Enhancing RNAi activity through 4'-O-hexadecyl-2'-deoxythymidine modification in the passenger strand

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Delivery

Background:

• Developing lipid-based strategies for siRNA delivery to extrahepatic organs is crucial.
• siRNA stability and efficient gene silencing are key challenges in therapeutic applications.

Purpose of the Study:

• To investigate the synergistic effects of 4 ac-O-C16-dT and phosphorothioate modifications on siRNA stability and gene silencing.
• To evaluate the potential of these modifications for carrier-free siRNA therapeutics.

Main Methods:

• Thermal melting studies to assess duplex stability.
• Nuclease resistance assays to determine metabolic stability (half-life).
• Dose-dependent gene silencing studies, Western blot, gel retardation assay, and molecular modeling.

Main Results:

• 4 ac-O-C16-dT modification improved siRNA duplex stability and metabolic stability (approx. 2-fold increase in half-life).
• Enhanced RNAi activity (approx. 4-fold increase) was observed with modifications at specific passenger strand positions.
• Modified siRNAs showed good binding to HSA protein, suggesting improved serum stability and biodistribution.

Conclusions:

• 4 ac-O-C16-dT modification significantly enhances siRNA stability and gene silencing efficacy.
• These modifications represent a promising strategy for developing carrier-free siRNA-based therapeutics for extrahepatic delivery.