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lanyu270

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[资源] RNAi技术

1.在设计RNAi实验时，可以先在以下网站进行目标序列的筛选：
http://www.ambion.com/techlib/misc/siRNA_finder.html
http://katahdin.cshl.org:9331/RNAi/
http://www.ic.sunysb.edu/Stu/shilin/rnai.html

2.RNAi目标序列的选取原则：
（1）siRNAs with lower G/C content (35-55%) are more active than those with G/C content higher than 55%；
（2）Beginning with the AUG start codon of your transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl, et al. recommend against designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
（3）Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.

3.siRNA的合成：
(1)IN VITRO: siRNAs generated in vitro by transcription with T7 DNA polymerase;
(2)IN VIVO: Short hairpin RNAs that are transcribed in vivo from vectors containing the human U6 promoter.
Selection of siRNA duplexes from the target mRNA sequence

Using Drosophila melanogaster lysates (Tuschl et al. 1999), we have systematically analyzed the silencing efficiency of siRNA duplexes as a function of the length of the siRNAs, the length of the overhang and the sequence in the overhang (Elbashir et al. 2001c). The most efficient silencing was obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair. 2'-Deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant. We used to select siRNA sequences with TT in the overhang.

The targeted region is selected from a given cDNA sequence beginning 50 to 100 nt downstream of the start codon. Initially, 5' or 3' UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. More recently, however, we have targeted 3'-UTRs and have not experienced any problems in knocking down the targeted genes. In order to design a siRNA duplex, we search for the 23-nt sequence motif AA(N19)TT (N, any nucleotide) and select hits with approx. 50% G/C-content (30% to 70% has also worked in our hands). If no suitable sequences are found, the search is extended using the motif NA(N21). The sequence of the sense siRNA corresponds to (N19)TT or N21 (position 3 to 23 of the 23-nt motif), respectively. In the latter case, we convert the 3' end of the sense siRNA to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. The antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. Because position 1 of the 23-nt motif is not recognized sequence-specifically by the antisense siRNA, the 3'-most nucleotide residue of the antisense siRNA, can be chosen deliberately. However, the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23-nt motif) should always be complementary to the targeted sequence. For simplifying chemical synthesis, we always use TT. More recently, we preferentially select siRNAs corresponding to the target motif NAR(N17)YNN, where R is purine (A, G) and Y is pyrimidine (C, U). The respective 21-nt sense and antisense siRNAs therefore begin with a purine nucleotide and can also be expressed from pol III expression vectors without a change in targeting site; expression of RNAs from pol III promoters is only efficient when the first transcribed nucleotide is a purine.

We always design siRNAs with symmetric 3' TT overhangs, believing that symmetric 3' overhangs help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs (Elbashir et al. 2001b; Elbashir et al. 2001c). Please note that the modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition. In summary, no matter what you do to your overhangs, siRNAs should still function to a reasonable extent. However, using TT in the 3' overhang will always help your RNA synthesis company to let you know when you accidentally order a siRNA sequences 3' to 5' rather than in the recommended format of 5' to 3'. You may think this is funny, but it has happened quite a lot.

Compared to antisense or ribozyme technology, the secondary structure of the target mRNA does not appear to have a strong effect on silencing. We say that, because we have already knocked-down more than 20 genes using a single, essentially randomly chosen siRNA duplex (Harborth et al. 2001). Only 3 siRNA duplexes have been ineffective so far. In one or two other cases, we have found siRNAs to be inactive because the targeting site contained a single-nucleotide polymorphism. We were also able to knock-down two genes simultaneously (e.g. lamin A/C and NuMA) by using equal concentrations of siRNA duplexes.

We recommend to blast-search (NCBI database) the selected siRNA sequence against EST libraries to ensure that only one gene is targeted. In addition, we also recommend to knock-down your gene with two independent siRNA duplexes to control for specificity of the silencing effect. If selected siRNA duplexes do not function for silencing, please check for sequencing errors of the gene, polymorphisms, and whether your cell line is really from the expected species. Our initial studies on the specificity of target recognition by siRNA duplexes indicate that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA degradation (Elbashir et al. 2001c). Furthermore, it is unknown if targeting of a gene by two different siRNA duplexes is more effective than using a single siRNA duplex. We think that the amount of siRNA-associating proteins is limiting for silencing rather than the target accessibility.
最近出版的综述和部分文章（部分已下载OK）
1: Chiu YL, Rana TM.
RNAi in Human Cells. Basic Structural and Functional Features of Small
Interfering RNA
Mol Cell. 2002 Sep;10(3):549-61..（OK）

2: Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J.
Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent
modulation of its stability.
EMBO J. 2002 Nov 1;21(21):5911-5920.

3: Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W.
Human Dicer preferentially cleaves dsRNAs at their termini without a requirement
for ATP.
EMBO J. 2002 Nov 1;21(21):5875-5885..（OK）

4: Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Radmark O.
Ribonuclease activity and RNA binding of recombinant human Dicer.
EMBO J. 2002 Nov 1;21(21):5864-5874..（OK）

5: Dernburg AF, Karpen GH.
A Chromosome RNAissance.
Cell. 2002 Oct 18;111(2):159-62..（OK）

6: Carmell MA, Xuan Z, Zhang MQ, Hannon GJ.
The Argonaute family: tentacles that reach into RNAi, developmental control,
stem cell maintenance, and tumorigenesis.
Genes Dev. 2002 Nov 1;16(21):2733-2742. .（OK）

7: Schwarz DS, Hutvagner G, Haley B, Zamore PD.
Evidence that siRNAs Function as Guides, Not Primers, in the Drosophila and
Human RNAi Pathways.
Mol Cell. 2002 Sep;10(3):537-48..（OK）

