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Engineering a Color Palette of FRET‐BRET ATP Sensors

ATP, a key player in energy metabolism, is involved in various cellular processes and is shown to be an indicator of cellular health. There are several methods to measure ATP levels, and genetically‐encoded sensors are excellent tools for visualizing energy metabolism in live cells. Currently, however, there has been a lack of color options. By building new variants of hybrid FRET‐BRET (Förster or bioluminescence resonance energy transfer) ATP sensors, our goal is to increase the flexibility and utility of these tools with different experimental formats. For example, differently colored sensors could be used to simultaneously monitor ATP levels in different individual cells or cell types as well as in different compartments within a single cell. Here, we have developed ATP sensors which employ different fluorescent protein FRET pairs in conjunction with the NanoLuc luciferase. We characterized the both the fluorescence and bioluminescence purified sensor proteins in response to ATP. Furthermore, we carried out live‐cell imaging experiments to validate that our sensors can be used for ratiometric quantification of ATP levels. These new color variants of hybrid FRET‐BRET ATP sensors will be an effective method for studying energy metabolism both in vitro and in vivo.