Propiolates-Based Selective Labeling and Affinity Capture Enables High-Fidelity Transcriptome-Wide Profiling of A-to-I RNA Editing

Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that regulates various biological processes and has been implicated in neurological diseases, cancer, and autoimmune diseases. However, current methods for detecting A-to-I sites, including inosine chemical erasing and acrylonitrile-derivative labeling, suffer from compromised sensitivity and specificity due to two critical limitations: cross-reactivity with pseudouridine and suboptimal enrichment efficiency. Here, we introduce a novel chemical labeling strategy using propiolates as selective inosine-binding agents, coupled with biotin-streptavidin enrichment, enabling precise transcriptome-wide profiling of A-to-I editing sites. Through screening a range of propiolates and optimizing the reaction conditions, we demonstrated that <i>tert</i>-butyl propiolate functions as a highly selective probe, achieving 6-fold higher specificity for I compared to pseudouridine (Ψ) in RNA editing detection. This scaffold represents the first application of propiolates in RNA editing detection. Subsequent RT-qPCR analysis revealed that the optimized protocol achieved a 55-fold enrichment efficiency of inosine-containing RNAs through copper-free click chemistry conjugation and streptavidin magnetic bead pulldown. Compared to acrylonitrile-derivative labeling methods, this protocol represents a 3.7-fold improvement in enrichment efficiency. Applied to human cellular RNA, this method robustly identified A-to-I editing sites with enhanced accuracy and coverage. By reducing pseudouridine cross-reactivity and enabling efficient RNA enrichment, our strategy provides a universal platform for studying RNA editing dynamics in development, disease, and therapeutic contexts, thereby opening new avenues for epitranscriptomic biomarker discovery. This work advances the molecular toolbox for epitranscriptomics, offering broad utility in dissecting the functional roles of A-to-I editing in health and pathology.