Open AccessOriented Soft DNA Curtains for Single-Molecule Imaging
Author(s) -
Aurimas Kopūstas,
Šaru̅nė Ivanovaitė,
Tomas Rakickas,
Ernesta Pocevičiu̅tė,
Justė Paksaitė,
Tautvydas Karvelis,
Mindaugas Zaremba,
E. Manakova,
Marijonas Tutkus
Publication year - 2021
Publication title -
langmuir
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.042
H-Index - 333
eISSN - 1520-5827
pISSN - 0743-7463
DOI - 10.1021/acs.langmuir.1c00066
Subject(s) - dna , template , biotinylation , biophysics , biomolecule , chemistry , nanotechnology , nucleic acid , molecule , combinatorial chemistry , biochemistry , materials science , biology , organic chemistry
Over the past 20 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 a 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 a 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 by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end 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 the 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 the binding location and position of individual fluorescently labeled proteins on DNA.