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

조회수 : 429 등록일 : 2020.03.30 00:00

Biotechnology and Bioengineering
Jo, Kyubong 2020.03.12

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

Kang, Y (Kang, Yujin)1 ] ; Cheon NY (Cheon, Na Young )1 ];  Cha, J (Cha, Jongjin)[ 2  ]
Kim, A (Kim, Ayoung)1 ] ; Kim, H (Kim, Hyung-il)[ 3 ] ; Lee, L (Lee, Luda)[ 3 ] ; Kim, KO (Kim, Kang O)[ 3 ] ; Jo, K (Jo, Kyubong)[ 4 ] ; Lee, JY (Lee, Ja Yil)[ 1.5 ] 

[1] School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, 44919, Republic of Korea 
[2] Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea 
[3] UNIST Central Research Facilities (UCRF), Ulsan National Institute of Science and Technology, Ulsan, 44919, Republic of Korea 
[4] Department of Chemistry and Integrated Biotechnology, Sogang University, Seoul, 04107, Republic of Korea 
[5] Center for Genomic Integrity, Institute for Basic Science, Ulsan, 44919, Republic of Korea † These authors contributed equally to this work

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 DNA curtain is highly suitable for a biochip. In the DNA curtain, DNA molecules are aligned along chromium nano-barriers, 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 nano-patterned slide, upon which carved nano-trenches serve as diffusion barriers. The nano-trenches 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, FP-DBP (Fluorescent Protein-DNA Binding Peptide). FPDBP was slowly photobleached and did not cause DNA photocleavage. This new DNA curtain system enables more stable and repeatable investigation of real-time protein-DNA interactions and will serve as a good platform for lab-on-a-chip.

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