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

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-molecule manipulation methods often infer protein-DNA interactions from DNA extension changes.
• Directly measuring protein-DNA association typically requires fluorescently labeling the protein, which is challenging.

Purpose of the Study:

• To develop a new, high-throughput method for visualizing unlabelled protein binding on DNA.
• To overcome the limitations of current single-molecule techniques in studying protein-DNA interactions.

Main Methods:

• Utilizing protein-induced fluorescence enhancement (PIFE) on sparsely Cy3-labeled DNA molecules.
• Monitoring DNA length changes under buffer flow in a microfluidic flow cell.
• Visualizing unlabelled protein binding through changes in DNA conformation.

Main Results:

• Successfully visualized unlabelled protein binding on DNA by detecting PIFE.
• Demonstrated a method that is not limited by the low protein concentrations typically required for single-molecule fluorescence imaging.
• Developed a relatively high-throughput assay for studying protein-DNA interactions.

Conclusions:

• The developed approach enables direct visualization of unlabelled protein binding to DNA.
• This method offers a broadly applicable, high-throughput alternative for studying diverse protein-DNA interactions.
• Overcomes the challenge of protein fluorescent labeling in single-molecule studies.