Abstract
Click chemistry is a powerful concept that refers to a set of covalent bond-forming reactions with highly favorable properties. In this Perspective, I outline the analogous concept of click biology as a set of reactions derived from the regular building blocks of living cells, rapidly forming covalent bonds to specific partners under cell-friendly conditions. Click biology using protein components employs canonical amino acids and may react close to the diffusion limit, with selectivity in living cells amid thousands of components generated from the same building blocks. I discuss how the criteria for click chemistry can be applied or modified to fit the extra constraints of click biology and achieve favorable characteristics for biological research. Existing reactions that may be described as click biology include split intein reconstitution, spontaneous isopeptide bond formation by SpyTag and SpyCatcher and suicide enzyme reaction with small-molecule ligands (HaloTag and SNAP-tag). I also describe how click biology has created new possibilities in fields including molecular imaging, mechanobiology, vaccines and engineering cellular intelligence.
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References
Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. Engl. 40, 2004–2021 (2001). The pioneering treatise setting out philosophy, practice and potential of click chemistry.
Patterson, D. M., Nazarova, L. A. & Prescher, J. A. Finding the right (bioorthogonal) chemistry. ACS Chem. Biol. 9, 592–605 (2014).
Bertozzi, C. A special virtual issue celebrating the 2022 Nobel Prize in Chemistry for the development of click chemistry and bioorthogonal chemistry. ACS Cent. Sci. 9, 558–559 (2023).
Oliveira, B. L., Guo, Z. & Bernardes, G. J. L. Inverse electron demand Diels–Alder reactions in chemical biology. Chem. Soc. Rev. 46, 4895–4950 (2017).
Bohacek, R. S., McMartin, C. & Guida, W. C. The art and practice of structure-based drug design: a molecular modeling perspective. Med. Res. Rev. 16, 3–50 (1996).
Bauer, D., Cornejo, M. A., Hoang, T. T., Lewis, J. S. & Zeglis, B. M. Click chemistry and radiochemistry: an update. Bioconjug. Chem. 34, 1925–1950 (2023).
Srinivasan, S. et al. SQ3370, the first clinical click chemistry-activated cancer therapeutic, shows safety in humans and translatability across species. Preprint at bioRxiv https://doi.org/10.1101/2023.03.28.534654 (2023).
Sun, F. & Zhang, W.-B. Genetically encoded click chemistry. Chin. J. Chem. 38, 894–896 (2020).
Kraut, D. A., Carroll, K. S. & Herschlag, D. Challenges in enzyme mechanism and energetics. Annu. Rev. Biochem. 72, 517–571 (2003).
Shah, N. H. & Muir, T. W. Inteins: nature’s gift to protein chemists. Chem. Sci. 5, 446–461 (2014).
Wu, H., Hu, Z. & Liu, X.-Q. Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc. Natl Acad. Sci. USA 95, 9226–9231 (1998). The discovery of how a DNA polymerase component from a cyanobacterium is expressed in two fragments, which reconstitute and splice together the functional protein, leading on to the future identification of many split inteins and powerful tools for click biology.
Eryilmaz, E., Shah, N. H., Muir, T. W. & Cowburn, D. Structural and dynamical features of inteins and implications on protein splicing. J. Biol. Chem. 289, 14506–14511 (2014).
Mills, K. V., Johnson, M. A. & Perler, F. B. Protein splicing: how inteins escape from precursor proteins. J. Biol. Chem. 289, 14498–14505 (2014).
Anastassov, S., Filo, M. & Khammash, M. Inteins: a Swiss army knife for synthetic biology. Biotechnol. Adv. 73, 108349 (2024).
Bhagawati, M. et al. In cellulo protein semi-synthesis from endogenous and exogenous fragments using the ultra-fast split Gp41-1 intein. Angew. Chem. Int. Ed. Engl. 59, 21007–21015 (2020).
Pinto, F., Thornton, E. L. & Wang, B. An expanded library of orthogonal split inteins enables modular multi-peptide assemblies. Nat. Commun. 11, 1529 (2020).
Carvajal-Vallejos, P., Pallisse, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–28696 (2012).
Gramespacher, J. A., Stevens, A. J., Thompson, R. E. & Muir, T. W. Improved protein splicing using embedded split inteins. Protein Sci. 27, 614–619 (2018).
