Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Abstract

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

“…Due to the flow-stretched DNA molecules being in the vicinity of the glass surface, DNA flow-stretch assays are perfectly compatible with total internal reflection fluorescence (TIRF) microscopy. This assay enables the selective visualization of protein-DNA interactions at the SM level with a low background even when fluorescently labelled protein is diffusing in a solution [53]. Individual DNA molecules are usually labeled with fluorescent intercalating dyes, as this makes it possible to observe the full contour length of the extended DNA.…”