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

Abstract

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.

Figure 1.

Structures of RNA modifications to improve metabolic stability and on-target activity of siRNAs.

Figure 2.

Sequences of PIK3CB mRNA (on-target), AM1-modified siRNA guide strands, YY1 and FADD off-target mRNAs (off-target), and phosphoramidite building blocks to introduce AM1 modification. ‘G’ denotes guide strand targeting PIK3CB and the number denotes the position of AM1 modification; ‘p’ denotes phosphodiester and ‘a’ denotes amide internucleoside linkages; the mismatched nucleotides in YY1 and FADD sequences are highlighted in pink.

Figure 3.

Dual-luciferase assay IC₅₀ values of unmodified (G0) and AM1-modified (G2-G20) siRNAs targeting PIK3CB (A), YY1 (B), and FADD (C). # indicates that IC₅₀ values for G3 targeting YY1 and G2 and G3 targeting FADD could not be calculated because the expression did not go below 60% at the maximum concentration (20 nM); when comparing the data with G0, * and ** indicate two-tailed P values of less than 0.05 and 0.01, respectively, as calculated using Student’s t-test.

Figure 4.

IC₅₀ curves of dual luciferase assay of PIK3CB, YY1, and FADD silencing by G3. Lack of YY1 and FADD silencing below 70% even at 20 nM G3 indicates strong suppression of off-target activity.

Figure 5.

Normalized activity ratios show fold change in siRNA specificity compared to unmodified siRNA. # at G2 and G3 indicates that the high selectivity cannot be precisely calculated because of absence of IC₅₀ values for YY1 and FADD.