Single-molecule level DNA-protein interaction studies

DNA-protein interactions play a key role in many biological processes. One unifying aspect of these interactions is target search process. It involves different mechanisms that are different for each protein, but can be separated into these classes: 3D diffusion (i.e., jumping), sliding, and hopping. Those interactions can differ in number of DNA target sites that protein can bind. DNA-protein interactions can occur, when one protein interacts with two targets either on a single DNA molecule (i.e. in Cis interaction) or on two separate molecules (i.e. in Trans interaction).

DNA-protein interaction occurs on a nanometer length scale at millisecond-to-second timescales. To monitor interactions occurring on the seconds time scale surface immobilization of one of interacting partners is often required. These interactions can be visualized at great detail through: force spectroscopy, fluorescence, Forster resonance energy transfer (FRET), atomic force microscopy (AFM), nanopores, DNA flow-stretch assays, etc [A. Kopūstas, et al. Applied Nano, 2022]. Single-molecule FRET (smFRET) approach worked very well for many different protein-DNA interaction studies.

Restriction endonucleases (REases) in bacteria and archaea function as defense against invading foreign DNA systems. Over the past 10 years we studied different REases using smFRET approach: Ecl18kI protein [M. Tutkus, et al. Biophysical J. 2017] , and NgoMIV [M. Tutkus, et al. Biopolymers. 2017]. Since both proteins recognize a palindromic DNA target sequence, DNA fragment in our DNA looping assay could adopt two type of loops (“U”- or “Phi”-shaped) depending on how each copy of protein was oriented on the target site (see figure below this paragraph). In our assay we monitored events of DNA looping that occurred after proteins’ binding onto target sites, and events of DNA loop release that occurred upon proteins dissociation from target sites.



Recently our team developed an alternative assay to the original DNA Curtains that we termed the Soft DNA Curtains. We fabricated streptavidin or traptavidin patterns (i.e. line-features) on the modified coverslip surface that can be utilized to assemble stably immobilized biotinylated DNA arrays. The application of hydrodynamic buffer flow allows extension of the immobilized DNA molecules along the surface of the flowcell channel. This year we published a follow-up article in Langmuir on the Oriented Soft DNA Curtains. In this work, we fabricated the uniformly oriented double-tethered DNA Curtains using heterologous labeling of the DNA by biotin and digoxigenin. In addition to that we increased stability of the immobilized DNA molecules using a more stable alternative to sAv called traptavidin as an ink for the fabrication of protein templates. The main goal of our research is to apply the developed platform for studies of DNA targeting mechanisms of diverse CRISPR-Cas (Clustered regulatory interspaced short palindromic repeats) systems family, novel molecular-tools – prokaryotic Argonaute (pAgo) proteins, and various restriction endonucleases. Since 2017 for these studies we received two grants from the Lithuanian research council. Also, we received funding for a post-doc project dedicated for pAgo studies.
our developed Igor Pro software for fluorescent spot intensity extraction and analysis

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