Nature Protocols最新文献

Protein-protein interactions underpin nearly all biological processes, and understanding the molecular mechanisms that govern these interactions is crucial for the progress of biomedical sciences. The emergence of artificial intelligence-driven computational tools can help reshape the methods of structural biology; however, model data often require empirical validation. The large scale of predictive modeling data will therefore benefit from optimized methodologies for the high-throughput biochemical characterization of protein-protein interactions. Biolayer interferometry is one of very few approaches that can determine the rate of biomolecular interactions, called kinetics, and, of the commonly available kinetic measurement techniques, it is the most suitable for high-throughput experimental designs. Here we provide step-by-step instructions on how to perform kinetics experiments using biolayer interferometry. We further describe the basis and execution of competition and epitope binning experiments, which are particularly useful for antibody and nanobody screening applications. The procedure requires 3 h to complete and is suitable for users with minimal experience with biochemical techniques.

RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.

Glycosaminoglycans (GAGs) are linear, unbranched heteropolysaccharides whose structural complexity determines their function. Accurate quantification of GAGs in biofluids at high throughput is relevant for numerous biomedical applications. However, because of the structural variability of GAGs in biofluids, existing protocols require complex pre-analytical procedures, have limited throughput and lack accuracy. Here, we describe the extraction and quantification of GAGs by using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-MS/MS). Designed for 96-well plates, this method enables the processing of up to 82 study samples per plate, with the remaining 14 wells used for calibrators and controls. Key steps include the enzymatic depolymerization of GAGs, their derivatization with 2-aminoacridone and their quantification via UHPLC-MS/MS. Each plate can be analyzed in a single UHPLC-MS/MS run, offering the quantitative and scalable analysis of 17 disaccharides from chondroitin sulfate, heparan sulfate and hyaluronic acid, with a level of precision and reproducibility sufficient for their use as biomarkers. The procedure from sample thawing to initiating the UHPLC-MS/MS run can be completed in ~1.5 d plus 15 min of MS runtime per sample, and it is structured to fit within ordinary working shifts, thus making it a valuable tool for clinical laboratories seeking high-throughput analysis of GAGs. The protocol requires expertise in UHPLC-MS/MS.

Superstructures with architectural complexity and unique functionalities are promising for a variety of practical applications in many fields, including mechanics, sensing, photonics, catalysis, drug delivery and energy storage/conversion. In the past five years, a number of attempts have been made to build superparticles based on amphiphilic polymeric micelle units, but most have failed owing to their inherent poor stability. Determining how to stabilize micelles and control their superassembly is critical to obtaining the desired mesoporous superparticles. Here we provide a detailed procedure for the preparation of ultrastable polymeric monomicelle building units, the creation of a library of ultrasmall organic-inorganic nanohybrids, the modular superassembly of monomicelles into hierarchical superstructures and creation of novel multilevel mesoporous superstructures. The protocol enables precise control of the number of monomicelle units and the derived mesopores for superparticles. We show that ultrafine nanohybrids display enhanced mechanical antipressure performance compared with pristine polymeric micelles, and describe the functional characterization of mesoporous superstructures that exhibit excellent oxygen reduction reactivity. Except for the time (4.5 d) needed for the preparation of the triblock polystyrene-block-poly(4-vinylpyridine)-block-poly(ethylene oxide) PS-PVP-PEO or the polystyrene-block-poly(acrylic acid)-block-poly(ethylene oxide) (PS-PAA-PEO) copolymer, the synthesis of the ultrastable monomicelle, ultrafine organic-inorganic nanohybrids, hierarchical superstructures and mesoporous superparticles require ~6, 30, 8 and 24 h, respectively. The time needed for all characterizations and applications are 18 and 10 h, respectively.

The ability to screen the reactivity of T cell receptors (TCRs) is essential to understanding how antigen-specific T cells drive productive or dysfunctional immune responses during infections, cancer and autoimmune diseases. Methods to profile large numbers of TCRs are critical for characterizing immune responses sustained by diverse T cell clones. Here we provide a medium-throughput approach to reconstruct dozens to hundreds of TCRs in parallel, which can be simultaneously screened against primary human tissues and broad curated panels of antigenic targets. Using Gibson assembly and miniaturized lentiviral transduction, individual TCRs are rapidly cloned and expressed in T cells; before screening, TCR cell lines undergo combinatorial labeling with dilutions of three fluorescent dyes, which allows retrieval of the identity of individual T cell effectors when they are organized and tested in pools using flow cytometry. Upon incubation with target cells, we measure the upregulation of CD137 on T cells as a readout of TCR activation. This approach is scalable and simultaneously captures the reactivity of pooled TCR cell lines, whose activation can be deconvoluted in real time, thus providing a path for screening the reactivity of dozens of TCRs against broad panels of synthetic antigens or against cellular targets, such as human tumor cells. We applied this pipeline to systematically deconvolute the antitumoral and antiviral reactivity and antigenic specificity of TCRs from human tumor-infiltrating lymphocytes. This protocol takes ~2 months, from experimental design to data analysis, and requires standard expertise in cloning, cell culture and flow cytometry.

