RESUMEN
Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA interactions in vitro. We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al.1.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética , Ensayo de Cambio de Movilidad Electroforética/métodos , ARN/metabolismo , ARN/genética , ADN/metabolismo , ADN/genética , Unión Proteica , Proteínas de Unión al ADN/metabolismo , Ácidos Nucleicos/metabolismo , HumanosRESUMEN
Plant long non-coding RNAs (lncRNAs) have been implicated in many biological processes, including responses to abiotic stresses, yet their detailed functions and especially their modes of action are still underexplored. lncRNAs often interact with proteins to participate in multiple levels of gene regulation. Therefore, identifying the RNA-binding proteins and validating their interaction with lncRNAs will be instrumental in revealing the functions of lncRNAs. Here, we describe two major methods to determine the interaction between lncRNA and proteins in vitro, RNA pull-down, and RNA EMSA.
Asunto(s)
ARN Largo no Codificante , Proteínas de Unión al ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Estrés Fisiológico/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , ARN de Planta/genética , ARN de Planta/metabolismo , Unión ProteicaRESUMEN
Nanoparticle carriers enable the multivalent delivery of nucleic acids to cells and protect them from degradation. In this chapter, we present a comprehensive overview of four methodologies: electrophoretic mobility shift assay (EMSA), alamarBlue/CFDA-AM cell viability dyes, fluorescence microscopy, and antiviral assays, which collectively are tools to explore interactions between nucleic acids and nanoparticles, and their biological efficacy. These assays provide insights into binding potential, cytotoxicity, and antiviral efficacy of nucleic acid-based nanoparticle treatments furthering the development of effective antiviral therapeutics.
Asunto(s)
Antivirales , Nanopartículas , Ácidos Nucleicos , Nanopartículas/química , Antivirales/farmacología , Humanos , Ácidos Nucleicos/química , Ensayo de Cambio de Movilidad Electroforética/métodos , Cationes/química , Supervivencia Celular/efectos de los fármacos , Microscopía Fluorescente , Portadores de Fármacos/química , AnimalesRESUMEN
BACKGROUND: Detecting antidrug antibodies (ADAs) against infliximab or adalimumab is useful for therapeutic drug monitoring. Various ADA detection methods exist, and antibody titer is an output in some algorithms. Homogenous mobility shift assay (HMSA) measures relative ADA concentration and determines drug-ADA complex size in vitro. However, the relevance of complex size determination in drug monitoring remains unclear. Hence, the association between complex size, ADA concentration, and sample detectable neutralizing activity was evaluated. METHODS: Sera from infliximab-treated and adalimumab-treated patients who tested positive for ADA in the National Screening Service were analyzed using 3 ADA assays. HMSA determined the relative ADA concentrations and complex sizes, competitive ligand-binding assay evaluated the sample neutralizing capacity, and enzyme-linked immunosorbent assay detected immunoglobulin (Ig)G4 ADA. RESULTS: Most ADA-positive samples (>80%) formed drug-ADA dimer complexes, whereas 17% had dimer and multimer complexes, and 3% had multimeric complexes. Multimer presence had 100% positive predictive value for detectable neutralizing activity. ADA concentration and detectable neutralizing activity were moderately correlated (r = 0.65) in adalimumab-treated patients and strongly correlated (r = 0.81) in infliximab-treated patients. In adalimumab-treated patients, multimer presence was a stronger predictor of neutralizing activity than ADA concentration was, but not in infliximab-treated patients. However, in infliximab-treated patient samples, multimer presence revealed a distinct subset with high ADA concentrations, neutralizing activity, and IgG4 ADA. CONCLUSIONS: Multimers detected using HMSA had a strong positive predictive value for competitive ligand-binding assay detectable neutralizing activity. Multimeric IgG4-containing ADA-drug complexes revealed a distinct subset of infliximab-treated patient samples, whose clinical relevance merits further investigation.
