Overview of presentation

(1)

RNAi在研究實務上的 整合應用技術

岑祥股份有限公司

技術專員費軫尹

20100803

(2)

Overview of presentation

Basic Biology of RNA interference

Application of siRNA for gene function ?

How to study miRNA？

How to deliver siRNA and miRNA?

New prospects on RNAi research

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RNAi: A Historical Perspective

Flies: RNAi 1998

Kennerdell et al. 1998. Cell 95:1017 Hammond et al. 2000. Nature 404:293

Zamore et al. 2000. Cell 101:25

Mammals: RNAi 2001

Elbashir et al. (2001) Nature 411:494

Fungi: quelling 1992

Romano (1992) Mol Micro 6:3343

Plants: co-suppression 1990

Napoli et al. 1990. Plant Cell 2:279

Worms: RNAi 1998

Fire et al. (1998) Nature 391:806

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Discovery of microRNA

1993

2000 2001

2002 2003

2004 2005

2006

2007

(5)

Mechanism of RNAi

1. miRNA gene is transcribed into pri-miRNA.

2. Pri-miRNA is processed into hairpin pre-miRNA.

3. Pre-miRNAs are

transported to cytoplasm.

4. Pre-miRNAs are processed into short, mature miRNA duplexes.

5. Mature miRNAs complex with RISC-like structure.

6. miRNA/RISC complex binds to target mRNA.

7. mRNA is translationally repressed or cleaved.

(6)

siRNA -- A tool to elucidate gene

function

(7)

only ~20% of randomly selected siRNAs knock-down target with efficiency 90% and higher

0 20 40 60 80 100 120

Activity of Randomly Selected siRNAs

(8)

siRNA Properties Correlate with Functionality

Stability at terminus of each strand

Overall G/C content of siRNA molecule

Base preferences at specific positions

Nature Biotechnology (2004) 22:326-330

0 10 20 30 40 50 60 70 80 90 100

0 1 2 3 4 5 6 7 8 9 10

8 compound algorithm score

Silencing efficiency

Cell (2003) 115: 209-216

8 parameter algorithm score

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Naturally Generated siRNA Pools in Model Organisms

Long dsRNA

siRNA Pool Dicer

Adapted from Sioud (2006) Nature Biotechnology, 24: 521-522.

Long dsRNA

Mammalian cell

Different activities Different specificities Different length

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Synthetic siRNA Pools Ensure Robust Silencing

Target mRNA AAAAA

Target sequences avoid

–known SNPs

–highly redundant sequences –highly homologous sequences

Controlled Length- 21mers Highly functional

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Optimal design results in uniformly high functionality

Validation of SMARTpool

^™

Technology

0 20 40 60 80 100 120

random individual siRNAs

Collaboration with Joan Brugge’s laboratory, Harvard Medical School

Bioinformatics Chemical Modifications

Pooling

(>300 kinase targets)

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Functional siRNAs Induce Off-target Effects

Adapted from Jackson et al., (2003) Nature Biotechnology, 21: 635-638.

on-target activity off-target

signature

Functionality ≠ Specificity

0.2 0.4 0.6 0.8

0 1.0

Fraction MAPK14 remaining

protein RNA

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miRNA Gene Targeting

Nature (2005) 433:769

mRNA down-regulated

miRNA can down-regulate multiple gene targets

miRNA

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Functional siRNAs Induce Off-target Effects

Adapted from Jackson et al., (2003) Nature Biotechnology, 21: 635-638.

Functionality ≠ Specificity

AUG UAA AAAAAAA

cleavage

AUG UAA AAAAAAA

“seed” binding

0.2 0.4 0.6 0.8

0 1.0

Fraction MAPK14 remaining

protein RNA

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Seed Region is Responsible for Off-target Effects

RNA (2006) 21:635

P-value = 2.82 x 10^-16<<<0.05 (using χ²test of independence)

Number of Genes with 3' UTR Hexamer Matches

0 20 40 60 80 100

0 1 2 More

Number of Matches per Gene

Number of Genesnumber of genes

Number of genes with 3’UTR matches

number of matches per gene

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RISC Associated Helicase Activity

Conventional siRNA RISC Process

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Modification Applied to ON-TARGETplus™ siRNA Reagents

Chemical modifications to both the sense and antisense strands of the siRNA duplex:

