HTS using siRNA libraries

Although synthetic siRNA libraries are becoming more available, most high throughput siRNA library-based screening was carried out with siRNA libraries encoded by different vectors. In this article, siRNA library construction methods and HTS applications are summarised.

RNA interference (RNAi) is a process of gene expression silencing induced by double stranded RNA1,2. It has become widely used in the functional study of genes immediately after the discovery that small interfering RNA (siRNA) was found to be the intermediate effector molecule of the RNA interference process3. siRNA are short double stranded RNA molecules that have a length between 19-23 bp, which upon entering cells, will be incorporated into a protein complex named RNA induced gene silence complex (RISC). One of the two strands of RNA is then destructed and the other strand will remain in the RISC complex as a guide sequence for target mRNA recognition and cleavage.

Choice of siRNA libraries

The first challenge for constructing an siRNA library is to design siRNA sequences covering a large number of genes. It has been clear from the beginning that only a fraction of siRNA can silence genes that have corresponding sequences. The underlying mechanism is still unclear. Initially people have proposed a model of free energy asymmetry, in which the difference of siRNA efficacy was attributed to differential loading of the two strands into RISC due to such free energy asymmetry on the siRNA ends4,5. However two recent papers suggested that discrimination occurs after both strands enter the RISC complex and one of the strands was cleaved before it was removed from the RISC6,7, but it is still unclear how the RISC complex cleaves one strand but not the other. With this note, it comes with no surprise that although there are many software packages that can be used for siRNA design, none can predict siRNA efficacy with anything approaching 100 per cent accuracy. In general, such software can only filter out siRNA that have obvious disadvantages such as a high degree of off-target highs, or other nucleotide patterns that are not compatible with known active siRNA8-13.

In general there are three types of siRNA libraries: siRNA chemically synthesised, siRNA formed by enzymatic reactions and siRNA encoded by vectors.

siRNA libraries formed through chemical synthesis are regarded to be better defined in composition and easier to use, but inherently more expensive material-wise. The designed siRNA can be directly fed into the high throughput RNA synthesis system. Synthetic siRNA is available from three of the major siRNA suppliers; Qiagen, Ambion and Dharmacon as shown on their respective Web sites. These libraries obviously are more popular among big pharma or biotech companies. Examples include Novartis, Intradigm, Affymetrix by Qiagen, Abbot and Millennium by Dharmacon and Rossetta by Ambion. Publication of screening results using these libraries generally seems to be delayed due to commercial reasons. Takeover of Proligo by Sigma might help to foster more of such deals in the future. It is however plausible that recent pricing developments has made synthetic siRNA much more affordable than before. More academic institutions start to gain access to synthetic siRNA libraries of different scale, as exemplified by the recent deal between Ambion and Max Planck Institute for MolecularCell Biology and Genetics in Dresden, Germany.

Immediately after the siRNA mechanism was discovered people have created a number of methods to generate siRNA through vectors based siRNA expression14-27 or Dicer-mediated cleavage of long double stranded siRNA28-30. Formation and management of such siRNA libraries involves a considerable amount of in-house work that is very demanding, but budget-wise appears to be much more suitable for most academic labs. Some early reports have focused mainly on the development of resources using the vector-based approaches31-33, but several extensive screening applications have also been executed with promising proof-of concepts as shown below.

Screening using siRNA libraries

Although cell array based screening methods have attracted significant attention, most of the siRNA library based screening was achieved using microtitre plates. It should be stressed that using the siRNA libraries for HTS is at an early stage, with only a handful of cases reported. Therefore it is still too early to be more generalised than a case presentation34-36.

It is natural that early screening has focused on a mature model system. Apoptosis is one such preferred assay system. One study has examined genes that could affect TRAIL-induced apoptosis. TRAIL is a TNF superfamily member that induces selective cytotoxicity of tumour cells when bound to its cognate receptors. In addition to detecting well-characterised genes in the apoptosis pathway, several modulator genes responsible for progression of the apoptotic signal through the intrinsic mitochondrial cell death pathway, or acting to limit TRAIL-induced apoptosis have been identified and novel functions of MYC and the WNT pathway in maintaining susceptibility to TRAIL was also highlighted37.

