Trends in Medical Research -

Transcription

1 RNA Interference Tel : / kimys@inje.ac.kr, RNAi (RNA or RNA silencing) ( ) DNA small RNA (srnas) mrna,. DNA small RNA. DNA RNA small RNA. small RNA mirna (microrna), RNA RNAi small RNA strna (small temporal RNA) mrna 80-90% small size RNA strna RNA mirna ( 4,000 mirna primates, rodents, birds, fish, worms, flies, plants and viruses. sequences/index.shtm). mirna, mirna, mirna RNA ( 1) RNA 1990

2 Korean Society of Medical Biochemistry and Molecular Biology News 2. RNA. mrna RNA (dsrna). mrna RNA. ( : ) 1. The RNAi and strna (mirna) pathways intersect. In both pathways, Dicer acts to generate the active small RNA regulator: sirnas from dsrna and mirnas from 70 nt strna precursors. sirnas trigger destruction of a perfectly complementary target RNA; mirnas repress the translation of targets with which they pair imperfectly (adapted from Current Opinion in Genetics & Development 2002, 12:225232).. 10 mrna DNA (antisense oligonucleotide) anti-sense RNA antisense RNA sense RNA, antisense RNA sense RNA RNA (double-strand RNA:dsRNA) RNA ( 1, 2). RNAi,. RNAi. RNAi ( ) RNAi sirna (small interfering RNA), long dsrna Dicer sirna(21~23 nt), sirna RISC (RNA-induced Silencing Complex) ( RISC ) target mrna ( 3). RNA /Vol.14/No.1

3 3. RNA. RNA. ( : ),. 3. RNA RNA (mirna: mirna) RNA ( 4B). RNA dsrna RNA ( 4C). 4. RNA. (A) RNA RNA RNA. (B) mirna. 3 40% mirna RNA. (C) dsrna,. ( : ), ( 4A), 1, RNA RNA. RNA 1998 Nature RNA, 10., ( ),, 2006

4 Korean Society of Medical Biochemistry and Molecular Biology News RNA RNA, 10~15 RNA, small RNA RNAi (RNA interference:rna ) sirna (small interfering RNA). RNAi MIT Technology Review RNAi. RNAi RNAi RNAi. RNAi.. RNA.,,, , 3. RNA? RNA. RNA /. DNA, RNA dsrna (sirna: small interfering RNA; ~21 nt RNA), ( 5) RNA 5. DNA RNA, /Vol.14/No.1

5 (genome-wide knockdown screening) ( 6), ( Biotech company ). 6. sirna library bar-code screens. (a) Schematic outline of the bar-code technology. A large population of cells is infected with the NKI shrna library and divided into two populations. One is treated (or selected), whereas the other population serves as a reference for the hybridization. From each population, genomic DNA is isolated and shrna cassettes are recovered by PCR. The PCR products are used as templates for an in vitro linear amplification reaction to generate RNA probes that are subsequently labeled with fluorescent dyes. The probes from the selected cells and reference population are hybridized to a DNA microarray containing the complementary sequences from the complete NKI shrna library. (b) An integrated shrna vector also delivers a gene-specific identifier in the form of a unique 19-mer molecular bar code that can be used to identify which cell carries any given knockdown allele in a large population of cells. (c) An example of the result of a barcode hybridization. The graph depicts the log2 of the average intensity versus the log2 of the fluorescence intensity ratio. shrna vectors that are enriched by the selection method are represented by spots with an increased ratio (adapted from Nat. Chem. Biol. 2, , 2006) (Sirna). (: AMD) ( 7) (FDA)... RNA sirna Sirna. Sirna SiRNA % 5.. (knock-out library) RNA genome scale knock-down library ( RNA, shrna cassette in plasmid, retroviral vector, and lentiviral vector) 7. Sirna RNA. sirna ( ) ( ) 60%. ( : Nature Biotech. 2003)

6 Korean Society of Medical Biochemistry and Molecular Biology News. sirna RNA. sirna, HIV, HCV,,,. RNA hurdle. RNAi target validation functional genomics. RNAi knock-down target gene RNAi., RNAi. RNAi-based drug (1): target validation (2): in vitro drug candidate (3): in vivo (2) drug candidate (4): (3) candidate., drug,,. RNAi, RNA-based drug 4 {stability ( ), delivery (), pharmacokinetics ( ), potency ()} (serum stablility: 17 half-life, circulation time : 4, tissue targeting :liver& endothelium, cellular stability : 21 ). RNAi-based drug, RNAi-based antisense RNA ribozyme celluar process 8. sirna. ( : Nature Biotech. 2003) RNA-derived sirna biogenesis RNAi-based drug. shrna shrna mirna perturbation. sirna RNAi mammalian cell 5. sirna in vitro sirna, transfection (in vitro preparation of sirna) sirna shrna(short hairpin RNA) ( ) PCR-derived sirna expression cassette transfection infection (in vivo expression of shrna), /Vol.14/No.1

