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A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions.

Spencer Schreier1,2, Bhanu Prakash Petla1, Tao Lin3

  • 1Department of Agronomy, Horticulture, and Plant Science, South Dakota State University, Brookings, SD, 57007, USA.

Plant Molecular Biology
|October 18, 2019
PubMed
Summary

A new, affordable assay quantifies DNA-protein interaction strength using tagged proteins and SYBR Gold stain. This method reveals distinct binding affinities of auxin response factor (ARF) proteins to DNA, impacting cellular auxin responses.

Keywords:
Auxin response element (AuxRE)Auxin response factor (ARF)DNA binding proteinDNA–protein interaction

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Quantifying DNA-protein interactions is crucial for understanding gene regulation.
  • Existing methods can be expensive, complex, or require specialized reagents.

Purpose of the Study:

  • To develop a simple, accessible, and inexpensive assay for quantifying DNA-protein interaction strength.
  • To utilize this assay to compare the binding affinities of different auxin response factor (ARF) transcription factors to DNA targets.

Main Methods:

  • Developed an assay using affinity resin to capture tagged recombinant proteins (e.g., 6xHis tag).
  • Employed sequential washes and centrifugation to remove non-specific binding.
  • Quantified captured DNA using SYBR Gold nucleic acid stain and fluorescence intensity.

Main Results:

  • The assay accurately quantifies DNA-protein complex amounts, reflecting interaction strength.
  • Demonstrated the assay's utility by comparing binding capacities of different ARF proteins to DNA targets, including AuxRE motifs.
  • Observed distinct comparative affinities of ARF proteins for AuxRE variants.

Conclusions:

  • The developed assay provides a versatile and quantitative method for assessing DNA-protein binding.
  • Specific ARF-DNA binding strengths likely play a role in the intricate cellular auxin response.
  • The assay's compatibility with standard laboratory equipment and reagents makes it broadly applicable.