mRNA knockdown by single strand RNA is improved by chemical modifications

Abstract

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

Figure 1.

Comparison of ssRNA and dsRNA oligos containing pyrimidine (Y) and purine (R) 2' modifications. ApoB (8786) sequence shown. In vitro cell-based evaluation of ApoB mRNA knockdown (error bars represent standard deviation of four replicates). Unmodified ribose (‘r’) is compared to 2'-fluoro (‘f’), 2'-methoxy (‘m’), and 2'-deoxy (‘d’) in all combinations on purines (R) and pyrimidines (Y). Oligos do not contain a 5' phosphate. Guide strand oligo sequence and color-coded modification patterns are depicted. Five other siRNAs were tested and a similar requirement of 2'F for ssRNA knockdown was observed (Supplementary Figure S1).

Figure 2.

Comparison of purine versus pyrimidine 2′ modifications for ApoB (8786) in the presence of 5′ phosphorylation of the guide strand. In vitro cell-based evaluation of ApoB mRNA knockdown (error bars represent standard deviation of four replicates). Unmodified ribose (‘r’) is compared to 2′-fluoro (‘f’), 2′-methoxy (‘m’), and 2′-deoxy (‘d’) in all combinations on purines (R) and pyrimidines (Y). All RNA oligos contain 5′ phosphate (5′p). Guide strand oligo sequence and color-coded modification patterns are depicted. Five other siRNAs with were also tested with 5′ phosphorylation (Supplementary Figure S2) and a similar requirement for 5′ phosphorylation for optimal in vitro ssRNA knockdown was observed (compare to Supplementary Figure S1).

Figure 3.

In vitro potency of single strands compared to duplex siRNAs. (A) ApoB (8786) and (B) ApoB (6981) compared as 2′OH and 2′F guide strands with 5′ phosphorylation. Single strands are ∼20- to 80-fold less potent than the corresponding duplex siRNAs containing identical guide strands. Potency and mRNA knockdown values listed in Supplementary Table S1.

Figure 4.

In vivo knockdown of single strand versus duplex siRNA demonstrates ssRNAi activity in vivo. RNAs formulated in a lipid nanoparticle (LNP) delivery vehicle. Modified guide strands (2'F) are compared to unmodified (2'OH). All oligos contain 5' phosphorylation. Comparison of Day 3 ApoB mRNA knockdown after an LNP delivered 3 mg/kg dose of dsRNA or ssRNA for target sites (A) ApoB (8786) and (B) ApoB (6981). Knockdown of 60–75% was observed for phosphorylated single strands containing 2'F modifications. Duration of mRNA knockdown after an LNP delivered 6 mg/kg dose was measured over 2 weeks (Day 2, Day 7, Day 14 time points) for (C) ApoB (8786) and (D) ApoB (6981). Knockdown values shown in Supplementary Table S2.

Figure 5.

Inclusion of three abasic residues at positions 1–3 (3aba) of the guide strand decreases the in vitro mRNA knockdown of both duplex and single strands of ApoB siRNAs. Ribose (2'OH, ‘r’) and fluoro (2'F, ‘f’) oligos with phosphate (5'p) are compared to those containing 3aba for (A) ApoB (8786) and (B) ApoB (6981). Abasic (3aba) data for additional ApoB and SSB siRNAs are shown in Supplementary Figure S3.

Figure 6.

5'RACE demonstrates duplex RNA, but not ssRNA, cleaves ApoB mRNA producing a cleavage product size consistent with the ApoB (8786) cleavage site. (A) Agarose gel of two different 5' RACE products (rev1n and rev2n amplicons; see map of primers) from mRNA purified from mouse livers treated with duplex or ssRNA (Figure 4). ApoB (8786) cleavage compared from in vivo mouse livers treated with unmodified (2'OH) or 2'F-modified duplex and single strands. (B) Representative sequence data from duplex siRNA cleavage products showing cleavage at the hallmark site indicative of Ago2 slicer activity and the presence of 5' RACE primer.