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Chapter 7:

RNAi and miRNA regulation

Chapter 7:

RNAi and miRNA regulation

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Outlines1. The background and

discovery of miRNAs2. RNAi discovery and

mechanism3. miRNA biogenesis and

regulation4. miRNA roles in

development, cell differentiation and virus

5. miRNA in cancer6. siRNA application

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Topic 1: The background Topic 1: The background and discovery of miRNAsand discovery of miRNAsTopic 1: The background Topic 1: The background and discovery of miRNAsand discovery of miRNAs

一、miRNA发现的背景和miRNA发现一、miRNA发现的背景和miRNA发现

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后基因组时代的基因调控： RNA RNA 调控调控

Most of the RNA transcribed from your genome doesn’t make protein. Carina Dennis talks to the revolutionaries who believe that it functions in gene-regulatory networks that underlie the complexity of higher organisms.

Most of the RNA transcribed from your genome doesn’t make protein. Carina Dennis talks to the revolutionaries who believe that it functions in gene-regulatory networks that underlie the complexity of higher organisms.

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1. 含有 30 亿对碱基的人类基因组仅含有 2 － 3万个蛋白质基因，是果蝇的两倍，啤酒酵母的4 倍。显而易见，生物的复杂性不由编码蛋白生物的复杂性不由编码蛋白质的数目决定质的数目决定。

2. 人类基因组的蛋白质编码区的总和占总基因组长度为 1 － 2 ％，那么其他 9898 ％的基因组有％的基因组有什么功能什么功能呢？当然，在这 98 ％的非蛋白质编码基因组序列里，约 24 ％为插入编码序列的内含子序列；人类基因平均每个基因有 7 个内含子。但这么冗长的内含子序列有什么生物学功能呢？

人类基因组草图带给科学家们的困惑

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人类基因组绝大部分都被转录成 RNA ，细胞内非编码 RNA的数量是编码 RNA 的上百倍。这促使许多科学家认为生物体复杂性被隐藏在它们所输出的非编码 RNA 内，而非编码序列内。

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The discovery of miRNAs

• miRNA was first discovered in 1993 by Victor Ambros at miRNA was first discovered in 1993 by Victor Ambros at Harvard (Harvard (lin-4lin-4))• The second miRNA The second miRNA Let-7Let-7 was discovered in 2000 by Frank was discovered in 2000 by Frank Slack as a postdoc at HarvardSlack as a postdoc at Harvard (Ruvkun lab)(Ruvkun lab)

Victor AmbrosVictor Ambros Gary Ruvkun

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RNAi研究—— Hot topics in the world 2000年， RNAi的研究进展被 Science杂志评为重大科技突破；

2001年“ RNAi”作为当年最重要的科学研究成果之一，再次入选“十大科技突破”；

2002 年 12 月 20日， Science杂志将“ Small RNA & RNAi”评为 2002年度最耀眼的明星。同时， Nature杂志亦将 Small RNA评为年度重大科技成功之一。

2003年，microRNA研究第四次入选“十大科技突破”，排在第四位。

2005年，microRNA研究第五次入选“十大科技突破”。

RNA研究的突破性进展，是生物医学领域近 20年来，可与 HGP相提并论的最重大成果之一。

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The first discoered miRNA: lin-4

Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell. 2004 Jan 23;116 (2 Suppl):S93-6.Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell. 2004 Jan 23;116 (2 Suppl):S89-92

Thought to be an oddity not a general phenomenon

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Breakthrough with BlastN of the second miRNA (stRNA) let-7

Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P,Davidson E, Ruvkun G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000 Nov 2;408(6808):86-9.

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microRNAs had been neglected for so many years because of their small size. OOPs!

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The number of the identified The number of the identified miRNAs is growing rapidly in recent miRNAs is growing rapidly in recent yearsyears. Over >10000Over >10000 miRNAs have been found until the August of this year (The miRBase Sequence Database). These miRNAs are from primates, rodents, birds, fish, worms, flies, plants and viruses. The data are freely available to all through the web interface at http://microrna.sanger.ac.uk/sequences/ and in flatfile form from ftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.

