and inter-individual variability of the human gut microbiota. To address this, we have developed a detailed protocol for establishing and analyzing a Simplified Human Intestinal Microbiota (SIHUMI)—a defined, in vitro bacterial consortium composed of seven fully sequenced and anaerobically culturable human gut commensals. This model enables highly reproducible and controlled experiments, in which the individual growth of each member can be quantitatively tracked over time (up to 48 h) via species-specific qPCR. The protocol outlines optimized and standardized steps, including consortium setup, time-resolved sample collection, DNA extraction and qPCR analysis. It can be used to evaluate community dynamics in response to interventions such as nutrients, antimicrobials or other xenobiotics. The system is readily adaptable: additional strains can be incorporated, including pathogens (e.g., Clostridioides difficile), to transform it into an infectious disease model. In addition, we describe two optional rapid methods for assessing interspecies interactions and provide an open-source web app for generating interaction network plots. This enables exploration of ecological mechanisms and potential off-target effects. The entire workflow—from setup to data acquisition—can be completed within 1 week. This qPCR-based protocol offers a validated and accessible platform for gut microbiome research, providing a standardized, strain-level and time-resolved alternative to 16S- or fluorescence-based workflows and enabling quantitative, scalable analysis of defined microbial communities. This protocol enables users to establish a defined, in vitro consortium composed of seven fully sequenced and anaerobically culturable human gut bacteria and follow the growth of individual members via strain-specific qPCR to evaluate community dynamics in response to interventions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4300-4330"},"PeriodicalIF":18.4,"publicationDate":"2026-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147486385","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":"Establishing a long-term versatile culture system for functional mouse mammary organoids from single cells","authors":"Lei Yuan, Ruolan Tang, Shaofang Xie, Zuobao Lin, Pei Cai, Shang Cai","doi":"10.1038/s41596-026-01333-1","DOIUrl":"10.1038/s41596-026-01333-1","url":null,"abstract":"The mammary gland serves as a pivotal model for studying stem cell dynamics and breast cancer, the most prevalent malignancy worldwide. Developing a long-term organoid culture system to study the normal physiology and pathophysiology of mammary glands in vitro is of paramount importance. However, current organoid systems lack the morphological and functional fidelity required to model its complex physiology. Here we present a detailed Protocol to establish a long-term, dynamic three-dimensional culture system for mouse mammary organoids, which we call ‘mini-glands’, that recapitulates in vivo morphogenesis and functional cycles. This method uses basal stem cells to generate organoids through sequential phases: sphere formation, polarity induction, symmetry breaking, branching morphogenesis and pseudoestrous cycle simulation. The resulting ‘mini-glands’ replicate the natural gland’s branched architecture and undergo developmental stages mimicking puberty, pregnancy, lactation and involution. Furthermore, the system enables lineage tracing of cell fate transitions and oncogenic transformation studies via genetic manipulation. By bridging the gap between in vitro models and in vivo complexity, this platform advances studies in mammary gland biology, breast cancer initiation and therapeutic screening. The Protocol can be readily performed by researchers with basic experience in mammalian cell culture and requires no specialized instrumentation. A full culture cycle typically takes ~2 weeks to produce mature, highly branched ‘mini-glands’. This Protocol generates mouse mammary organoids with the tree-like architecture and cellular heterogeneity of native tissue, which recapitulate mammary physiology across the estrous cycle, pregnancy, lactation and involution.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4217-4249"},"PeriodicalIF":18.4,"publicationDate":"2026-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147486810","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":"Ligand-assisted interfacial monomicelle assembly to incorporate intermetallic nanoparticles into mesoporous carbon nanostructures","authors":"Pengpeng Qiu, Guihua Zhu, Minghao Li, Weichao Bao, Ying Jiang, Lianjun Wang, Wan Jiang, Wei Luo","doi":"10.1038/s41596-025-01326-6","DOIUrl":"10.1038/s41596-025-01326-6","url":null,"abstract":"Intermetallic nanoparticles (iNPs) exhibit ordered superlattice structures characterized by unique properties, for example, long-range ordering, robust metallic bonding and site-isolation effects. Multicomponent (>2) iNPs are particularly interesting for the development of advanced metallic catalysts for electrochemical applications. Integration of iNPs within mesoporous carbon nanostructures enhances mass and electron transfer during electrolysis and provides a protective mesoporous confinement that prevents iNP sintering and loss during operation. Here we describe a generalized two-step strategy to integrate iNPs with up to eight metal components