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mRNA knockdown by single strand RNA is improved by chemical modifications.

Haringsma HJ, Li JJ, Soriano F, Kenski DM, Flanagan WM, Willingham AT - Nucleic Acids Res. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Sirna Therapeutics, 1700 Owens Street, Fourth Floor, San Francisco, CA 94158, USA.

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.

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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.
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gkr1301-F6: 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.

Mentions: Conventional Ago2-mediated cleavage of target mRNA occurs between positions 9–10 of the region hybridizing to the guide strand. The PCR-based rapid amplification of cDNA ends (RACE) methodology is routinely applied to measure the 5′-end of the predicted siRNA cleavage product. PCR primers are designed to produce an amplicon of predicted size based on the site of siRNA cleavage of the target mRNA. Two primers are ‘nested’ together in subsequent PCR amplifications to confer specificity to the 5′RACE. Two separate PCR amplicons were designed (Supplementary Table S5) and used for 5′RACE from mouse liver mRNA isolated from animals treated with ApoB (8786) dsRNA and ssRNA (Figure 4). The rev1n amplicon should be 319 nt and the rev2n amplicon should be 659 nt in size based on the predicted site for ApoB (8786) cleavage. Both amplicons produce distinct bands of expected size for samples treated with the duplex siRNAs (Figure 6A). However, ssRNA treated samples did not produce a cleavage product despite yielding 74–91% ApoB knockdown in vivo. Since the ssRNA and dsRNA samples were amplified side by side, we are confident that the failure to isolate ssRNA cleavage products is not a technical artifact. Purification and sequencing of a subset of the rev1n amplicons from the duplex siRNA samples confirmed the predicted Ago2 cleavage site and the presence of the 5′RACE primer (Figure 6B).Figure 6.


mRNA knockdown by single strand RNA is improved by chemical modifications.

Haringsma HJ, Li JJ, Soriano F, Kenski DM, Flanagan WM, Willingham AT - Nucleic Acids Res. (2012)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351186&req=5

gkr1301-F6: 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.
Mentions: Conventional Ago2-mediated cleavage of target mRNA occurs between positions 9–10 of the region hybridizing to the guide strand. The PCR-based rapid amplification of cDNA ends (RACE) methodology is routinely applied to measure the 5′-end of the predicted siRNA cleavage product. PCR primers are designed to produce an amplicon of predicted size based on the site of siRNA cleavage of the target mRNA. Two primers are ‘nested’ together in subsequent PCR amplifications to confer specificity to the 5′RACE. Two separate PCR amplicons were designed (Supplementary Table S5) and used for 5′RACE from mouse liver mRNA isolated from animals treated with ApoB (8786) dsRNA and ssRNA (Figure 4). The rev1n amplicon should be 319 nt and the rev2n amplicon should be 659 nt in size based on the predicted site for ApoB (8786) cleavage. Both amplicons produce distinct bands of expected size for samples treated with the duplex siRNAs (Figure 6A). However, ssRNA treated samples did not produce a cleavage product despite yielding 74–91% ApoB knockdown in vivo. Since the ssRNA and dsRNA samples were amplified side by side, we are confident that the failure to isolate ssRNA cleavage products is not a technical artifact. Purification and sequencing of a subset of the rev1n amplicons from the duplex siRNA samples confirmed the predicted Ago2 cleavage site and the presence of the 5′RACE primer (Figure 6B).Figure 6.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Sirna Therapeutics, 1700 Owens Street, Fourth Floor, San Francisco, CA 94158, USA.

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.

Show MeSH
Related in: MedlinePlus