DNA Curtains technology

DNA–protein interactions are fundamental to biology and play a key role in disease mechanisms and drug development. DNA curtains technology is a high-throughput single-molecule method that enables direct visualization of these interactions.

DNA curtains are formed within microfluidic flow channels, where many individual DNA molecules are organized into parallel arrays. This arrangement allows simultaneous imaging of hundreds of single DNA strands.

By combining single-molecule resolution with high-throughput data acquisition, the method enables quantitative analysis of binding kinetics and provides insight into underlying molecular mechanisms.

Nanofabrication

DNA curtains can be configured as either single- or double-tethered systems, depending on the nanofabrication design.
 In single-tethered DNA curtains, one end of each DNA molecule is anchored to a nanofabricated barrier, allowing the DNA to extend under flow. In double-tethered configurations, both ends of the DNA are immobilized between nanobarriers, resulting in more constrained and defined DNA positioning.

These two geometries provide complementary experimental conditions, enabling the investigation of a broad range of DNA-protein interactions.

Lipid bilayer formation

The surface of the microfluidic flow channel is passivated using liposomes, which spread and form a supported lipid bilayer (SLB). Passivation using an SLB minimizes non-specific binding of proteins to the surface, allowing for reliable imaging of just DNA-protein interactions of interest.

DNA tethering

In both single- and double-tethered systems, DNA is tethered to the SLB via a biotin–streptavidin interaction. By applying a small flow, the tethered DNA molecules migrate toward the nanofabricated barriers, which capture and align the DNA at their leading edges. From this organized arrangement, individual molecules can be extended, visualized, and subjected to controlled biochemical conditions to study DNA–protein interactions of interest.

In double-tethered systems, the free end of the DNA is additionally tethered via a digoxigenin/anti-digoxigenin interaction, enabling imaging of DNA molecules in the absence of flow.

Capturing DNA-protein interactions

DNA Curtains enables real-time single-molecule observation of how proteins search, bind, translocate along, and remodel DNA across many molecules in parallel [1].

The method has been used to directly resolve distinct biological mechanisms, including DNA repair proteins switching between search modes during damage detection [2], and motor proteins moving along DNA and encountering obstacles such as R-loops [3].

It has also been applied to map stepwise target interrogation prior to nuclease cleavage [4], and to reveal genome-organization mechanisms such as protein clustering, RNA-mediated recruitment, and distinct DNA-bound conformational states [5,6].