Driving Forces for Single DNA Stretching Assessed by In Situ TIRFM

Herein, single DNA molecule stretching in a hydrodynamic flow was monitored by in situ total internal reflection fluorescence microscopy (TIRFM). Different driving forces for DNA stretching were assessed systematically. The binding force between the substrate and the DNA molecule significantly affected the dynamic stretching behavior of coiled DNA, where DNA stretched only on the substrate modified by -NH₂ due to electrostatic adsorption. Proper flow force from the fluid was favorable for DNA stretching, which was in stark contrast to DNA stretching restricting at a lower flow rate or washing off DNA molecules at a higher flow rate of fluid. Moreover, DNA stretching was restricted in low pH solution but improved in high pH solution. This is due to the confrontation between contractive and repulsion forces in coiled DNA molecules in different pH, which is related to the Donnan equilibrium in a single condensed DNA molecule. Additionally, external Na⁺ would break this Donnan equilibrium, leading to a restriction for DNA stretching. This study will guide quantitative dynamics studies of DNA stretching in the future.