A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

Abstract

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-λ-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to >80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

Single-particle trajectories reveal two-state diffusion-kinetics of hOGG1 proteins on DNA

“…We finally conclude that more single-molecule tracking measurements should be done, with hOGG1 on DNA and with other diffusers. New experimental protocols ease large-scale single-molecule data collection ( 41 ), and optimized tools of analysis are now available and promise novel insights, as demonstrated here with old, suboptimal data. The increased resolution and much higher throughput will enable rigorous exploration of a larger range of pH and salt concentrations and may help integrate results from single-molecule and ensemble experiments.…”

“…Flow-stretch assays like the one studied here and in ( 41 ) trade the precision of optical tweezers for a simpler experimental setup and the possibility to record on multiple DNA molecules in parallel, providing much more data. Here we recommend to use a strong flow to stretch the DNA strands in order to drive their fluctuations as fast as possible relative to the time-lapse of recordings, while assuring that this does not lead to drift of the attached proteins.…”

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

“…Given that long DNA molecules are tethered and stretched along the surface, it then becomes really convenient to visualize and track the movement of fluorescently labeled proteins of interest along the DNA at the SM level in real time [56]. This also allows one to determine protein-binding profiles on DNA molecules and dwell times, assess the preferential binding sites of the protein, and thoroughly characterize the real-time dynamics of the ongoing individual protein-DNA interactions [57]. Nevertheless, long DNA molecules are more prone to self-entanglement into knots, especially under the conditions of a rapid linearization force, which can induce the formation of DNA knots [58].…”

Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

“…Preparation of flow channels and tethering and stretching of dsDNA by laminar flow was carried out according to previously published methods 12,21 . The 49 kB -DNA was used for λ sliding experiments and a 7kb DNA with an Osym operator in the middle and Cy3 fluorophores in each end (created by PCR) was used for operator binding experiments.…”