A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

Sci Rep. 2016 Dec 23;6:39653. doi: 10.1038/srep39653.

Abstract

Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anisotropy
  • DNA / chemistry
  • DNA Repair
  • DNA Replication
  • Deoxyribonuclease BamHI / metabolism
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Humans
  • Ions
  • Ku Autoantigen / chemistry
  • Microscopy, Fluorescence
  • Nucleic Acids / chemistry*
  • Protein Binding
  • Protein Biosynthesis
  • Proteins / chemistry*
  • RNA / chemistry
  • Spectrometry, Fluorescence*
  • Transcription, Genetic

Substances

  • Fluorescent Dyes
  • Ions
  • Nucleic Acids
  • Proteins
  • RNA
  • DNA
  • Deoxyribonuclease BamHI
  • Ku Autoantigen