into mesoporous carbon nanostructures that allows control over the ordering degree, phases and morphology. Ligand-assisted interfacial assembly of monomicelles on diverse metal substrates (using a laboratory-made amphiphilic copolymer as a structure-directing agent, with dopamine acting as both carbon precursor and metal-coordinating ligand) results in mesostructured metal–organic superstructures. All of the examples described have at least one noble metal (Pt or Pd) combined with transition metal elements (for example, Fe, Co, among others). Thermal processing of these metal–organic superstructures in an ammonia (NH3) atmosphere induces the formation of chemically ordered iNPs while simultaneously creating the mesoporous structure. The Protocol also includes procedures for two example electrochemical applications: the oxygen reduction reaction and nitrate reduction reaction for NH3 production. The entire synthetic procedure takes ~5 d, while physical characterization via electron microscopy, X-ray diffraction and nitrogen sorption isotherms require ~2 d. Investigating the catalytic mechanisms, utilizing in situ Fourier-transform infrared spectroscopy and online differential electrochemical mass analysis typically take 4–6 h for electrocatalytic reactions. It is useful to have materials that contain multiple catalytic sites for electrochemical reactions with complex mechanisms. This Protocol describes the incorporation of intermetallic nanoparticles with up to eight metal elements in carbon nanostructures.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"3996-4033"},"PeriodicalIF":18.4,"publicationDate":"2026-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147486406","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 HOST assay: an automated video-based evaluation of mosquito attraction to thermal infrared","authors":"Nicolas A. DeBeaubien, Avinash Chandel, Vincent L. Salgado, Craig Montell","doi":"10.1038/s41596-026-01335-z","DOIUrl":"10.1038/s41596-026-01335-z","url":null,"abstract":"Female mosquitoes transmit pathogens to people during blood feeding, leading to ~700 million infections annually. Females navigate toward humans by integrating multiple sensory cues while flying. Here we describe a behavioral assay system using automated video analysis to measure mosquito landing and walking on target areas emitting human body temperature, elevated CO2, and human skin odor, and quantify preference indices for such behaviors. This host seeker tracking (HOST) assay can be adapted to measure landing and movement on a target of interest under different combinations and intensities of sensory stimuli. It is distinct from other host-seeking assays in that it allows the impact of thermal infrared radiation and convection to be analyzed individually. Mated female mosquitoes are transferred to specialized cages, allowed to recover, and then assayed. During each HOST experiment, an assay cage is lowered into an arena where females are presented with host-associated cues, and their landing and walking behaviors on targets of interest are video-recorded. After completing the assays, the videos are analyzed using custom scripts (V-HOST), which automatically determine landing and movement on the target areas, and preference indices. The HOST protocol uses interchangeable cages rather than direct release of mosquitoes into the behavior arena, which increases assay throughput, and prevents release of mosquitoes. The HOST assay employs commonly available materials and equipment, making it a readily accessible technique. Using this approach, an experimenter can perform ~24 individual technical replicates in a 4 h period (ZT0–ZT4) and quickly perform the video analysis (10–15 min). Here the authors describe an automated video-based protocol that enables the evaluation of mosquito landing and walking behaviors on targets of interest (for example, a fictive host) in response to thermal infrared in combination with other host cues.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4278-4299"},"PeriodicalIF":18.4,"publicationDate":"2026-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147474386","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 ENBSe-based photoredox catalysts for O2-independent phototherapy in living systems","authors":"Yingying Zhang \u0000 (, ), Yingnan Wu \u0000 (, ), Zehao Jing \u0000 (, ), Xiaoqiang Chen \u0000 (, ), Mingle Li \u0000 (, ), Xiaojun Peng \u0000 (, )","doi":"10.1038/s41596-025-01328-4","DOIUrl":"10.1038/s41596-025-01328-4","url":null,"abstract":"Photodynamic therapy using an appropriate photocatalyst results in the production of cytotoxic reactive oxygen species for tumor ablation. However, the inherent O2 dependence of conventional photodynamic therapy limits its clinical translation. To overcome this challenge, here we developed a selenium-substituted Nile blue derivative (ENBSe) as a versatile O2-independent photocatalyst. Under near-infrared light irradiation, ENBSe can drive the biological oxidation of NADH to NAD+ while simultaneously triggering the cascade reduction of cytochrome c (Fe³⁺ to Fe²⁺), even in the absence of O2. To improve tumor specificity and targeting, we further developed a conditionally activatable photoredox catalysis (ConAPC) system. ENBSe is covalently attached to 4-nitrobenzyl chloride via a carbonic anhydride bond, wherein the