Synthesis and Properties of RNA Modified with Thioamide Internucleoside Linkage

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Recent advancements in RNA therapeutics highlight the importance of chemical modifications.
• Modifications to the RNA sugar-phosphate backbone are impactful but underexplored.
• Phosphorothioates are an example of successful backbone modifications.

Purpose of the Study:

• To synthesize and characterize RNA molecules containing thioamide linkages in the sugar-phosphate backbone.
• To evaluate the thermal stability of RNA duplexes with thioamide modifications.
• To assess the RNA interference (RNAi) activity, including target cleavage, specificity, and duplex stability, of modified short interfering RNAs (siRNAs).

Main Methods:

• Synthesis of RNA oligonucleotides incorporating thioamide linkages.
• Thermal denaturation studies (UV-Vis spectroscopy) to determine melting temperatures (Tm) of RNA duplexes.
• In vitro assays to measure siRNA-mediated target RNA cleavage and assess specificity.

Main Results:

• Thioamide linkages significantly destabilized short, self-complementary RNA model duplexes compared to unmodified RNA.
• In the context of short interfering RNAs (siRNAs), thioamide modifications had a comparable effect on duplex stability as amide modifications.
• Both amide and thioamide modifications demonstrated similar efficacy in target RNA cleavage activity and specificity within siRNAs.

Conclusions:

• Thioamide linkages represent a novel backbone modification for RNA.
• While destabilizing in simple duplexes, thioamides are well-tolerated by the RNA interference machinery in siRNAs.
• Thioamides can be considered a valuable addition to the chemical biologist's toolkit for developing RNA-based therapeutics.