Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies
| Autor: | Aurimas Kopu Stas, Marijonas Tutkus, Oskaras Venckus, Mindaugas Zaremba, Ramunas Valiokas, Šaru Nė Ivanovaitė, Elena Manakova, Vytautas Navikas, Tomas Rakickas |
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| Rok vydání: | 2019 |
| Předmět: |
Streptavidin
Materials science Microfluidics Immobilized Nucleic Acids Biotin Nanotechnology 02 engineering and technology 010402 general chemistry 01 natural sciences Proof of Concept Study Soft lithography chemistry.chemical_compound Molecular recognition Lab-On-A-Chip Devices Electrochemistry General Materials Science Protein–DNA interaction Organic Chemicals Deoxyribonucleases Type II Site-Specific Spectroscopy Fluorescent Dyes Total internal reflection fluorescence microscope Surfaces and Interfaces DNA 021001 nanoscience & nanotechnology Condensed Matter Physics 0104 chemical sciences Restriction enzyme Template chemistry Microscopy Fluorescence 0210 nano-technology Protein Binding |
| Zdroj: | Langmuir : the ACS journal of surfaces and colloids. 35(17) |
| ISSN: | 1520-5827 |
| Popis: | The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices. |
| Databáze: | OpenAIRE |
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