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Mature miRNA 21-25nt ssRNA Highly conserved A more restricted spatial and

temporal expression pattern Slightly regulation About 1 thousand of human miRNAs

regulate 1/3 human genes

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Homology BetweenC. elegans and Homo sapiens miRNAs

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Predicted miRNA Precursors

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Topic 2: RNA interference Topic 2: RNA interference and its mechanismand its mechanism

Topic 2: RNA interference Topic 2: RNA interference and its mechanismand its mechanism

二、 RNA干扰及其机制二、 RNA干扰及其机制

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1 Double-stranded RNA inhibits expression of genes homologous to that RNA. [phenomena- 现象 ]双链 RNA 抑制含其同源序列含其同源序列基因的表达

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2006年的诺贝尔生理学奖获得者：Andrew Z. FireAndrew Z. FireCraig C. MelloCraig C. MelloAndrew Z. FireAndrew Z. FireCraig C. MelloCraig C. Mello

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Fig 2. Analysis of RNA-interference effects Fig 2. Analysis of RNA-interference effects in individual cells.in individual cells. Fluorescence micrographs show progeny of injected animals from GFP-GFP-reporter strain PD4251 (a C. elegans strain reporter strain PD4251 (a C. elegans strain expressing GFP fluorescence protein) (expressing GFP fluorescence protein) ( 使用外源导使用外源导入的报告基因入的报告基因 )).

Young larva ( 幼虫 )

Adult ( 成虫 )

adult body wall at high magnification ( 高放大倍数的成虫体壁 )

Control dsRNA ds-gfp RNA

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The discovery of RNAi explains the virus-The discovery of RNAi explains the virus-induced gene silencing in plants (induced gene silencing in plants ( 植物病植物病毒引起的基因沉默毒引起的基因沉默 ).).

Most plant viruses have single-stranded RNA genomes, which are released from the protein coat of their virus particles as they enter a cell.Their genomic RNA is then replicated by the virus encoded RNA-dependent RNA polymerase to produce sense and antisense RNA, which can hybridize to form dsRNA and trigger an RNAi response against their own sequences.

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2. Short interfering RNA (siRNAs) are produced from dsRNA and direct machinery that switch off genes in various way. [Mechanism- 机制 ]

从双链 RNA 产生的小干扰 RNA 可以指导用不同机制关闭基因的细胞机器

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Terms1. RNAi: RNA interference. Degradation of mRNA molecules

specified by complementary double-stranded RNA 2. dsRNA: double-stranded RNA. Complementary RNA

molecules which form a double helix 3. siRNA: small interfering RNA: RNA duplexes of 21-23

nucleotide pairs with 2 unpaired nucleotides at each 3’ end. Identify mRNA molecules for degradation in RNAi.

4. RISC: RNA-induced silencing complex. Including proteins and siRNAs and degrades mRNA molecules

5. PTGS: posttranscriptional gene silencing: A means of gene regulation in plants which occurs after the production of mRNA.

6. PPD: PAX and PIWI domain. Proteins, including those involved in RNAi, PTGS and quelling, that contain the above two protein domains.

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RNA Interference Post-transcriptional gene silencing

First discovered in c. elegans and plants Protective role: parasitic and viral

resistance

Mammals RNAi occurs –

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Mechanism of siRNA RNAi

2 step mechanistic model:

1. Initiation step– Generation of siRNAs

2. Effector step– Degradation of target mRNA

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Initiation StepATP

ATP

ADP + ppi

ADP + ppi

DICERDICER

KINASEKINASE

Effector Step

• siRNA bindingsiRNA binding• siRNA unwindingsiRNA unwinding• RISC activationRISC activation

Mechanism of RNAi

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Processing the dsRNA precursorsWhen dsRNAs are processed by Dicer, RNA duplexes with a length of about 21 nt are produced.RNA silencing effector complex assemblyThe siRNAs are subsequently incorporated into a multiprotein complex. This complex is known as the RNA-induced silencing complex (RISC). mRNA cleavage and repression of translation Once the RISC complex has been formed, the siRNAs in the RISC complex guide degradation that is sequence-specific, of the complementary or near complementary mRNAs. The RISC works by cleaving the mRNA in the middle of its complementary region.

