Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices
| Autor: | Mattia Fontana, Klaus Mathwig, Dolf Weijers, Elmar van der Wijk, Simon Lindhoud, Willy A. M. van den Berg, Šarunė Ivanovaitė, Johannes Hohlbein |
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| Přispěvatelé: | Pharmaceutical Analysis |
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
single-molecule biophysics
Response element Biomedical Engineering Biophysics Oligonucleotides microfluidics Biochemie Biochemistry General Biochemistry Genetics and Molecular Biology Biomaterials chemistry.chemical_compound single-molecule fluorescence detection Fluorescence Resonance Energy Transfer Nanotechnology single-molecule Förster resonance energy transfer Transcription factor auxin response factor chemistry.chemical_classification lab-on-a-chip Oligonucleotide total-internal reflection fluorescence microscopy Biomolecule DNA-binding domain DNA Single-molecule experiment Förster resonance energy transfer Biofysica chemistry EPS DNA Probes Transcription Factors |
| Zdroj: | Advanced Biology, 6(4):2100953. WILEY-V C H VERLAG GMBH Advanced Biology, 6(4) Advanced Biology 6 (2022) 4 |
| ISSN: | 2701-0198 |
| Popis: | Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled 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. |
| Databáze: | OpenAIRE |
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