Work

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

Year: 2022

Type: article

Abstract: Synthetic siRNA guide strands are typically designed with perfect complementarity to the passenger strand and the target mRNA. We examined whether siRNAs with intentional guide-strand bulges are functional in vitro and in vivo . Importantly, this was done by systematic shortening of the passenger strand, evaluating identical 19-mer guide-strand sequences but forcing them into conformations with 1- to 4-nt bulges after annealing. We demonstrate that guide-strand bulges can be well tolerated at several positions of unmodified and modified siRNAs. Beyond that, we show that GalNAc-conjugated siRNAs with bulges at certain positions of the guide strand repress transthyretin in murine primary hepatocytes and in vivo in mice. In vivo , a GalNAc-conjugated siRNA with a 1-nt bulge at position 14 of the guide strand was as active as the perfectly complementary siRNA. Finally, in a luciferase reporter system, mRNA target sequences were systematically shortened so that RNA-induced silencing complex activity could only occur with a guide-strand bulge. Here, luciferase reporters were repressed when 1- and 2-nt deletions of the reporter were applied to the edges of the sequence. We conclude that some guide-strand bulges versus target transcript can result in target repression and therefore should be evaluated as off-target risks.

Source: Molecular Therapy — Nucleic Acids

DOI: 10.1016/j.omtn.2022.02.004

Authors: Judith Hauptmann, Vivien Hehne, Melanie Balzer, Lucas Bethge, Marie Wikstrom Lindholm

Institution: Silence Therapeutics (Germany)

Language: English

Topic: RNA Interference and Gene Delivery

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,900