Described herein is a protocol for producing a synthetic extracellular matrix that can be modified in situ during three-dimensional cell culture. The hydrogel platform is established using modular building blocks employing bio-orthogonal tetrazine (Tz) ligation with slow (norbornene, Nb) and fast (trans-cyclooctene, TCO) dienophiles. A cell-laden gel construct is created via the slow, off-stoichiometric Tz/Nb reaction. After a few days of culture, matrix properties can be altered by supplementing the cell culture media with TCO-tagged molecules through the rapid reaction with the remaining Tz groups in the network at the gel-liquid interface. As the Tz/TCO reaction is faster than molecular diffusion, matrix properties can be modified in a spatiotemporal fashion simply by altering the identity of the diffusive species and the diffusion time/path. Our strategy does not interfere with native biochemical processes nor does it require external triggers or a second, independent chemistry. The biomimetic three-dimensional cultures can be analyzed by standard molecular and cellular techniques and visualized by confocal microscopy. We have previously used this method to demonstrate how in situ modulation of matrix properties induces epithelial-to-mesenchymal transition, elicits fibroblast transition from mesenchymal stem cells and regulates myofibroblast differentiation. Following the detailed procedures, individuals with a bachelor's in science and engineering fields can successfully complete the protocol in 4-5 weeks. This protocol can be applied to model tissue morphogenesis and disease progression and it can also be used to establish engineered constructs with tissue-like anisotropy and tissue-specific functions.

Durable and functional regeneration of the airway epithelium in vivo with transplanted stem cells has the potential to reconstitute healthy tissue in diseased airways, such as in cystic fibrosis or primary ciliary dyskinesia. Here, we present detailed protocols for the preparation and culture expansion of murine primary and induced pluripotent stem cell-derived airway basal stem cells (iBCs) and methods for their intra-airway transplantation into polidocanol-conditioned murine recipients to achieve durable in vivo airway regeneration. Reconstitution of the airway tissue resident epithelial stem cell compartment of immunocompetent mice with syngeneic donor cells leverages the extensive self-renewal and multipotent differentiation properties of basal stem cells (BCs) to durably generate a broad diversity of mature airway epithelial lineages in vivo. Engrafted donor-derived cells re-establish planar cell polarity as well as functional ciliary transport. By using this same approach, human primary BCs or iBCs transplanted into NOD-SCID gamma recipient mice similarly display engraftment and multilineage airway epithelial differentiation in vivo. The time to generate mouse or human iBCs takes ~60 d, which can be reduced to ~20 d if previously differentiated cells are thawed from cryopreserved iBC archives. The tracheal conditioning regimen and cell transplantation procedure is completed in 1 d. A competent graduate student or postdoctoral trainee should be able to perform the procedures listed in this protocol.

Unbiased forward genetic screens have been extensively employed in biological research to elucidate functional genomics. In pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation screens, various genetically encoded gain-of-function or loss-of-function mutations are introduced into a heterogeneous population of cells. Subsequently, these cells are screened for phenotypes, perturbation-associated genotypes are analyzed and a connection between genotype and phenotype is determined. CRISPR screening techniques enable the investigation of important biological questions, such as how bacterial toxins kill cells and cause disease. However, the broad spectrum of effects caused by diverse toxins presents a challenge when selecting appropriate screening strategies. Here, we provide a step-by-step protocol for a genome-wide pooled CRISPR perturbation screen to study bacterial toxins. We describe technical considerations, pilot experiments, library construction, screen execution, result analysis and validation of the top enriched hits. These screens are applicable for many different types of toxins and are anticipated to reveal a repertoire of host factors crucial in the intoxication pathway, such as receptors, trafficking/translocation factors and substrates. The entire protocol takes 21-27 weeks and does not require specialized knowledge beyond basic biology.