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RNAi
Technical Knock Down of Gene
Expression
RNA Silencing – RNA Interference
• a new method for silencing specific genes
• a potent method requiring only a few molecules per cell to be effective
• a systemic method spreading out through the whole organism
• to some extent hereditary and can be transmitted through the germline for
several generations
• epigenetic (transmission of phenotypes by mechanisms other than DNA
sequence changes)
Chul Geun Kim ([email protected])
Laboratory of Molecular Genetics
Hanyang University
Dogmatic View of Gene Expression
DNA
RNA
Protein
Post-transcriptional Control: Knock down
Quantitative Control:
Levels of mRNA not proportional to levels of mRNA
synthesized or protein produced
Qualitative Control:
More than one protein from a single gene
(e.g. Differential RNA Processing or RNA editing)
An “RNA-Centric” View of Gene Expression
DNA
RNA
Protein
RNA: A Diverse Class of Molecules
DNA
rRNA
tRNA
Vault
Y RNAs
7SK
Xist, H19
RNA
snRNAs
snoRNAs
Guide RNA
Introns
5’ UTR
3’ UTR
Catalytic:
Ribozymes
Telomerase
Viral RNAs
Retrotransposons
MicroRNAs
Non Coding RNAs: ‘Ribo Regulators’
(~97% of RNAs Present in Human Cells are Non-Coding)
Non Coding RNAs:
SnoRNAs
• Large Family
• Intron-encoded
• Guide RNA Modification
Telomerase RNA
• Component of telomerase
• Provides template for
telomere synthesis
• Role in Cancer and Aging
Kill the messenger!
mRNA:
cap
AAAA...
in vitro transcribe an antisense RNA:
cap
AAAA...
mRNA can no longer be translated into a protein
•
•
•
Antisense technology was used in worms...
Difficult to explain: sense and antisense RNA preparations are each
sufficient to cause interference.
Perhaps, the interfering RNA populations include some molecules with
double-stranded character.
Synthetic Antisense Oligos
Procedure of Morphoino Synthesis
Antisense Strategy
Direct injection or ingestion by cell
Transfection of
expression vector
Translation
Block
dsRNA-specific
RNase
RNA
interference
Transcription
Historically Important Discoveries
1990
cosuppression of purple color in plants
1998
dsRNA injection in worms
1999
short RNAs
identified in
plants
2000
RISC activity partially purified
2001
2002
siRNAs identified
RNAi shown
in vitro
Dicer identified
RNAi used against HIV
genome-wide RNAi
screens begin
RNA interference – The Beginning
- Fire et al.: "Potent and specific genetic interference by double-stranded RNA
in Caenorhabditis elegans " Nature 391: 806-11 (1998)
- Introduction of RNA into cells to interfere with function of an endogeneous gene
- Investigation of the requirements for structure and delivery of interference RNA
mex-3 RNA
Double-stranded RNA
control: not stained
wt
sense
antisense
inject
wt + antisense RNA
wt + ds RNA
C. elegans
• ds mixture causes potent and specific interference
• ds RNA substancially more effective than antisence
• effect were evident in both the injected animals and their progeny
RNAi in Mammalian Cells
• Long dsRNA triggers global (non-specific) gene-silencing (i.e. interferon response)
• Breakthrough: Short dsRNA (~22 nt) induces RNAi
Silencing of lamin proteins in human cells by dsRNA transfection
Nature 2001 411: 494-498
Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs
dsRNA
processing
~22nt siRNAs
amplification
target
mRNA
degradation
spreading
recognition
copying
+
processing
secondary
siRNAs
RNA Interference (RNAi)
•
•
Double stranded RNA is responsible for post-transcriptional gene silencing of the gene
from which it was derived. SPECIFIC
NATURAL BIOLOGICAL MECHANISM IN PLANTS, INSECTS AND MAMMALS
- RNA interference (RNAi) represents an evolutionary conserved cellular defense mechanism for controlling the expression
of alien genes in filamentous fungi, plants, and animals.
- dsRNA is often a byproduct of viral replication or is formed by aberrant transcription from genetic elements after random
integration in the host genome.
