Target-Specific Dyes with Responsive Long-Lived Fluorescence Lifetimes for Biological Interrogation

/Project Summary The fluorescence lifetime of a dye can inform about cellular signaling, protein-protein interactions, and membrane microenvironments with fluorescence lifetime imaging microscopy (FLIM) and time-resolved flow cytometry (TR- Flow Cytometry). These techniques primarily use changes in the fluorescence lifetime from Föster-Resonance Energy Transfer (FRET) dye pairs to convey biological information occurring within a 10-nm distance. In addition, FLIM is often used to circumvent biological autofluorescence by producing an image based on the differences in the fluorescence lifetimes of a dye. However, the fluorescence lifetimes of archetypal dyes are short (< 3 ns), and their minimal lifetime changes upon biological stimuli cannot be resolved with these instruments, limiting the extent and precision of biological interrogation during their operation. The goal of our program is to synthesize, characterize, and implement a palette of target-specific organic dyes with biologically-responsive long-lived fluorescence lifetimes, primarily by incorporating thermally activated delayed fluorescence (TADF) photophysics. Of biological relevance is that these organic dyes can yield two long-lived fluorescence lifetimes that are mechanoresponsive, thermoresponsive, and sensitive to voltage. Therefore, we hypothesize that concomitant changes in the fluorescence lifetime of our optical reporting dye technology will be easily resolved with FLIM and TR-Flow Cytometry and inform us of viscosity, polarity, and voltage changes associated with biological function. In addition, this TADF dye technology can potentially extend the Föster resonance distances of the FRET dye pairs, opening new frontiers for interrogating biomolecules separated by distances > 10 nm. Of relevance is sensing cholesterol, as its levels impact membrane viscosity. Our preliminary data demonstrate that a TADF dye can sense cholesterol levels in liposomes, as its fluorescence lifetime increased from 9.4 ns to 13 ns when cholesterol levels were increased from 0% to 20%. Contrarily, a constant fluorescence lifetime of ~ 2.6 ns was obtained for DI-8-ANEPPS, a cholesterol-sensing dye. Notably, a long-lived fluorescence lifetime of 711 ns was detected for the TADF dye in giant multilamellar vesicles (GMLVs) that was not clearly detected in liposomes. These results are significant, as these lifetime increments are easily resolved with fluorescence lifetime measurements and are a goal for dye synthesis. A plausible explanation is that the lifetime differences between GMLVs and liposomes are due to membrane curvature differences, confirming that TADF dyes can be used to inform membrane morphology. Building on these findings, we are synthesizing a palette of TADF dyes that fully intercalate into membranes, target the mitochondria and important organelles, and serve as bioconjugates for biological interrogation with FLIM and TR-Flow Cytometry. To achieve this objective, our program combines previous success in organic dye synthesis, photophysical characterization of organic TADF dyes, and biophysical interrogation in artificial and living systems. Our program can have applications in medical diagnostics, cellular trafficking, and other biological research beyond the immediate scope of this initial work. 1 Project Number: 1R35GM157146-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Ricardo Vázquez | Institution: TRUSTEES OF INDIANA UNIVERSITY, BLOOMINGTON, IN | Award Amount: $382,026 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-F (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11020097