A force-based, parallel assay for the quantification of protein-DNA interactions

PLoS One. 2014 Feb 27;9(2):e89626. doi: 10.1371/journal.pone.0089626. eCollection 2014.

Abstract

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • DNA / metabolism*
  • DNA-Binding Proteins / metabolism*
  • High-Throughput Screening Assays / methods*
  • Protein Binding
  • Transcription Factors / metabolism
  • Zinc Fingers

Substances

  • DNA-Binding Proteins
  • Transcription Factors
  • DNA

Grants and funding

Funding provided by German Science Foundation SFB 1032-A01 and a European Research Council Advanced Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.