Driving Forces for Single DNA Stretching Assessed by In Situ TIRFM

Area of Science:

• Biophysics
• Molecular Biology
• Physical Chemistry

Background:

• Understanding DNA molecule dynamics is crucial for molecular biology and nanotechnology.
• Single-molecule manipulation techniques provide insights into DNA behavior under various conditions.

Purpose of the Study:

• To systematically investigate the effects of different driving forces on single DNA molecule stretching.
• To elucidate the influence of substrate binding, hydrodynamic flow, pH, and ionic strength on DNA stretching dynamics.

Main Methods:

• Utilized in situ total internal reflection fluorescence microscopy (TIRFM) to monitor single DNA molecule stretching.
• Applied controlled hydrodynamic flow and varied substrate modifications, pH, and sodium ion (Na+) concentrations.

Main Results:

• DNA stretching was observed only on amine (-NH2)-modified substrates due to electrostatic adsorption.
• Optimal fluid flow facilitated DNA stretching, while low or high flow rates hindered or removed DNA.
• Low pH restricted DNA stretching, whereas high pH improved it, linked to Donnan equilibrium.
• External Na+ ions disrupted the Donnan equilibrium, restricting DNA stretching.

Conclusions:

• Substrate modification, fluid dynamics, pH, and ionic strength are critical parameters controlling single DNA molecule stretching.
• The findings provide a foundation for future quantitative studies on DNA dynamics.