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Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation

Nature Chemistry volume 15, pages 694–704 (2023)Cite this article

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Abstract

Proteins provide essential functional regulation of many bioprocesses across all scales of life; however, new techniques to specifically modulate protein activity within living systems and in engineered biomaterials are needed to better interrogate fundamental cell signalling and guide advanced decisions of biological fate. Here we establish a generalizable strategy to rapidly and irreversibly activate protein function with full spatiotemporal control. Through the development of a genetically encoded and light-activated SpyLigation (LASL), bioactive proteins can be stably reassembled from non-functional split fragment pairs following brief exposure (typically minutes) to cytocompatible light. Employing readily accessible photolithographic processing techniques to specify when, where and how much photoligation occurs, we demonstrate precise protein activation of UnaG, NanoLuc and Cre recombinase using LASL in solution, biomaterials and living mammalian cells, as well as optical control over protein subcellular localization. Looking forward, we expect that these photoclick-based optogenetic approaches will find tremendous utility in probing and directing complex cellular fates in both time and three-dimensional space.

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Fig. 1: LASL affords complete spatiotemporal control over protein activation within living systems.
Fig. 2: pSC provides user control of SpyLigation in solution.
Fig. 3: LASL enables site-specific patterned protein localization in 3D biomaterials and living cells.
Fig. 4: Assembly of UnaG and NanoLuc through LASL of split protein fragments in solution and biomaterials.
Fig. 5: Photoactivation of split UnaG with spatiotemporal precision in living cells through intracellular LASL.
Fig. 6: Spatially controlled photoactivation of primary cell genome editing via LASL.

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Data availability

All pertinent experimental and characterization data are available within this manuscript and its associated Supplementary Information. Plasmids generated during the current study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We recognize T. Rapp and R. Francis for synthetic advice, J. Shadish for helpful discussion on molecular cloning, R. Gharios for forward-looking conversations, R. Bretherton for providing the SpyTag peptide and transgenic dermal fibroblasts, J. Davis for gifting the HEK-293T cells (originally acquired from the American Type Culture Collection; CRL-3216) and S. Edgar (in memoriam) for assistance with mass spectrometry. This work was supported by a CAREER Award (DMR 1652141 to C.A.D.) and grants (DMR 1807398 and CBET 1803054 to C.A.D.) from the National Science Foundation, as well as a Maximizing Investigators’ Research Award (R35GM138036 to C.A.D.) from the National Institutes of Health. Student fellowship support was provided by the Institute for Stem Cell and Regenerative Medicine (to B.G.M.-R.) and Mary Gates Endowment for Students (to A.C.S., C.H.B. and S.K.) at the University of Washington. Part of this work was conducted with instrumentation provided by the Joint Center for Deployment and Research in Earth Abundant Materials. The Thorlabs multiphoton microscope was acquired with and operated under support from the Washington Research Foundation and the University of Washington College of Engineering, Institute for Stem Cell and Regenerative Medicine and Departments of Chemical Engineering, Bioengineering, Chemistry and Biology.

Author information

Author notes

  1. These authors contributed equally: Emily R. Ruskowitz, Brizzia G. Munoz-Robles.

Authors and Affiliations

  1. Department of Chemical Engineering, University of Washington, Seattle, WA, USA

    Emily R. Ruskowitz, Sebastian Kurniawan, Jeremy R. Filteau & Cole A. DeForest

  2. Department of Bioengineering, University of Washington, Seattle, WA, USA

    Brizzia G. Munoz-Robles & Cole A. DeForest

  3. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Alder C. Strange

  4. Department of Biology, University of Washington, Seattle, WA, USA

    Carson H. Butcher

  5. Department of Chemistry, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  6. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  7. Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  8. Institute for Protein Design, University of Washington, Seattle, WA, USA

    Cole A. DeForest

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Contributions

E.R.R. and C.A.D. conceived of and designed the experiments. E.R.R., B.G.M.-R., A.C.S., C.H.B., S.K. and J.R.F. performed the experiments. E.R.R., B.G.M.-R., A.C.S., S.K., C.H.B. and C.A.D. analysed the data and prepared the figures. E.R.R., B.G.M.-R. and C.A.D. wrote the paper.

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Correspondence to Cole A. DeForest.

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Nature Chemistry thanks Cecile Echalier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Photoactivation of split UnaG with spatiotemporal precision in living cells through intracellular LASL.

a-c, Mask-based photolithography spatiotemporally directs UnaG reassembly within HEK-293T cell culture. a, Fluorescent images of culture dish with inlays of exposure boundary magnified. b, Individual cell UnaG/mCh signal quantified radially outwards from the photomask’s center, normalized to the average UnaG/mCh ratio in unexposed cells. Dashed line indicates exposure edge. c, Violin scatter plots of normalized UnaG/mCh ratios in light-(un)exposed regions. Light treatments, λ = 365 nm, 20 mW cm−2, 20 min. Asterisks denote conditions with statistically significant differences in signal (p < 0.0001, two-tailed unpaired t-tests). Similar results were independently achieved in 3 experimental replicates. Scale bars, 1 mm (a).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–16, methods 1–28 and Tables 1 and 2.

Reporting Summary

Supplementary Data 1

Statistical data for Supplementary Figs. 1, 3, 4, 6, 9–11 and 14–16.

Source data

Source Data Fig. 2

Source data for Fig. 2a,d.

Source Data Fig. 3

Source data for Fig. 3d,e,h,i.

Source Data Fig. 4

Source data for Fig. 4c–e,k.

Source Data Fig. 5

Source data for Fig. 5c,e,f.

Source Data Extended Data Fig. 1

Source data for Extended Fig. 1b,c.

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Ruskowitz, E.R., Munoz-Robles, B.G., Strange, A.C. et al. Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation. Nat. Chem. 15, 694–704 (2023). https://doi.org/10.1038/s41557-023-01152-x

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