All Publications

  • Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells. Nature methods Lee, S. Y., Cheah, J. S., Zhao, B., Xu, C., Roh, H., Kim, C. K., Cho, K. F., Udeshi, N. D., Carr, S. A., Ting, A. Y. 2023


    The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

    View details for DOI 10.1038/s41592-023-01880-5

    View details for PubMedID 37188954

    View details for PubMedCentralID 2766670

  • Cell-Type-Specific Intracellular Protein Delivery with Inactivated Botulinum Neurotoxin. Journal of the American Chemical Society Roh, H., Dorner, B. G., Ting, A. Y. 2023


    The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms, but existing approaches are limited by immunogenicity, lack of cell-type-specificity, or their multi-component nature. Here we explore inactivated botulinum neurotoxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain can be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3 to 55 kDa in size, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a mixed culture. These explorations suggest that BoNT may be a versatile platform for targeted protein and peptide delivery into mammalian cells.

    View details for DOI 10.1021/jacs.3c01145

    View details for PubMedID 37098770

  • A Single-Component Luminescent Biosensor for the SARS-CoV-2 Spike Protein. Journal of the American Chemical Society Ravalin, M., Roh, H., Suryawanshi, R., Kumar, G. R., Pak, J. E., Ott, M., Ting, A. Y. 2022


    Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of Escherichia coli and Saccharomyces cerevisiae. This biosensor is deployed in multiple homogeneous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an unaugmented cell phone camera. Binding-activated tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.

    View details for DOI 10.1021/jacs.2c04192

    View details for PubMedID 35876794