DNA curtain is a single-molecule imaging technique used to visualize protein–DNA interactions in real time with high-throughput. This method arranges long double-stranded or single-stranded DNA molecules on a supported lipid bilayer within a microfluidic chamber, where hydrodynamic flow aligns them against nanofabricated diffusion barriers. Traditional single-molecule techniques typically observe one molecule at a time, which limits throughput. In contrast, DNA curtains allow hundreds to thousands of DNA molecules to be monitored simultaneously using total internal reflection fluorescence microscope (TIRFM). This high-throughput capability enables direct observation of how proteins interact with DNA, translocate along it, and search for target sequences.
History The DNA curtains technique was first introduced by Eric C. Greene and colleagues at Columbia University. Early methods used supported lipid bilayers and nanofabricated diffusion barriers to align long DNA molecules into parallel arrays for high-throughput single-molecule imaging. Subsequent work incorporated nanoscale diffusion barriers, and Greene’s group later standardized DNA curtain protocols for TIRFM. During this period, double-tethered DNA curtains were developed, allowing DNA molecules to remain extended without continuous buffer flow. In 2012, single-stranded DNA curtains were introduced, extending the technique to ssDNA substrates. In 2015, Gallardo at Cornell University introduced a photolithography-based method for large-scale fabrication of chromium diffusion barriers, allowing the assembly of hundreds of DNA arrays within a single flow cell for high-throughput single-molecule studies. In 2021, Kopuštas and co-workers introduced Soft DNA Curtains, a lithography-free method.
DNA curtains have continuously evolved through improvements in nanofabrication, surface patterning, and tethering strategies. The technique has become a widely used high-throughput platform for observing DNA–protein interactions across diverse biological systems.
Principles Flow cell assembly
DNA curtain experiment typically begins with the assembly of a microfluidic flow cell composed of a fused-silica slide and a borosilicate glass coverslip. A narrow channel is formed between the two surfaces using double-sided adhesive tape, which creates the internal chamber for buffer and sample exchange. Inlet and outlet ports are glued over the drilled holes in the flow cell, and syringes are attached to these ports to support bilayer deposition. This configuration creates a stable, sealed, and optically clear microfluidic environment that is well suited for supported lipid bilayer formation, DNA tethering, and regulated buffer flow.
Diffusion barriers Diffusion barriers play an important role in the DNA curtains technique by organizing lipid-tethered DNA molecules into orderly arrays, stopping each strand at one end as it diffuses across the membrane. The barrier patterns can be designed in various shapes depending on the experimental requirements. Early versions included scratched quartz barriers, later replaced by chromium-based diffusion barriers. Zig-zag designs became standard because they prevent lateral diffusion. Discontinuous patterns were later introduced to improve the ability to distinguish individual DNA molecules, particularly at higher DNA densities. Regardless of design, the barriers function to halt the lateral diffusion of DNA within the supported lipid bilayer. Under hydrodynamic flow, tethered DNA migrates until it encounters the barrier, where it becomes trapped and extended. By controlling DNA concentration, individual molecules can be isolated within each barrier feature.
Lipid bilayer formation Supported lipid bilayers (SLBs) provide a passivated and fluid surface for organizing tethered DNA molecules. The bilayer is typically formed by adsorbing vesicles onto the glass flow cell surface. It creates a membrane-like layer that minimizes nonspecific adsorption of proteins and nucleic acids. Biotinylated DNA is attached to the SLB through biotin–streptavidin binding and can diffuse laterally within the two-dimensional lipid layer while remaining close to the surface for imaging. Under hydrodynamic flow, the tethered DNA moves across the bilayer until it reaches a diffusion barrier, where it becomes aligned and extended. These properties of SLBs make it versatile and effective platform for organizing DNA molecules in real-time single-molecule experiments. DNA tethering DNA molecules are anchored to a supported lipid bilayer for controlled imaging and observation. In DNA curtains, both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) can be used. Lambda phage DNA is commonly used as a dsDNA substrate because it is commercially available and contains regions with varying adenine-thymine (AT) content, allowing studies of sequence-dependent protein binding. For ssDNA curtains, long single-stranded molecules are produced via rolling circle replication. Tethered DNA remains near the surface and diffuses within the lipid bilayer, facilitating alignment at diffusion barriers. This enables real-time observation of protein–DNA interactions.
Visualization Visualization of proteins in DNA curtains experiments is achieved by labeling them with an inorganic dye, attaching quantum dots, or creating a fusion protein that links the protein of interest to a fluorescent tag. DNA curtains containing dsDNA are visualized using intercalating dyes such as YOYO-1, while curtains containing ssDNA are typically visualized using replication protein A–eGFP. Once the DNA curtains are formed, the labeled proteins are introduced into the microfluidic flow cell and both proteins and DNA molecules are simultaneously imaged with TIRFM. This method allows for direct, time-resolved observation of protein–DNA interactions, including diffusion, binding events, and target search processes.
