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Published December 23, 2020 | Supplemental Material + Accepted Version
Journal Article Open

Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning


Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

Additional Information

© 2020 Elsevier. Received 23 November 2019, Revised 22 June 2020, Accepted 20 November 2020, Available online 16 December 2020. We would like to thank Drs. Liqun Luo (Stanford University) and Jing Ren (MRC) for their critical reading and feedback. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (P30-GM124165). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10-RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. This work was supported by funding to L.T. (BRAIN Initiative U01NS090604, U01NS013522, and DP2MH107056 from NIH), to E.K.U. (Mistletoe Foundation Research Fellowship), and to G.O.M. (ARCS Scholarship), as well as by the Howard Hughes Medical Institute. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute, the NIH (BRAIN RF1MH117069), the Center for Molecular and Cellular Neuroscience of the Chen Institute, and the Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research. V.A.A. is funded by Intramural Programs of NIAAA and NINDS ZIA-AA000421 and Innovation Award from NIH-DDIR. T.L.K. is funded by the NIH (R01AA019454, P60AA011605, and U24AA025475), and O.J.H. is funded by the NIH (5T32NS007431-20). Author Contributions. E.K.U., J.P.K., M.A., L.L.L., and L.T. conceived of and designed the study. E.K.U. designed the machine-learning method, screened and optimized sensors, and characterized them in purified protein, mammalian cells, cultured neurons, and brain slice, with significant contribution from C.D., D.A.J., and J.S. J.P.K., S.S., and G.R. designed OSTA and stopped-flow experiments, and J.P.K. performed them. M.A. and V.G. designed and performed in vivo fiber photometry and EEG/EMG recording in BLA and mPFC in fear learning and sleep/wake cycles. O.J.H., M.E.F., and T.L.K. designed and performed in vivo fiber photometry experiments in BLA, OFC, and BNST during social interaction. R.L. and V.Y.-Y. designed and performed computational Rosetta modeling. Z.Y. and J.A.P. provided luciferase experimental data for establishing machine-learning methods. J.C. and D.T.L. provided significant insight for the machine-learning methods. J.S. characterized the sensor in acute slice using two-photon imaging. A.M. and V.A.A. designed and performed photometry imaging in acute slice. S.H. and A.J.F. performed crystallography. J.S.M., P.M.B., A.V.S., H.A.L., and L.L.L. provided iAChSnFR0.6 and performed preliminary experiments on serotonin binding. S.B. and L.D.L. synthesized caged serotonin. G.O.M. provided dissociated neuronal cultures. L.P.C. and D.E.O. produced chemical reagents. S.M.U. and S.G.A. provided SSRIs and guidance in cell-assay design. E.K.U., J.P.K., L.L.L., and L.T. wrote the manuscript with significant input from other authors. Declaration of Interests. L.T. and G.O.M. are co-founders of Seven Biosciences. D.E.O. is a founder of Delix.

Attached Files

Accepted Version - nihms-1654804.pdf

Supplemental Material - 1-s2.0-S0092867420316123-mmc1.xlsx

Supplemental Material - 1-s2.0-S0092867420316123-mmc2.pdf


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Additional details

August 22, 2023
December 22, 2023