Asunto(s)
Adalimumab , Monitoreo de Drogas , Infliximab , Infliximab/inmunología , Infliximab/uso terapéutico , Infliximab/sangre , Humanos , Adalimumab/inmunología , Adalimumab/sangre , Adalimumab/uso terapéutico , Monitoreo de Drogas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Masculino , Persona de Mediana Edad , Ensayo de Cambio de Movilidad Electroforética/métodos , Adulto , Anticuerpos Neutralizantes/sangreRESUMEN
DNA-binding proteins perform diverse functions, including regulating cellular growth and orchestrating chromatin architecture. Here, we present a protocol to discover proteins specifically interacting with a hexanucleotide repeat DNA, the expansion of which is known as the most frequent genetic cause of familial C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. We describe steps to fish out DNA-binding proteins recognizing double-stranded repeat DNAs using a SILAC (stable isotope labelling by amino acids in cell culture)-based approach and validate the results using electrophoretic mobility shift assay. For complete details on the use and execution of this protocol, please refer to Liu et al.1.
Asunto(s)
Proteínas de Unión al ADN , ADN , ADN/metabolismo , ADN/genética , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Marcaje Isotópico/métodosRESUMEN
RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA-RBP pairs from natural components remains challenging while such synthetic RNA-RBP pairs could significantly expand the RNA-RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 1012 unique RNA sequences and 4.0 × 108 unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA-RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA-RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.
Asunto(s)
Bioensayo/métodos , Biblioteca de Genes , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Aptámeros de Nucleótidos , Bioensayo/normas , Ensayo de Cambio de Movilidad Electroforética/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Moleculares , ARN/química , Proteínas de Unión al ARN/química , Proyectos de Investigación , Técnica SELEX de Producción de Aptámeros , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie/métodosRESUMEN
The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Vesículas Extracelulares/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Gases , Humanos , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
One of the most commonly described biological feature of processed pseudogenes is the ability to influence the expression of their parental coding genes. As evidenced in several studies, the high sequence similarity between these RNA pairs sets up a certain level of competition for posttranscriptional regulators, including, among others, RNA-binding proteins (RBPs). RBPs may affect, positively or negatively, the stability of bound mRNAs, so that, if an overexpressed pseudogene competes with its homologous coding gene, the downstream protein synthesis would change, with potential pathological consequences. Given these premises, a rigorous and comprehensive understanding of interactions between pseudogene-parental gene RNA pairs and RBPs could provide further insights into the biological bases of complex diseases, such as cancer, cardiovascular disease, and type 2 diabetes, identifying novel predictive and/or prognostic biomarkers.Herein, we detail easily adaptable protocols of plasmid-based molecular cloning and RNA-electrophoretic mobility shift assay (EMSA) used in our laboratory for determining the interaction between a cytoplasmatic stabilizing protein (αCP1) and the pseudogene-parental gene RNA pair HMGA1-p /HMGA1. We also offer a general overview of RNA immunoprecipitation procedures and present novel bioinformatic tools for predicting RBPs binding sites on pseudogene transcripts.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Inmunoprecipitación/métodos , Seudogenes/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Sitios de Unión , Unión Competitiva , Biotinilación , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Proteína HMGA1a/genética , Humanos , Mediciones Luminiscentes , Unión Proteica , Sondas ARN , Estabilidad del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , TransfecciónRESUMEN
RNA-protein (RNP) complexes are promising biomaterials for the fields of nanotechnology and synthetic biology. Protein-responsive RNA sequences (RNP motifs) can be integrated into various RNAs, such as messenger RNA, short-hairpin RNA, and synthetic RNA nanoobjects for a variety of purposes. Direct observation of RNP interaction in solution at high resolution is important in the design and construction of RNP-mediated nanostructures. Here we describe a method to construct and visualize RNP nanostructures that precisely arrange a target protein on the RNA scaffold with nanometer scale. High-speed AFM (HS-AFM) images of RNP nanostructures show that the folding of RNP complexes of defined sizes can be directly visualized at single RNP resolution in solution.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Nanoestructuras/química , Ribonucleoproteínas/química , Secuencias de Aminoácidos , Ensayo de Cambio de Movilidad Electroforética/métodos , Motivos de Nucleótidos , Pliegue de ProteínaRESUMEN
Electrophoretic mobility shift assays (EMSAs) are among the most frequently used and straightforward experiments for studying protein-nucleic acid interactions. EMSAs rely on the principle that protein-nucleic acid complexes have reduced electrophoretic mobility in a native gel matrix compared to free nucleic acid due to their larger size and reduced negative charge. Therefore, bands for the protein-nucleic acid complexes are shifted in a gel and can be distinguished from free nucleic acids. EMSAs remain a popular technique since they do not require specialist equipment and the complexes formed are easily visualized. Furthermore, the technique can be adapted to enable various aspects of protein-nucleic acid interactions to be investigated, including sequence specificity, estimated binding affinity, and binding stoichiometry.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Ácidos Nucleicos/análisis , Proteínas/análisis , Resinas Acrílicas , Fenómenos Biofísicos , ADN/análisis , ADN/metabolismo , Ácidos Nucleicos/metabolismo , Unión Proteica , Proteínas/metabolismo , ARN/análisis , ARN/metabolismoRESUMEN
Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Animales , Antígeno B7-2/metabolismo , Antígenos CD28/metabolismo , Línea Celular , Ensayo de Cambio de Movilidad Electroforética/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Imagen Molecular , Unión Proteica , Reproducibilidad de los Resultados , Imagen Individual de MoléculaRESUMEN
Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.
