Split intein vectors permit oversize FLIM-FRET biosensor neuronal expression

Lee, J. O.; Puhl, H.; Holder, A.; Sheridan, A.; Darden, C.; Shah, M.; Nguyen, T.; Hines, K.; Augustin, S. M.; Kim, Y.; Vogel, S. S.; Lovinger, D. M.

doi:10.64898/2026.06.02.725428

bioRxiv · 10.64898/2026.06.02.725428

Split intein vectors permit oversize FLIM-FRET biosensor neuronal expression

Lee, J. O.·Puhl, H.·Holder, A.·Sheridan, A.·Darden, C.·Shah, M.·Nguyen, T.·Hines, K.·Augustin, S. M.·Kim, Y.·Vogel, S. S.·Lovinger, D. M.

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Abstract

Adeno-associated virus (AAV) packaging limits constrain the design, performance, and in vivo application of genetically encoded biosensors. We developed a split intein-mediated reconstitution strategy enabling modular delivery and reassembly of oversized fluorescence lifetime-based biosensors. Using this platform, we engineered an oversized cAMP sensor compatible with one-photon fluorescence lifetime measurements, enabling monitoring of intracellular signaling dynamics in distinct neuronal subtypes in freely behaving mice.

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Lee, J. O., Puhl, H., Holder, A., Sheridan, A., Darden, C., Shah, M., Nguyen, T., Hines, K., Augustin, S. M., Kim, Y., Vogel, S. S., Lovinger, D. M.. 2026-06-05. Split intein vectors permit oversize FLIM-FRET biosensor neuronal expression. https://doi.org/10.64898/2026.06.02.725428

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