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 tert-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.