A proximity labeling platform for high-resolution mapping of biomolecular condensate microenvironments and interactomes

Background

Biomolecular condensates are dynamic, membrane-less compartments within eukaryotic cells that form through liquid-liquid phase separation. These structures play vital roles in organizing cellular biochemistry, concentrating proteins and nucleic acids, and regulating processes such as stress response, gene expression, and protein homeostasis. Understanding the precise molecular interactions within these condensates is crucial for elucidating their functions and for identifying how their dysfunction contributes to diseases like Alzheimer’s, Parkinson’s, and ALS. High-resolution mapping of the local interactome within condensates can reveal key regulatory mechanisms and potential therapeutic targets, making it a significant area of interest in cell biology and biomedical research.

Current proximity labeling approaches for profiling condensate microenvironments face several limitations that hinder their effectiveness. Traditional enzyme-based labeling methods generate reactive intermediates that often diffuse too far from their site of origin, leading to poor spatial resolution and labeling of unintended targets. The large size of labeling enzymes can also disrupt the native structure of condensates or fail to access confined regions. Additionally, these methods typically depend on the presence of specific amino acids, restricting their applicability and potentially missing important interactions. A lack of precise temporal control further complicates efforts to capture dynamic changes during condensate assembly or disassembly. Collectively, these challenges prevent accurate, high-resolution mapping of the immediate molecular environment within biomolecular condensates.

Technology

This technology is a proximity-based labeling system designed for high-resolution mapping of the local interactome within biomolecular condensates—membrane-less cellular compartments critical for organizing proteins and nucleic acids. The system features a composite of a biomolecular condensate and a transition metal catalyst, such as an iridium complex, which is coupled to the condensate via covalent, electrostatic, or van der Waals interactions. Upon activation, typically by light, the catalyst transfers energy to a protein labeling agent (like a diazirine or azide), generating a highly reactive intermediate (carbene or nitrene) with a nanosecond-scale half-life and a diffusion radius of just 2 to 10 nanometers. This ensures that only biomolecules in the immediate vicinity of the condensate are labeled. The labeling agents can be further functionalized with detection tags (e.g., biotin, fluorophores), enabling downstream proteomic analysis and visualization. The system also allows for precise temporal control, making it possible to profile interactions at specific stages of condensate assembly or disassembly.

This solution is differentiated by its ability to overcome the major limitations of traditional proximity labeling methods, which often suffer from excessive diffusion of reactive intermediates, large enzyme size, dependency on specific amino acids, and lack of temporal precision. By leveraging a small, tunable transition metal catalyst and generating ultra-short-lived intermediates, this approach confines labeling to the nanometer-scale microenvironment of the condensate, achieving unparalleled spatial resolution. The modular design allows for adaptation to diverse condensate types and cellular locations, and the temporal control enables dynamic studies of condensate biology. Experimental validation has shown that this system can capture both known and novel interactors, track real-time changes during condensate lifecycle transitions, and identify potential disease-relevant regulatory proteins, making it a powerful and versatile tool for cell biology and disease research.

Benefits

• Enables high-resolution, nanometer-scale proximity labeling of biomolecular condensate microenvironments
• Provides precise temporal control to study condensate dynamics during specific lifecycle stages
• Generates highly reactive intermediates with short half-life and limited diffusion radius for localized labeling
• Applicable to diverse biomolecular condensates across various cellular compartments
• Facilitates identification of both known and novel protein and nucleic acid interactors within condensates
• Supports downstream detection using versatile labeling agents functionalized with markers like biotin or fluorophores
• Overcomes limitations of existing methods such as excessive diffusion, enzyme size, and amino acid dependency
• Offers a modular and generalizable platform for studying condensate biology and disease-related protein interactions

Applications

• High-resolution mapping of protein interactomes
• Temporal analysis of condensate dynamics
• Disease mechanism discovery in neurodegeneration
• Drug target identification in condensates
• Profiling nucleic acid interactions in situ

Additional info

This system utilizes a transition metal photocatalyst, such as an iridium complex, coupled to biomolecular condensates via covalent or non-covalent interactions. Upon activation, the catalyst facilitates Dexter energy transfer to labeling agents, generating highly reactive carbene or nitrene intermediates. With a 1–5 nanosecond half-life and 2–10 nanometer diffusion radius, these intermediates enable high-resolution, proximity-based mapping of local interactomes with precise temporal control during specific cellular lifecycle stages.