Sense strand is deterred from entering RISC

Antisense strand requires more rigorous complementarity with target mRNA

Jackson AL et al. (2006) RNA 12: 1197-1205.

patent pending

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Chemical Modifications Eliminate Sense Strand-Mediated RNAi and Enhance Antisense Strand Specificity

patent pending

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Off-target Phenotype is Independent of Target Silencing

0 20 40 60 80 100 120 140

MAP2K1-1 MAP2K1-2 MAP2K1-3 MAP2K1-4 MAP2K2-1 MAP2K2-2 MAP2K2-3 MAP2K2-4 Lipid Untreated Lipid (MAP2K1) Untreated (MAP2K1) Lipid (MAP2K2) Untreated (MAP2K2)

Cell survival (%) __Target/GAPDH mRNA ratio (%)

- cell survival - target knockdown

Adapted from Fedorov et al., (2006) RNA, 12:1188

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Off-target Phenotype is Eliminated in the Context of siRNA Pools

0 20 40 60 80 100 120 140

MAP2K1-1 MAP2K1-2 MAP2K1-3 MAP2K1-4 MAP2K2-1 MAP2K2-2 MAP2K2-3 MAP2K2-4 Lipid Untreated Lipid (MAP2K1) Untreated (MAP2K1) Lipid (MAP2K2) Untreated (MAP2K2)

Cell survival (%) __Target/GAPDH mRNA ratio (%)

- cell survival - target knockdown

MAP2K1 pool MAP2K2 pool

Adapted from Fedorov et al., (2006) RNA, 12:1188

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ON-TARGETplus

^™SMARTpool^®

Reagents Provide Maximal Specificity

Collaboration with Agilent Technologies

(22)

How to study miRNA?

Functional Assay

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miRIDIAN™ MicroRNA Mimics and Inhibitors:

What Are The Biological Outcomes?

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Product Details: miRIDIAN microRNA Mimic

Double-stranded synthetic RNA oligonucleotide

• Intended to mimic function of endogenous miRNA , chemically modified

Chemically modified to:

• Enter miRNA pathway with active strand

• Exclude passenger strand from loading

• Minimize interferon response

• Improve target binding specificity & efficiency

miRIDIAN Mimic designs now updated to Sanger miRBase 13.1

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Applications of miRIDIAN microRNA Mimics

Supplement miRNA activity to study gain-of-function effects

Screen for miRNAs that regulate gene expression and affect cellular pathways

Elucidate miRNA involvement in normal biological and disease pathways

Identify and validate miRNA targets

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miRIDIAN microRNA Mimic Positive Controls

Endogenous miRNA positive control

miR-122 Aldolase A

• Validated miRIDIAN microRNA Mimic that targets Aldolase A in human, mouse, and rat

• Provides the ability to optimize assay conditions by monitoring mimic function on an endogenous gene target (Aldolase A) with a conserved miR-122 binding site

Many cells lines express low to moderate levels of miR-122. Aldolase A is a predicted target of miR-122 and the 3' UTR is conserved in human, mouse and rat at the 8-mer miR-122 predicted seed site.

miRIDIAN microRNA Mimics designed to modulate endogenous miR- 122 was transfected at 50 nM (Huh-7 at 40 nM) using DharmaFECT 1 into the indicated cell lines and assessed for their ability to decrease AldoA mRNA levels. AldoA downregulation was determined at 3 days (HepG2 at 5 days) post-transfection.

(幾乎多數細胞都有表現的內 源性miRNA)

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Product Details: miRIDIAN microRNA Hairpin Inhibitors

Most effective inhibition of endogenous mature microRNA function by means of proprietary, innovative design

Patent-pending molecule combines chemical modifications and completely novel secondary structure motif

Superior potency and longevity in comparison to any other synthetic product offered commercially

Enhanced potency and longevity allows for multiplexed microRNA inhibition at very low nanomolar concentrations with minimal toxicity

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Applications of miRIDIAN microRNA HP Inhibitors

Suppress miRNA activity to study loss-of-function effects

Screen for miRNAs that regulate gene expression and affect cellular processes

Elucidate miRNA involvement in normal biological and disease pathways

Identify and validate miRNA targets

(29)

miRIDIAN microRNA HP Inhibitor Positive Controls

Targets miR-16 in human, mouse, and rat cells resulting in reduced miR-16 activity

(30)

Delivery of siRNA or miRNA &

experimental tips

(31)

Transfection: Definition

A method for delivering DNA or RNA into mammalian cells, using a

complexing reagent such as cationic lipids.

