Biology subjects

Ivanovaite, S.

Publications and source records attributed to Ivanovaite, S..

3 recordsLinked to original sources

bioRxiv2023

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

Monitoring of DNA-protein interactions is essential in understanding many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and the mechanisms for target search differ across proteins. Revealing temporal interactions with two target sites, both in Cis and in Trans, is crucial in target search mechanisms studies. Here, we present two single-molecule Forster resonance energy transfer (smFRET)-based assays to study BfiI-DNA interactions. The first assay, smFRET-based DNA looping assay, detects both "Phi" and "U"-shaped DNA looping events. We modified it to only allow in Trans BfiI-target DNA interactions to improve specificity and reduce limitations in the observation time. Our TIRF microscopy measurements directly observe the on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binding events last longer on target sites and that the BfiI rarely changes conformations during binding. This newly developed assay could be useful for other two-targets-binding DNA-interacting proteins and could be employed for dsDNA substrate BfiI-PAINT, which is useful for DNA stretch-assays and other super-resolution fluorescence microscopy studies.

biophysics↗

bioRxiv2021

Probing DNA - transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, we combine nanofluidic devices and camera-based, single-molecule Forster resonance energy transfer (smFRET) detection to study the interactions between plant transcription factors of the auxin response family (ARF) and DNA oligonucleotides that contain target DNA response elements. In particular, we show that the binding of the unlabelled ARF DNA binding domain (ARF-DBD) to donor and acceptor labelled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, our data on fluorescently labelled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

biophysics↗

bioRxiv2020

Oriented Soft DNA Curtains for Single Molecule Imaging

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.

biophysics↗