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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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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).
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gkr1301-F1: 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).

Mentions: The mouse hepatocyte derived cell line Hepa1-6 was transfected with single strand and duplex siRNAs at 100 and 10 nM concentrations. For dsRNAs, 10 nM is likely a saturating concentration as 100 nM did not result in significant improvements in observed target knockdown. Therefore, two siRNAs were also screened at 1 nM. Target knockdown was measured 24 h later and compared to unmodified controls. Figure 1 shows the results for ApoB (8786), though a similar trend was observed for the other five siRNAs tested (Supplementary Figure S1). Generally, dsRNAs were broadly tolerant of modifications though there was notable sensitivity to 2′-deoxy which is consistent with Ago2 preference for RNA (rather than DNA) substrates (13,14). High percentage methoxy modification of the guide strand was tolerated within dsRNAs though a loss in knockdown was seen at the submaximal 1 nM concentration. Such tolerance of methoxy modification is likely due to our inclusion of 2′OH at position 14 of the guide strand which has previously been reported as highly sensitive to methoxy modifications (17).Figure 1.


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC3351186&req=5

gkr1301-F1: 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).
Mentions: The mouse hepatocyte derived cell line Hepa1-6 was transfected with single strand and duplex siRNAs at 100 and 10 nM concentrations. For dsRNAs, 10 nM is likely a saturating concentration as 100 nM did not result in significant improvements in observed target knockdown. Therefore, two siRNAs were also screened at 1 nM. Target knockdown was measured 24 h later and compared to unmodified controls. Figure 1 shows the results for ApoB (8786), though a similar trend was observed for the other five siRNAs tested (Supplementary Figure S1). Generally, dsRNAs were broadly tolerant of modifications though there was notable sensitivity to 2′-deoxy which is consistent with Ago2 preference for RNA (rather than DNA) substrates (13,14). High percentage methoxy modification of the guide strand was tolerated within dsRNAs though a loss in knockdown was seen at the submaximal 1 nM concentration. Such tolerance of methoxy modification is likely due to our inclusion of 2′OH at position 14 of the guide strand which has previously been reported as highly sensitive to methoxy modifications (17).Figure 1.

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