In another apoptosis-related screening effort, the apoptosis inducing reagent was actually double stranded RNA poly(I-C). Mammalian cells respond to long double stranded RNA through a PKR triggered antiviral process that could lead to cellular apoptosis. The authors have utilised an original vector library that allowed them to avoid the confounding effects of the interferon response. With the aim to identify the genes involved in apoptosis that was induced by double-stranded RNA the screening led to the identification of two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. The authors were able to further prove that MST2 and protein kinase Calpha both activated the pro-apoptotic signal mediated by ERK238.

Proliferation and/or cell division is another favourite cellular model for siRNA based HTS. In a recent study, a set of retroviral vectors encoding 23, 742 distinct siRNAs were constructed to target 7,914 different human genes. Using this resource, the authors identified several genes that can modulate p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest39.

In yet another example, a genome-scale library of endoribonuclease-prepared short interfering siRNA library was generated from a sequence-verified complementary DNA collection representing 15,497 human genes. From this library 5, 305 siRNAs were used in a test run to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, 37 genes were found to be required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown40.

siRNA expressed from PCR cassettes has also been successfully used at a large scale for HTS41. Schultz group at Scripps have generated a siRNA expression cassette library that targets >8000 genes with two siRNA sequences per gene using a dual promoter system. A high-throughput cell-based screen of an arrayed siRNA expression cassette library identified known components of the NF-kB signaling pathways as well as genes that may have previously unrecognised roles in the regulation the NF-kB transcription activity.

Although inhibition of viral genes could be a straight forward way of containing viral inhibition by siRNA drugs, it has been demonstrated that knock-down of host factors such as receptors can also result in reduced viral load. In an effort to identify novel proviral host factors involved in human immunodeficiency virus (HIV) infection, a screen of a small interfering RNA (siRNA) library targeting 5,000 potential genes was performed42. Several novel factors whose knockdown inhibited infection were identified, including Pak3, a member of the serine/threonine group I PAK kinases.

Unleashing the potential

One of the hurdles for HTS is the wide spread off-target effect of siRNA, which seems to far exceed people’s expectations. One recent survey suggested that mutations in a majority of the positions along the siRNA targeting site does not abolish the inhibitory activity of the siRNA43. More astonishingly, it was found that off-target effect could rise from a stretch of complementation as short as 7 nts!44. It was further discovered that consensus sequences of 5 or 9 nt can induce specific cellular responses in a manner that is not dependent on the sequences outside the consensus region through pathways not related to RNAi45,46. All these complications call for vigorous confirmation of any phenotype changes induced by a particular siRNA in HTS, in order to verify that the intended target gene is really relevant.

During the last years there has been a plethora of new technologies and methods emerging in the field of siRNA applications and this trend is expected to continue. Improvements in new technologies, in the areas of siRNA formation, validation and high throughput screening systems47-49 will help to unleash the tremendous potential of siRNA libraries in high throughput target discovery and validation.