Bhagawati, M. et al. A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions. Proc. Natl Acad. Sci. USA 116, 22164–22172 (2019). This study overcomes the obstacle of many split inteins requiring reducing conditions, optimizing a bacteriophage split intein that contains key reactive serines instead of cysteines, allowing molecular surgery at the cell surface.
Burton, A. J., Haugbro, M., Parisi, E. & Muir, T. W. Live-cell protein engineering with an ultra-short split intein. Proc. Natl Acad. Sci. USA 117, 12041–12049 (2020). Lake Vida has ice all year round and contains water with exceptional salinity. From this frigid landscape, a split intein platform was shown to react with outstanding speed and efficiency, allowing precision editing on histones within living cells.
Neugebauer, M., Böcker, J. K., Matern, J. C. J., Pietrokovski, S. & Mootz, H. D. Development of a screening system for inteins active in protein splicing based on intein insertion into the LacZα-peptide. Biol. Chem. 398, 57–67 (2017).
Popa, M. P., McKelvey, T. A., Hempel, J. & Hendrix, R. W. Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. J. Virol. 65, 3227–3237 (1991).
Wikoff, W. R. et al. Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289, 2129–2133 (2000).
Kang, H. J. & Baker, E. N. Intramolecular isopeptide bonds: protein crosslinks built for stress? Trends Biochem. Sci. 36, 229–237 (2011).
Zakeri, B. & Howarth, M. Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting. J. Am. Chem. Soc. 132, 4526–4527 (2010).
Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).
Keeble, A. H. et al. Evolving accelerated amidation by SpyTag/SpyCatcher to analyze membrane dynamics. Angew. Chem. Int. Ed. Engl. 56, 16521–16525 (2017).
Keeble, A. H. et al. Approaching infinite affinity through engineering of peptide–protein interaction. Proc. Natl Acad. Sci. USA 116, 26523–26533 (2019).
Zhang, S. et al. One-step construction of circularized nanodiscs using SpyCatcher–SpyTag. Nat. Commun. 12, 5451 (2021).
Veggiani, G. et al. Programmable polyproteams built using twin peptide superglues. Proc. Natl Acad. Sci. USA 113, 1202–1207 (2016).
Keeble, A. H. et al. DogCatcher allows loop-friendly protein–protein ligation. Cell Chem. Biol. 29, 339–350 (2022).
Fan, R. & Aranko, A. S. Catcher/Tag toolbox: biomolecular click-reactions for protein engineering beyond genetics. ChemBioChem 25, e202300600 (2024).
Dicks, M. D. J. et al. Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2. Mol. Ther. 30, 3639–3657 (2022).
Fantoni, N. Z., El-Sagheer, A. H. & Brown, T. A hitchhiker’s guide to click-chemistry with nucleic acids. Chem. Rev. 121, 7122–7154 (2021).
Driscoll, C. L., Keeble, A. H. & Howarth, M. R. SpyMask enables combinatorial assembly of bispecific binders. Nat. Commun. 15, 2403 (2024).
Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008). A tour de force in enzymology converts a bacterial dehalogenase into a self-labeling enzyme, including extensive semi-rational optimization and demonstration of the power of the covalent labeling across a range of methods.
Encell, L. P. et al. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr. Chem. Genomics 6, 55–71 (2012).
Deprey, K. & Kritzer, J. A. HaloTag forms an intramolecular disulfide. Bioconjug. Chem. 32, 964–970 (2021).
Frei, M. S. et al. Engineered HaloTag variants for fluorescence lifetime multiplexing. Nat. Methods 19, 65–70 (2022).
Chen, W., Younis, M. H., Zhao, Z. & Cai, W. Recent biomedical advances enabled by HaloTag technology. Biocell 46, 1789–1801 (2022).
Liu, D. S., Phipps, W. S., Loh, K. H., Howarth, M. & Ting, A. Y. Quantum dot targeting with lipoic acid ligase and HaloTag for single-molecule imaging on living cells. ACS Nano 6, 11080–11087 (2012).
Neklesa, T. K. et al. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543 (2011).
Liu, X., Long, M. J. C. & Aye, Y. Proteomics and beyond: cell decision-making shaped by reactive electrophiles. Trends Biochem. Sci. 44, 75–89 (2019).
Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003). Conversion of a DNA repair enzyme into a powerful approach for cellular labeling: the establishment of SNAP-tag chemistry.
Dreyer, R., Pfukwa, R., Barth, S., Hunter, R. & Klumperman, B. The evolution of SNAP-tag labels. Biomacromolecules 24, 517–530 (2023).
Sun, X. et al. Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. ChemBioChem 12, 2217–2226 (2011).
Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).
Zimmermann, M. et al. Cell-permeant and photocleavable chemical inducer of dimerization. Angew. Chem. Int. Ed. Engl. 53, 4717–4720 (2014).
Nakamura, H., Yoshikawa, M., Oda-Ueda, N., Ueda, T. & Ohkuri, T. A comprehensive analysis of novel disulfide bond introduction site into the constant ___domain of human Fab. Sci. Rep. 11, 12937 (2021).
Rossi, E. A., Goldenberg, D. M. & Chang, C. H. Complex and defined biostructures with the dock-and-lock method. Trends Pharmacol. Sci. 33, 474–481 (2012).
Liu, X. et al. Structure-based reactivity profiles of reactive metabolites with glutathione. Chem. Res. Toxicol. 33, 1579–1593 (2020).
Chiu, J. & Hogg, P. J. Allosteric disulfides: sophisticated molecular structures enabling flexible protein regulation. J. Biol. Chem. 294, 2949–2960 (2019).
Garrido Ruiz, D., Sandoval-Perez, A., Rangarajan, A. V., Gunderson, E. L. & Jacobson, M. P. Cysteine oxidation in proteins: structure, biophysics, and simulation. Biochemistry 61, 2165–2176 (2022).
Dent, M. R. & DeMartino, A. W. Nitric oxide and thiols: chemical biology, signalling paradigms and vascular therapeutic potential. Br. J. Pharmacol. https://doi.org/10.1111/bph.16274 (2023).
Griffin, B. A., Adams, S. R. & Tsien, R. Y. How FlAsH got its sparkle: historical recollections of the biarsenical-tetracysteine tag. Methods Mol. Biol. 1266, 1–6 (2015).
Mo, J. et al. Third-generation covalent TMP-tag for fast labeling and multiplexed imaging of cellular proteins. Angew. Chem. Int. Ed. Engl. 61, e202207905 (2022).
Liu, W., Samanta, S. K., Smith, B. D. & Isaacs, L. Synthetic mimics of biotin/(strept)avidin. Chem. Soc. Rev. 46, 2391–2403 (2017).
Green, N. M. Avidin and streptavidin. Methods Enzymol. 184, 51–67 (1990).
Beckett, D., Kovaleva, E. & Schatz, P. J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999).
Howarth, M., Takao, K., Hayashi, Y. & Ting, A. Y. Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc. Natl Acad. Sci. USA 102, 7583–7588 (2005).
Chivers, C. E. et al. A streptavidin variant with slower biotin dissociation and increased mechanostability. Nat. Methods 7, 391–393 (2010).
Christeller, J. T., Markwick, N. P., Burgess, E. P. J. & Malone, L. A. The use of biotin-binding proteins for insect control. J. Econ. Entomol. 103, 497–508 (2010).
Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).
Demonte, D., Drake, E. J., Lim, K. H., Gulick, A. M. & Park, S. Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins 81, 1621–1633 (2013).
Fairhead, M. et al. SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly. J. Am. Chem. Soc. 136, 12355–12363 (2014).
Narayanan, K. B. & Han, S. S. Peptide ligases: a novel and potential enzyme toolbox for catalytic cross-linking of protein/peptide-based biomaterial scaffolds for tissue engineering. Enzyme Microb. Technol. 155, 109990 (2022).
Pishesha, N., Ingram, J. R. & Ploegh, H. L. Sortase A: a model for transpeptidation and its biological applications. Annu. Rev. Cell Dev. Biol. 34, 163–188 (2018).
Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl Acad. Sci. USA 102, 15815–15820 (2005).
Wu, P. et al. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proc. Natl Acad. Sci. USA 106, 3000–3005 (2009).
Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D. & Bertozzi, C. R. A Pictet–Spengler ligation for protein chemical modification. Proc. Natl Acad. Sci. USA 110, 46–51 (2013).
Sun, B. & Kumar, S. Protein adsorption loss—the bottleneck of single-cell proteomics. J. Proteome Res. 21, 1808–1815 (2022).
Guo, J. et al. The development of proximity labeling technology and its applications in mammals, plants, and microorganisms. Cell Commun. Signal. 21, 269 (2023).
Seckute, J. & Devaraj, N. K. Expanding room for tetrazine ligations in the in vivo chemistry toolbox. Curr. Opin. Chem. Biol. 17, 761–767 (2013).
Uematsu, M. & Baskin, J. M. Chemical approaches for measuring and manipulating lipids at the organelle level. Cold Spring Harb. Perspect. Biol. 15, a041407 (2023).
Johnson, J. A., Lu, Y. Y., Van Deventer, J. A. & Tirrell, D. A. Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Chem. Biol. 14, 774–780 (2010).
Dube, D. H. & Bertozzi, C. R. Metabolic oligosaccharide engineering as a tool for glycobiology. Curr. Opin. Chem. Biol. 7, 616–625 (2003).
Salic, A. & Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl Acad. Sci. USA 105, 2415–2420 (2008).
de la Torre, D. & Chin, J. W. Reprogramming the genetic code. Nat. Rev. Genet. 22, 169–184 (2021).
Hodneland, C. D., Lee, Y.-S., Min, D.-H. & Mrksich, M. Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands. Proc. Natl Acad. Sci. USA 99, 5048–5052 (2002).
Mizukami, S., Hori, Y. & Kikuchi, K. Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications. Acc. Chem. Res. 47, 247–256 (2014).
Minoshima, M. et al. Development of a versatile protein labeling tool for live-cell imaging using fluorescent β-lactamase inhibitors. Angew. Chem. Int. Ed. Engl. 62, e202301704 (2023).
Whitesides, G. M. Cool, or simple and cheap? Why not both? Lab Chip 13, 11–13 (2013). An important philosophical perspective on the value of simplicity, in contrast to the frequent academic tendency to promote expensive and intricate solutions.
Keeble, A. H. & Howarth, M. Insider information on successful covalent protein coupling with help from SpyBank. Methods Enzymol. 617, 443–461 (2019).
McLachlan, G. et al. Progress in respiratory gene therapy. Hum. Gene Ther. 33, 893–912 (2022).
Borkowski, O. et al. Cell-free prediction of protein expression costs for growing cells. Nat. Commun. 9, 1457 (2018).
Nikić, I., Kang, J. H., Girona, G. E., Aramburu, I. V. & Lemke, E. A. Labeling proteins on live mammalian cells using click chemistry. Nat. Protoc. 10, 780–791 (2015).
Zimmerman, J. B., Anastas, P. T., Erythropel, H. C. & Leitner, W. Designing for a green chemistry future. Science 367, 397–400 (2020).
Paloheimo, M., Haarmann, T., Mäkinen, S. & Vehmaanperä, J. Production of industrial enzymes in Trichoderma reesei. In Gene Expression Systems in Fungi: Advancements and Applications (eds Schmoll, M. & Dattenböck, C.) 23–57 (Springer Nature, 2016).
Tyndall, S. M., Maloney, G. R., Cole, M. B., Hazell, N. G. & Augustin, M. A. Critical food and nutrition science challenges for plant-based meat alternative products. Crit. Rev. Food Sci. Nutr. 64, 638–653 (2024).
Way, J. C., Collins, J. J., Keasling, J. D. & Silver, P. A. Integrating biological redesign: where synthetic biology came from and where it needs to go. Cell 157, 151–161 (2014).
Keeble, A. H. & Howarth, M. Power to the protein: enhancing and combining activities using the Spy toolbox. Chem. Sci. 11, 7281–7291 (2020).
Rahikainen, R. et al. Overcoming symmetry mismatch in vaccine nanoassembly through spontaneous amidation. Angew. Chem. Int. Ed. Engl. 60, 321–330 (2021).
Balchin, D., Hayer-Hartl, M. & Hartl, F. U. Recent advances in understanding catalysis of protein folding by molecular chaperones. FEBS Lett. 594, 2770–2781 (2020).
Radanović, T. & Ernst, R. The unfolded protein response as a guardian of the secretory pathway. Cells 10, 2965 (2021).
Jain, T. et al. Biophysical properties of the clinical-stage antibody landscape. Proc. Natl Acad. Sci. USA 114, 944–949 (2017).
Houk, K. N., Leach, A. G., Kim, S. P. & Zhang, X. Binding affinities of host–guest, protein–ligand, and protein-transition-state complexes. Angew. Chem. Int. Ed. Engl. 42, 4872–4897 (2003).
Yen, C. et al. The development of global vaccine stockpiles. Lancet Infect. Dis. 15, 340–347 (2015).
Minutolo, N. G. et al. Quantitative control of gene-engineered T-cell activity through the covalent attachment of targeting ligands to a universal immune receptor. J. Am. Chem. Soc. 142, 6554–6568 (2020).
Brune, K. D. & Howarth, M. New routes and opportunities for modular construction of particulate vaccines: stick, click, and glue. Front. Immunol. 9, 1432 (2018).
Merrill, R. A. et al. A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins. J. Biol. Chem. 294, 16164–16171 (2019).
Lin, D. et al. Time-tagged ticker tapes for intracellular recordings. Nat. Biotechnol. 41, 631–639 (2023).
Donovan, D. A. et al. Engineered chromatin remodeling proteins for precise nucleosome positioning. Cell Rep. 29, 2520–2535 (2019).
Vester, S. K. et al. SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly. Nat. Commun. 13, 3714 (2022).
Kompa, J. et al. Exchangeable HaloTag ligands for super-resolution fluorescence microscopy. J. Am. Chem. Soc. 145, 3075–3083 (2023).
Moran, U., Phillips, R. & Milo, R. SnapShot: key numbers in biology. Cell 141, 1262 (2010).
Wilhelm, J. et al. Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag, and CLIP-tag. Biochemistry 60, 2560–2575 (2021).
Krämer, K. How click conquered chemistry. Chem World www.chemistryworld.com/features/how-click-conquered-chemistry/4016366.article (2022).
van den Bosch, S. M. et al. Evaluation of strained alkynes for Cu-free click reaction in live mice. Nucl. Med. Biol. 40, 415–423 (2013).
Sasmal, P. K. et al. Catalytic azide reduction in biological environments. ChemBioChem 13, 1116–1120 (2012).
Versteegen, R. M., Rossin, R., ten Hoeve, W., Janssen, H. M. & Robillard, M. S. Click to release: instantaneous doxorubicin elimination upon tetrazine ligation. Angew. Chem. Int. Ed. Engl. 52, 14112–14116 (2013).
Darko, A. et al. Conformationally strained trans-cyclooctene with improved stability and excellent reactivity in tetrazine ligation. Chem. Sci. 5, 3770–3776 (2014).
Swift, J. L., Heuff, R. & Cramb, D. T. A two-photon excitation fluorescence cross-correlation assay for a model ligand–receptor binding system using quantum dots. Biophys. J. 90, 1396–1410 (2006).
Pabis, A., Risso, V. A., Sanchez-Ruiz, J. M. & Kamerlin, S. C. Cooperativity and flexibility in enzyme evolution. Curr. Opin. Struct. Biol. 48, 83–92 (2018).
Clarke, A. et al. A low temperature limit for life on earth. PLoS ONE 8, e66207 (2013).
Kashefi, K. & Lovley, D. R. Extending the upper temperature limit for life. Science 301, 934 (2003).
Linser, P. J., Smith, K. E., Seron, T. J. & Neira Oviedo, M. Carbonic anhydrases and anion transport in mosquito midgut pH regulation. J. Exp. Biol. 212, 1662–1671 (2009).
Choi, J. J. et al. Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans. J. Mol. Biol. 356, 1093–1106 (2006).
Perugino, G. et al. Activity and regulation of archaeal DNA alkyltransferase. J. Biol. Chem. 287, 4222–4231 (2012).
Wang, Y., Shi, Y., Hellinga, H. W. & Beese, L. S. Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics. Nucleic Acids Res. 51, 5883–5894 (2023).
Courchesne, N. M. D., Duraj-Thatte, A., Tay, P. K. R., Nguyen, P. Q. & Joshi, N. S. Scalable production of genetically engineered nanofibrous macroscopic materials via filtration. ACS Biomater. Sci. Eng. 3, 733–741 (2017).
Schoene, C., Bennett, S. P. & Howarth, M. SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries. Sci. Rep. 6, 21151 (2016).
Mouysset, B., Le Grand, M., Camoin, L. & Pasquier, E. Poly-pharmacology of existing drugs: how to crack the code? Cancer Lett. 588, 216800 (2024).
England, C. G., Luo, H. & Cai, W. HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986 (2015).
Liu, X. et al. Split chimeric antigen receptor-modified T cells targeting glypican-3 suppress hepatocellular carcinoma growth with reduced cytokine release. Ther. Adv. Med. Oncol. 12, 1758835920910347 (2020).
Roy, A. & Gauld, J. W. Sulfilimine bond formation in collagen IV. Chem. Commun. 60, 646–657 (2024).
Alibardi, L. Vertebrate keratinization evolved into cornification mainly due to transglutaminase and sulfhydryl oxidase activities on epidermal proteins: an immunohistochemical survey. Anat. Rec. 305, 333–358 (2022).
Rajendran, A. K. et al. Trends in mechanobiology guided tissue engineering and tools to study cell–substrate interactions: a brief review. Biomater. Res. 27, 55 (2023).
Otomo, T., Teruya, K., Uegaki, K., Yamazaki, T. & Kyogoku, Y. Improved segmental isotope labeling of proteins and application to a larger protein. J. Biomol. NMR 14, 105–114 (1999).
Modica, J. A., Skarpathiotis, S. & Mrksich, M. Modular assembly of protein building blocks to create precisely defined megamolecules. ChemBioChem 13, 2331–2334 (2012). Demonstration of the power of high-yield irreversible covalent coupling with HaloTag and cutinase for generating novel classes of protein architecture.
Fryer, T. et al. Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering. ACS Cent. Sci. 8, 1182–1195 (2022).
Khairil Anuar, I. N. A. et al. Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox. Nat. Commun. 10, 1734 (2019).
Sun, F., Zhang, W. B., Mahdavi, A., Arnold, F. H. & Tirrell, D. A. Synthesis of bioactive protein hydrogels by genetically encoded SpyTag–SpyCatcher chemistry. Proc. Natl Acad. Sci. USA 111, 11269–11274 (2014).
Wang, R., Yang, Z., Luo, J., Hsing, I. M. & Sun, F. B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release. Proc. Natl Acad. Sci. USA 114, 5912–5917 (2017).
Scott, C. P., Abel-Santos, E., Jones, A. D. & Benkovic, S. J. Structural requirements for the biosynthesis of backbone cyclic peptide libraries. Chem. Biol. 8, 801–815 (2001). Given the power of cyclization to reduce peptide flexibility and degradation, showing how split intein cyclization allows selection of potential drug leads.
Sohrabi, C., Foster, A. & Tavassoli, A. Methods for generating and screening libraries of genetically encoded cyclic peptides in drug discovery. Nat. Rev. Chem. 4, 90–101 (2020).
Schoene, C., Bennett, S. P. & Howarth, M. SpyRings declassified: a blueprint for using isopeptide-mediated cyclization to enhance enzyme thermal resilience. Methods Enzymol. 580, 149–167 (2016).
Zhang, W. B., Sun, F., Tirrell, D. A. & Arnold, F. H. Controlling macromolecular topology with genetically encoded SpyTag–SpyCatcher chemistry. J. Am. Chem. Soc. 135, 13988–13997 (2013). Establishing a simple route to prepare a panel of programmed nonlinear protein topologies through efficient isopeptide bond formation.
Li, T., Zhang, F., Fang, J., Liu, Y. & Zhang, W.-B. Rational design and cellular synthesis of proteins with unconventional chemical topology. Chin. J. Chem. 41, 2873–2880 (2023).
Zhang, F., Liu, Y., Da, X.-D. & Zhang, W.-B. Toward selective synthesis of protein olympiadanes via orthogonal active templates in one step. CCS Chem. 6, 1047–1059 (2023). Integrating supramolecular chemistry with protein engineering, this paper harnesses multiple genetically directed couplings to achieve intricate mechanically interlocked architectures.
Hong, H. et al. HaloTag: a novel reporter gene for positron emission tomography. Am. J. Transl. Res. 3, 392–403 (2011).
