Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level

Abstract

The fluorescence microscopy has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb λ DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified λ DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast

“…Single-molecule experiments were performed in the flow cell. Flow cell assembly and λDNA biotinylation were modified from our existing procedure ( 90 ). Streptavidin of 10 µL (0.5 mg/mL, Sigma-Aldrich) was injected into the flow cell and incubated for 2–3 minutes at room temperature.…”

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

“…All of these approaches not only enable the surface immobilization of individual DNA molecules, but they also passivate the exposed surface of a glass coverslip against non-specific biomolecules adsorption. PEGylation of the surface is the most widely utilized procedure in these kinds of assays, as the glass surface is at first functionalized with methoxy-PEG and biotin-PEG molecules and then streptavidin is deposited on such a surface to allow the immobilization of a biotinylated DNA [45,46]. Upon the application of a buffer flow, single-tethered DNA molecules are stretched along the surface due to the hydrodynamic force.…”