A novel BRET-based genetically encoded biosensor for functional imaging of hypoxia.

Optical imaging methods, such as fluorescence, have greatly increased its impact as a monitoring technique with the development of a broad range of fluorescent proteins used to visualize many types of biological processes, such as cancer biology. Although the most popular of these proteins is the green fluorescent protein (GFP), autofluorescence due to the absorption of the exciting radiation by endogenous fluorophores and signal dispersion raises doubts about its suitability as an in vivo tracer. In the last years a number of groups have developed several NIR fluorescent proteins that enables real-time imaging to take place without interference from autofluorescence events allowing at the same time to take a deep view into the tissues. We therefore have outlined fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1alpha, which is upregulated in growing tumors. At the same time, by fusing a fluorescent to a bioluminescent protein, we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor.