In a groundbreaking advance that pushes the boundaries of protein engineering and cell-surface targeting, researchers have unveiled a highly modular platform capable of programmable protein ligation directly on living cells. This innovative system, known as SMART-SpyCatcher, leverages a diverse array of targeting modalities, including antibody fragments, mimetics, peptides, and small molecules, to enable precise, logic-gated control of proximity labeling. The implications for this versatile technology are vast, promising unprecedented capabilities in cellular diagnostics, targeted therapeutics, and proximity-dependent biochemical analyses.
The core strength of the SMART platform lies in its remarkable modularity. Early iterations utilized designed ankyrin repeat proteins (DARPins) fused to SpyN and SpyC modules for cell-surface receptor targeting. Building on this, the team demonstrated seamless incorporation of single-domain antibodies (sdAbs) and single-chain fragment variables (scFvs) through recombinant bacterial expression, greatly expanding the targetable protein repertoire. This modular compatibility underscores the adaptability of the SMART system, facilitating rapid deployment across varied cellular contexts with minimal protocol alteration.
Notably, the researchers introduced a strategic carboxy-terminal cysteine residue into the eNrdJ-1C^cage component, which naturally lacks cysteines, to create a reactive handle for chemical conjugation. This clever modification enabled straightforward installation of synthetic ligands, including peptidyl and small-molecule antagonists. Exemplifying this approach, SpyC was chemically conjugated to BKT140, a cyclic peptide antagonist targeting CXCR4, a key chemokine receptor implicated in cancer metastasis and immune modulation. Similarly, the small-molecule antagonist SCH58261 was attached to SpyC to engage ADORA2A, a G-protein-coupled receptor with roles in immunosuppression and tumor microenvironment regulation.
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These functionalized SpyC conjugates sharply broaden the toolkit for engaging diverse cell-surface receptors beyond the conventional antibody landscape. The team validated this expanded targeting capability across multiple cell lines, including K562 cells engineered to express HER2 and EGFR, as well as the OE19 gastric carcinoma line exhibiting high HER2 and EpCAM levels but low CXCR4 and ADORA2A expression. Further sophistication was achieved through the development of OE19 derivatives with doxycycline-inducible expression of either CXCR4 or ADORA2A receptors, enabling precise control of receptor presentation in live-cell experiments.
Remarkably, the integration of these varied targeting vectors did not necessitate modifications to the original SMART-SpyCatcher protocol. The platform’s intrinsic compatibility allowed consistent execution of AND-gated proximity labeling assays, yielding expected fluorescence outputs in flow cytometry analyses. This finding attests to the robust design of SMART, wherein disparate molecular recognition elements can be interchanged without compromising functional performance. Such flexibility is of paramount importance for applications requiring adaptable targeting strategies in heterogeneous cellular environments.
At the mechanistic heart of the SMART approach lies an AND-gated system that requires simultaneous engagement of two distinct cell-surface markers for effective protein proximity labeling. When combined with SpyTag003 conjugates carrying enzymatic catalysts such as APEX2 peroxidase, the system facilitates spatially resolved biotinylation of proximal proteins upon activation with hydrogen peroxide and biotin-phenol substrates. Western blot analyses of treated K562, OE19, A431, and induced OE19^CXCR4DOX cells confirmed the specificity and efficiency of this logic-gated labeling, with signal intensities correlating strongly with predicted AND-gate receptor expressions.
Expanding beyond enzymatic labeling, the team innovatively harnessed photocatalytic proximity labeling to confer further spatial and temporal control. Employing a SpyTag003 molecule conjugated to an iridium photocatalyst, they demonstrated selective cell-surface labeling under 450-nanometer light irradiation. When combined with a biotin-diazirine chemical probe, this approach enabled targeted covalent modification of proteins in live-cell mixtures, as evidenced by flow cytometry evaluation of varied K562 cell subpopulations. This light-dependent modality represents a powerful complement to enzymatic methods, allowing precise control over labeling windows and minimizing background noise.
This spectrum of targeting modalities and labeling strategies places SMART at the forefront of programmable protein ligation technologies. By integrating recombinant antibody fragments, synthetic peptides, and small-molecule ligands within a unified SpyTag/SpyCatcher framework, researchers can tailor proximity labeling to almost any cell-surface receptor constellation. Such an approach holds promise for dissecting complex signaling networks, identifying cell subtypes within heterogeneous tissues, and engineering bespoke therapeutics predicated on highly selective receptor engagement.
Beyond the proof-of-concept studies presented, the modularity of SMART suggests broad utility across biomedical research and clinical applications. For instance, the ability to chemically tether ligands to SpyC components opens pathways to interface with previously ‘undruggable’ targets or to incorporate novel synthetic chemistry strategies for receptor engagement. Meanwhile, the facile bacterial expression of sdAb and scFv fusions streamlines production pipelines, enhancing accessibility for diverse research laboratories.
The compatibility of SMART components with existing molecular biology tools and cell lines further amplifies its appeal. Inducible receptor expression systems enable dynamic interrogation of target protein functions in situ, while the multiplexing capacity fostered by modular targeting expands possibilities for combinatorial logic gating—allowing for highly selective interrogation of cell states defined by multiple markers. Consequently, SMART stands as a versatile platform for both fundamental biological discovery and translational medicine.
Moreover, the integration of photocatalytic labeling techniques exemplifies how SMART transcends traditional biochemical methods, embracing advances in photochemistry to impart additional layers of control. Light-triggered protein ligation and labeling afford unmatched temporal precision, potentially enabling real-time tracking of receptor interactions, dynamic signaling events, and spatiotemporal mapping of protein neighborhoods in living systems. Such capabilities will undoubtedly catalyze new insights into cell biology as well as innovative therapeutic strategies.
In essence, SMART represents a leap in programmable biotechnology, harmonizing recombinant protein engineering, chemical conjugation, and photochemistry within a single, adaptable system. Its ability to target diverse receptors using multiple molecular modalities, coupled with logic-gated proximity labeling, sets the stage for transformative advances in both research and clinical contexts. As the platform matures, it will be exciting to witness its deployment in complex biological settings, including in vivo applications, where precision targeting is paramount.
Ultimately, this study redefines the landscape of cell-surface receptor targeting and protein ligation. By harnessing modular components that can be mixed and matched according to experimental needs—be it antibodies, peptides, or small molecules—and combining them with innovative labeling mechanisms, SMART opens new avenues for understanding and manipulating cellular environments with unparalleled specificity and control.
Subject of Research: Programmable protein ligation on cell surfaces using diverse targeting modalities and logic-gated proximity labeling.
Article Title: Programmable protein ligation on cell surfaces.
Article References: Kofoed, C., Erkalo, G., Tay, N.E.S. et al. Programmable protein ligation on cell surfaces. Nature (2025). https://doi.org/10.1038/s41586-025-09287-2
Image Credits: AI Generated
Tags: cell surface engineering technologycellular diagnostics advancementschemical conjugation methodsmodular protein engineeringprogrammable protein ligationprotein engineering breakthroughsprotein-targeting modalitiesproximity labeling techniquesscFvs in protein targetingsingle-domain antibodies applicationSMART-SpyCatcher platformtargeted therapeutics innovations
