Label‐Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein‐RNA Complexes

Abstract

The electrophoretic mobility shift assay (EMSA) is a well‐established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein–nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (Kd) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol specifically to determine Kd values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modified RNA oligonucleotides. EMSA‐based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein‐RNA interactions, and a troubleshooting guide is provided. © 2018 by John Wiley & Sons, Inc.

Assays and Technologies for Developing Proteolysis Targeting Chimera Degraders

“…In addition, the size of a molecule is not an issue for the ITC assay, whereas the sensitivity of other biophysical techniques such as SPR and bio-layer interferometry (BLI) is largely dependent on the molecular weight of their analytes. ITC has been increasingly [65] used in determining protein-protein [66,67], protein-nucleic acid [68][69][70] and proteinsmall molecule interactions [71,72]. In recent years, ITC has also been applied in profiling the thermodynamic parameters and cooperativities of PROTAC molecules, providing invaluable insights into the interplay between a PROTAC molecule and its target protein and E3 ligase [25,29,34,73,74].…”

Amino Acid Composition in Various Types of Nucleic Acid-Binding Proteins

“…Identification of nucleic acid-binding proteins is one of the most important tasks in molecular biology. Currently, nucleic acid-binding proteins can be identified and further characterized by several experimental techniques, including pull-down assays [ 2 , 3 ], yeast one-hybrid system [ 4 , 5 ], electrophoretic mobility shift assays [ 6 , 7 ], chromatin immunoprecipitation [ 8 , 9 ], and by other specialized techniques [ 10 , 11 ]. However, it is time-consuming and expensive to identify nucleic acid-binding proteins by experimental approaches [ 12 ].…”

Physico-chemical principles of HDL-small RNA binding interactions

“…Based on HDL titrations, the binding affinity of HDL to tDR-GlyGCC-30-AF546 was calculated ( Fig. S2B ), as previously described [32], and results from EMSA confirmed that HDL harbors strong affinity towards tDR-GlyGCC-30. These results support that HDL preferentially bind to ssRNAs and possess strong affinity to tDRs based on length and sequence.…”

“…EMSA were performed as previously described [32]. Briefly, AF546-tDR-GlyGCC-30 (1µM) was incubated with HDL (serial dilutions) and crosslinked (UV 254nm) for 15min on ice.…”