Oriented Soft DNA Curtains for Single-Molecule Imaging

Abstract

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.

Advancing Understanding of DNA-BfiI Restriction Endonuclease Cis and Trans Interactions through smFRET Technology

“…While the Argonaute protein can be loaded with a guide molecule of desired sequence (thus allowing more controlled labeling), the dsDNA substrate should contain at least one BfiI target sequence for successful labeling. We believe that this dsDNA labeling approach could prove useful for DNA Curtains experiments 38,[42][43][44][45] and other DNA stretch-assays. 11,46 Besides dsDNA labeling, it is likely that our developed assay will be useful for other two targets-binding DNA-interacting proteins.…”

smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease

“…While the Argonaute protein, employed in the protein-assisted PAINT approach, can be loaded with a guide molecule of desired sequence (thus allowing more controlled labeling), the dsDNA substrate should contain at least one BfiI target sequence for successful labeling. We believe that this dsDNA labeling approach could prove useful for DNA Curtains experiments ,− and other DNA stretch assays. , Besides dsDNA labeling, it is likely that our developed assay will be useful for mechanistic studies of target search mechanisms and interaction dynamics of other important DNA binding proteins such as transposon systems or even artificially formed dimers of nucleases.…”

The miEye: Bench-top super-resolution microscope with cost-effective equipment

“…Both excitation schemes work well for SMLM super-resolution imaging, whether in vitro or in cells. The only exception is that the TIRF scheme works better for DNA-PAINT [35] or similar methods [36] , [37] , [38] aiming to visualize reversibly binding fluorescent molecules while containing diffusing fluorescent molecules in solution.…”