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1.
Methods Mol Biol ; 2566: 233-240, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152256

RESUMO

Terbium citrate staining represents the method of choice for RNA visualization at transmission electron microscopy. Because of its affinity for guanosines in RNA rather than in DNA, terbium citrate gives precise results being a selective staining. However, difficulties often arise when performing this technique, especially in crucial and delicate steps such as rinses, when it is not uncommon to excessively reduce the already feeble contrast. For these reasons, we developed a straightforward and secure approach to overcome such complications. Here we report a new method for RNA single molecule localization by means of terbium citrate vapors, viable for every type of fixation and embedding.


Assuntos
RNA , Térbio , Ácido Cítrico , DNA , Microscopia Eletrônica
2.
Methods Mol Biol ; 2570: 13-38, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156771

RESUMO

Oligonucleotide ligands (DNA, RNA, or XNA), also known as aptamers, are selected against various target molecules using an iterative, evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). To select aptamers against complex cell surface proteins in their native state, a variant of SELEX termed ligand-guided selection (LIGS) was recently introduced. The significance of LIGS is rooted in its strategy of exploiting the selection step in SELEX to identify highly specific aptamers against known cell surface markers. Thus, in LIGS, a higher-affinity secondary ligand, such as a monoclonal antibody (mAb) to a whole-cell bound to an evolved SELEX library, is introduced to outcompete sequences against the mAb targeting cell surface protein or induce a conformational switch to destabilize the aptamer-surface cell surface protein resulting in elution of the sequences. Here, we describe the detailed method of LIGS utilized in identifying aptamers against T-cell receptor cluster of differentiation three complex (TCR-CD3) expressed in human T-cells and T-cell leukemia.


Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Anticorpos Monoclonais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Humanos , Ligantes , RNA , Receptores de Antígenos de Linfócitos T , Técnica de Seleção de Aptâmeros/métodos
3.
Methods Mol Biol ; 2570: 45-61, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156773

RESUMO

Aptamers are single-stranded DNA or RNA molecules which bind with high specificity to the molecular target for which they have been selected. Through a number of unnatural modifications, the diversity of interactions available for this class of molecules can be greatly expanded, potentially leading to better binding properties. Herein we describe a method to prepare an initial chemically modified DNA library for SELEX. It comprises the synthesis of a modified nucleotide in the CuAAC reaction and its purification by an adapted HPLC method. Subsequent stage is the incorporation of the prepared modified nucleotide into a DNA library in a primer extension reaction and a quick and easy method of its purification using an electroelution device. This allows the recovery of the resulting chemically modified ssDNA library with high efficiency to kick off the aptamer selection process.


Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples , Biblioteca Gênica , RNA , Técnica de Seleção de Aptâmeros/métodos
4.
Methods Mol Biol ; 2570: 63-71, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156774

RESUMO

SELEX has enabled the selection of aptamers, nucleic acids that can bind a defined ligand, in some cases with exceptionally high affinity and specificity. The SELEX protocol has been adapted many times to fit a variety of needs. This protocol describes such an adaptation, namely, RNA-Capture SELEX that we have used to successfully develop small molecule-binding RNA aptamers. Our proposed method specifically selects not only for excellent binding but also for conformational switching. In consequence, we found this SELEX method to be particularly suitable for identifying aptamers for further application in synthetic riboswitch engineering.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Aptâmeros de Nucleotídeos/química , Ligantes , Fenômenos Magnéticos , RNA , Técnica de Seleção de Aptâmeros/métodos , Estreptavidina/metabolismo
5.
Methods Mol Biol ; 2570: 155-173, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156781

RESUMO

Fluorogenic RNA aptamers are synthetic RNAs that have been evolved by in vitro selection methods to bind and light up conditionally fluorescent organic ligands. Compared with other probes for RNA detection, they are less invasive than hybridization-based methods (FISH, molecular beacons) and are considerably smaller than fluorescent protein-recruiting systems (MS2, Pumilio variants). Fluorogenic aptamers have therefore found widespread use as genetically encodable tags for RNA detection in live cells and have also been used in combination with riboswitches to construct versatile metabolite sensors for in vitro use. Their success builds on a fundamental understanding of their three-dimensional structure to explain the mechanisms of ligand interaction and to rationally design functional aptamer devices. In this protocol, we describe a supramolecular FRET-based structure probing method for fluorogenic aptamers that exploits distance- and orientation-dependent energy transfer efficiencies between site-specifically incorporated fluorescent nucleoside analogs and non-covalently bound ligands, exemplified by 4-cyanoindol riboside (4CI) and the DMHBI+-binding RNA aptamer Chili. This method yields structural restraints that bridge the gap between traditional low-resolution secondary structure probing methods and more elaborate high-resolution methods such as X-ray crystallography and NMR spectroscopy.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Aptâmeros de Nucleotídeos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ligantes , Nucleosídeos , RNA/genética
6.
Methods Mol Biol ; 2570: 223-234, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156786