nitro group can be specifically cleaved by nitroreductase (NTR), an enzyme overexpressed in hypoxic tissues. Such ConAPC design prevents reaction with NADH and quenches the fluorescences of ENBSe, which means that the drug molecule, ENBSe–NTR, is catalytically inactive. ENBSe–NTR is, to our knowledge, the first tumor microenvironment-responsive ConAPC molecule that enables O2-free, tumor-specific catalytic therapy. By replacing the 4-nitrobenzyl chloride group, it might be possible to target cells with different microenvironmental conditions. This protocol presents a standardized workflow encompassing the synthesis of ENBSe and its application for photocatalytic modulation of cellular electron flow in the mitochondrial electron transport chain via an O2-independent mechanism of action. The outlined protocol specifies a synthesis period of ~4 d for ENBSe, ~4 h for photoredox spectroscopic characterization and 4–5 weeks for photodiagnostic assessment in cancer cell and mice models. Photodynamic therapy is more effective if the photocatalyst works in hypoxic conditions. This protocol describes ENBSe, a photoredox catalyst driving NADH oxidation and initiating cytochrome c reduction, establishing a method for cancer phototherapy.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4062-4092"},"PeriodicalIF":18.4,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147468761","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":"Quantitative imaging of apoplastic pH in plant roots via confocal microscopy","authors":"Ann-Kathrin Rößling, Niklas Mayle, Laurent Guerard, Elke Barbez","doi":"10.1038/s41596-026-01337-x","DOIUrl":"10.1038/s41596-026-01337-x","url":null,"abstract":"The regulation of apoplastic pH is critical for plant growth and development, affecting processes such as nutrient uptake, cell wall expansion and intercellular signaling. Conventional methods for measuring apoplastic pH, including pH indicators in growth media and ion-selective electrodes, often fall short of providing the spatial resolution and accuracy needed for detailed studies. Here we present a protocol for the quantitative imaging of apoplastic pH in Arabidopsis thaliana roots using confocal microscopy combined with the fluorescent pH probe 8-hydroxy-pyrene-1,3,6-trisulfonic acid trisodium salt, also called pyranine. This approach addresses the limitations of genetic sensors and traditional pH measurement techniques by offering a nontoxic, cost-effective and precise method for pH assessment at cellular resolution via ratiometric confocal imaging. In addition, we introduce an updated Fiji plugin for ratiometric image conversion. The new plugin enhances workflow efficiency by automating image processing while offering several options for customization, thereby ensuring reliable and reproducible results. The full procedure, from staining to image analysis, can be completed within ~2–4 h, depending on the number of samples and imaging depth. This protocol provides a robust tool for plant physiologists to investigate apoplastic pH dynamics with high spatial resolution and accuracy in plant tissues. This protocol details the quantitative imaging of apoplastic pH in Arabidopsis thaliana roots using confocal microscopy combined with the fluorescent pH probe 8-hydroxy-pyrene-1,3,6-trisulfonic acid trisodium salt, enabling apoplastic pH dynamics to be assessed with high spatial resolution and accuracy.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4331-4346"},"PeriodicalIF":18.4,"publicationDate":"2026-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147434312","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":"FuzDrop: sequence-based prediction of the propensity of proteins for liquid–liquid phase separation and aggregation","authors":"Michele Vendruscolo, Monika Fuxreiter","doi":"10.1038/s41596-025-01267-0","DOIUrl":"10.1038/s41596-025-01267-0","url":null,"abstract":"Proteins exhibit complex phase behavior as they convert between the native state, the liquid condensate (or droplet) state and the solid condensate (or amyloid) state. To facilitate the study of these processes, we describe the FuzDrop method of predicting the condensation propensity of proteins to undergo liquid–liquid phase separation and to subsequently form amyloid aggregates. The method is based on the principle that liquid condensations reflect a balance between enthalpic and entropic contributions; FuzPred is an algorithm that provides sequence-based estimates for these contributions in stoichiometric complexes ( https://fuzpred.bio.unipd.it/predictor ). FuzDrop extends this algorithm to protein condensates, and enables prediction of the propensity for amyloid formation within liquid condensates, known as the condensation pathway to protein aggregation ( https://fuzdrop.bio.unipd.it/predictor ). This prediction is based on the principle that the sequence regions that promote aggregation within liquid condensates have a multiplicity of binding modes, because they have a strong propensity for both entropic-driven interactions to stabilize the droplet state and enthalpic-driven interactions to stabilize the amyloid state. The time required for FuzDrop predictions on the web server scales linearly with protein length and is typically ~30 s for a protein of 500 residues. By enabling predictions of protein phase behavior, FuzDrop may facilitate experimental studies directed at the development of therapies for protein condensation diseases. FuzDrop predicts the condensation propensity of proteins on the basis of their amino acid sequences. This protocol describes the underlying theory and how to use the results to understand liquid-liquid phase separation and amyloid aggregate formation.