Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.     

Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays

We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect the formation of protein-RNA complexes. The EMSA that we describe avoids the use not only of radioactivity but also of neurotoxic acrylamide by using agarose as the gel matrix in which the free nucleic acid is separated from protein-nucleic acid complexes. After separation of free from complexed RNA in agarose, the RNA is electroblotted to positively charged nylon. The biotin-labeled RNA is readily bound by a streptavidin-alkaline phosphatase conjugate, allowing for very sensitive chemiluminescent detection ( approximately 0.1-1.0 fmol limit). Using our system, we were able to purify both known iron-responsive proteins (IRPs) from rat liver and assess their binding affinity to RNA containing the iron-responsive element (IRE) using the same batch of biotinylated RNA. We show data indicating that agarose is especially useful for cases when large complexes are formed, although smaller complexes are even better resolved.     

Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA

Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid-protein interactions. When nucleic acid is bound by protein, its mobility during gel electrophoresis is reduced. Similarly, the final position of protein within a complex is shifted when compared to its free state. Here we provide a protocol for a simple approach that uses these mobility differences to identify nucleic acid-binding proteins. Following EMSA, denaturing gel electrophoresis is implemented to provide a second dimension of separation. Protein that binds a specific nucleic acid is identified as a spot(s) whose presence at a particular position(s) is dependent on nucleic acid within the initial binding reaction. The polypeptide in a spot can be subsequently identified by mass spectrometry. As EMSAs can be performed using partially purified or cell extracts, this approach substantially reduces the need for protein purification. It should facilitate the identification of a nucleic acid-binding protein within approximately 4 d.     

Mass spectrometric identification of RNA binding proteins from dried EMSA gels

Electrophoretic mobility shift assays (EMSA) are commonly employed for the analysis of nucleic acid/ protein interactions with a native gel system. Here, we report a method to identify RNA binding proteins from a dried EMSA gel by mass spectrometry following autoradiography. Compared to wet gel exposure, our approach resulted in an improved protein identification sensitivity and RNA/protein complex isolation accuracy. The method described here is useful for the large scale characterization of RNA- or DNA-protein complexes.     

Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions

Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5–10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.     

Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager.     

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.     

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.     

Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction

The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants. A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction. Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates. For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction. A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given. The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.     

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Mth10b, a unique member of the Sac10b family, does not bind nucleic acid

The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.     

Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II.

A rapid and simple method has been developed which allows detection and isolation of covalent DNA/protein adducts. The method is based upon the use of an ionic detergent, SDS, to neutralize cationic sites of weakly bound proteins thereby resulting in their dissociation off the helix. Proteins tightly or covalently bound to DNA that are not dissociable by SDS, result in the precipitation of the DNA fragment by the addition of KCl; however, free nucleic acid does not precipitate. The method is particularly useful as an analytical tool to titrate the binding of prototypic covalent binding proteins, topoisomerase I and II; thus, quantitation of topoisomerase activity is possible under defined conditions. As an analytical tool the method can be used as a general assay in the purification of as yet unidentified topoisomerases or other activities that bind DNA covalently. Moreover, the technology can be adapted for use in a preparative mode to separate covalent complexes from free DNA in a single step.     

Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA

Helicases are molecular motors that unwind double-stranded DNA or RNA. In addition to unwinding nucleic acids, an important function of these enzymes seems to be the disruption of protein-nucleic acid interactions. Bacteriophage T4 Dda helicase can displace proteins bound to DNA, including streptavidin bound to biotinylated oligonucleotides. We investigated the mechanism of streptavidin displacement by varying the length of the oligonucleotide substrate. We found that a monomeric form of Dda catalyzed streptavidin displacement; however, the activity increased when multiple helicase molecules bound to the biotinylated oligonucleotide. The activity does not result from cooperative binding of Dda to the oligonucleotide. Rather, the increase in activity is a consequence of the directional bias in translocation of individual helicase monomers. Such a bias leads to protein-protein interactions when the lead monomer stalls owing to the presence of the streptavidin block.     

Thermodynamic database for protein-nucleic acid interactions (ProNIT).

MOTIVATION: Protein-nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data. Thus, we have developed a Thermodynamic Database for Protein-Nucleic Acid Interactions (ProNIT). RESULTS: We have collected experimentally observed binding data from the literature. ProNIT contains several important thermodynamic data for protein-nucleic acid binding, such as dissociation constant (K(d)), association constant (K(a)), Gibbs free energy change (DeltaG), enthalpy change (DeltaH), heat capacity change (DeltaC(p)), experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data are integrated into a relational database system together with structural and functional information to provide flexible searching facilities by using combinations of various terms and parameters. A www interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. AVAILABILITY: ProNIT is freely accessible at the URL http://www.rtc.riken.go.jp/jouhou/pronit/pronit.html.     

Effects of a protecting osmolyte on the ion atmosphere surrounding DNA duplexes

Osmolytes are small, chemically diverse, organic solutes that function as an essential component of cellular stress response. Protecting osmolytes enhance protein stability via preferential exclusion, and nonprotecting osmolytes, such as urea, destabilize protein structures. Although much is known about osmolyte effects on proteins, less is understood about osmolyte effects on nucleic acids and their counterion atmospheres. Nonprotecting osmolytes destabilize nucleic acid structures, but effects of protecting osmolytes depend on numerous factors including the type of nucleic acid and the complexity of the functional fold. To begin quantifying protecting osmolyte effects on nucleic acid interactions, we used small-angle X-ray scattering (SAXS) techniques to monitor DNA duplexes in the presence of sucrose. This protecting osmolyte is a commonly used contrast matching agent in SAXS studies of protein-nucleic acid complexes; thus, it is important to characterize interaction changes induced by sucrose. Measurements of interactions between duplexes showed no dependence on the presence of up to 30% sucrose, except under high Mg(2+) conditions where stacking interactions were disfavored. The number of excess ions associated with DNA duplexes, reported by anomalous small-angle X-ray scattering (ASAXS) experiments, was sucrose independent. Although protecting osmolytes can destabilize secondary structures, our results suggest that ion atmospheres of individual duplexes remain unperturbed by sucrose.