Imaging Total internal reflection fluorescence microscopy (TIRFM) is widely used in DNA curtain experiments to visualize fluorescently labeled DNA molecules and their associated proteins at single-molecule resolution. In TIRFM, a laser beam is directed through a microscope slide and reflected at an angle exceeding the critical angle, generating an evanescent electromagnetic wave at the interface between the slide and the aqueous buffer. This wave penetrates a few hundred nanometers into the sample, producing a highly restricted excitation volume that significantly reduces background fluorescence. DNA curtain assays commonly use prism-based TIRFM. This optical configuration enables real-time imaging of nanofabricated DNA arrays and supports detailed analysis of protein–DNA interactions under physiologically relevant conditions. Procedure In DNA curtains, the surface of a flow cell is coated with a supported lipid bilayer (SLB), with a small fraction of lipids functionalized with biotin. The bilayer is then treated with neutravidin or streptavidin, which allows biotin-labeled DNA molecules to attach. Because the lipid bilayer is fluid, the DNA can move freely in two dimensions. When a hydrodynamic flow is applied, the DNA is directed toward lipid diffusion barriers on the surface. These barriers, positioned perpendicular to the flow, prevent lateral movement of the lipids, causing the DNA to accumulate along the leading edges of the barriers. Under continuous flow, hundreds or even thousands of DNA molecules align in parallel and stretch across the surface. This arrangement enables high-resolution imaging of DNA using total internal reflection fluorescence microscopy (TIRFM).
Types of DNA Curtains Conventional DNA curtains
Single-tethered DNA curtains
This is the earliest and simplest configuration used in DNA curtain experiments. In single-tethered curtains, each DNA molecule is attached by one end to a supported lipid bilayer through a biotin–streptavidin linkage, while the rest of the DNA extends freely into the solution. Because each molecule is anchored only at one end, continuous flow is required to maintain extension. When the flow is stopped, the DNA recoils and diffuses away from the surface. The main advantages of this configuration are its simplicity and rapid assembly, making it well-suited for experiments examining binding and dissociation kinetics, such as transient protein–DNA interactions under applied tension. Double-tethered DNA Curtains
This configuration was developed to overcome the limitation of continuous buffer flow required in single-tethered DNA curtains. In double-tethered curtains, both ends of each DNA molecule are attached to the surface, typically one end to a supported lipid bilayer through a biotin–streptavidin linkage, and the other end is anchored to a fixed pedestal coated with anti-digoxigenin antibodies that bind to a digoxigenin (DIG) tag on the DNA. Once both ends are anchored, the DNA remains stretched and visible even when the buffer flow is stopped, allowing the entire DNA length to be imaged without hydrodynamic tension. This feature is particularly useful for experiments that require flow-free observation, such as those involving costly reagents or proteins sensitive to shear forces. Single-stranded DNA Curtains
ssDNA curtains are a specialized configuration of the DNA curtain platform designed for visualizing long single-stranded DNA (ssDNA) molecules at the single-molecule level. Long ssDNA substrates are generated by rolling circle replication. The resulting ssDNA molecules are anchored to a supported lipid bilayer through biotin–streptavidin linkages, leaving the rest of each molecule free in solution. ssDNA curtains have been applied to study numerous DNA-processing proteins and complexes. This method has provided key insights into the mechanisms of presynaptic complex assembly, filament remodeling, homology search, and regulation of recombination intermediates.
Spatially patterned DNA curtains
Parallel array of double-tethered isolated DNA curtains Parallel Array of Double-Tethered Isolated (PARDI) patterns are microfabricated structures used to produce sparsely arranged DNA curtains for single-molecule studies. This configuration minimizes interactions between neighboring strands and maintains a low local DNA concentration. The large spacing between molecules reduces steric and kinetic interference, enabling direct observation of individual protein binding events. Thus, this configuration is well suited for studying protein–DNA interactions, particularly protein association kinetics, under conditions with controlled, low DNA concentration.
Crisscrossed DNA curtains Crisscrossed DNA curtains are a specialized arrangement of double-tethered DNA molecules designed to generate controlled regions of locally high DNA concentration. In this configuration, individual DNA strands are organized so that they intersect at approximately 90° angles. Crisscrossed DNA curtains have been used to study how proteins move between two closely spaced DNA substrates, providing a controlled system for studying inter-DNA transfer and dynamic protein exchange.
U-shaped DNA curtains U-shaped DNA curtains represent a more complex configuration in which both ends of individual DNA molecules are initially immobilized on a supported lipid bilayer through biotin–streptavidin linkages. When hydrodynamic buffer flow is applied, the DNA bends into a “U” shape, and the double-tethered molecules align along zigzag barriers that restrict lipid diffusion. U-shaped curtains are especially useful for studying DNA compaction mechanisms.
Advanced DNA curtains Suspended DNA curtains