Abstract 1664 An Oligomeric Platform for Fluorescent Protein Homo-FRET Anisotropy Protease Sensors

Genetically encoded Förster resonance energy transfer (FRET) biosensors have been extensively developed using two different fluorophores, referred to as heteroFRET. However, two-color heteroFRET takes up substantial color space that can cause spectral cross-talk when using multiple sensors, or prevent the use of multiple sensors all together. Alternatively, homoFRET overcomes this problem by using a single-colored fluorophore in which homoFRET efficiency can be measured by fluorescence anisotropy. Here we developed a homo-FRET protease sensor that utilizes mVenus, a yellow fluorescent protein, and paired it with an oligomerization toolbox as a proof of principle. The oligomeric mVenus sensor is stable over a wide range of pH values, and its response to protease activity was minimally affected by photobleaching. In addition, response of the protein was independent of sensor concentration because of the inherently ratiometric nature of anisotropy measurements. Furthermore, we show that these sensors can be used to detect protease activity in live bacteria. The oligomer platform we have developed can be easily modified to detect activity of other proteases of interest and shows great promise for use in the cellular environment. This work was supported by NIH Grant R01GM134380 to M.T. including Diversity Supplements for C.T. and A.S.