Amide Internucleoside Linkages Suppress the MicroRNA-like Off-Target Activity of Short Interfering RNA

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• RNA interference (RNAi) is a promising therapeutic strategy utilizing short interfering RNAs (siRNAs).
• Chemical modifications are crucial for siRNA clinical success, but challenges remain in extrahepatic delivery, durability, and reducing off-target effects.
• Improving siRNA specificity is key to minimizing unintended gene silencing.

Purpose of the Study:

• To synthesize novel amide-linked guanosine dimers (G AM1 G and G AM1 A) for incorporation into siRNAs.
• To systematically evaluate the impact of replacing internucleoside phosphate linkages with amide bonds on siRNA performance.
• To assess the potential of amide linkages to enhance siRNA specificity by reducing microRNA-like off-target activity.

Main Methods:

• Synthesis of amide-linked guanosine dimers (G AM1 G and G AM1 A).
• Systematic "amide walk" replacing internucleoside phosphates in a guide strand targeting PIK3CB.
• Dual-luciferase and RT-qPCR assays in HeLa cells to evaluate gene silencing and off-target effects.

Main Results:

• The amide linkage at position 3 (between nucleosides 3 and 4) effectively suppressed the cleavage of off-target YY1 and FADD mRNAs in a model system.
• This suppression was comparable to the performance of glycol nucleic acid (GNA), an industry gold standard modification.
• The study demonstrated the feasibility of synthesizing and incorporating amide linkages into siRNAs.

Conclusions:

• Amide linkages, particularly in the seed region of siRNAs, show strong potential for improving therapeutic specificity.
• These modifications can mitigate microRNA-like off-target effects, enhancing the safety profile of siRNA therapeutics.
• Further development of amide-linked siRNAs could lead to more effective and targeted gene silencing therapies.