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Family of RNase III enzymes Recognize and process dsRNA into siRNA Each dicer enzyme has an amino terminal helicase

domain, 2 RNase III catalytic domains, dsRNA binding domain and a PAZ domain

Dicer is thought to act as a dimer of 2 enzymes

Dicer

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The question to be addressed is The question to be addressed is “Why exogenous dsRNA can “Why exogenous dsRNA can inhibit expression of genes inhibit expression of genes homologous to that RNA?” homologous to that RNA?”

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Exogenous dsRNA外源双链 RNA

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The targets of the RNAi-directed gene silencing

1.Degradation of the target mRNA ( 引起靶标 mRNA 的降解 ),

2.Inhibition of translation of the target mRNA ( 抑制靶标 mRNA 的翻译 ),

3.Silencing the gene transcription from the target promoter ( 引起靶标启动子的转录沉默 ).

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The heart of the RNAi mechanism1.1. Dicer:Dicer: an RNaseIII-like multidomain

ribonuclease that first processes input dsRNA into small fragments called short interfering RNAs (siRNAs) or microRNAs (miRNA). Dicer then helps load its small RNA products into RISC.

2.2. RISCRISC (RNA induced silencing (RNA induced silencing complexes) complexes) (RNA 诱导的沉默复合体 ): a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.

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Dicer:Dicer:

Structural organization:---A PAZ domain, binds the end of the dsRNA---Two RNase III domains---Other non-conserved domains.

贾第鞭毛虫

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The crystal structure of the Giardia intact Dicer enzyme shows that the PAZ domainthe PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic RNase III domainstwo catalytic RNase III domains by a flat, positively charged surface.

The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.

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RISC: RISC: the key component is Argonaute (AGO)the key component is Argonaute (AGO)

Argonaute (AGO)Argonaute (AGO): A large protein family that constitutes key components of RISCs. ---AGO proteins are characterized by two unique two unique domains, PAZ and PIWI,domains, PAZ and PIWI, whose functions are not fully understood. Current evidence suggests that the PAZPAZdomain binds the 3’-end two-nucleotide overhangs of the siRNA duplex, whereas the PIWI PIWI domain of some AGO proteins confers slicer activity. PAZ and PIWIdomains are both essential to guide the interaction between the siRNA and the target mRNA for cleavage or translational repression. ---Distinct AGO members have distinct functionsDistinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave themRNA target, whereas AGO1 and AGO3 do not.

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A model for siRNA-guidedmRNA cleavage by Argonaute

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The multiple functions of RNAi

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Topic 3: miRNA biogenesis Topic 3: miRNA biogenesis and regulationand regulation

Topic 3: miRNA biogenesis Topic 3: miRNA biogenesis and regulationand regulation

三、miRNA生成和调控三、miRNA生成和调控

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1. MicroRNA (miRNA) & its processing

微小 RNA 及其加工

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MicroRNA (miRNA):MicroRNA (miRNA): A type of non-coding small RNA (~21–23 nucleotides) produced by Dicer from a stem-loop structured RNA precursor (~70-90 nts ong) ( 结构和来源 ). miRNAs are widely expressed in animal and plant cells and functions in the form of RNA–protein complexes, termed miRISCs. miRNAs have been implicated in the control of development because they lead to the destruction or translational suppression of target mRNAs with homology to the miRNA ( 生物学功能和机制 ).

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The miRNA genes and Structure The miRNA genes and Structure of pri-miRNAsof pri-miRNAs

Pri-miRNAs bear the 5’ cap and 3’ poly(A) tails

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miRNA processingmiRNA processing

Pri-miRNA(miRNA 初级转录产物 )

DroshaDrosha(1)(1)

pre-miRNA(miRNA 前体 )

DicerDicer(2)(2)

miRNA

Exportin 5 (Exp5) transports pre-miRNA to the cytoplasm

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1. A typical metazoan pri-miRNApri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments.

