TY  - GEN
ID  - heidok35060
AV  - public
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/35060/
Y1  - 2024///
CY  - Heidelberg
A1  - Hellweg, Lars
TI  - Development of Chemogenetic Biosensors with Large Dynamic Ranges and Spectral Flexibility
N2  - Understanding cellular processes at the molecular level is fundamental for gaining insights into biological mechanisms and their implications in health and disease. Fluorescent biosensors have emerged as powerful tools in this regard, as they enable real-time monitoring of these processes with high spatiotemporal resolution using live-cell imaging. Despite significant progress in the development of biosensors, limitations still exist. While intensiometric biosensors often exhibit large dynamic ranges, they are susceptible to changes in sensor concentration and photobleaching, potentially resulting in skewed experimental results. In contrast, ratiometric FRET-based biosen-sors offer a more robust quantification, but often suffer from a low dynamic range and occupy a substantial part of the spectral space, hindering their multiplexing capability with other fluorescent tools.
To address these limitations, I developed a chemogenetic FRET system called ChemoX, based on a fluorescent protein FRET donor and rhodamine-based FRET acceptor, which is conjugated to the self-labeling protein HaloTag7. Engineering of a specific interface between the fluorescent protein and HaloTag7, enabled the generation of high efficiency FRET pairs. The spectral prop-erties of ChemoX can be modified by either replacing the FRET donor with a fluorescent protein of different color, or by labeling HaloTag7 with spectrally distinct fluorophores, all while ensuring that a high FRET efficiency is maintained. The utilization of FRET pairs with large spectral sep-aration combined with the ability to fine-tune their FRET efficiency, allowed for the generation of ChemoX biosensors with different colors and large dynamic ranges. I demonstrated the gener-alizability of this approach by developing biosensors for Ca2+, ATP and NAD+ with improved dynamic ranges compared to established FRET-based biosensors. Furthermore, the increased multiplexing capacity of ChemoX sensors enabled real-time monitoring of free NAD+ at different subcellular localizations within the same cell, representing a valuable addition to existing tools for the investigation of compartmentalized NAD+ dynamics.
Moreover, I leveraged the ChemoX interface to modulate the fluorescence intensity of bright rho-damine-based fluorophores and develop single-channel fluorescence lifetime-based ChemoX sensors with far-red emission wavelengths. The spectral properties of these sensors hold promise for their use in in vivo applications. Finally, I generated bioluminescent ChemoX biosensors through simple fusion of the luciferase NanoLuc to the N-terminus of fluorescent ChemoX bio-sensors and demonstrated their compatibility for high-throughput screenings using microplate reader systems.
In summary, I developed a versatile chemogenetic system that allows for the development of bio-sensors with large dynamic ranges and spectral flexibility, providing the scientific community with a complemented toolbox for the investigation of important cell metabolites.
ER  -