siRNA carrying an (E)-vinylphosphonate moiety at the 5΄ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Abstract

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5΄- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5΄- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5΄-(E)-vinylphosphonate (5΄-E-VP) guide RNA at 2.5-Å resolution. The structure demonstrates how the 5΄ binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5΄-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5΄-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.

5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo

“…We synthesized the phosphoramidite of 5΄-( E )-vinylphosphonate 2΄- O –Me-uridine and incorporated it into the guide strand as a last coupling during the 3΄-5΄ oligonucleotide chemical synthesis. Since 5΄ terminal base is not involved in RISC - target mRNA interaction (25,32), the 5΄-( E )-vinylphosphonate 2΄- O –Me-uridine phosphoramidite can be used to incorporate 5΄-( E )-vinylphosphonate in any siRNA independently of target gene and sequence (Figure 2 and Supplementary Figure S3). When tested in vitro by passive uptake, 5΄-( E )-vinylphosphonate hsiRNAs were just as effective as 5΄-phosphate hsiRNAs, confirming that the 5΄-( E )-vinylphosphonate modification is well tolerated by the RNA-induced silencing complex (RISC) assembly (Figure 2B).…”

“…We have identified two underlying mechanisms: resistance to phosphatases and resistance to 5΄-to-3΄ exonucleases. We and others have shown that 5΄-( E )-vinylphosphonate is recognized as a phosphate mimic by AGO2 (a nuclease in the RNA-induced silencing complex, RISC) and allows or even facilitates loading of siRNA guide strand into RISC (23,25). However, our data suggests that 5΄-( E )-vinylphosphonate is not recognized as a phosphate mimic by the main cytoplasmic 5΄-to-3΄ exonuclease, XRN1.…”

“…Among phosphonates tested, 5΄-( E )-vinylphosphonate appears to be the most effective phosphate analog in siRNAs (18–21). Indeed, both single stranded siRNAs and GalNAc-conjugated double stranded siRNAs benefit from 5΄-( E )-vinylphosphonate modification (22–25). …”

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates

“…Samples were then centrifuged at 21000 × g for 5 min at 4°C. The siRNA-containing supernatants were transferred to new vials; sense and antisense strand levels were quantified by stem loop reverse transcription followed by Taqman PCR (SL-RT qPCR) based on previously published methods ( 30 , 31 ). Primers and probe used for SL-RT qPCR assay are shown in Supplementary Table S2 .…”

Multivalent Recruitment of Human Argonaute by GW182

“…(Figure 1C). Since guide RNAs bind very tightly to hAgo2, on the order of 1 nM (Elkayam et al, 2016), the contribution of RNA-hAgo dissociation would be negligible compared to the equilibrium between hAgo and the GW/WG motifs, and was therefore not taken into account. We should note that binding of hGW182 (TNRC6A) motif-1 to hAgo2-RNA is approximately 16 times tighter than a previously measured binding constant for the homologous region of TNRC6B (Pfaff et al, 2013) (with 72% identity) (Figure S1A) binding to hAgo2-RNA (118 nM vs 1.87 μM).…”