Engineering miRNA features into siRNAs: Guide-strand bulges are compatible with gene repression

Area of Science:

• Molecular Biology
• RNA Interference

Background:

• Synthetic small interfering RNAs (siRNAs) are crucial for gene silencing.
• Standard siRNA design emphasizes perfect complementarity between guide strands, passenger strands, and target mRNA.

Purpose of the Study:

• To investigate the functionality of siRNAs with intentional guide-strand bulges.
• To assess the tolerance and efficacy of bulges in both in vitro and in vivo settings.

Main Methods:

• Systematic shortening of passenger strands to create 1- to 4-nucleotide (nt) guide-strand bulges.
• Evaluation of unmodified and modified siRNAs, including GalNAc-conjugated versions.
• Testing in murine primary hepatocytes and in vivo mouse models.
• Utilizing a luciferase reporter system with modified target sequences.

Main Results:

• Guide-strand bulges are well-tolerated at multiple positions in siRNAs.
• GalNAc-conjugated siRNAs with specific guide-strand bulges effectively repressed transthyretin expression in hepatocytes and mice.
• A 1-nt bulge at position 14 of a GalNAc-conjugated siRNA showed in vivo activity comparable to perfectly complementary siRNA.
• Reporter assays confirmed target repression mediated by guide-strand bulges.

Conclusions:

• Intentional guide-strand bulges can be functional and tolerated in siRNA design.
• Certain bulges in GalNAc-conjugated siRNAs maintain high gene-silencing efficacy in vivo.
• The potential for guide-strand bulges to cause target repression should be considered during off-target risk assessments.