Large-Scale,High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

Abstract

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

Keywords

RNAi high-throughput screening target validation shRNA reporter assay

References

Elbashir SM , Lendeckel W , Tuschl T : RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev 2001;15:188-200.

Elbashir SM , Harborth J , Lendeckel W , Yalcin A , Weber K , Tuschl T : Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494-498.

Pasquinelli AE , Ruvkun G : Control of developmental timing by micrornas and their targets. Annu Rev Cell Dev Biol 2002;18:495-513.

Hannon GJ , Rossi JJ : Unlocking the potential of the human genome with RNA interference. Nature 2004;431:371-378.

Dorsett Y , Tuschl T : siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 2004;3:318-329.

Paddison PJ , Caudy AA , Hannon GJ : Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA 2002;99:1443-1448.

Brummelkamp TR , Bernards R , Agami R : A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-553.

Kim DH , Behlke MA , Rose SD , Chang MS , Choi S , Rossi JJ : Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol 2005;23:222-226.

Siolas D , Lerner C , Burchard J , Ge W , Linsley PS , Paddison PJ , et al: Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 2005;23:227-231.

10.

Meister G , Tuschl T : Mechanisms of gene silencing by double-stranded RNA. Nature 2004;431:343-349.

11.

Murchison EP , Hannon GJ : miRNAs on themove: miRNA biogenesis and the RNAi machinery. Curr Opin Cell Biol 2004;16:223-229.

12.

Reynolds A , Leake D , Boese Q , Scaringe S , Marshall WS , Khvorova A : Rational siRNA design for RNA interference. Nat Biotechnol 2004;22:326-330.

13.

Chalk AM , Wahlestedt C , Sonnhammer EL : Improved and automated prediction of effective siRNA. Biochem Biophys Res Commun 2004;319:264-274.

14.

Elbashir SM , Martinez J , Patkaniowska A , Lendeckel W , Tuschl T : Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 2001;20:6877-6888.

15.

Pancoska P , Moravek Z , Moll UM : Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA. Nucleic Acids Res 2004;32:1469-1479.

16.

Schwarz DS , Hutvagner G , Du T , Xu Z , Aronin N , Zamore PD : Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003;115:199-208.

17.

Ui-Tei K , Naito Y , Takahashi F , Haraguchi T , Ohki-Hamazaki H , Juni A , et al: Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 2004;32:936-948.

18.

Hohjoh H : Enhancement of RNAi activity by improved siRNA duplexes. FEBS Lett 2004;557:193-198.

19.

Amarzguioui M , Prydz H : An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun 2004;316:1050-1058.

20.

Paddison PJ , Caudy AA , Sachidanandam R , Hannon GJ : Short hairpin activated gene silencing in mammalian cells. Methods Mol Biol 2004;265:85-100.

21.

Rubinson DA , Dillon CP , Kwiatkowski AV , Sievers C , Yang L , Kopinja J , et al: A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003;33:401-406.

22.

Gupta S , Schoer RA , Egan JE , Hannon GJ , Mittal V : Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci USA 2004;101:1927-1932.

23.

Tiscornia G , Tergaonkar V , Galimi F , Verma IM : CRE recombinase-inducible RNA interference mediated by lentiviral vectors. Proc Natl Acad Sci USA 2004;101:7347-7351.

24.

van de Wetering M , Oving I , Muncan V , Pon Fong MT , Brantjes H , van Leenen D , et al: Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep 2003;4:609-615.

25.

Wiznerowicz M , Trono D : Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 2003;77:8957-8961.

26.

Czauderna F , Santel A , Hinz M , Fechtner M , Durieux B , Fisch G , et al: Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res 2003;31:e127-e127.

27.

Miyagishi M , Taira K : U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002;20:497-500.

28.

Paul CP , Good PD , Winer I , Engelke DR : Effective expression of small interfering RNA in human cells. Nat Biotechnol 2002;20:505-508.

29.

Mathews DH , Sabina J , Zuker M , Turner DH : Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 1999;288:911-940.

30.

Lobo SM , Ifill S , Hernandez N : cis-acting elements required for RNA polymerase II and III transcription in the human U2 and U6 snRNA promoters. Nucleic Acids Res 1990;18:2891-2899.

31.

Kunkel GR , Maser RL , Calvet JP , Pederson T : U6 small nuclear RNAis transcribed by RNA polymerase III. Proc Natl Acad Sci USA 1986;83:8575-8579.

32.

Kunkel GR , Pederson T : Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an upstream TATATA box. Nucleic Acids Res 1989;17:7371-7379.

33.

Markova SV , Golz S , Frank LA , Kalthof B , Vysotski ES : Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa: a novel secreted bioluminescent reporter enzyme. J Biol Chem 2004;279:3212-3217.

34.

Breaker RR , Joyce GF : A DNA enzyme with Mg(2+)-dependent RNA phosphoesterase activity. Chem Biol 1995;2:655-660.

35.

Joyce GF : Directed evolution of nucleic acid enzymes. Annu Rev Biochem 2004;73:791-836.

36.

Doench JG , Petersen CP , Sharp PA : siRNAs can function as miRNAs. Genes Dev 2003;17:438-442.

37.

Kumar R , Conklin DS , Mittal V : High-throughput selection of effective RNAi probes for gene silencing. Genome Res 2003;13:2333-2340.

38.

Ziauddin J , Sabatini DM : Microarrays of cells expressing defined cDNAs. Nature 2001;411:107-110.

39.

Silva JM , Mizuno H , Brady A , Lucito R , Hannon GJ : RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. Proc Natl Acad Sci USA 2004;101:6548-6552.

40.

Zhao X , Vainshtein I , Gellibolian R , Shu Y , Dotimas H , Wang XM , et al: Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling. Assay Drug Dev Technol 2003;1:823-833.

41.

Eglen RM : Enzyme fragment complementation: a flexible high throughput screening assay technology. Assay Drug Dev Technol 2002;1:97-104.

42.

Eglen RM , Singh R : Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screening. Comb Chem High Throughput Screen 2003;6:381-387.

43.

Paddison PJ , Silva JM , Conklin DS , Schlabach M , Li M , Aruleba S , et al: A resource for large-scale RNA-interference-based screens in mammals. Nature 2004;428:427-431.

44.

Berns K , Hijmans EM , Mullenders J , Brummelkamp TR , Velds A , Heimerikx M , et al: Alarge-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 2004;428:431-437.

45.

Silva JM , Li MZ , Chang K , Ge W , Golding MC , Rickles RJ , et al: Secondgeneration shRNA libraries covering the mouse and human genomes. Nat Genet 2005;37:1281-1288.

46.

Castanotto D , Li H , Rossi JJ : Functional siRNA expression from transfected PCR products. RNA 2002;8:1454-1460.

47.

Lamesch P , Milstein S , Hao T , Rosenberg J , Li N , Sequerra R , et al: C. elegans ORFeome version 3.1: increasing the coverage of ORFeome resources with improved gene predictions. Genome Res 2004;14:2064-2069.

48.

Reboul J , Vaglio P , Rual JF , Lamesch P , Martinez M , Armstrong CM , et al: C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat Genet 2003;34:35-41.

49.

Rual JF , Hirozane-Kishikawa T , Hao T , Bertin N , Li S , Dricot A , et al: Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 2004;14:2128-2135.

50.

Rual JF , Hill DE , Vidal M : ORFeome projects: gateway between genomics and omics. Curr Opin Chem Biol 2004;8:20-25.