Endogenous and exogenous short interfering RNAs (siRNAs) require a 5′-phosphate for loading into Ago2 and cleavage of the target mRNA. We applied a synthetic 5′-phosphate to siRNA guide strands to evaluate if phosphorylation in vivo is rate limiting for maximal siRNA knockdown and duration. We report, for the first time, an in vivo evaluation of siRNAs with a synthetic 5′-phosphate compared to their unphosphorylated versions. siRNAs that contained a 5′-phosphate had the same activity in vivo compared with unphosphorylated siRNAs, indicating phosphorylation of an siRNA is not a rate limiting step in vivo.
Get full access to this article
View all access options for this article.
References
1.
BOLANDA., HUNTZINGERE., SCHMIDTS., IZAURRALDEE., WEICHENRIEDERO.2011. Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein. Proc. Natl. Acad. Sci. U. S. A., 108:10466–10471.
2.
BOUTLAA., DELIDAKISC., LIVADARASI., TSAGRISM., TABLERM.2001. Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila. Curr. Biol., 11:1776–1780.
3.
CAMPOCHIAROP. A.2006. Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders. Gene Ther., 13:559–562.
4.
CARTHEWR. W., SONTHEIMERE. J.2009. Origins and Mechanisms of miRNAs and siRNAs. Cell, 136:642–55.
CHIUY. L., RANAT. M.2003. siRNA function in RNAi: a chemical modification analysis. RNA, 9:1034–1048.
7.
COREYD. R.2007. RNA learns from antisense. Nat. Chem. Biol., 3:8–11.
8.
DE FOUGEROLLESA., VORNLOCHERH. P., MARAGANOREJ., LIEBERMANJ.2007. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov., 6:443–453.
9.
DJURANOVICS., ZINCHENKOM. K., HURJ. K., NAHVIA., BRUNELLEJ. L., ROGERSE. J., GREENR.2010. Allosteric regulation of Argonaute proteins by miRNAs. Nat. Struct. Mol. Biol., 17:144–150.
DORSETTY., TUSCHLT.2004. siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov., 3:318–329.
12.
ELBASHIRS. M., HARBORTHJ., LENDECKELW., YALCINA., WEBERK., TUSCHLT.2001a. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411:494–8.
13.
ELBASHIRS. M., LENDECKELW., TUSCHLT.2001b. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev., 15:188–200.
14.
FISHERM., ABRAMOVM., VAN AERSCHOTA., XUD., JULIANOR. L., HERDEWIJNP.2007. Inhibition of MDR1 expression with altritol-modified siRNAs. Nucleic Acids Res., 35:1064–1074.
15.
FRANKF., SONENBERGN., NAGARB.2011. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature, 465:818–822.
16.
HARBORTHJ., ELBASHIRS. M., BECHERTK., TUSCHLT., WEBERK.2001. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci., 114:4557–4565.
17.
JACKSONA. L., BURCHARDJ., LEAKED., REYNOLDSA., SCHELTERJ., GUOJ., JOHNSONJ. M., LIML., KARPILOWJ., NICHOLSK.et al.2006. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA, 12:1197–1205.
18.
KAWAMATAT., YODAM., TOMARIY.2011. Multilayer checkpoints for microRNA authenticity during RISC assembly. EMBO Rep., 12:944–949.
19.
KENSKID. M., COOPERA. J., LIJ. J., WILLINGHAMA. T., HARINGSMAH. J., YOUNGT. A., KUKLINN. A., JONESJ. J., CANCILLAM. T., MCMASTERSD. R.et al.2009. Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5′-terminal phosphorylation both in vitro and in vivo. Nucleic Acids Res., 38:660–671.
20.
LAYZERJ. M., MCCAFFREYA. P., TANNERA. K., HUANGZ., KAYM. A., SULLENGERB. A.2004. In vivo activity of nuclease-resistant siRNAs. RNA, 10:766–771.
21.
MAJ. B., YUANY. R., MEISTERG., PEIY., TUSCHLT., PATELD. J.2005. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature, 434:666–670.
SCHWARZD. S., HUTVAGNERG., HALEYB., ZAMOREP. D.2002. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell, 10:537–548.
29.
SENG. L., BLAUH. M.2006. A brief history of RNAi: the silence of the genes. Faseb. J., 20:1293–1299.
30.
WANGY., SHENGG., JURANEKS., TUSCHLT., PATELD. J.2008. Structure of the guide-strand-containing argonaute silencing complex. Nature, 456:209–213.
31.
WATTSJ. K., CHOUBDARN., SADALAPUREK., ROBERTF., WAHBAA. S., PELLETIERJ., PINTOB. M., DAMHAM. J.2007. 2′-fluoro-4′-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity. Nucleic Acids Res., 35:1441–1451.
32.
WEIJ., JONESJ., KANGJ., CARDA., KRIMMM., HANCOCKP., PEIY., ASONB., PAYSONE., DUBININAN.et al.2010. RNA-induced silencing complex-bound small interfering RNA is a determinant of RNA interference-mediated gene silencing in mice. Mol. Pharmacol., 79:953–963.
33.
WEITZERS., MARTINEZJ.2007. The human RNA kinase hClp1 is active on 3′ transfer RNA exons and short interfering RNAs. Nature, 447:222–226.
34.
WINCOTTF. E.2001. Strategies for oligoribonucleotide synthesis according to the phosphoramidite method. Curr. Protoc. Nucleic Acid Chem., 3:3.5.1–3.5.12.
35.
YUANY. R., PEIY., CHENH. Y., TUSCHLT., PATELD. J.2006. A potential protein-RNA recognition event along the RISC-loading pathway from the structure of A. aeolicus Argonaute with externally bound siRNA. Structure, 14:1557–1565.
36.
ZOUY., TILLERP., CHENI. W., BEVERLYM., HOCHMANJ.2008. Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry. Rapid Commun. Mass Spectrom., 22:1871–1881.
