switchSENSE: A new technology to study protein-RNA interactions

Methods. 2017 Apr 15;118-119:137-145. doi: 10.1016/j.ymeth.2017.03.004. Epub 2017 Mar 9.

Abstract

Characterization of RNA-binding protein interactions with RNA became inevitable to properly understand the cellular mechanisms involved in gene expression regulation. Structural investigations bring information at the atomic level on these interactions and complementary methods such as Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are commonly used to quantify the affinity of these RNA-protein complexes and evaluate the effect of mutations affecting these interactions. The switchSENSE technology has recently been developed and already successfully used to investigate protein interactions with different types of binding partners (DNA, protein/peptide or even small molecules). In this study, we show that this method is also well suited to study RNA binding proteins (RBPs). We could successfully investigate the binding to RNA of three different RBPs (Fox-1, SRSF1 and Tra2-β1) and obtained KD values very close to the ones determined previously by SPR or ITC for these complexes. These results show that the switchSENSE technology can be used as an alternative method to study protein-RNA interactions with KD values in the low micromolar (10-6) to nanomolar (10-7-10-9) and probably picomolar (10-10-10-12) range. The absence of labelling requirement for the analyte molecules and the use of very low amounts of protein and RNA molecules make the switchSENSE approach very attractive compared to other methods. Finally, we discuss about the potential of this approach in obtaining more sophisticated information such as structural conformational changes upon RBP binding to RNA.

Keywords: Interaction; K(D); Kinetics; Protein; RNA; switchSENSE.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Calorimetry / methods
  • DNA, Single-Stranded / genetics*
  • DNA, Single-Stranded / metabolism
  • Humans
  • Kinetics
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Nucleic Acid Hybridization / methods*
  • Protein Array Analysis / instrumentation
  • Protein Array Analysis / methods*
  • Protein Binding
  • RNA / genetics*
  • RNA / metabolism
  • RNA Splicing Factors / genetics
  • RNA Splicing Factors / metabolism
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Serine-Arginine Splicing Factors / genetics
  • Serine-Arginine Splicing Factors / metabolism
  • Surface Plasmon Resonance / methods
  • Thermodynamics
  • Transcription, Genetic

Substances

  • DNA, Single-Stranded
  • Nerve Tissue Proteins
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • SRSF1 protein, human
  • TRA2B protein, human
  • Serine-Arginine Splicing Factors
  • RNA