RESUMO

RNA aptamers can be genetically encoded in cells to probe and manipulate cellular function. The usefulness of aptamers in mammalian cells is limited by low accumulation and degradation by ribonucleases. Expression of circular RNA aptamers using the Tornado expression system achieves high stability and an abundance of intracellular RNA aptamers. With this method, RNA aptamers with otherwise minimal activity become potent inhibitors. Here, we describe protocols to characterize circular RNA aptamers expressed using Tornado. Included are methods to assess stability, abundance, subcellular localization, and target binding by circular RNA aptamers.


Assuntos
Aptâmeros de Nucleotídeos , Animais , Aptâmeros de Nucleotídeos/química , Mamíferos/genética , RNA/química , RNA Circular , Ribonucleases/metabolismo
7.
Methods Mol Biol ; 2570: 243-269, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156788

RESUMO

Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Ligantes , Microfluídica/métodos , RNA/genética
8.
Methods Mol Biol ; 2577: 229-240, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173577

RESUMO

The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.


Assuntos
Endonucleases , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Mamíferos/metabolismo , Camundongos , RNA , Ativação Transcricional
9.
Methods Mol Biol ; 2576: 349-359, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152201

RESUMO

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a major development in PCR technology, is a powerful and sensitive gene analysis technique that has revolutionized the field of gene expression assays. In this chapter, we describe in detail RNA extraction, reverse transcription (RT), and relative quantification of genes forming the endocannabinoid system in different experimental models. In particular, we here provide specific and sensitive assays to be used to assess gene expression of the endocannabinoid system components in mouse, rat, or human samples.


Assuntos
Endocanabinoides , Transcrição Reversa , Animais , Humanos , Camundongos , RNA/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Methods Mol Biol ; 2563: 117-133, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227470

RESUMO

A vast number of intracellular membraneless bodies also known as biomolecular condensates form through a liquid-liquid phase separation (LLPS) of biomolecules. To date, phase separation has been identified as the main driving force for a membraneless organelles such as nucleoli, Cajal bodies, stress granules, and chromatin compartments. Recently, the protein-RNA condensation is receiving increased attention, because it is closely related to the biological function of cells such as transcription, translation, and RNA metabolism. Despite the multidisciplinary efforts put forth to study the biophysical properties of protein-RNA condensates, there are many fundamental unanswered questions regarding the mechanism of formation and regulation of protein-RNA condensates in eukaryotic cells. Major challenges in studying protein-RNA condensation stem from (i) the molecular heterogeneity and conformational flexibility of RNA and protein chains and (ii) the nonequilibrium nature of transcription and cellular environment. Computer simulations, bioinformatics, and mathematical models are uniquely positioned for shedding light on the microscopic nature of protein-RNA phase separation. To this end, there is an urgent need for innovative models with the right spatiotemporal resolution for confronting the experimental observables in a comprehensive and physics-based manner. In this chapter, we will summarize the currently emerging research efforts, which employ atomistic and coarse-grained molecular models and field theoretical models to understand equilibrium and nonequilibrium aspects of protein-RNA condensation.


Assuntos
Organelas , RNA , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Corpos Enovelados/metabolismo , Organelas/metabolismo , RNA/metabolismo
11.
Methods Mol Biol ; 2563: 149-160, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227472

RESUMO

Many biomolecular condensates, including nucleoli and stress granules, form via dynamic multivalent protein-protein and protein-RNA interactions. These molecular interactions nucleate liquid-liquid phase separation (LLPS) and determine condensate properties, such as size and fluidity. Here, we outline the experimental procedures for single-molecule fluorescence experiments to probe protein-RNA interactions underlying LLPS. The experiments include single-molecule Förster (Fluorescence) resonance energy transfer (smFRET) to monitor protein-induced conformational changes in the RNA, protein-induced fluorescence enhancement (PIFE) to measure protein-RNA encounters, and single-molecule nucleation experiments to quantify the association and buildup of proteins on a nucleating RNA. Together, these experiments provide complementary approaches to elucidate a molecular view of the protein-RNA interactions that drive ribonucleoprotein condensate formation.