2. The terminal loop is unessential, whereas the the flanking ssRNA segments are flanking ssRNA segments are critical for processingcritical for processing.

3. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction.

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Han et al., Cell 125, 887–901, June 2, 2006

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Human Drosha and Dicer share Human Drosha and Dicer share the same RNase III domains the same RNase III domains and dsRNA binding domain.and dsRNA binding domain.

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2. MicroRNA (miRNA) targets and regulation.

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A comparison between miRNA and siRNA

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Three strategies of miRNA and Three strategies of miRNA and target recognition (targets are target recognition (targets are locating in 3’ UTRs).locating in 3’ UTRs).

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Topic 4: miRNA roles in Topic 4: miRNA roles in development, cell development, cell

differentiation and virusdifferentiation and virus

Topic 4: miRNA roles in Topic 4: miRNA roles in development, cell development, cell

differentiation and virusdifferentiation and virus

四、MicroRNA在发育中的调控作用，及其他作用四、MicroRNA在发育中的调控作用，及其他作用

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Victor R. Ambros 秀丽线虫 C. elegans

1. miRNA in C. elegans development1. miRNA in C. elegans development

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lin-4lin-4 and and let-7let-7 miRNAs miRNAs control the developmental time of C. elegans.

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Expression of lin-4Expression of lin-4 allows C. elegans to proceed to the late developmental stage

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lin-4lin-4 binds its target mRNAsbinds its target mRNAs by imperfect base pairing.

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2. miRNAs in vertebrate development: There are a lot unknown because the the lack of efficient methods to uncover the targets of miRNAs.

2. miRNAs in vertebrate development: There are a lot unknown because the the lack of efficient methods to uncover the targets of miRNAs.

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Figure 2. Expression of Expression of miR-124amiR-124a and and miR-1miR-1 in in Zebrafish, Medaka, Mouse, and Fly.Zebrafish, Medaka, Mouse, and Fly. miR-124a is restrictedly expressed in the brain and the spinal cord in fish and mouse or to the ventral nerve cord in the fly. The expression of miR-1 is restricted to the muscles and the heart in the mouse.

青鳉斑马鱼 小鼠 果蝇

Learning the miRNA function from its expression patternLearning the miRNA function from its expression pattern

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3. miRNA controls plant phenotypes (控制植物表型特征 )

Jaw-miRNA 控制拟南芥叶形变化

(Nature, 2003)

61( Science 2004)3种 miRNA控制造血干细胞向淋巴细胞的分化过程

4. miRNA controls the differentiation of the hematopoietic stem cell (调控造血干细胞的分化 )

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5. Some viruses encode miRNAs (有些病毒编码miRNAs)

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Topic 5: miRNA in cancerTopic 5: miRNA in cancerTopic 5: miRNA in cancerTopic 5: miRNA in cancer

五、微小 RNA在癌症发生中的作用五、微小 RNA在癌症发生中的作用

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miRNAs in human:miRNAs in human: There are >1000 miRNAs from human have been found and annotated. They are named as has-miRhas-miRx.

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miRNA expression pattern changes during oncogenesis, and is unique for each cancer.微小 RNA在癌症发生中表达谱的变化

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Figure 3, Comparison between normal and tumor samples reveals global changes in miRNA expression.

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One mechanism of miRNA controlling oncogene expression 微小 RNA调控癌基因表达的一种机制。

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1.1. c-Mycc-Myc is a helix–loop–helix leucine zipper transcription factor that regulates an estimated 10–15% of genes in the human and Drosophila genomes.

2.2. c-Mycc-Myc activates expression of a cluster of six miRNAs on human chromosome 13. (Figure 1)

3.3. E2F1E2F1 is the transcription factor, which is a target of c-Myc that promotes cell cycle progression.

4.4. Expression of E2F1 is negatively Expression of E2F1 is negatively regulated by two miRNAs in this regulated by two miRNAs in this cluster, miR-17-5p and miR-20a.cluster, miR-17-5p and miR-20a. (Figure 1)

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Used 2’-O-methyl Antisense oligonucleotides to downregulate the level of miR-17-5p and miR-20a, and then analyzed the protein (B-Western) and mRNA levels (C-Northen) of E2F1.