Asunto(s)
Electroforesis Capilar/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Fluorometría/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Técnicas Analíticas Microfluídicas/métodos , Sulfotransferasas/análisis , Proteínas Bacterianas/análisis , Proteínas Bacterianas/antagonistas & inhibidores , Bacteroides thetaiotaomicron/enzimología , Compuestos de Boro/análisis , Conformación de Carbohidratos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Sistemas de Computación , Colorantes Fluorescentes/análisis , Glicosaminoglicanos/metabolismo , Cinética , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/análisis , Especificidad por Sustrato , Sulfotransferasas/antagonistas & inhibidoresRESUMEN
RNA editing by cytidine (C) to uridine (U) conversions frequently occurs in land plant mitochondria and plastids. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins in a sequence-specific manner. In the moss Physcomitrella patens, all PPR editing factors possess the DYW-deaminase domain at the C-terminus. Here, we describe methods for the direct sequencing of cDNA to detect RNA editing events and the RNA electrophoresis mobility shift assay (REMSA) to analyze the specific binding of PPR editing factors to their target RNA.
Asunto(s)
Bryopsida/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , Mitocondrias/genética , Proteínas de Plantas/genética , Plastidios/genética , Edición de ARN/genética , ARN de Planta/genética , Bryopsida/metabolismo , Citidina/química , Citidina/genética , ADN Complementario/genética , Mitocondrias/metabolismo , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , ARN de Planta/metabolismo , Uridina/química , Uridina/genéticaRESUMEN
RNA can bind within the major groove of purine-rich DNA via Hoogsteen base pairing and form a triple helical RNA-DNA structure that anchors the RNA to specific DNA sequences, thereby targeting RNA-associated regulatory proteins to distinct genomic sites. Here we present methods to analyze the potential of a given RNA to form triplexes in vitro and to validate these structures in vivo.
Asunto(s)
ADN/química , Ensayo de Cambio de Movilidad Electroforética/métodos , ARN/química , Células HeLa , Humanos , Conformación de Ácido NucleicoRESUMEN
A significant fraction of non-coding RNAs (ncRNAs) is associated with chromatin, shown to regulate gene expression and to organize nuclear architecture. Mechanisms of direct and indirect RNA-chromatin interactions have been described, including the sequence-specific formation of triple helix structures. Triplexes are formed by the sequence-specific binding of RNA to the bases located in the major groove of DNA. We recently showed that triplexes do exist in the context of cellular chromatin and that these structures are stabilized by the histone H3 tail of adjacent nucleosomes. The in vitro characterization of the specificity and binding affinity of triplex sequences next to nucleosomes are essential parameters to identify potential sites of RNA-chromatin interaction in vivo. Here we provide a detailed protocol to determine the influence of nucleosome positioning on triple helix formation. This assay allows the comparative quantification of triplex formation and specificity for triplex targeting sequences relative to the spatial nucleosome position.
Asunto(s)
ADN/química , Ensayo de Cambio de Movilidad Electroforética/métodos , Nucleosomas/metabolismo , ARN no Traducido/química , Animales , Línea Celular , Células Cultivadas , Humanos , Nucleosomas/química , Unión Proteica , ARN no Traducido/metabolismoRESUMEN
Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.
