Bioorthogonal Enzyme-Probe System For Spatially Specific Biomolecule Labeling

SUMMARY

Novel proximity labeling technology utilizes a bioorthogonal protecting group, impervious to endogenous esterases, paired with evolved BS2 esterase mutants. The enzyme-mediated unmasking creates a reactive acid chloride intermediate that rapidly labels adjacent RNA, ensuring high specificity and sensitivity for biomolecule mapping within living cells.

The Unmet Need: Techniques for mapping biomolecules within living cells with spatial specificity and signaling sensitivity

• Proximity labeling is a vital technique in cell biology, allowing researchers to map the spatial organization and interactions of biomolecules, including RNA and proteins, within living cells. This capability is crucial for deciphering complex cellular processes, understanding disease mechanisms, and identifying novel therapeutic targets. There is a significant need for methods that can precisely identify and label molecules in their native environment with high spatial resolution and fidelity, providing accurate insights into the intricate architecture of the cell.

• However, current proximity labeling technologies encounter substantial limitations. A major challenge is the poor sensitivity for RNA labeling, as many existing methods generate radical intermediates that are inefficient at tagging RNA molecules. Furthermore, a pervasive problem is non-specific labeling. This arises because endogenous esterases, naturally present in cells, can prematurely unmask the probe substrates, leading to unintended and widespread labeling throughout the cell rather than exclusively at the desired location. Even more advanced approaches, while improving RNA tagging, still struggle with this pervasive non-specific activity, particularly in cell types with high endogenous esterase expression.

The Proposed Solution: A bioorthogonal protecting group–enzyme system paired with evolved BS2 esterase mutants to achieve precise spatial labeling of biomolecules in living cells

• The faculty inventor developed a novel method for spatially specific labeling of biomolecules, particularly RNA, within living cells. It employs a bioorthogonal protecting group–esterase pair, featuring a novel protecting group that masks substrate molecules and resists cleavage by endogenous cellular esterases. Substrates are unmasked only by genetically evolved Bacillus subtilis (BS2) mutants, which possess enhanced hydrolysis activity. Upon unmasking, a reactive acid chloride intermediate is formed, rapidly labeling nearby RNA with high specificity and minimal diffusion. The development of these highly active BS2 variants was facilitated by an ultrahigh-throughput directed evolution platform utilizing yeast surface display, enabling the screening of millions of enzyme variants.

• The technology is highly differentiated by directly addressing critical limitations of previous proximity labeling techniques. Unlike prior methods that suffered from non-specific labeling due to endogenous esterase activity, the unique protecting group ensures that unmasking and subsequent labeling occur exclusively where the engineered BS2 enzyme is present. Furthermore, the generation of an acid chloride intermediate significantly improves RNA labeling efficiency and spatial resolution compared to less effective radical intermediates used previously. The adaptable directed evolution platform also provides a robust tool for future enzyme engineering.

ADVANTAGES

ADVANTAGES

• Significantly improves specificity and sensitivity for proximity labeling, particularly for RNA

• Prevents non-specific labeling by resisting cleavage from endogenous cellular esterases

• Enables highly spatially specific labeling of biomolecules within living cells

• Ultrahigh-throughput directed evolution platform for rapid enzyme discovery and optimization

APPLICATIONS

• Map biomolecules in cells

• Enzyme-activated pro-drugs

• High-throughput enzyme discovery

• Selective biorthogonal chemistry

• Enzyme-based diagnostic tools