PhD Student Medical University, Sofia Sofia, Sofiya, Bulgaria
Abstract: The objective of this study was to evaluate the cytotoxicity of newly developed fluorescent dyes with specific binding properties to RNA. The nucleic acid-binding dye was tested for its specificity toward RNA and/or DNA. Differences in structure, charge, and hydrophilicity were anticipated to influence cellular permeability and toxicity.
A three-step protocol was implemented to assess the novel dye. Mesenchymal stem cells from the apical papilla were isolated and cultured until reaching 80% confluence. The cells were then trypsinized using 0.05% trypsin-EDTA (Gibco) for 5 minutes at 37°C. Subsequently, the cells were divided into two groups: one seeded in a 48-well plate and the other in a 25 cm² plastic flask.
The dye was administered to the cells in the 48-well plate, which were immediately observed using the InCell Analyzer 6000 (GE Healthcare). In the flask group, the dye was added 24 hours prior to flow cytometry analysis. Cellular penetration was monitored and recorded with the InCell Analyzer 6000 at 30-minute intervals over a six-hour period.
Apoptotic and viable cells were differentiated using the Annexin V kit for flow cytometry on the Navios system (Bio-Rad). High-throughput fluorescent cellular analysis visualized apoptotic cells with an FITC-conjugated anti-annexin V antibody, while nuclei were counterstained with DAPI.
Results indicated that the tested dye exhibited minimal cytotoxicity and demonstrated specific binding to RNA, particularly within the nucleus. High-throughput fluorescent cellular analysis revealed a strong signal from the nuclei of living cells that were not in an apoptotic state.
The development of new, specific dyes is crucial for advancing our understanding of cellular processes. Currently, only one RNA-specific binding dye is commercially available. Creating novel, non-toxic dyes capable of distinguishing between RNA and DNA or binding specific protein regions will provide valuable tools for studying transmembrane transport, RNA translation, protein folding, and interactions.
Funding Source: Grant - Bulgarian Science Fund (Sofia, Bulgaria, BG) GRANT NUMBER: КП-06-Н73/12
