RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3' Nuclease-resistant...

Area of Science:

• Molecular Biology
• Gene Silencing Technologies
• Nucleic Acid Therapeutics

Background:

• RNA interference (RNAi) utilizes small interfering RNA (siRNA) for gene silencing.
• siRNA processing involves Ago2 and C3PO within the RNA-induced silencing complex (RISC).
• The 3' end of siRNA passenger strands can be modified with DNA without compromising RNAi function.

Purpose of the Study:

• To evaluate the efficacy of novel 3'-DNA/siRNA constructs for RNA interference.
• To compare the nuclease resistance and gene silencing activity of different DNA-modified siRNA designs.
• To assess the impact of these constructs on SIRT1 gene expression and cancer cell viability.

Main Methods:

• Synthesis and testing of four distinct siRNA constructs with 3'-DNA modifications: guide-3'-DNA/ds siRNA, passenger-3'-DNA/ds siRNA, guide-3'-DNA/ss siRNA, and passenger-3'-DNA/ss siRNA.
• Assessing SIRT1 mRNA and protein knockdown in noncancer and cancer cells.
• Evaluating cancer cell apoptosis induction.
• Testing nuclease resistance of constructs in serum.

Main Results:

• Constructs I (guide-3'-DNA/ds siRNA) and II (passenger-3'-DNA/ds siRNA) effectively silenced SIRT1 mRNA and protein, inducing apoptosis in cancer cells.
• Construct IV (passenger-3'-DNA/ss siRNA), lacking a guide strand, showed no effect.
• The 3'-DNA mini-hairpin provided significant nuclease resistance to double-stranded siRNA constructs (I and II).
• Single-stranded guide-3'-DNA/ss siRNA (construct III) was susceptible to nucleases, losing RNAi activity.

Conclusions:

• 3'-DNA/siRNA constructs, particularly passenger-modified double-stranded siRNA, offer enhanced stability and potent gene silencing capabilities.
• The 3'-DNA modification confers nuclease resistance, crucial for therapeutic applications of RNAi.
• These modified siRNAs demonstrate potential for selective gene targeting and cancer therapy.