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bioRxiv · 10.1101/2020.06.15.151662

Oriented Soft DNA Curtains for Single Molecule Imaging

Kopustas, A.·Ivanovaite, S.·Rakickas, T.·Poceviciute, E.·Paksaite, J.·Karvelis, T.·Zaremba, M.·Manakova, E.·Tutkus, M.
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Abstract

Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single- or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

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Kopustas, A., Ivanovaite, S., Rakickas, T., Poceviciute, E., Paksaite, J., Karvelis, T., Zaremba, M., Manakova, E., Tutkus, M.. 2020-06-15. Oriented Soft DNA Curtains for Single Molecule Imaging. https://doi.org/10.1101/2020.06.15.151662

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