Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo

Abstract

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Figure 1.

UNAs at position 1, 2 and 3 of the guide strand impair mRNA degradation. (A) Structure of the nucleotides used for oligonucleotide synthesis. (B) ApoB siRNA sequence used in this study. (C) Percent mRNA degradation of ApoB mRNA as determined by qPCR. Unmodified refers to the ApoB siRNA sequence containing only ribonucleotides and dTdT overhangs. The negative control is a scrambled siRNA sequence. Individual UNAs were substituted at the indicated position on the guide strand of the duplex. A paired t-test was conducted to evaluate UNA modifications to the unmodified control. The asterisk indicates a significant change in mRNA degradation from unmodified siRNA (P < 0.001).

Figure 2.

Clp1 kinase failed to phosphorylate the 5′-termini of siRNAs containing UNAs at position 1 or 2. (A) PNK was used as a control kinase to evaluate phosphorylation of the siRNAs and to ensure the integrity of the synthesized siRNAs. siRNAs were incubated with PNK and samples were run on the mass spectrometer as described in Materials and Methods section. Mass spectrometric analysis of the siRNAs indicated that the siRNAs were intact. (B) Clp1 siRNA kinase assay was performed to evaluate the role of Clp1 in phosphorylation of UNA modified siRNA as described in (A). Unmodified refers to the ApoB siRNA sequence containing only ribonucleotides and dTdT overhangs.

Figure 3.

Chemical phosphorylation of siRNAs containing UNAs at position 1, but not position 2 or 3, restores biological activity in cell based assays. (A) 5′-terminal phosphorylation of the guide strand restores activity when UNAs are at position 1. Unmodified refers to the ApoB sequence containing only ribonucleotides and dTdT overhangs. The negative control is a scrambled sequence. Individual UNAs are placed at the indicated position on the guide strand. Oligonucleotides with a 5′-terminal hydroxyl group on the guide strand are shown in black and oligonucleotides with a 5′-terminal phosphate on the guide strand are shown in white (all passenger strands contained a 5′-terminal hydroxyl group). (B) Binding of oligonucleotides to epitope-tagged Ago2 in cell lysates. Ago2 binding studies were performed as described in Materials and Methods section. The percent of the UNA modified oligonucleotides bound to Ago2 was normalized to unmodified siRNA. The negative control refers to a sequence that is mismatched to the stem loop PCR primers. (C) Cleavage by purified Ago2 of a Cy3 target oligo that mimics the cellular mRNA target. The gel is on the right and quantification of the cleavage is on the left. Ago2 cleavage studies were performed as described in ‘Materials and Methods’ section.

Figure 4.

UNAs demonstrate in vivo activity at 24 h post-IV injection. (A) Unmodified siRNA, siRNA with an UNA at position 1 (UNA1), and siRNA containing a 5′-terminal phosphate with UNA at position 1 (UNA1P) were formulated into lipid nanoparticles and injected in mice at 6 mg/kg. The livers were harvested and ApoB mRNA levels were evaluated by qPCR 24 h after injection (student’s t-test, P < 0.0001). Bars represent the mean and standard deviation of five mice per group. (B) UNA1 and UNA1P function via an Ago2 mechanism. 5′RACE was used to detect the ApoB mRNA cleavage site. Mouse liver RNA samples were evaluated for each sample group. 5′RACE products are 150 bp. The precise Ago2-mediate cleavage site on ApoB was mapped by sequencing and is designated by an arrow. The yellow box indicates the 5′-RACE linker sequence.