Dynamic Control of RNA Structure and Function through Bioorthogonal Staudinger Chemistry

Area of Science:

• Chemical Biology
• Molecular Biology
• RNA Therapeutics

Background:

• RNA molecules play critical roles in gene expression and regulation.
• Controlling RNA function chemically offers precise tools for biological studies and therapeutics.
• Existing methods for RNA modification and regulation have limitations in reversibility and scope.

Purpose of the Study:

• To develop a reversible chemical strategy for modulating RNA structure and function.
• To investigate the use of a novel bifunctional azide reagent (DAPIC) for RNA postsynthetic modification.
• To demonstrate the application of this platform in regulating CRISPR-Cas9 gene editing systems.

Main Methods:

• Design and synthesis of a bifunctional azide reagent, 1,3-diazidopropan-2-yl 1H-imidazol-1-carboxylate (DAPIC).
• Chemical modification of RNA's 2'-hydroxyl group using DAPIC, leading to functional disruption.
• Reactivation of modified RNA via Staudinger reduction using 2-diphenylphosphinoethylamine (DPPEA).
• Application of the system to guide RNAs in CRISPR-Cas9 gene editing, assessing DNA cleavage activity.

Main Results:

• DAPIC specifically modifies the 2'-hydroxyl of RNA, reversibly inhibiting its structure and function.
• Staudinger reduction with DPPEA efficiently restores RNA activity, demonstrating reversibility.
• DAPIC modification abrogates CRISPR-Cas9 DNA cleavage, which is restored in a DPPEA-dependent manner in vitro and in cells.
• DAPIC shows enhanced reactivity and reduced reagent needs compared to monoazide analogs.

Conclusions:

• A robust and generalizable chemical platform for reversible RNA regulation based on Staudinger chemistry has been established.
• This method provides a powerful tool for conditional gene editing and studying RNA functions in complex biological systems.
• The reversible chemical control of RNA function opens new avenues for RNA-based therapeutics and molecular diagnostics.