High‐throughput single‐molecule imaging system using nanofabricated trenches and fluorescent DNA‐binding proteins

DNA curtain is a high‐throughput system, integrating a lipid bilayer, fluorescence imaging, and microfluidics to probe protein–DNA interactions in real‐time and has provided in‐depth understanding of DNA metabolism. Especially, the microfluidic platform of a DNA curtain is highly suitable for a biochip. In the DNA curtain, DNA molecules are aligned along chromium nanobarriers, which are fabricated on a slide surface, and visualized using an intercalating dye, YOYO‐1. Although the chromium barriers confer precise geometric alignment of DNA, reuse of the slides is limited by wear of the barriers during cleaning. YOYO‐1 is rapidly photobleached and causes photocleavage of DNA under continuous laser illumination, restricting DNA observation to a brief time window. To address these challenges, we developed a new nanopatterned slide, upon which carved nanotrenches serve as diffusion barriers. The nanotrenches were robust under harsh cleaning conditions, facilitating the maintenance of surface cleanliness that is essential to slide reuse. We also stained DNA with a fluorescent protein with a DNA‐binding motif, fluorescent protein–DNA binding peptide (FP–DBP). FP–DBP was slowly photobleached and did not cause DNA photocleavage. This new DNA curtain system enables a more stable and repeatable investigation of real‐time protein–DNA interactions and will serve as a good platform for lab‐on‐a‐chip. We develop a new DNA curtain platform, which is a high‐throughput single‐molecule imaging system, using nanotrenches and fluorescent protein‐DNA binding peptides (FP‐DBP). Nanotrenches, which are carved on the slides, not only improve the optical properties for fluorescence imaging but also allow us to clean the slides under harsh conditions, facilitating permanent reuse. The FP‐DBPs dramatically elongate the measurement time period due to slow photobleaching and no photocleavage, and the reversible binding of FP‐DBPs to DNA according to pH or ionic strength do not disturb investigation of protein‐DNA interactions.