Assuntos
Condensados Biomoleculares , RNA , Transferência Ressonante de Energia de Fluorescência/métodos , Nanotecnologia , RNA/metabolismo , Ribonucleoproteínas/metabolismo
12.
Methods Mol Biol ; 2563: 199-213, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227474

RESUMO

Liquid-liquid phase separation of protein and RNA complexes into biomolecular condensates has emerged as a ubiquitous phenomenon in living systems. These protein-RNA condensates are thought to be involved in many biological functions in all forms of life. One of the most sought-after properties of these condensates is their dynamical properties, as they are a major determinant of condensate physiological function and disease processes. Measurement of the diffusion dynamics of individual components in a multicomponent biomolecular condensate is therefore routinely performed. Here, we outline the experimental procedure for performing in-droplet fluorescence correlation spectroscopy (FCS) measurements to extract the diffusion coefficient of individual molecules within a biomolecular condensate in vitro. Unlike more common experiments such as fluorescence recovery after photobleaching (FRAP), where data interpretation is not straightforward and strictly model dependent, FCS offers a robust and more accurate way to quantify biomolecular diffusion rates in the dense phase. The small observation volume allows FCS experiments to report on the local diffusion coefficient within a spatial resolution of <1 µm, making it ideal for probing spatial inhomogeneities within condensates as well as variable dynamics within subcompartments of multiphasic condensates.


Assuntos
Ácidos Nucleicos , Condensados Biomoleculares , Recuperação de Fluorescência Após Fotodegradação , RNA , Análise Espectral
13.
Methods Mol Biol ; 2563: 371-381, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227483

RESUMO

Biomolecular condensates, first discovered in eukaryotic cells, were recently found in bacteria. The small size of these organisms presents unique challenges for identifying and characterizing condensates. Here, we describe a single-molecule approach for studying biomolecular condensates in live bacterial cells. Specifically, we outline a protocol to quantify the mobility of RNA polymerase in E. coli using HILO (highly inclined and laminated optical sheet) illumination with the photoconvertible fluorophore mMaple3. Our analysis classifies the trajectories of individual molecules by their local density, enabling a comparison of molecular mobilities between different subcellular compartments.


Assuntos
Escherichia coli , Imagem Individual de Molécula , RNA Polimerases Dirigidas por DNA , Escherichia coli/genética , Células Eucarióticas , RNA
14.
Methods Mol Biol ; 2568: 1-12, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227558

RESUMO

Recent technological developments such as cryogenic electron microscopy (Cryo-EM) and X-ray free electron lasers (XFEL) have significantly expanded the available toolkit to visualize large, complex noncoding RNAs and their complexes. Consequently, the quality of the RNA sample, as measured by its chemical monodispersity and conformational homogeneity, has become the bottleneck that frequently precludes effective structural analyses. Here we describe a general RNA sample preparation protocol that combines cotranscriptional RNA folding and RNA-RNA complex assembly, followed by native purification of stoichiometric complexes. We illustrate and discuss the utility of this versatile method in overcoming RNA misfolding and enabling the structural and mechanistic elucidations of the T-box riboswitch-tRNA complexes.


Assuntos
Riboswitch , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Dobramento de RNA , RNA de Transferência/genética
15.
Methods Mol Biol ; 2568: 13-23, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227559

RESUMO

Structural analyses of large, complex noncoding RNAs continue to lag behind their rapid discovery and functional descriptions. Site-specifically incorporated, minimally invasive fluorescent probes such as 2-aminopurine (2AP) and pyrrolo-cytosine (PyC) have provided essential complementary information about local RNA structure, conformational dynamics, and interactions. Here I describe a protocol that benchmarks and correlates local RNA conformations with their respective fluorescence lifetimes, as a general technique that confers key advantages over fluorescence intensity-based methods. The observation that fluorescence lifetimes are more sensitive to local structures than sequence contexts suggests broad utility across diverse RNA and ribonucleoprotein systems.