Strauch, R. C. et al. Reporter protein-targeted probes for magnetic resonance imaging. J. Am. Chem. Soc. 133, 16346–16349 (2011).
Li, Y. et al. Conformable self-assembling amyloid protein coatings with genetically programmable functionality. Sci. Adv. 6, eaba1425 (2020).
Williams, D. M. et al. Facile protein immobilization using engineered surface-active biofilm proteins. ACS Appl. Nano Mater. 1, 2483–2488 (2018).
Ma, W. et al. Modular assembly of proteins on nanoparticles. Nat. Commun. 9, 1489 (2018).
Wang, F. et al. General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM. Proc. Natl Acad. Sci. USA 117, 24269–24273 (2020).
Ramlaul, K. et al. A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate. J. Struct. Biol. 215, 107999 (2023).
Patel, A. et al. Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors. EMBO J. 43, 132–150 (2024).
Wang, X.-J. et al. Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum. PLoS ONE 9, e99651 (2014).
Free, K., Nakanishi, H. & Itaka, K. Development of synthetic mRNAs encoding split cytotoxic proteins for selective cell elimination based on specific protein detection. Pharmaceutics 15, 213 (2023).
Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W. & Muir, T. W. Intein zymogens: conditional assembly and splicing of split inteins via targeted proteolysis. J. Am. Chem. Soc. 139, 8074–8077 (2017).
Zhao, Y. et al. SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein–RNA interactions with high accuracy. Nucleic Acids Res. 47, e33 (2019).
Gu, J. et al. GoldCLIP: gel-omitted ligation-dependent CLIP. Genomics Proteomics Bioinformatics 16, 136–143 (2018).
Taniguchi, Y. & Kawakami, M. Application of HaloTag protein to covalent immobilization of recombinant proteins for single molecule force spectroscopy. Langmuir 26, 10433–10436 (2010).
Rivas-Pardo, J. A. et al. A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues. Nat. Commun. 11, 2060 (2020).
Napierski, N. C. et al. A novel ‘cut and paste’ method for in situ replacement of cMyBP-C reveals a new role for cMyBP-C in the regulation of contractile oscillations. Circ. Res. 126, 737–749 (2020).
Tornabene, P. et al. Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Sci. Transl. Med. 11, eaav4523 (2019).
Muik, A. et al. Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer. Biomaterials 144, 84–94 (2017).
Sabin, L. et al. Viral particles retargeted to skeletal muscle. US Patent WO/2023/081850 (2023).
Bachmann, M. F. & Jennings, G. T. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787–796 (2010).
Hills, R. A. & Howarth, M. Virus-like particles against infectious disease and cancer: guidance for the nano-architect. Curr. Opin. Biotechnol. 73, 346–354 (2022).
Wang, Q. et al. Bioconjugation by copper(I)-catalyzed azide–alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 3192–3193 (2003).
Li, X. & Xiong, Y. Application of ‘click’ chemistry in biomedical hydrogels. ACS Omega 7, 36918–36928 (2022).
van den Berg van Saparoea, H. B., Houben, D., de Jonge, M. I., Jong, W. S. P. & Luirink, J. Display of recombinant proteins on bacterial outer membrane vesicles by using protein ligation. Appl. Environ. Microbiol. 84, e02567-17 (2018).
Brune, K. D. et al. Plug-and-Display: decoration of virus-like particles via isopeptide bonds for modular immunization. Sci. Rep. 6, 19234 (2016).
Thrane, S. et al. Bacterial superglue enables easy development of efficient virus-like particle based vaccines. J. Nanobiotechnology 14, 30 (2016).
King, L. D. W. et al. Preclinical development of a stabilized RH5 virus-like particle vaccine that induces improved anti-malarial antibodies. Cell Rep. Med. 5, 101654 (2024).
Smit, M. J. et al. First-in-human use of a modular capsid virus-like vaccine platform: an open-label, non-randomised, phase 1 clinical trial of the SARS-CoV-2 vaccine ABNCoV2. Lancet Microbe 4, e140–e148 (2023). Entry of click biology into human clinical trials through modular decoration of VLPs.
Baker, D. What has de novo protein design taught us about protein folding and biophysics? Protein Sci. 28, 678–683 (2019).
