Chinese Bulletin of Life Sciences. Progress in the preparation of sirnas. ZHANG Zhong-Hua, HOU Yong-Tai*

Transcription

1 Chinese Bulletin of Life Sciences Vol. 16, No. 4 Aug., (2004) (small interfering ) sh Q522; Q813 A RNase Progress in the preparation of s ZHANG Zhong-Hua, HOU Yong-Tai* (Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of CAS, Shanghai , China) Abstract: Recently, s (small interfering s) have been widely used to induce interference in mammalian systems. Till now, there are five methods to produce s, such as chemical synthesis, in vitro transcriptions, RNase III-family enzymatic digestion, expression vectors and expression cassettes. It is essential to choose an effective sequence in those methods. Furthermore, with the developments of drug discovery and genomics, the s preparation should be developed and consummated in several ways, such as high-throughput screening of effective s, stability of s, efficiently gene transfer and regulation of the expression of s. Key words: preparation; ; sh; interference 1 ( interference i) ds(double-stranded ) (small interfering ) [1] 20nt [2~4] 5' 3' (1977 ) (1961 ) *

2 232 (accessibility) [5~6] (1) cdna 50~100nt 5' 3' UTR(untranslated regions) (2) cdna 5'-AA(N19)TT N GC% 50% 32%~79%GC GC% G G G (G-quartet) 5'-AA(N19)TT 5'-AA (N21) 5'-NA(N21) EST BLAST (3) mfold structure GENETYX-Mac8.0 mfold Michael Zuker mfold ( forml.cgi) (4) GFP luciferase CAT (5) 3~4 Invivogen DNA Designer [5~6] Ambion Iris Genetics (chemical synthesis) [2] transcription) [7] ds RNase III (in vitro (Dicer, E. coli RNase III in vitro RNase IIIfamily enzymatic digestion of ds) [8~9] ( expression vectors) [10~12] ( expression cassettes, SECs) [13] [2] Elbashir -TT- 3' -UU- 21nt 1 21nt 29nt 200~1 000bp 55~75nt 50nt ( T7 70nt ) 4 ~ ~6 + RNase RNase RNase ( ( ) ) ( ) /

3 µmol 1mg (24 ) ( ) 10~100nmol/L 3~4 2.2 T7 T7 [9] 5' - AA- 3' leader T ' [7] 2.3 RNase ds RNase 200~1 000nt ds RNase III [9] Dicer [8] ds (cocktail) 2.4 sidna ( Pol ) ( Pol ) [14] Pol III/ Pol III U6 sn H1 5' U6 H1 [10~12] Pol ti Met t Val 5' [14] 1 (1) ( 2A) [10~11] (2) 5~9nt

4 234 2 A: ; B: sh ; C: sh PCR (stem-loop) sh(short hairpin )( 2B) [12] Pol / Shinagawa Ishii [14] P CMV pdecap (Deletion of Cap structure and PolyA) ski ds Dicer sh Pol SEC 3 SEC PCR DNA 2.5 ( expression cassettes SECs) SEC PCR [13] SEC Pol sh Pol ( 2C) SEC SEC T 3.1 [5~6] His6 Myc HA Flag GFP luciferase

5 235 (microarray) [15] 3' 3.2 Czauderna [4] ib(inverted deoxy abasic) ( NH 2 ) 2'-O- 2'-O Boden [16] sh Pol U6 27 t Met sh sh 3.3 [17~18] ttr(the tetracycline repressor) 4 [18~19] [1] Tuschl T, Borkhardt A. Small interfering s: a revolutionary tool for the analysis of gene function and gene therapy. Mol Intervent, 2002, 2(3): 158~167 [2] Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21- nucleotide s mediate interference in cultured mammalian cells. Nature, 2001, 411: 494~498 [3] Amarzguioui M, Holen T, Babaie E, et al. Tolerance for mutations and chemical modifications in a. Nucl Acid Res, 2003, 31(2): 589~595 [4] Czauderna F, Fechtner M, Dames S, et al. Structural variations and stabilising modifications of synthetic s in mammalian cells. Nucl Acid Res, 2003, 31(11): 2705~2716 [5] Far R KK, Sczakiel G. The activity of in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucl Acid Res, 2003, 31 (15): 4417~4424 [6] Bohula E A, Salisbury A J, Sohail M, et al. The efficacy of small interfering s targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem, 2003, 278 (18): 15991~15997 [7] Donzé O, Picard D. interference in mammalian cells using s synthesized with T7 polymerase. Nucl Acid Res, 2002, 30(10): e46 [8] Myers J W, Jones J T, Meyer T, et al. Recombinant Dicer efficiently converts large dss into s suitable for gene silencing. Nat Biotech, 2003, 21: 324~328 [9] Yang D, Buchholz F, Huang Z D, et al. Short duplexes produced by hydrolysis with Escherichia coli RNase III mediated effective interference in mammalian cells. Proc Natl Acad Sci USA, 2002, 99(15): 9942~9947 [10] Miyagishi M, Taira K. U6 promoter-driven s with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotech, 2002, 19: 497~500 [11] Lee N S, Dohjima T, Bauer G, et al. Expression of small interfering s targeted against HIV-1 rev transcripts in

6 236 human cells. Nat Biotech, 2002, 19: 500~505 [12] Paul C P, Good P D, Winer I, et al. Effective expression of small interfering in human cells. Nat Biotech, 2002, 19: 505~508 [13] Castanotto D, Li H T, Rossi J J. Functional expression from transfected PCR products., 2002, 8: 1454~1460 [14] Shinagawa T, Ishii S. Generation of Ski-knockdown mice by expressing a long double-strand from an polymerase II promoter. Genes Devel, 2003, 17: 1340~1345 [15] Kumar R, Conklin D S, Mittal V. High-throughput selection of effective i probes for gene silencing. Genome Res, 2003, 13: 2333~2340 [16] Boden D, Pusch O, Lee F, et al. Promoter choice affects the potency of HIV-1 specific interference. Nucl Acid Res, 2003, 31(17): 5033~5038 [17] Barton G M, Medzhitov R. Retroviral delivery of small interfering into primary cells. Proc Natl Acad Sci USA, 2002, 99(23): 14943~14945 [18] Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible interference. J Virology, 2003, 77(16): 8957~8961 [19] Wang J, Tekle E, Oubrahim H, et al. Stable and controllable interference: investigating the physiological function of glutathionylated actin. Proc Natl Acad Sci USA, 2003, 100(9): 5103~5106