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Some microRNAs are potential oncogenes 有些微小 RNA可能是致癌基因。

74B-细胞淋巴瘤

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Figure 1. The mir-17–92 cluster shows increased Figure 1. The mir-17–92 cluster shows increased expression in B-cell lymphoma samples and cell expression in B-cell lymphoma samples and cell lines.lines. The level of mir-17–92 pri-miRNA was determined by real-time quantitative RT-PCR in 46 lymphomas and 47 colorectal carcinomas, and compared to levels found in corresponding normal tissues from five individuals.

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Figure 2. Overexpression of the mir-17–19b Figure 2. Overexpression of the mir-17–19b cluster accelerates c-myc-induced cluster accelerates c-myc-induced lymphomagenesis in mice.lymphomagenesis in mice.

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Topic 6: siRNA applicationTopic 6: siRNA applicationTopic 6: siRNA applicationTopic 6: siRNA application

六、 siRNA的应用六、 siRNA的应用

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5’5’

3’3’ 5’5’

3’3’

siRNA

• Around 21 base pairs long• Have 5’ phosphates and 3’ hydroxyl groups and 2 to 3 nucleotide overhands on the 3’ end• All that helps with binding to RISC• siRNA is unwound by RISC activity and antisense strand is left to bind to target mRNA

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Tools Needed to Effectively Study RNAi

•In Vitro Approach (Dicer)

•Ready-to-use synthetic siRNA

•Pol III generated shRNA (Vectors)

siRNAPreparation

Sequence Design

•Long dsRNA

•siRNA oligos

•dsDNA oligos

Assays, Analysis,

and Verification•Reporters

•Cell Based Assays

•Antibodies

•LUX™ Primers

•Transfection (Transient)

•Viral Delivery (Transient or Stable)

Delivery

siRNA/shRNA generation

Transfect exogenous siRNA into cellsTransfect exogenous siRNA into cells (transient expression)

Chemical synthesis In vitro transcription of shRNA with T7 promoter. In vitro transcription of long dsRNA by that are then

cleaved by E. coli RNase III or RNase III-like DICER.Expression of shRNA in cultured cells or in animal modelsExpression of shRNA in cultured cells or in animal models shRNA produced with pol III promoter from the

transfected DNA plasmids or viral vector

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Plasmid-based vectors Transient nature Low and variable transfection efficiency

Viral-based vectors Highly efficient Better than plasmid-based

Pol II promoter-based plasmid vectors Tissue specific

siRNA/shRNA generation

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Gou DM, Zhang HH, Baviskar PS, and Liu L. Primer-Extension Based Method for the generation of siRNA/miRNA expression vector. Physiol. Genomics 2007 14; 31(3):554-62

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1. Transcription from RNAP III promoters of U6 and H1 are well characterized. RNAP III transcription uses a well-defined termination signal (TTTTT) and the products have no extra sequence.

2. Transcription from these promoters is very efficient in various tissues.

Expression of hairpin RNA (shRNA) using Pol III promoters

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A mammalian expression vector designed to direct the intracellular synthesis of siRNAs.

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siRNA Delivery in vitro

Chemical transfection (Lipofectamine, Oligofectamine, TransIT-TKO, Siport Amine, Siport

Electroporation into parasites in vivo

Intramuscular injection Hydrodynamic transfection into mammals

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Research Applications of RNAi: A new strategy of reverse genetics & a novel way of gene knock-out

It can be used in reverse genetics ( 反向遗传学 ) to identify the cellular or biological function of a gene.

It can be combined with genomics to perform large-scale genetic screens aimed at gene discovery.

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Therapeutic Applications of RNAi:A new strategy to invitation of new drugs and gene therapy

siRNAs can be used to counter viral infection by specifically destroying the mRNAs of the pathogenic viruses, such as HIV and HBV.

siRNAs can be applied to counter cancers by specifically down-regulate the expression of genes related to oncogenesis.