Du, J., Kong, Y., Wen, Y., Shen, E. & Xing, H. HUH endonuclease: a sequence-specific fusion protein tag for precise DNA–protein conjugation. Bioorg. Chem. 144, 107118 (2024).
Sacca, B. et al. Orthogonal protein decoration of DNA origami. Angew. Chem. Int. Ed. Engl. 49, 9378–9383 (2010).
Komiya, E. et al. Exploration and application of DNA-binding proteins to make a versatile DNA–protein covalent-linking patch (D-Pclip): the case of a biosensing element. J. Am. Chem. Soc. 146, 4087–4097 (2024).
Mashimo, Y., Maeda, H., Mie, M. & Kobatake, E. Construction of semisynthetic DNA–protein conjugates with Phi X174 Gene-A* protein. Bioconjug. Chem. 23, 1349–1355 (2012). Pioneering the site-specific covalent linkage of protein to DNA through engineering of a phage ligation system.
Bernardinelli, G. & Högberg, B. Entirely enzymatic nanofabrication of DNA–protein conjugates. Nucleic Acids Res. 45, e160 (2017).
Lovendahl, K. N., Hayward, A. N. & Gordon, W. R. Sequence-directed covalent protein–DNA linkages in a single step using HUH-tags. J. Am. Chem. Soc. 139, 7030–7035 (2017). Establishment of a panel of efficient modules, engineered from viruses and bacteria, to empower spontaneous covalent coupling of DNA to protein.
Silverman, S. K. Catalytic DNA: scope, applications, and biochemistry of deoxyribozymes. Trends Biochem. Sci. 41, 595–609 (2016).
Sun, P., Gou, H., Che, X., Chen, G. & Feng, C. Recent advances in DNAzymes for bioimaging, biosensing and cancer therapy. Chem. Commun. 60, 10805–10821 (2024).
Baum, D. A. & Silverman, S. K. Deoxyribozyme-catalyzed labeling of RNA. Angew. Chem. Int. Ed. Engl. 46, 3502–3504 (2007).
Kong, L.-Z. et al. Understanding nucleic acid sensing and its therapeutic applications. Exp. Mol. Med. 55, 2320–2331 (2023).
Hoffmann, M. D. et al. Multiparametric ___domain insertional profiling of adeno-associated virus VP1. Mol. Ther. Methods Clin. Dev. 31, 101143 (2023).
Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
Weill, U. et al. Genome-wide SWAp-Tag yeast libraries for proteome exploration. Nat. Methods 15, 617–622 (2018).
Eiamthong, B. et al. Discovery and genetic code expansion of a polyethylene terephthalate (PET) hydrolase from the human saliva metagenome for the degradation and bio-functionalization of PET. Angew. Chem. Int. Ed. Engl. 61, e202203061 (2022).
Mican, J. et al. Exploring new galaxies: perspectives on the discovery of novel PET-degrading enzymes. Appl. Catal. B Environ. 342, 123404 (2024).
Lin, Z. et al. Evolutionary-scale prediction of atomic-level protein structure with a language model. Science 379, 1123–1130 (2023).
Lu, H. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022).
Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011).
Lovelock, S. L. et al. The road to fully programmable protein catalysis. Nature 606, 49–58 (2022).
Yin, Z. et al. Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anticancer vaccine. ACS Chem. Biol. 10, 2364–2372 (2015).
Ortiz de Montellano, P. R. Acetylenes: cytochrome P450 oxidation and mechanism-based enzyme inactivation. Drug Metab. Rev. 51, 162–177 (2019).
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Funding was provided by the Engineering and Physical Sciences Research Council (EPSRC EP/T030704/1).
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M.R.H. is an inventor on a patent on traptavidin (UK Intellectual Property Office 0919102.4), a SpyBiotech cofounder and shareholder, and an inventor on patents on spontaneous amide bond formation (UK Intellectual Property Office 2117283.8, 2104999.4, 1915905.2, 1903479.2, 1819850.7, 1706430.4, 1705750.6, 1509782.7, 1002362.0).
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Howarth, M.R. Click biology highlights the opportunities from reliable biological reactions. Nat Chem Biol 21, 991–1005 (2025). https://doi.org/10.1038/s41589-025-01944-x
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DOI: https://doi.org/10.1038/s41589-025-01944-x
