A modular photocatalyst-based labeling system for high-resolution mapping of biological microenvironments and interactions

Background

Proximity-based labeling technologies have become essential tools in molecular and cellular biology for mapping the spatial organization of proteins and their interactions within living systems. These methods enable researchers to identify and profile protein-protein interactions and microenvironmental composition in situ, providing insights into cellular processes that are otherwise difficult to capture. The ability to resolve these interactions with high spatial and temporal precision is particularly valuable for understanding dynamic biological events, such as signaling at cellular membranes or within organelles. As biological systems are highly compartmentalized and complex, there is a growing need for labeling techniques that can operate effectively in live cells or tissues, offering nanometer-scale resolution and minimal perturbation to native cellular functions.

Current proximity labeling approaches predominantly rely on enzyme-based systems, such as peroxidases or biotin ligases, to generate reactive intermediates that covalently tag nearby proteins. However, these enzyme-generated intermediates often have relatively long half-lives and large diffusion radii, which can result in significant off-target labeling and reduced spatial specificity. Additionally, the bulky size of enzymes, their limited substrate specificity, and the lack of precise temporal control over labeling reactions further constrain their utility, especially in densely packed or highly dynamic biological environments. These limitations hinder efforts to achieve accurate mapping of protein interactions within confined microenvironments, underscoring the need for new systems that offer finer spatial control, tunable reactivity, and compatibility with live-cell or in vivo applications.

Technology

This proximity-based labeling system is a highly modular platform designed for high-resolution mapping of biological microenvironments, particularly for identifying protein-protein interactions in living cells and tissues. The system utilizes tetrapyrrole or transition metal photocatalysts that are conjugated to targeting ligands, enabling precise localization to specific cellular environments. Upon activation by long-wavelength light (650–1100 nm), these photocatalysts transfer energy to protein labeling agents—such as azides, diazirines, or phenols—through Dexter energy transfer or single electron transfer mechanisms. This process generates highly reactive intermediates (e.g., carbenes, nitrenes, or radicals) with extremely short half-lives and minimal diffusion radii, ensuring that only proteins in the immediate vicinity are covalently labeled. The labeled proteins can be functionalized with affinity tags like biotin or fluorophores, facilitating their isolation, enrichment, or visualization through downstream analytical techniques.

What sets this technology apart is its unprecedented spatial and temporal precision, overcoming the limitations of traditional enzyme-based labeling methods. Unlike enzymes, which often produce reactive species with longer lifetimes and broader diffusion, this system’s intermediates are confined to nanometer-scale regions, drastically reducing off-target labeling. The use of small-molecule photocatalysts allows for fine-tuning of reactivity, absorption properties, and solubility, making the system adaptable for both intracellular and extracellular applications. Additionally, the platform supports multiplexed analysis by employing photocatalysts with distinct light absorption profiles, enabling simultaneous mapping of multiple environments. Its modular design, compatibility with live-cell and in vivo applications, and the ability to target virtually any biological microenvironment make it a powerful and differentiated tool for proteomics, interactome mapping, and molecular imaging.

Benefits

• Enables high spatial resolution protein labeling with nanometer-scale precision (diffusion radius <10 nm)
• Provides precise temporal control of labeling via light activation (650–1100 nm)
• Uses modular photocatalysts and labeling agents for tunable reactivity and multiplexed analysis
• Compatible with live-cell and in vivo applications due to long-wavelength light penetration
• Allows targeted labeling through conjugation of photocatalysts to biomolecular binding agents
• Generates highly reactive intermediates with extremely short half-lives, minimizing off-target effects
• Facilitates downstream protein isolation, enrichment, and imaging via functional affinity tags
• Adaptable to diverse biological environments with customizable catalyst solubility and permeability

Applications

• High-resolution interactome mapping
• Live-cell protein localization imaging
• Multiplexed cellular compartment analysis
• Targeted proteomic enrichment

Additional info

This technology utilizes tetrapyrrole or transition metal photocatalysts conjugated to targeting ligands for high-resolution mapping of biological microenvironments. Long-wavelength light triggers energy transfer to labeling agents, generating reactive intermediates with sub-5-nanosecond half-lives and diffusion radii under 10 nanometers. These intermediates covalently bond to neighboring proteins, enabling precise spatial profiling. The modular design supports tunable reactivity and multiplexed analysis of protein interactions in vivo.