A generic platform for the development of fluorescent biosensors based on modified recombinant binders.

Genetically-encoded fluorescent biosensors are essential tools for the in situ visualization and study of chemical processes occurring inside live cells and more complex organisms. However, few well-working biosensors are currently available because they require protein domains that show large conformational changes in response to the stimulus of interest, of which comparatively few are known. We propose to develop a strategy to develop biosensors against a much broader variety of targets, by modifying nanobodies, specific variants of antibodies, such that a strong conformational change results upon binding to the target. This conformational change is then used to modulate the emission of a fluorescent moiety, thus delivering a fluorescent biosensor that is fully genetically-encoded and that can be readily expressed inside cells. The envisioned strategy can be combined with any nanobody available now or in the future, opening up in situ biosensing to an unprecedented range of targets. The proposed systems will be highlighted by imaging intracellular processes and in visualizing the response of tumors to immunotherapy. This work will drastically expand our opportunities for visualizing live-cell processes and also form the basis for extensive follow-up research.