RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins

Methods Enzymol. 2015:558:465-493. doi: 10.1016/bs.mie.2015.02.007. Epub 2015 May 12.


RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered.

Keywords: Binding affinity; CLIP; Dissociation constant; EMSA; High-throughput sequencing; RNA motif; RNA secondary structure; RNA splicing; Surface plasmon resonance.

MeSH terms

  • Algorithms
  • Base Sequence
  • Binding Sites
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / metabolism
  • Gene Expression Regulation
  • Gene Library
  • Humans
  • Kinetics
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA / chemistry*
  • RNA / genetics
  • RNA / metabolism
  • RNA Recognition Motif Proteins / chemistry*
  • RNA Recognition Motif Proteins / genetics
  • RNA Recognition Motif Proteins / metabolism
  • RNA Splicing Factors
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Repressor Proteins / chemistry*
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Software*
  • Thermodynamics
  • Transcription, Genetic


  • Drosophila Proteins
  • Oligodeoxyribonucleotides
  • RBFOX2 protein, human
  • RNA Recognition Motif Proteins
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • Repressor Proteins
  • Sxl protein, Drosophila
  • RNA