Clickable RNA via 4'-C-Ethynyl Cytidine─A Novel Design for Metabolically Stable Guide RNAs in RNA Editing.

Area of Science:

• Oligonucleotide chemistry
• RNA therapeutics
• Chemical biology

Background:

• Chemical modifications are crucial for improving RNA therapeutics' stability, delivery, and efficacy.
• Existing modifications face challenges in versatility and direct integration for specific functions.

Purpose of the Study:

• To synthesize and incorporate novel 4"-C-ethynyl cytidine (4"-C-EthC) modifications into oligonucleotides.
• To evaluate the utility of these modifications as a "click handle" for further functionalization.
• To assess the impact of these modifications on RNA duplex stability, nuclease resistance, and gene editing efficiency.

Main Methods:

• Synthesis of 4"-C-EthC and 4"-C-EthC-2"-OMe phosphoramidite building blocks.
• Incorporation of modified building blocks into oligonucleotides.
• Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) for triazole formation.
• Assessment of thermodynamic stability (A-form RNA duplexes).
• Nuclease degradation assays for metabolic stability.
• Application in guide RNAs (gRNAs) for A-to-I editing in mammalian cells.

Main Results:

• Successfully synthesized and incorporated 4"-C-EthC and its derivatives into oligonucleotides.
• Demonstrated that triazole residues formed via CuAAC enhance thermodynamic stability of RNA duplexes.
• Showcased increased resistance of modified oligonucleotides to nuclease degradation.
• Validated the use of these modified gRNAs for site-directed A-to-I editing in mammalian cells, showing comparable performance to phosphorothioate modifications.

Conclusions:

• 4"-C-ethynyl cytidine modifications offer a versatile platform for creating stable and functional oligonucleotides.
• The "click handle" enables straightforward functionalization, enhancing RNA therapeutic potential.
• These modifications show promise for advanced RNA-based therapeutics and gene editing applications.