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 5","pages":"2016-2042"},"PeriodicalIF":16.0,"publicationDate":"2026-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147434283","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 placenta-tropic mRNA lipid nanoparticles for pregnancy disorders","authors":"Kelsey L. Swingle, Michael J. Mitchell","doi":"10.1038/s41596-025-01325-7","DOIUrl":"10.1038/s41596-025-01325-7","url":null,"abstract":"Lipid nanoparticles (LNPs) have garnered tremendous enthusiasm in preclinical and clinical settings for the delivery of nucleic acids such as mRNA. With applications in protein replacement therapies, vaccines and gene editing, mRNA LNPs have only recently been explored in the context of pregnancy disorders. There is a significant need for the design of novel therapeutic technologies such as mRNA LNPs to treat obstetric disorders like pre-eclampsia that are associated with placental pathology and detrimental effects on maternal and fetal health. Here, we present a step-by-step procedure for the preparation and evaluation of placenta-tropic mRNA LNPs for researchers from varied disciplines to explore their application in treating pregnancy disorders. In this Protocol, we describe steps for synthesizing and purifying the key ionizable lipid excipient of the placenta-tropic LNP formulation (4 d) before preparing mRNA LNPs using microfluidic mixing (1 d). Then, we detail in vitro mechanistic evaluations of the effect of protein adsorption on LNP-mediated mRNA transfection to placental trophoblasts (3 d). Finally, we outline methods for isolating reproductive tissues from time-dated pregnant mice to assess in vivo LNP biodistribution and mRNA transfection to the murine placenta (16 d). Compared to alternative LNP formulation procedures, this Protocol focuses on delivering mRNA LNPs to the placenta with a workflow that can be applied for a range of obstetric disorders. This Protocol seeks to increase interdisciplinary work at the interface of nanomedicine, gene modulation and reproductive health. We present a Protocol for the preparation of placenta-tropic mRNA lipid nanoparticles (LNPs), evaluation of the effects of protein adsorption on mRNA transfection in vitro and assessment of LNP biodistribution and mRNA transfection in time-dated pregnant mice.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"3967-3995"},"PeriodicalIF":18.4,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147434269","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 targeted lipid nanoparticles for precision nucleic acid delivery","authors":"Hannah C. Geisler, Elisa Battistini, Ajay S. Thatte, Marshall S. Padilla, Michael J. Mitchell","doi":"10.1038/s41596-025-01330-w","DOIUrl":"10.1038/s41596-025-01330-w","url":null,"abstract":"Intravenous administration of lipid nanoparticles for the delivery of nucleic acid therapeutics remains constrained by passive uptake mechanisms in the liver, often necessitating high doses to achieve meaningful transfection in specific cells of interest. Targeted LNPs (tLNPs) can overcome these challenges by (i) enabling receptor-mediated endocytosis in difficult-to-transfect cells, thereby reducing passive clearance; (ii) increasing the proportion of LNPs reaching their intended target; and (iii) enabling comparable protein expression at lower doses. Here, we provide a step-by-step guide for formulating tLNPs functionalized with whole antibodies or antibody fragments using traditional laboratory equipment. We outline procedures for antibody preparation and labeling (0.5–1 d), antibody–LNP conjugation (1–2 d), tLNP purification and characterization (1 d) and in vivo and ex vivo targeting evaluation (3–4 d). To demonstrate the versatility of this protocol, we validate in vivo targeting to two mouse tissues: we show that anti-platelet endothelial cell adhesion molecule 1 antibody conjugation to lung-tropic LNPs enhances lung transfection by five times compared to nontargeted LNPs, and anti-epidermal growth factor receptor antibody conjugation to liver-tropic LNPs enhances liver transfection by 20 times. We also demonstrate ex vivo targeting to primary human T cells, where anti-CD5 antibody conjugation to LNPs boosts uptake by 4.5 times and significantly increases mRNA transfection. Importantly, this modular strategy is compatible with any LNP formulation or antibody. In outlining these procedures, we seek to deliver a robust and reproducible workflow for the manufacturing of tLNPs, with the ultimate goal of advancing their therapeutic potential and facilitating clinical translation. This protocol outlines the steps to prepare functionalized lipid nanoparticles covalently conjugated with whole antibodies or antibody fragments for targeted nucleic acid delivery to different tissues or cell types.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 9","pages":"4133-4168"},"PeriodicalIF":18.4,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147390211","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}