Propiolates-Based Selective Labeling and Affinity Capture Enables High-Fidelity Transcriptome-Wide Profiling of...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Adenosine-to-inosine (A-to-I) RNA editing is a crucial post-transcriptional process impacting gene expression and implicated in various diseases.
• Existing detection methods for A-to-I sites lack sensitivity and specificity due to cross-reactivity with pseudouridine and inefficient RNA enrichment.

Purpose of the Study:

• To develop a novel, highly sensitive, and specific chemical labeling strategy for transcriptome-wide profiling of A-to-I RNA editing sites.
• To overcome the limitations of current methods, particularly pseudouridine cross-reactivity and suboptimal enrichment efficiency.

Main Methods:

• A new chemical labeling strategy employing propiolates as selective inosine-binding agents was developed.
• Optimization involved screening propiolates and reaction conditions, identifying tert-butyl propiolate as a highly specific probe.
• Biotin-streptavidin enrichment was coupled with copper-free click chemistry and magnetic bead pulldown for efficient RNA enrichment.

Main Results:

• The novel propiolate probe demonstrated 6-fold higher specificity for inosine over pseudouridine.
• The optimized protocol achieved a 55-fold enrichment efficiency for inosine-containing RNAs, a 3.7-fold improvement over acrylonitrile-derivative methods.
• The method successfully identified A-to-I editing sites in human cellular RNA with enhanced accuracy and coverage.

Conclusions:

• This propiolate-based chemical labeling strategy offers a significant advancement for precise A-to-I RNA editing detection.
• The enhanced specificity and enrichment efficiency provide a universal platform for studying RNA editing dynamics in biological and pathological contexts.
• This work expands the epitranscriptomic toolbox, facilitating biomarker discovery and understanding the functional roles of A-to-I editing.