References

1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 19, 806-811
2. Hannon, G.J. and Rossi, J.J. 2004. Unlocking the potential of the human genome with RNA interference. Nature 431: 371–378
3. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498
4. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. 2003 Asymmetry in the assembly of the RNAi enzyme complex. Cell. 115, 199-208
5. Khvorova A, Reynolds A, Jayasena SD. 2003 Functional siRNAs and miRNAs exhibit strand bias. Cell. 115, 209-216
6. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD. 2005 Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell. 123, 607-620
7. Rand TA, Petersen S, Du F, Wang X. 2005 Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell. 123, 621-629
8. Henschel A, Buchholz F, Habermann B. 2004. DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Res. 32, W113-120
9. Wang L, Mu FY. 2004. A Web-based design center for vector-based siRNA and siRNA cassette. Bioinformatics. 20, 1818-1820
10. Sandy P, Ventura A, Jacks T. 2005 Mammalian RNAi: a practical guide. Biotechniques. 39, 215-224
11. Huesken D, Lange J, Mickanin C, Weiler J, Asselbergs F, Warner J, Meloon B, Engel S, Rosenberg A, Cohen D, Labow M, Reinhardt M, Natt F, Hall J. 2005 Design of a genome-wide siRNA library using an artificial neural network. Nat Biotechnol.23, 995-1001
12. Jagla B, Aulner N, Kelly PD, Song D, Volchuk A, Zatorski A, Shum D, Mayer T, De Angelis DA, Ouerfelli O, Rutishauser U, Rothman JE. 2005 Sequence characteristics of functional siRNAs. RNA. 11, 864-72
13. Arziman Z, Horn T, Boutros M. 2005 E-RNAi: a web application to design optimized RNAi constructs. Nucleic Acids Res. 33, W582-588
14. Barton, G.M. and Medzhitov, R. 2002. Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. 99:14943–14945
15. Brummelkamp, T.R., Bernards, R., and Agami, R. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553
16. Calegari, F., Haubensak, W., Yang, D., Huttner, W.B., and Buchholz, F. 2002. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc. Natl. Acad. Sci. 99: 14236–14240
17. Castanotto D, Li H, Rossi JJ. 2002. Functional siRNA expression from transfected PCR products. RNA. 8, 1454-1460
18. Lee NS, Dohjima T, Bauer G et al. 2002. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol. 20, 500-505
19. Miyagishi M, Taira K. 2002. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol. 20, 497-500
20. Sui G, Soohoo C, Affar el B et al. 2002. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA. 16, 5515-5520
21. Xia H, Mao Q, Paulson HL, Davidson BL. 2002. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol. 20, 1006-1010
22. Yu JY, DeRuiter SL, Turner DL. 2002. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA. 99, 6047-6052
23. Boden D, Pusch O, Lee F, Tucker L, Shank PR, Ramratnam B. 2003. Promoter choice affects the potency of HIV-1 specific RNA interference. Nucleic Acids Res. 31, 5033-8
24. Li MJ, Bauer G, Michienzi A et al. 2003. Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. Mol Ther 8, 196-206
25. Holle L, Hicks L, Song W, Holle E, Wagner T, Yu X. 2004. Bcl-2 targeting siRNA expressed by a T7 vector system inhibits human tumor cell growth in vitro. Int J Oncol. 24, 615-621
26. Hosono T, Mizuguchi H, Katayama K et al. 2004. Adenovirus vector-mediated doxycycline-inducible RNA interference. Hum Gene Ther. 15, 813-819
27. van de Wetering M, Oving I, Muncan V, Pon Fong MT, et al. 2003. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609-615
28. Sen G, Wehrman TS, Myers JW, Blau HM. 2004. Restriction enzyme-generated siRNA (REGS) vectors and libraries. Nat Genet. 36, 183-189
29. Luo B, Heard AD, Lodish HF. 2004. Small interfering RNA production by enzymatic engineering of DNA (SPEED). Proc Natl Acad Sci USA. 101, 5494-5499
30. Shirane D, Sugao K, Namiki S, Tanabe M, Iino M, Hirose K. 2004. Enzymatic production of RNAi libraries from cDNAs. Nat Genet. 36, 190-196