Asunto(s)
Encéfalo/metabolismo , Ensayo de Cambio de Movilidad Electroforética/métodos , Proteínas de la Membrana/metabolismo , Animales , Lipoilación/fisiología , RatonesRESUMEN
The FinO family of proteins constitutes a group of RNA chaperones that interacts with small RNAs (sRNAs) to regulate gene expression in many bacterial species. Here we describe detailed protocols for the biochemical analysis of the RNA chaperone activity of these proteins. Methods are described for preparation of RNA, RNA 5' end labeling with radioisotope and modified EMSA protocols to test the ability of these proteins to catalyze RNA strand exchange and RNA duplex formation.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Técnicas de Sonda Molecular , ARN Pequeño no Traducido/química , Proteínas de Unión al ARN/química , Proteínas Represoras/química , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Estabilidad del ARN , ARN Pequeño no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Diverse types of RNA-binding proteins chaperone the interactions of noncoding RNAs by increasing the rate of RNA base pairing and by stabilizing the final RNA duplex. The E. coli protein Hfq facilitates interactions between small noncoding RNAs and their target mRNAs. The chaperone and RNA annealing activity of Hfq and other RNA chaperones can be evaluated by determining the kinetics of RNA base pairing in the presence and absence of the protein. This chapter presents protocols for measuring RNA annealing kinetics using electrophoretic gel mobility shift assays (EMSA), stopped-flow fluorescence, and fluorescence anisotropy. EMSA is low cost and can resolve reaction intermediates of natural small RNAs and mRNA fragments, as long as the complexes are sufficiently long-lived (≥10 s) to be trapped during electrophoresis. Stopped-flow fluorescence can detect annealing reactions between 1 ms and 30 s and is best suited for measuring the rapid annealing of oligoribonucleotides. Fluorescence anisotropy reports the physical size of the complex and is well-suited for monitoring the association and dissociation of RNA from Hfq during the chaperone cycle.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Chaperonas Moleculares/metabolismo , ARN/metabolismo , Animales , Polarización de Fluorescencia/métodos , Humanos , Chaperonas Moleculares/química , ARN/química , Estabilidad del ARNRESUMEN
RNA and DNA hairpin formation and disruption play key regulatory roles in a variety of cellular processes. The 59-nucleotide transactivation response (TAR) RNA hairpin facilitates the production of full-length transcripts of the HIV-1 genome. Yet the stability of this long, irregular hairpin becomes a liability during reverse transcription as 24 base pairs must be disrupted for strand transfer. Retroviral nucleocapsid (NC) proteins serve as nucleic acid chaperones that have been shown to both destabilize the TAR hairpin and facilitate strand annealing with its complementary DNA sequence. Yet it has remained difficult to elucidate the way NC targets and dramatically destabilizes this hairpin while only weakly affecting the annealed product. In this work, we used optical tweezers to measure the stability of TAR and found that adding NC destabilized the hairpin and simultaneously caused a distinct change in both the height and location of the energy barrier. This data was matched to an energy landscape predicted from a simple theory of definite base pair destabilization. Comparisons revealed the specific binding sites found by NC along the irregular TAR hairpin. Furthermore, specific binding explained both the unusual shift in the transition state and the much weaker effect on the annealed product. These experiments illustrate a general method of energy landscape transformation that exposes important physical insights.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Chaperonas Moleculares/metabolismo , Pinzas Ópticas , Estabilidad del ARN , ARN Interferente Pequeño/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1 , Secuencias Invertidas Repetidas , Chaperonas Moleculares/química , Unión Proteica , ARN Interferente Pequeño/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
It is well established that the RNA-binding protein La has RNA chaperone activity. Recent work suggests that the La protein has two distinct RNA chaperone domains (RCD-A and RCD-B) assisting structural changes in diverse groups of RNA molecules such as RNA polymerase III transcripts (e.g., pre-tRNA, U6 snRNA), cellular messenger, and viral RNAs. In this protocol we focus on the RNA chaperone domain RCD-B, which is located in the carboxy-terminal domain of La. It has been shown that this RNA chaperone domain assists structural changes in predicted RNA hairpins folded in the 5'-untranslated regions of cyclin D1 and Bcl2 mRNAs. Besides RNA helicases, which are implicated in melting RNA hairpin structures in an ATP-dependent manner, RNA chaperones fulfil a similar function in an ATP-independent manner. Aiming to study the RNA chaperon activity of La, we established a La-dependent molecular beacon-based RNA chaperone assay and systematically tested the various salt conditions. Herein we describe the assay format and design to study the salt dependency of RNA chaperones. This protocol can be easily adapted to test the RNA chaperone activity of other RNA-binding proteins and to optimize assay conditions.