Single-molecule DNA-flow stretching assay as a versatile hybrid tool for investigating DNA-protein interactions

Abstract

Single-molecule techniques allow researchers to investigate individual molecules and obtain unprecedented details of the heterogeneous nature of biological entities. They play instrumental roles in studying DNA-protein interactions due to the ability to visualize DNA or proteins and to manipulate individual DNA molecules by applying force or torque. Here, we describe single-molecule DNA-flow stretching assays as hybrid tools that combine forces with fluorescence. We also review how widely these assays are utilized in elucidating working mechanisms of DNA-binding proteins. Additionally, we provide a brief explanation of various efforts to prepare DNA substrates with desired internal protein-binding sequences. More complicated needs for DNA-protein interaction research have led to improvements in single-molecule DNA flow-stretching techniques. Several DNA flow-stretching variants such as DNA curtain, DNA motion capture assays, and protein-induced fluorescence enhancement (PIFE) are introduced in this mini review. Singlemolecule DNA flow-stretching assays will keep contributing to our understanding of how DNA-binding proteins function due to their multiplexed, versatile, and robust capabilities. [BMB Reports 2025; 58(1): 41-51].

Fig. 1

Single-molecule DNA flow-stretching assay. (A) Schematic of the assay. Laser light is incident at the critical angle, generating the evanescent field. A syringe pump pulls a syringe plunger to generate flow. Air spring dampens high-frequency flow fluctuations. (B) A biotinylated DNA is tethered to the surface of a microfluidic flow cell through streptavidin-biotin interaction. Flow stretches the DNA. QD: quantum dot. (C) Images of flow-stretched single-tethered lambda DNA. While DNA-intercalated sytox orange dye visualizes the entire length of DNA (left), quantum dot-tagged DNA allows visualization of only the untethered DNA end (right). Flow direction: upward. Each figure: 3.04 × 14.4 μm. (D) Various ways to minimize or get around nonspecific DNA/protein surface adsorption. From left: adsorption of surface blocking proteins such as casein and bovine serum albumin (BSA), surface chemistry via polyethylene glycol (PEG), formation of lipid bilayer, and constructing DNA skybridge. Small molecules such as biotins were not shown for simplicity. All figures in Figure 1: not drawn to scale.

Fig. 2

Overview of DNA curtain assay. (A) Lipid bilayers formed around streptavidin passivate the surface of a microfluidic flow cell. Applying flow leads to DNA stretching while streptavidin remains still. (B) In a DNA curtain assay, a lipid-bound DNA migrates in the same direction as the buffer flow direction until it is stopped by a diffusion barrier. (C) The top view of flow-stretched singly-tethered DNA molecules shown in (B). (D) A double-tethered DNA in a DNA curtain assay. One end of the biotinylated DNA is tethered to streptavidin and the other digoxigenin-tagged DNA end binds to an anti-digoxigenin antibody on a pedestal. (E) DNA curtain assay with single-stranded (ss) DNA produced by a rolling circle replication. For efficient ssDNA stretching, fluorescently labeled replication protein A (RPA) can be introduced. All figures in Figure 2: not drawn to scale.

Fig. 3

DNA motion capture assay and protein-induced fluorescence enhancement (PIFE). (A) Under laminar flow, DNA experiences differential tension. Thus, different parts of the DNA exhibit different degrees of stretching. (B) In the DNA motion capture assay, five quantum dots are labeled at specific positions on λ-DNA. Upon flowing proteins, DNA bridgers and benders show different DNA compaction patterns. (C) When a protein is located in the vicinity of Cy3 dye, the fluorescence intensity increases. This phenomenon is called protein-induced fluorescence enhancement (PIFE). (D) PIFE can be combined with a single-molecule DNA flow-stretching assay. Here, Cy3 dyes are sparsely labeled on a DNA and proteins are introduced by flow. The example graph represents changes in DNA end-to-end length and integrated Cy3 intensity on the DNA over time.