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.

/pdf/electrophoretic-mobility-shift-assay-emsa-for-detecting-1asq4y5oju.pdf

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

Detection of double-stranded RNAs (dsRNAs) is a central mechanism of innate immune defense in many organisms. We here discuss several families of dsRNA-binding proteins involved in mammalian antivi...

Double-Stranded RNA Sensors and Modulators in Innate Immunity.

Molecular patterns in pathogenic RNAs can be recognized by the innate immune system, and a component of this response is the interferon-induced enzyme RNA-activated protein kinase (PKR). The major activators of PKR have been proposed to be long double-stranded RNAs. We report that RNAs with very limited secondary structures activate PKR in a 5'-triphosphate-dependent fashion in vitro and in vivo. Activation of PKR by 5'-triphosphate RNA is independent of RIG-I and is enhanced by treatment with type 1 interferon (IFN-alpha). Surveillance of molecular features at the 5' end of transcripts by PKR presents a means of allowing pathogenic RNA to be distinguished from self-RNA. The evidence presented here suggests that this form of RNA-based discrimination may be a critical step in mounting an early immune response.

https://science.sciencemag.org/content/sci/318/5855/1455.full.pdf

5'-Triphosphate-Dependent Activation of PKR by RNAs with Short Stem-Loops

Mechanism of PKR Activation by dsRNA.

Mechanism of PKR Activation: Dimerization and Kinase Activation in the Absence of Double-stranded RNA

Global analysis of non-specific protein–nucleic interactions by sedimentation equilibrium

The dsRNA-activated protein kinase (PKR) plays a major role in the cellular response to viral infection. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. The dsRBD consists of two tandem copies of a conserved double-stranded RNA binding motif, dsRBM1 and dsRBM2. dsRNA binding is believed to activate PKR by inducing dimerization and subsequent autophosphorylation reactions. We have characterized the function of the dsRBD by assessing the binding of dsRBM1 and dsRBD to a series of dsRNA sequences ranging from 15 to 45 bp. For dsRBM1, the binding stoichiometries agree with an overlapping ligand binding model where the motif binds to multiple faces of the dsRNA duplex and overlaps along the helical axis. Similar behavior is observed for a dsRBD containing both dsRBM1 and dsRBM2 for sequences up to 30 bp; however, the binding affinity is enhanced 30-fold. Longer dsRNA sequences exhibit lower-than-expected stoichiometries, indicating a change in binding mode. NMR spectrosc...

Mechanism of interaction of the double-stranded RNA (dsRNA) binding domain of protein kinase R with short dsRNA sequences.

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Global analysis of non-specific protein–nucleic interactions by sedimentation equilibrium

Mechanism of interaction of the double-stranded RNA (dsRNA) binding domain of protein kinase R with short dsRNA sequences.