31. Michiels F, van Es H, van Rompaey L, Merchiers P, Francken B, Pittois K, van der Schueren J, Brys R, Vandersmissen J, Beirinckx F, Herman S, Dokic K, Klaassen H, Narinx E, Hagers A, Laenen W, Piest I, Pavliska H, Rombout Y, Langemeijer E, Ma L, Schipper C, Raeymaeker MD, Schweicher S, Jans M, van Beeck K, Tsang IR, van de Stolpe O, Tomme P, Arts GJ, Donker J.. 2002. Arrayed adenoviral expression libraries for functional screening. Nat Biotechnol. 20, 1154-1157
32. Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M, Aruleba S, Balija V, O’Shaughnessy A, Gnoj L, Scobie K, Chang K, Westbrook T, Cleary M, Sachidanandam R, McCombie WR, Elledge SJ, Hannon GJ. 2004. A resource for large-scale RNA-interference-based screens in mammals. Nature. 428, 427-431
33. Chen M, Zhang L, Zhang HY, Xiong X, Wang B, Du Q, Lu B, Wahlestedt C, Liang Z. 2005 A universal plasmid library encoding all permutations of small interfering RNA. Proc Natl Acad Sci U S A. 102, 2356-2361
34. Akashi H, Matsumoto S, Taira K. Gene discovery by ribozyme and siRNA libraries. Nat Rev Mol Cell Biol. 6, 413-22
35. Ito M, Kawano K, Miyagishi M, Taira K. 2005 Genome-wide application of RNAi to the discovery of potential drug targets. FEBS Lett. 579, 5988-5995.
36. Liang Z. 2005 High-throughput screening using genome-wide siRNA libraries. IDrugs. 8, 924-926
37. Aza-Blanc, P., Cooper, C.L., Wagner, K., Batalov, S., Deveraux, Q.L., and Cooke, M.P. 2003. Identification of modulators of TRAIL induced apoptosis via RNAi-based phenotypic screening. Mol. Cell.12: 627–637
38. Matsumoto S, Miyagishi M, Akashi H, Nagai R, Taira K. 2005 Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response non-inducible small interfering RNA expression vector library. J Biol Chem. 280, 25687-25696
39. Berns K, Hijmans EM, Mullenders J et al. 2004. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature. 428, 431-437
40. Kittler R, Putz G, Pelletier L, Poser I, Heninger AK, Drechsel D, Fischer S, Konstantinova I, Habermann B, Grabner H, Yaspo ML, Himmelbauer H, Korn B, Neugebauer K, Pisabarro MT, Buchholz F. 2004 An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature. 432, 1036-40
41. Zheng L, Liu J, Batalov S, Zhou D, Orth A, Ding S, Schultz PG. 2004. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc Natl Acad Sci USA. 101, 135-40
42. Nguyen DG, Wolff KC, Yin H, Caldwell JS, Kuhen KL. 2006 “UnPAKing” Human Immunodeficiency Virus (HIV) Replication: Using Small Interfering RNA Screening To Identify Novel Cofactors and Elucidate the Role of Group I PAKs in HIV Infection. J Virol. 80, 130-137
43. Du Q, Thonberg H, Wang J, Wahlestedt C, Liang Z. 2005 A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 33,1671-7
44. Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE, Fesik SW, Shen Y. 2005 siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527-35
45. Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G. 2005 Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 11, 263-270
46. Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I. 2005 Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol. 23, 457-462
47. Ovcharenko D, Jarvis R, Hunicke-Smith S, Kelnar K, Brown D. 2005 High-throughput RNAi screening in vitro: from cell lines to primary cells. RNA. 11, 985-993
48. Xin H, Bernal A, Amato FA, Pinhasov A, Kauffman J, Brenneman DE, Derian CK, Andrade-Gordon P, Plata-Salaman CR, Ilyin SE. 2004 High-throughput siRNA-based functional target validation. J Biomol Screen. 9, 286-293
49. Brummelkamp TR, Berns K, Hijmans EM, Mullenders J, Fabius A, Heimerikx M, Velds A, Kerkhoven RM, Madiredjo M, Bernards R, Beijersbergen RL. 2005 Functional identification of cancer-relevant genes through large-scale RNA interference screens in mammalian cells. Cold Spring Harb Symp Quant Biol. 69, 439-445