Amide Modifications in the Seed Region of the Guide Strand Improve the On-Target Specificity of Short Interfering RNA.

RNA interference (RNAi) is a well-established research tool and is also maturing as a novel therapeutic approach. For the latter, microRNA-like off-target activity of short interfering RNAs (siRNAs) remains as one of the main problems limiting RNAi drug development. In this communication, we report that replacement of a single internucleoside phosphodiester in the seed region (nucleotides 2 to 7) of the guide strand with an amide linkage suppressed the undesired microRNA-like off-target activity by at least an order of magnitude. For the specific siRNA targeting the PIK3CB gene, an amide modification between the third and fourth nucleotides of the guide strand showed the strongest enhancement of specificity (completely eliminated off-target silencing) while maintaining high on-target activity. These results are important because off-target activity is one of the main remaining roadblocks for RNA based drug development.

Optimization of Automated Synthesis of Amide-Linked RNA.

The recent FDA approval of several antisense and siRNA drugs illustrates the utility of nucleic acid chemical modifications, but numerous challenges remain for generalized nucleic acid therapeutics, urging the exploration of new modification strategies. Replacing backbone phosphates with amides has shown promise for enhancing siRNA activity, specificity, and nuclease resistance; however, amide-linked RNA has not been fully explored due to lengthy and low yielding manual amide coupling procedures. We have addressed this by automating the assembly of amide-linked RNA using an Expedite 8909 nucleic acid synthesizer and optimizing solid-phase synthesis conditions to achieve 91-95% yields in just 5 min of coupling time. The optimized protocol allowed synthesis of a 21-nucleotide-long siRNA guide strand having six consecutive amide linkages at the 3'-end with an overall yield of ∼1%. Our results show that the steric hindrance caused by bulky 2'-O protecting groups and steric hindrance of the solid support are the key optimization variables for improving the amide couplings.

Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages.

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3'-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5'-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.