8: Ramaswamy G, Slack FJ.
siRNA. A Guide for RNA Silencing.
Chem Biol. 2002 Oct;9(10):1053-5..（OK）

9: Calegari F, Haubensak W, Yang D, Huttner WB, Buchholz F.
Tissue-specific RNA interference in postimplantation mouse embryos with
endoribonuclease-prepared short interfering RNA.
Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14236-40.（free）

10: Capodici J, Kariko K, Weissman D.
Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA
Interference.
J Immunol. 2002 Nov 1;169(9):5196-201..（OK）

11: Timms MW, Van Deursen FJ, Hendriks EF, Matthews KR.
Mitochondrial Development during Life Cycle Differentiation of African
Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control
Point.
Mol Biol Cell. 2002 Oct;13(10):3747-59.

12: Zhang Y, Chalfie M.
MTD-1, a touch-cell-specific membrane protein with a subtle effect on touch
sensitivity.
Mech Dev. 2002 Nov;119(1):3.

13: Fukumoto H, Deng A, Irizarry MC, Fitzgerald ML, Rebeck GW.
Induction of the cholesterol transporter ABCA1 in CNS cells by LXR agonists
increases secreted A{beta} levels.
J Biol Chem. 2002 Oct 15 [epub ahead of print]

14: Wojtkowiak A, Siek A, Alejska M, Jarmolowski A, Szweykowska-Kulinska Z,
Figlerowicz M.
RNAi And Viral Vectors As Useful Tools In The Functional Genomics Of Plants.
Construction Of BMV-Based Vectors For RNA Delivery Into Plant Cells.
Cell Mol Biol Lett. 2002;7(2A):511-22..（OK）

15: Sun Y, Cheng Z, Ma L, Pei G.
beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis and this is
mediated by its enhancement of p38 MAPK activation.
J Biol Chem. 2002 Oct 4 [epub ahead of print]

16: Caudy AA, Myers M, Hannon GJ, Hammond SM.
Fragile X-related protein and VIG associate with the RNA interference machinery.
Genes Dev. 2002 Oct 1;16(19):2491-6..（OK）

17: Reichhart JM, Ligoxygakis P, Naitza S, Woerfel G, Imler JL, Gubb D.
Splice-activated UAS hairpin vector gives complete RNAi knockout of single or
double target transcripts in Drosophila melanogaster.
Genesis. 2002 Sep-Oct;34(1-2):160-4..（OK）

18: Enerly E, Larsson J, Lambertsson A.
Reverse genetics in Drosophila: from sequence to phenotype using UAS-RNAi
transgenic flies.
Genesis. 2002 Sep-Oct;34(1-2):152-5.（OK）

19: Borkhardt A.
Blocking oncogenes in malignant cells by RNA interference-New hope for a highly
specific cancer treatment?
Cancer Cell. 2002 Sep;2(3):167..（OK）

20: Gaudilliere B, Shi Y, Bonni A.
RNA interference reveals a requirement for MEF2A in activity-dependent neuronal
survival.
J Biol Chem. 2002 Sep 13 [epub ahead of print](free)

21: Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T.
Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi.
Cell. 2002 Sep 6;110(5):563..（OK）

22: Tijsterman M, Okihara K, Thijssen K, Plasterk R.
PPW-1, a PAZ/PIWI Protein Required for Efficient Germline RNAi, Is Defective in
a Natural Isolate of C. elegans.
Curr Biol. 2002 Sep 3;12(17):1535..（OK）

23: Xu F, Gaggero C, Cohen S.
Polyadenylation can regulate ColE1 type plasmid copy number independently of any
effect on RNAI decay by decreasing the interaction of antisense RNAI with its
RNAII target.
Plasmid. 2002 Jul;48(1):49..（OK）

24: Hall IM, Shankaranarayana GD, Noma K, Ayoub N, Cohen A, Grewal SI.
Establishment and maintenance of a heterochromatin domain.
Science. 2002 Sep 27;297(5590):2232-7.(free)

25: Zentella R, Yamauchi D, Ho TH.
Molecular dissection of the gibberellin/abscisic Acid signaling pathways by
transiently expressed RNA interference in barley aleurone cells.
Plant Cell. 2002 Sep;14(9):2289-301..（OK）

26: Negeri D, Eggert H, Gienapp R, Saumweber H.
Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential
nuclear cofactor involved in Notch signal transduction.
Mech Dev. 2002 Sep;117(1-2):151.

27: Lassus P, Rodriguez J, Lazebnik Y.
Confirming specificity of RNAi in mammalian cells.
Sci STKE. 2002 Aug 27;2002(147)L13..（OK）

28: Morris JC, Wang Z, Drew ME, Englund PT.
Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi
library.
EMBO J. 2002 Sep 2;21(17):4429-38..（OK）

29: Allshire R.
Molecular biology. RNAi and heterochromatin--a hushed-up affair.
Science. 2002 Sep 13;297(5588):1818-9. (free)

30: Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA.
Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by
RNAi.
Science. 2002 Sep 13;297(5588):1833-7.(free)

31: Krichevsky AM, Kosik KS.
RNAi functions in cultured mammalian neurons.
Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11926-9.(free)

32: Oshiumi H, Begum NA, Matsumoto M, Seya T.
[RNA interference for mammalian cells]
Nippon Yakurigaku Zasshi. 2002 Aug;120(2):91-5. Japanese.

33: Coburn GA, Cullen BR.
Potent and specific inhibition of human immunodeficiency virus type 1
replication by RNA interference.
J Virol. 2002 Sep;76(18):9225-31..（OK）

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