Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells.

Autor: Sekhon H; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., Ha JH; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., Presti MF; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., Procopio SB; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., Jarvis AR; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., Mirsky PO; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., John AM; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA., Loh SN; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA. lohs@upstate.edu.
Jazyk: angličtina
Zdroj: Nature methods [Nat Methods] 2023 Dec; Vol. 20 (12), pp. 1920-1929. Date of Electronic Publication: 2023 Nov 09.
DOI: 10.1038/s41592-023-02065-w
Abstrakt: A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.
(© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE
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