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Boolean logic-gated protein presentation through autonomously compiled molecular topology

Nature Chemical Biology volume 21, pages 1981–1991 (2025)Cite this article

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Abstract

Stimulus-responsive materials have enabled advanced applications in biosensing, tissue engineering and therapeutic delivery. Although controlled molecular topology has been demonstrated as an effective route toward creating materials that respond to prespecified input combinations, prior efforts suffer from a reliance on complicated and low-yielding multistep organic syntheses that dramatically limit their utility. Harnessing the power of recombinant expression, we integrate emerging chemical biology tools to create topologically specified protein cargos that can be site-specifically tethered to and conditionally released from biomaterials following user-programmable Boolean logic. Critically, construct topology is autonomously compiled during expression through spontaneous intramolecular ligations, enabling direct and scalable synthesis of advanced operators. Using this framework, we specify protein release from biomaterials following all 17 possible YES/OR/AND logic outputs from input combinations of three orthogonal protease actuators, multiplexed delivery of three distinct biomacromolecules from hydrogels, five-input-based conditional cargo liberation and logically defined protein localization on or within living mammalian cells.

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Fig. 1: Autonomously compiled molecular topologies enable Boolean logic-based protein presentation.
Fig. 2: Autonomously assembled logic operators undergo preprogrammed cleavage from bead surfaces in response to actuator input combinations.
Fig. 3: Autonomously compiled molecular topologies enable the multiplexed and independently triggered release of distinct proteins from a hydrogel network.
Fig. 4: Genetic encodability enables seamless scale-up in logical response complexity.
Fig. 5: Boolean logic-based labeling of the mammalian extracellular membrane.
Fig. 6: Autonomous compilation and logic-defined localization within mammalian cells.

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Data availability

All pertinent experimental and characterization data are available within this paper and its associated Supplementary Information. Plasmids generated during the current study are listed in the Supplementary Information and are available through Addgene. Source data are provided with this paper.

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Acknowledgements

We thank R. Francis and A. Garcia for collaborating on designing and validating the SpyCatcher-azide, C. Yang and R. Brady for synthesizing and supplying PEG-tetraBCN and J. Davis for gifting HEK293T cells (previously obtained from the ATCC, CRL-3216). We acknowledge support from D. Whittington at the University of Washington MS Center. Part of this work was conducted with instrumentation provided by the Joint Center for Deployment and Research in Earth Abundant Materials. This work was supported by a grant (DMR 1807398, C.A.D.) from the National Science Foundation and a Maximizing Investigators’ Research Award (R35GM138036, C.A.D.) from the National Institutes of Health. Student support was further provided through a John C. Berg Endowed Fellowship (R.G.), the National Science Foundation (DGE-2140004, M.L.R.), a Goldwater Scholarship (A.L.), a Washington NASA Space Grant (A.L), a University of Washington MEM-C Academic-Year Research Accelerator Fellowship (A.L) and a University of Washington Mary Gates Endowment for Students (S.P.K.).

Author information

Author notes

  1. These authors contributed equally: Ryan Gharios, Murial L. Ross.

Authors and Affiliations

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

    Ryan Gharios, Annabella Li, Jack Hoye & Cole A. DeForest

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

    Murial L. Ross, Shivani P. Kottantharayil & Cole A. DeForest

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

    Cole A. DeForest

  4. Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  5. Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA, USA

    Cole A. DeForest

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

    Cole A. DeForest

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Contributions

R.G., M.L.R., and C.A.D. conceptualized and designed the experiments. R.G., M.L.R., A.L., S.P.K. and J.H. performed the experiments. R.G., M.L.R., A.L., S.P.K., J.H. and C.A.D. analyzed the data. C.A.D. prepared the main text figures with input from R.G. and M.L.R. C.A.D. and M.L.R. prepared the supplementary figures with input from R.G. R.G., M.L.R. and C.A.D. wrote the paper with input from all other authors.

Corresponding author

Correspondence to Cole A. DeForest.

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Competing interests

C.A.D., R.G. and M.L.R. have filed a patent application (PCT/US2025/031504) related to the work described in this article. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Mass spectrometric validation of logically releasable mGreenLantern.

a, YES-gated single-input tethers. b-c, Two-input (b) OR-gated and (c) AND-gated tethers. d-g, Three-input (d) OR/(AND)-, (e) AND/(OR)-, (f) OR/OR-, and (g) AND/AND-gated tethers. Plot titles correspond to the protein tether identity.

Source data

Extended Data Fig. 2 SDS-PAGE analysis of in-solution treatment of autonomously compiled mGreenLantern pendants.

a, mGreenLantern-A∧(S∨T) is differentially cleaved following the 8 possible input combinations of A, S, and T. Input conditions A, S, T, and ST are expected to linearize the product but not induce payload release, leading to a product with less electrophoretic mobility than the initial cyclic construct. Payload release is expected following input conditions AS, AT, and AST, accompanied with a product band that migrates further than the starting species. b, Changes in topology and/or molecular weight in response to a specific set of inputs leads to changes in protein electrophoretic mobility, which can be analyzed through gel densitometry. c, The response profiles of the YES-gated single-input tethers. d-e, The response profiles of the two-input (d) OR-gated and (e) AND-gated tethers. f-h, The response profiles of the three-input (f) OR/(AND)-, (g) AND/(OR)-, and (h) OR/OR-gated tethers. Plot titles correspond to the protein tether identity; the y-axis represents extent of cleavage as measured through migration on an SDS-PAGE with gel densitometry analysis; the x-axis indicates treatment conditions, wherein N indicates no treatment, A indicates eSrtA(2A9), S indicates eSrtA(4S9), and T indicates TEV. Green bars indicate conditions expected to yield tether cleavage, whereas red bars indicate conditions expected to keep the tether non-cleaved. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n = 3 experimental replicates.

Source data

Extended Data Fig. 3 SDS-PAGE analysis of in-solution treatment of autonomously compiled mCherry and mCerulean pendants.

a-c, Changes in topology and/or molecular weight in response to a specific set of inputs leads to changes in protein electrophoretic mobility, which can be analyzed through gel densitometry. Results are shown for species (a) mCherry-S, (b) mCerulean-T, and (c) mCerulean-S∧T. Plot titles correspond to the protein tether identity; the y-axis represents extent of cleavage as measured through migration on an SDS-PAGE with gel densitometry analysis; the x-axis indicates treatment conditions, wherein N indicates no treatment, A indicates eSrtA(2A9), S indicates eSrtA(4S9), and T indicates TEV. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n = 3 experimental replicates.

Source data

Extended Data Fig. 4 Multiplexed YES-gated release of proteins from hydrogel biomaterials.

a, mGreenLantern-A, mCherry-S, and mCerulean-T are tethered homogenously into an underlying PEG-based hydrogel network via SpyLigation, each exhibiting a different YES-gated response. b, Appropriate proteins are individually released when their corresponding input is present. Fully colored bars (green for mGreenLantern, red for mCherry, blue for mCerulean) indicate conditions expected to yield protein release, whereas opaque colored bars denote conditions not expected to result in release. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n = 3 experimental replicates.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–16 and Methods.

Reporting Summary

Supplementary Data 1

Source data for Supplementary Figs. 3, 4, 11 and 14.

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Gharios, R., Ross, M.L., Li, A. et al. Boolean logic-gated protein presentation through autonomously compiled molecular topology. Nat Chem Biol 21, 1981–1991 (2025). https://doi.org/10.1038/s41589-025-02037-5

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