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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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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.
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gkr1301-F3: 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.

Mentions: At the two concentrations tested, ssRNAs appear less potent than their corresponding duplex siRNAs. Two ApoB siRNAs were selected for dose response measurements and calculation of EC50 values (Figure 3). Unmodified and 2′F-modified RNAs were compared as single strands and duplexes over an eight-point dose–response ranging from 40 nM down to 0.002 nM (all oligos were phosphorylated). The value of 2′F modifications for ssRNA activity was immediately evident for both ApoB (8786) and (6981) sequences: unmodified ssRNAs essentially had no knockdown over the dose range while 2′F ssRNA exhibited a dose response. However, the duplex siRNAs were found to be 20- to 80-fold more potent than their corresponding single strands with picomolar potencies for dsRNAs compared to the nanomolar potencies measured for ssRNAs (Supplementary Table S1). ApoB (8786) duplexes possess 15 pM potencies (for both unmodified and 2′F) while the 2′F ssRNA EC50 is ∼1.2 nM. The ApoB (6981) 2′F duplex is ∼2-fold more potent than the unmodified duplex (2′OH); however, both are significantly more potent than the 2′F ssRNA (∼0.9 nM). Despite the lower overall potencies relative to duplexes, it is important to note that ssRNAs are still capable of maximal mRNA knockdown >90% and that this level of knockdown was enabled by chemical modifications.Figure 3.


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)

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

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

gkr1301-F3: 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.
Mentions: At the two concentrations tested, ssRNAs appear less potent than their corresponding duplex siRNAs. Two ApoB siRNAs were selected for dose response measurements and calculation of EC50 values (Figure 3). Unmodified and 2′F-modified RNAs were compared as single strands and duplexes over an eight-point dose–response ranging from 40 nM down to 0.002 nM (all oligos were phosphorylated). The value of 2′F modifications for ssRNA activity was immediately evident for both ApoB (8786) and (6981) sequences: unmodified ssRNAs essentially had no knockdown over the dose range while 2′F ssRNA exhibited a dose response. However, the duplex siRNAs were found to be 20- to 80-fold more potent than their corresponding single strands with picomolar potencies for dsRNAs compared to the nanomolar potencies measured for ssRNAs (Supplementary Table S1). ApoB (8786) duplexes possess 15 pM potencies (for both unmodified and 2′F) while the 2′F ssRNA EC50 is ∼1.2 nM. The ApoB (6981) 2′F duplex is ∼2-fold more potent than the unmodified duplex (2′OH); however, both are significantly more potent than the 2′F ssRNA (∼0.9 nM). Despite the lower overall potencies relative to duplexes, it is important to note that ssRNAs are still capable of maximal mRNA knockdown >90% and that this level of knockdown was enabled by chemical modifications.Figure 3.

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