Enzyme-Triggered Acylation Probes for Spatial RNA Labeling

SUMMARY

Enhanced spatial RNA labeling accuracy and efficiency through bioorthogonal acylating agents that enable precise proximity-dependent RNA modification and imaging, improving cellular RNA mapping for both research and clinical molecular diagnostics

The Unmet Need: Current RNA proximity-labeling methods lack specificity and efficiency for precise subcellular RNA mapping

• Traditional RNA labeling approaches predominantly use radicals or reactive oxygen species, which often require high reagent concentrations, extended reaction times, and may introduce artifacts or incomplete localization profiles, limiting their effectiveness in mapping RNA within distinct cellular compartments.

• There is a growing industry-wide demand for molecular tools that offer high specificity and controlled reactivity for RNA labeling, driven by advancements in spatial transcriptomics and the need for precise molecular interrogation of subcellular RNA dynamics to understand complex cellular processes.

The Proposed Solution: Bioorthogonal acylation reagents activated by targeted esterases enable controlled, proximity-dependent RNA labeling

• The faculty inventor developed ester probes that are selectively unmasked by a Bacillus subtilis esterase (BS2) localized to subcellular regions, releasing highly reactive acid chlorides that covalently modify RNA 2’-hydroxyl groups in proximity.

• This strategy differs from existing techniques by employing a controlled non-radical acylation reaction with tunable probe electrophilicity and incorporates alkyne handles for downstream imaging and enrichment via click chemistry. Experimental validation includes in vitro and cellular models demonstrating selective spatial RNA labeling in both membrane-bound and membrane-less organelles, with demonstrable mapping of mitochondrial, nuclear, and nucleolar RNAs, confirming the technology’s specificity and functional versatility.

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ADVANTAGES

• High specificity through esterase-targeted unmasking

• Enhanced spatial resolution in RNA labeling

• Bioorthogonal chemistry with non-radical acylation

• Compatibility with click chemistry for imaging and enrichment

• Versatile application across membrane-bound and membrane-less organelles

• Improved tunability of probe reactivity via ester electrophilicity

APPLICATIONS

• Subcellular RNA mapping in molecular and cellular biology research

• Spatial transcriptomics for cancer and neurodegenerative disease studies

• Development of RNA-targeted diagnostic and therapeutic platforms