7 . sirna, RNAi sirna 5 ( 1 4 ). (1) RNA oligonucleotide : sirna. RNAi 2-3 long-term study. Dharmacon, Ambion,. (2) in vitro transcription small RNA. (3) in vitro transcription long dsrna Rnase III family enzyme (e.g. Dicer, Rnase III) : long dsrna RNAi, PKR, sirna. long dsrna sirna processing RNase III (or Dicer) sirna. in vitro transcription kit sense anti-sense RNA annealing, RNase III sirna sirna sirna gene silencing., heterogenous sirna non-specific gene silencing. (4) shrna expression plasmid viral vector : transient gene silencing phenotypic change, gene silencing effect. sirna. RNA transcript RNA polymerase III (pol III) promoter ( pol III promoter U6 promoter H1 promoter. promoter ), promoter sirna target sequence sense antisense sequence (4-11 nt) loop small hairpin RNA (shrna). shrna sirna processing enzyme (Dicer or Rnase III) sirna target gene silencing.,,,. mirna pol II promoter system shrna. (5) PCR-derived sirna expression cassette. 5, 3 4 sirna knock-down

8 Korean Society of Medical Biochemistry and Molecular Biology News mrna sirna 21~23 nt target sequence (design). target sequence RNAi, sirna RNAi data base (secondary structure, GC contents, cisacting sequences binding to proteins, asymmetry of 5' and 3' sequences, and experimental education with sirna library ) functional sirna Web site ( wadsworth.org/sirna.pl, sirna target mrna degradation. 3-4 target site sirna. sirna, factor RNAi. mrna, RNAi machinery protein, genetic compensation mechanisms RNAi. sirna shrna consistent result, RNAi., RNAi knock-down sirna RNAi. RISC 21~23 nt RNA 80~90% mrna targeting (mirna action mechanism). sirna target mrna mrna non-specific knock-down, off-target effect. off-target effect sirna design algorithm sirna. RNAi RNA/DNA transfection viral infection. stress damage expression profile. misinterpretation. RNAi off-target effect expression profile change, negative- positive control. RNA, RNA,. ( RNA ),, /Vol.14/No.1

9 , 8. RNA RNA Birmingham, A. et al. 3 UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, (2006). 2. Dorsett, Y. & Tuschl, T. sirnas: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, (2004). 3. Dykxhoorn, D.M., Palliser, D. & Lieberman, J. The silent treatment: sirnas as small molecule drugs. Gene Ther. 13, (2006). 4. Echeverri, C.J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user s guide. Nat. Rev. Genet. 7, (2006). 5. Elbashir, S.M., Harborth, J., Weber, K. & Tuschl, T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, (2002). 6. Fedorov, Y. et al. Off-target effects by sirna can induce toxic phenotype. RNA 12, (2006). 7. Filipowicz, W., Jaskiewicz, L., Kolb, F.A. & Pillai, R.S. Posttranscriptional gene silencing by sirnas and mirnas. Curr. Opin. Struct. Biol. 15, (2005). 8. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, (2004). 9. Harborth, J. et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 13, (2003). 10. Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. & Prydz, H. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30, (2002). 11. Huesken, D. et al. Design of a genome-wide sirna library using an artificial neural network. Nat. Biotechnol. 23, (2005). 12. Ito, M., Kawano, K., Miyagishi, M. & Taira, K. Genomewide application of RNAi to the discovery of potential drug targets. FEBS Lett. 579, (2005). 13. Jackson, A.L. & Linsley, P.S. Noise amidst the silence: offtarget effects of sirnas Trends Genet. 20, (2004). 14. Jackson, A.L. et al. Widespread sirna ff-target transcript silencing mediated by seed region sequence complementarity. RNA 12, (2006). 15. Kasim, V., Taira, K. & Miyagishi, M. Screening of sirna target sequences by using fragmentized DNA. J. Gene Med. 8, (2006). 16. Lin, X. et al. sirna-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, (2005). 17. Reynolds, A. et al. Rational sirna design for RNA interference. Nat. Biotechnol. 22, (2004). 18. Saetrom, P. & Snove, O., Jr. A comparison of sirna efficacy predictors. Biochem. Biophys. Res. Commun. 321, (2004). 19. Sontheimer, E.J. Assembly and function of RNA silencing complexes. Nat. Rev. Mol. Cell Biol. 6, (2005). 20. Tomari, Y. & Zamore, P.D. Perspective: machines for RNAi. Genes Dev. 19, (2005). 21. Valencia-Sanchez, M.A., Liu, J., Hannon, G.J. & Parker, R. Control of translation and mrna degradation by mirnas and sirnas. Genes Dev. 20, (2006). 22. Zhao, H.F. et al. High-throughput screening of effective sir- NAs from RNAi libraries delivered via bacterial invasion. Nat. Methods 2, (2005).