The novel method herein proposed, aims to study Deoxyribonucleic acid (DNA) and Rad51 repair protein in its resting state after their interaction by using a combination of biological preparation and physical measures. Rad51 is a highly conserved protein; it is involved in eukaryotes genome stability and can interact with single strand (ss) and double strands (ds) DNA.
In our work, a droplet of the solution containing the dsDNA/Rad51 complexes was deposited on micro-fabricated super-hydrophobic substrates (SHS) to obtain self-organized and suspended fibers. The silicon-based SHS were designed to incorporate a regular circular array of pillars and to maintain a high contact angle with the drop. The samples were let dehydrate at controlled temperature and humidity conditions. At the end of the buffer evaporation process, non-suspended material and salt excess are concentrated on the top of a few micro-pillars in a limited area (drop residual) of the device while ordered and self-assembled DNA/Rad51 fibers are suspended between micro-pillars. To find the ideal conditions to obtain and suspend the nucleic acid/protein complexes, several parameters were investigated: saline buffer, DNA and protein concentrations were widely titrated and showed a significant effect on the biomolecule suspension on SHS.
The samples were then preliminarily checked by microscopy techniques and then described by the Raman spectra acquired. Several techniques were used: optical microscopy, Energy Dispersive X-Ray Spectroscopy (EDAX), Scanning Electron Microscopy (SEM) and Raman Spectroscopy. Protein expressions, DNA suspension, micro-fabrication and characterization were all performed in KAUST Core Labs and Structural Molecular Imaging Light Enhanced Spectroscopies (SMILEs) Lab.
The novel approach presented in this work is highly multidisciplinary and comprises physical measurements (Raman spectroscopy and EM imaging), chemistry and biology. In future the method can be used further expanded supporting the data with HRTEM direct imaging to elucidate the nucleic acids/proteins behavior in the multiple phases of the genome repair processes. Also, it and can serve as a fingerprint of the biological molecules involved in biological interactions, their localization and structural characterization, providing a new tool for structural analysis, screening and diagnostics.
| Identifer | oai:union.ndltd.org:kaust.edu.sa/oai:repository.kaust.edu.sa:10754/630025 |
| Date | 22 November 2018 |
| Creators | Morello, Maria Caterina |
| Contributors | Di Fabrizio, Enzo M., Physical Science and Engineering (PSE) Division, Anthopoulos, Thomas D., Fratalocchi, Andrea |
| Source Sets | King Abdullah University of Science and Technology |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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