A versatile and high-throughput flow-cell system combined with fluorescence imaging for simultaneous single-molecule...

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Flow-cells offer advantages for single-molecule studies but are often underestimated for quantitative applications.
• Accurate force measurement and simultaneous visualization are crucial for understanding biomolecular mechanics.

Purpose of the Study:

• To develop a fully calibrated, gas-pumped flow-cell system for simultaneous single-molecule force measurement and fluorescence imaging.
• To enable precise force application and measurement for studying biomolecular dynamics.

Main Methods:

• Developed a gas-pumped, fully calibrated flow-cell system.
• Integrated fluorescence imaging for simultaneous force measurement and visualization.
• Accounted for hydrodynamic drags for accurate force application and measurement.

Main Results:

• Achieved force measurement up to 100 pN with sub-pN precision, ultra-high stability (<0.01 pN/10 min), and tuning accuracy (~0.04 pN).
• Enabled parallel tracking of hundreds of protein motors and monitoring of biomolecular conformational changes in real time.
• Observed tight association of single-stranded binding (SSB) proteins with ssDNA and investigated reversed transcription.

Conclusions:

• The developed flow-cell system is a versatile, quantitative, and high-throughput tool for single-molecule manipulation and visualization.
• This system provides a foundation for advanced studies in biophysics and molecular biology.
• Enabled new insights into DNA dynamics, transcription, and protein-nucleic acid interactions.