A general approach to visualize protein binding and DNA conformation without protein labelling.

Dan Song,Thomas G W Graham,Joseph J Loparo

doi:10.1038/ncomms10976

Abstract

Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein–DNA interactions.

Highlights

Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate
Protein–DNA association is detected in the absence of protein labelling by exploiting protein-induced fluorescence enhancement (PIFE) of fluorescently labelled DNA that occurs on protein binding
20 kb double-stranded DNA (dsDNA) that were sparsely labelled with Cy3 dyes were immobilized at one end to a functionalized glass coverslip in a microfluidic flow cell (Fig. 1a)

Summary

Introduction

Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Ensemble approaches including surface plasmon resonance, white light interferometry and electrophoretic mobility shift assay are capable of measuring protein–DNA binding but are not sensitive to how these proteins change DNA conformation For this reason, single-molecule nanomanipulation methods have been widely used to study how DNA-binding proteins remodel DNA5. Our approach is a relatively simple yet a highly quantitative method for characterizing complex protein–DNA interactions on kilobase length scales

Methods

Results

Conclusion