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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 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).
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gkr1301-F2: 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).

Mentions: The same series of RNA modifications evaluated in Figure 1 were then tested in the presence of 5′ phosphorylation of the guide strand. Figure 2 shows that addition of a 5′ phosphate significantly improves the potency of ssRNA, resulting in significant knockdown even at a lower concentration of 10 nM. The overall effectiveness of 2′F-modified ssRNA activity for the other five siRNAs is also more evident when the ssRNAs are phosphorylated (Supplementary Figure S2). In the presence of 5′ phosphorylation, the beneficial impact of 2′F content is significantly more obvious (compare to Supplementary Figure S1). While the ssRNA knockdown at this lower concentration still lags that of the comparable duplex siRNAs, the combination of 2′F modifications and 5′ phosphorylation are key to improving the potency of ssRNA knockdown: improving knockdown from negligible levels to 65–95% (Supplementary Table S3).Figure 2.


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

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

gkr1301-F2: 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).
Mentions: The same series of RNA modifications evaluated in Figure 1 were then tested in the presence of 5′ phosphorylation of the guide strand. Figure 2 shows that addition of a 5′ phosphate significantly improves the potency of ssRNA, resulting in significant knockdown even at a lower concentration of 10 nM. The overall effectiveness of 2′F-modified ssRNA activity for the other five siRNAs is also more evident when the ssRNAs are phosphorylated (Supplementary Figure S2). In the presence of 5′ phosphorylation, the beneficial impact of 2′F content is significantly more obvious (compare to Supplementary Figure S1). While the ssRNA knockdown at this lower concentration still lags that of the comparable duplex siRNAs, the combination of 2′F modifications and 5′ phosphorylation are key to improving the potency of ssRNA knockdown: improving knockdown from negligible levels to 65–95% (Supplementary Table S3).Figure 2.

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