Assuntos
2-Aminopurina , RNA , 2-Aminopurina/química , Fluorescência , Corantes Fluorescentes/química , Conformação de Ácido Nucleico , RNA/química , Ribonucleoproteínas , Espectrometria de Fluorescência/métodos
16.
Methods Mol Biol ; 2568: 25-36, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227560

RESUMO

Fluorescent RNA aptamers are tools for studying RNA localization and interactions in vivo. The photophysical properties of these in vitro selected RNAs should be characterized prior to cellular imaging experiments. Here, we describe the process of determining the fluorophore affinity, fluorescence enhancement, and fluorescence lifetime(s) of the Mango-III fluorescence turn-on aptamer. Parameters determined through these protocols will aid in establishing conditions for live-cell imaging.


Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/genética , Fluorescência , Corantes Fluorescentes , RNA
17.
Methods Mol Biol ; 2568: 75-101, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227563

RESUMO

Recognition of the growing importance of RNA as a target for therapeutic or diagnostic ligands brings the importance of computational predictions of docking poses to such receptors to the forefront. Most docking programs have been optimized for protein targets, based on a relatively rich pool of known docked protein structures. Unfortunately, despite progress, numbers of known docked RNA complexes are low and the accuracy of the computational predictions trained on those inadequate samples lags behind that achieved for proteins. Compared to proteins, RNA structures generally have fewer docking pockets, have less diverse electrostatic surfaces, and are more flexible, raising the possibility of producing only transiently available good docking targets. We are presenting a docking prediction protocol that adds molecular dynamics simulations before and after the actual docking in order to explore the conformational space of the target RNA and then to reevaluate the stability of the predicted RNA-ligand complex. In this way we are attempting to overcome important limitations of the docking programs: the rigid (fully or mostly) target structure and imperfect nature of the docking scoring functions.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Sítios de Ligação , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas/química , RNA/metabolismo
18.
Methods Mol Biol ; 2568: 123-132, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227565

RESUMO

A capacity to detect the binding profiles of RNA targets for an RNA-binding protein (RBP) under different cellular conditions is essential to understand the functions of the RBP in posttranscriptional regulation. However, the prediction of RBP binding sites in vivo remains challenging. Tools that predict RBP-RNA interactions using sequence and/or predicted structures cannot reflect the exact state of RNA in vivo. PrismNet, which uses both sequences and in vivo RNA structure information from probing experiments, can accurately predict RBP binding under different cellular conditions by deep learning, and can be applied for functional studies of RBPs. Here, we provide a detailed protocol showing how to train a PrismNet model of RBP-RNA interactions for an RBP, and how to apply the model for predictions of the RBP binding under different conditions.


Assuntos
Proteínas de Ligação a RNA , RNA , Sítios de Ligação/genética , Regulação da Expressão Gênica , Ligação Proteica , RNA/química , Proteínas de Ligação a RNA/metabolismo
19.
Methods Mol Biol ; 2568: 133-145, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227566

RESUMO

Atomic force microscopy (AFM) is an important and versatile technique to investigate the structures and dynamics of biomolecules under physiologically relevant conditions at the single-molecule level. Recent progresses in high-resolution AFM imaging of nucleic acids have expanded this technique from simple characterization of double-stranded DNA or RNA to detailed analyses of the structure and dynamics of large functional RNAs with complex folds. Several technical developments, such as sharper probes and more stable instruments with novel imaging modes, AFM is capable of directly visualizing RNA conformational heterogeneity in solution in real time. Here, we introduce a comprehensive method for recording high-resolution images of RNA molecules, including sample preparation, instrument setup, data acquisition, and image processing.


Assuntos
DNA , Ácidos Nucleicos , DNA/química , Microscopia de Força Atômica/métodos , Conformação de Ácido Nucleico , RNA
20.
Methods Mol Biol ; 2568: 165-177, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227568

RESUMO

RNA-level regulation by riboswitches relies on the specific binding of small metabolites to the aptamer domain to trigger substantial conformational changes that affect transcription or translation. Although several biophysical methods have been employed to study such RNAs, the utility of any one single method is limited. Hybrid approaches, therefore, are essential to better characterize these intrinsically dynamic molecules and elucidate their regulatory mechanisms driven by ligand-induced conformational changes. This chapter outlines procedures for biochemical and biophysical characterization of RNA that employs a combination of solution-based methods: isothermal titration calorimetry (ITC), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Collectively, these tools provide a semi-quantitative assessment of the thermodynamics associated with ligand binding and subsequent conformational changes.


Assuntos
Riboswitch , Ligantes , Conformação de Ácido Nucleico , RNA/química , Espalhamento a Baixo Ângulo , Difração de Raios X
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