High‐throughput single‐molecule imaging system using nanofabricated trenches and fluorescent DNA‐binding proteins
| Autor: | Na Young Cheon, Ja Yil Lee, Hyung‐il Kim, Kang O. Kim, Yujin Kang, Jongjin Cha, Ayoung Kim, Luda Lee, Kyubong Jo |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
0301 basic medicine Fluorescence-lifetime imaging microscopy Materials science Lipid Bilayers Microfluidics Bioengineering Nanotechnology 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology A-DNA Lipid bilayer Biochip Fluorescent Dyes Benzoxazoles Quinolinium Compounds Optical Imaging DNA Single Molecule Imaging Fluorescence Nanostructures DNA-Binding Proteins 030104 developmental biology chemistry Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 117:1640-1648 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.27331 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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