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bioRxiv · 10.1101/2025.09.12.675751

Leveraging bioorthogonal conjugation for alpha synuclein fibril surveillance

Jenkins, R. A.·Wu, S.·Fujimura, G.·Heredia, A.·Flowers, C.·Sun, C.·Sawaya, M.·Loo, J.·Rodriguez, J. A.
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Abstract

Alpha synuclein (-syn) amyloid fibrils are associated with various neurodegenerative diseases. To better understand the molecular and cellular basis for -syn fibril persistence and spread, we implemented a fluorophore labeling strategy to surveil pre-formed -syn fibrils in solution and in cells. We leveraged amber codon mediated incorporation of a tetrazine-based artificial amino acid (TetV2.0) to install a cyclooctene-conjugated Janeliaflour, JF549, at four sites on human -syn: residues 4, 60, 96 and 136. Fast coupling occurred under mild buffer conditions and in the presence of the disease-associated cofactor and cytotoxic lipid, psychosine. Labeled fibrils retained their polymorphic features, seeded the growth of new fibrils in vitro, and induced the seeding of positive puncta in -syn FRET biosensor HEK293T cells. This allowed simultaneous tracking of exogenous and endogenous -syn aggregates in biosensor cells, and their localization within the cells. In doing so, our approach facilitates more detailed mechanistic investigation of -syn aggregates.

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Jenkins, R. A., Wu, S., Fujimura, G., Heredia, A., Flowers, C., Sun, C., Sawaya, M., Loo, J., Rodriguez, J. A.. 2025-09-17. Leveraging bioorthogonal conjugation for alpha synuclein fibril surveillance. https://doi.org/10.1101/2025.09.12.675751

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