DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Abstract

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

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

“…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.…”

“…Continuous real-time monitoring of these dynamic interactions typically requires immobilizing one of interacting partners on a surface, after which they can be visualized in great detail through force spectroscopy, fluorescence, Förster resonance energy transfer (FRET), atomic force microscopy (AFM), nanopores, and DNA flow-stretch assays. 11 Single molecule FRET (smFRET) approaches also work well for high-throughput format studies of many protein-DNA interactions 12 including CRISPR-Cas 13,14 , SSB protein 15 , RecA 16 , DNA polymerase 17,18 , RNA polymerase 19,20 and other proteins [21][22][23] . The smFRET experiments can follow different study designs including: A) both dyes of a FRET pair on a single DNA fragment, 24 B) one dye on DNA and the other on protein, 8 and C) both dyes on a single protein.…”

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.…”

“…We believe that this dsDNA labeling approach could prove useful for DNA Curtains experiments 44 , 49 − 52 and other DNA stretch assays. 17 , 53 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.…”

“…Alternatively, structural methods, such as X-ray crystallography or cryo-electron microscopy, are extremely precise and provide atom-level resolution, but they only provide snapshots of molecules at different stages of complex formation. Continuous real-time monitoring of dynamic protein–DNA interactions can be performed upon tethering molecules of interest through force spectroscopy, fluorescence, Förster resonance energy transfer (FRET), atomic force microscopy (AFM), nanopores, and DNA flow-stretch assays . Single-molecule FRET (smFRET) approaches work especially well for high-throughput studies of protein–DNA interactions including CRISPR-Cas, , SSB protein, RecA, DNA polymerase, , RNA polymerase, , and other proteins. − The smFRET experiments can employ different designs, including (A) both dyes of a FRET pair linked to a single DNA fragment, (B) one dye on the DNA and the other on the protein, and (C) both dyes on a single protein .…”