(683d) Forster Resonance Energy Transfer Probes for Measuring Biochemical Activities: A Flexible Cloning Library and Suitability for Quantitative Experiments

Numerous genetically-encoded, Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. In general, these probes are a single protein where an acceptor/donor fluorescent protein (FP) pair is connected by a “sensing unit” that is conformationally reactive to the biochemical activity of interest. Changes in this biochemical activity alter the distance and/or orientation between the acceptor and donor, and thus are observable as changes in FRET. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells, they are an attractive technology for developing quantitative models of spatiotemporal signaling dynamics. We are exploiting the general, modular FRET probe structure in combination with multi-site gateway cloning technology to create a library of sensing units and FPs with various localization tags. This library allows us to construct probes “on-demand” (within  ~1 week) for measuring biochemical activities of interest, in cellular compartments of interest, all while controlling which parts of the light spectrum are taken for FRET analysis (to the extent possible by existing FRET-compatible FPs). However, to be useful for quantitative modeling purposes, the observed FRET from such probes should also have a large linear range of responsiveness. Here we report that measuring FRET by intensity-based, ratiometric methods, as is currently standard practice, yields data that scales non-linearly with respect to the biological quantity of interest. A log transformation of such ratiometric data improves the effective linear range, and paradoxically, some overlap between donor and acceptor emission spectra, which is usually avoided, should be beneficial for increasing the effective linear range of log-transformed ratiometric data. An alternative way to measure FRET is by analyzing fluorescence lifetimes, and we find that measuring FRET via fluorescence lifetimes is inherently linear with respect to the biological quantity of interest. Overall, our results provide a rapid and flexible cloning system for creating custom FRET probes and also suggest that fluorescence lifetime imaging is the preferred technique for obtaining quantitative data from FRET probes.