A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs

Abstract

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.

Figure 1.

Sequences of amide-modified siRNA guide (GN) and passenger (PN) strands. N denotes the position number of the internucleoside phosphodiester linkage that has been replaced with an amide; 0 denotes the unmodified strands; * and # denotes the numbering in black and green sequences, respectively. NTC = non-targeting control siRNA.

Figure 2.

Comparison of silencing activity of the amide-modified siRNAs monitored separately for two different target RNAs, one complementary to the guide strand (darker colored bars) and one complementary to the passenger strand (lighter colored bars). (A) blue siRNA guide strands (GN) duplexed with the unmodified passenger strand (P0) or the ON-TARGETplus (OTP) modifications; (B) black siRNA guide strands (GN*) duplexed with the unmodified passenger strand (P0*) and the unmodified guide strand (G0*) duplexed with the amide-modified passenger strand (P1*); (C) green siRNA guide strands (GN#) duplexed with the unmodified passenger strand (P0#) and the unmodified guide strand (G0#) duplexed with the modified passenger strand (P1#); and (D) black siRNA guide strands (GN*) duplexed with the amide-modified passenger strand (P1*).

Figure 3.

Molecular modeling illustrates the difference in conformations and interactions for phosphate and amide linkages between the first and second nucleosides of the guide strand (5´-P, P1, P2 and P3 identify the guide strand phosphates). The structure of the simulated complex with amide (highlighted with yellow carbon atoms) replacing the bridging phosphate at G1 is colored by atom and superimposed on the structure of the reference complex (PDB ID 4F3T; all residues are colored in gray and phosphate P1 is colored in orange (P) and red (O)). Distances between amide NH and O and selected Ago2 amino acid side chains as well as K550 and Q548 and N551 are indicated with dashed black lines and H-bonds involving 5´-P, P1 and P2 in the reference structure are indicated with solid gray lines.