AAAAAAA

target

detect outcome deliver

silence

(32)

SMART tip : barrier tip ≠ filter tip

(33)

Transfection and Viability

Broad spectrum of cell types

Efficient delivery

Low toxicity

Robust silencing

DNA

Efficient siRNA Delivery with Minimal Effect on Cell Viability

siGLO Green

(34)

Applications of gene profiling

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35

Experimental design of Cytokine Protein Arrays

Cells treated with drugs/siRNA/miRNA of gene-of-interest or not

Collect supernatant or cell lysate Profile protein expression by Protein Array

Data analysis

Discover pathway-of-interest or biomarker

RNAi in Protein screening

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How to profiling gene expression by RNAi?

Antibody Array

• Semi-quantitative

• Quantitative

(37)

How to analyze?

(38)

Data presentation

Nature Vol 441|22 June 2006

Nature Medicine,Oct 2007

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39

Quantibody Array – Multiple ELISA platform

Sandwich-based ELISA

Quantibody Array使用三明治 法ELISA原理：利用固定於玻片載體上的專一 capture antibodies，依序加入待測檢 體、detection antibodies、和螢光標定物，最後以Laser Scanner的cy3波長判讀分析。

Alexa Fluor Dye-Streptavidin

Detection Antibody

Cytokine

Capture Antibody 10~40 cytokines for one sample

一張slide上有16個sub-array；

每個sub-array上有10到20個 cytokine以及positive control 、 negative control。每個target 都四重複，確保最佳實驗再現性。

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40

Format: Standard glass slide with 16 removable wells Sample Volume: 50-100 ul

Detection Range: 0.5-3000 pg/ml Standard Curve Range: 5-800 pg/ml Signal Duration: 6 hours

Reproducibility: <20% well-to-well CV Detection Method: Fluorescence. (Cy3)

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Data Performance

A549 cell expression levels of IL-8, IP-10 and RANTES are altered by TNFα stimulation and siRNA transfection.

(42)

Reverse phase: Profiling gene function by RNAi Library

(43)

MicroRNA expression profiling to biomarker discovery

(44)

• ELISA assay for downstream protein expression

Collect supernatant for determination of specific target protein Fix cell lysate on 96-well plate for In-Cell-ELISA

Detect phenotypes to get hints

(45)

• Staining of Specific Marker as phenotype (IHC/IF) -- cell survival or proliferation

-- cell morphology

-- cytotoxicity or apoptosis -- cell migration

-- cell stress or DNA damage

Detect phenotypes to get hints (continued)

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Overview of presentation

Overview of presentation

RNAi: A Historical Perspective

Discovery of microRNA

Mechanism of RNAi

siRNA -- A tool to elucidate gene

function

Activity of Randomly Selected siRNAs

siRNA Properties Correlate with Functionality

Naturally Generated siRNA Pools in Model Organisms

Synthetic siRNA Pools Ensure Robust Silencing

Validation of SMARTpool

Technology

Functional siRNAs Induce Off-target Effects

miRNA Gene Targeting

Functional siRNAs Induce Off-target Effects

Seed Region is Responsible for Off-target Effects

RISC Associated Helicase Activity

Off-target Phenotype is Independent of Target Silencing

Off-target Phenotype is Eliminated in the Context of siRNA Pools

ON-TARGETplus

Reagents Provide Maximal Specificity

miRIDIAN™ MicroRNA Mimics and Inhibitors:

What Are The Biological Outcomes?

Product Details: miRIDIAN microRNA Mimic

Applications of miRIDIAN microRNA Mimics

miRIDIAN microRNA Mimic Positive Controls

Product Details: miRIDIAN microRNA Hairpin Inhibitors

Applications of miRIDIAN microRNA HP Inhibitors

miRIDIAN microRNA HP Inhibitor Positive Controls

Delivery of siRNA or miRNA &

experimental tips

Transfection: Definition

SMART tip : barrier tip ≠ filter tip

Transfection and Viability

Applications of gene profiling

RNAi in Protein screening

How to profiling gene expression by RNAi?

How to analyze?

Data presentation

Data Performance

Reverse phase: Profiling gene function by RNAi Library

Detect phenotypes to get hints

Detect phenotypes to get hints (continued)

Thank you for attention!