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

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.