•
RNAi FUNCTIONS
–
–
–
–
regulates expression of protein coding genes (e.g. microRNA)
mediates resistance to both exogenous parasitic and exogenous pathogenic nucleic acid
response to aberrant RNAs
used experimentally to block gene expression
RNA interference – Mechanism
DICER
- RNAse III, ds-specific endonuclease
- Dimer, 2 catalytic domains, helicase and PAZ motif
- produce 2-3 nt 3´ overhangs
- ATP-dependent ribonuclease
RISC
- RNA-induced silencing complex
- RISC contains siRNA
- precurser activated by ATP
- find and destroy mRNA of complementary sequence
- contains endo- and exonuclease, cleaves the hybrid in
the middle followed by degradation
- ARO: PAZ domain (assembly)
RNA-Mediated Gene Silencing
- RNA Interference
- ‘Cosuppression’ by transgenes in plants
- ‘Quelling’ in Fungi
- Transcriptional Gene Silencing (TGS)
Common Trigger:
RNA-Mediated Gene Silencing
Science 2002 296:1263-1265
PTGS - Post-Transcriptional Gene Silencing
MicroRNAs (also known as Small-temporal RNAs)
- let7 and lin4 (from worm) were first examples; expanding family of ‘RiboRegulators’
- negative regulator of genes
- 70 nt precursor, processed by DICER, results not in dsRNA
- bind target and prevent ribosomal elongation
RNAi by siRNAs
~22nt
siRNAs
Developmental regulation
by stRNAs (µ RNAs)
processing
~22nt
lin-4
target
recognition
mRNA
lin-14
mRNA
lin-41
mRNA
processing
~22nt
let-7
target
recognition
3’UTR
3’UTR
degradation
Translational repression
TGS - Transcriptional Gene Silencing
plant:
- methylation in promotor regions leads to gene silencing
- MET as a part of RISC
C.elegance:
- polycomb-dependent mechanism
- polycomb proteins ass. with RISC
- chromatin remodeling: open – close transition
How does the RNAi machinery aid
in the formation of silent chromatin?
•
Possibility that siRNAs bring methyltransferases to
the target loci, where they are important in histone tail
modification
– i.e. Drosophila targets acteyltransferase with
RNA binding chromodomain to histone H4
siRNA and Silent Chromatin - Model
- RNA homologous to centromeric repeats are processed:
siRNAs
- siRNAs may recruit Clr4 histone H3 methylase
- result in methylation of H3 Lys9
- Swi6 binds chromatin
- Gene silencing
Related Gene Silencing Mechanisms May Function in Mammals
•
•
•
X chromosome inactivation in mammals
– Xist RNA coating of inactive X chromosome, but no data yet suggests that
Xist is processed by RNAi machinery
– Mouse – X inactivation and Igf2r imprinting are mediated by noncoding
antisense RNA
Possibly in organisms with DNA methylation; Histone protein modification similar to
S. pombe would in turn cause DNA methylation and subsequent gene silencing
regulation
Future work using RNAi introduced in experiments should include study of
chromatin structure or modifications at the locus of the affected gene
Science 297:1833-1837
Science 297:2215-2218
RNA interference - Mechanism
RNAi Applications
GENETIC TOOL
Probing Gene Function
GENE THERAPY
Combat Viral Infection
Treat Genetic Diseases (New expression strategies)
RNAi – Advantages
- dsRNA is the interfering agent (stability)
- it is highly specific
- it is remarkably potent (only a few dsRNA molecules per cell are required
for effective interference)
- the interfering activity can cause interference in cells and tissues far removed
from the site of introduction
RNAi for analysis of gene function and as therapeutic
- duplexes of 21-nt small interfering RNAs (siRNAs)
- guide sequence-specific degradation of the homologous mRNA
- degradation of targeted mRNAs, "knock-down"
- targeting of essential genes causes growth arrest or triggers apoptosis
siRNAs can be produced by:
• Chemical synthesis
• Enzymatic synthesis
• RNase III/Dicer cleavage of long dsRNA
• Plasmid based in vivo expression
• siRNA Expression Cassettes (SECs)
siRNA Design
siRNA has UU 3′ termini; target must start with AA:
1. Scan mRNA for AA dinucleotide sequences.
2. Record the occurrence of each AA and the 3′ adjacent 19 nucleotides.
3. G/C content < 50% is preferable.
4. BLAST search candidates, eliminating those with significant homology to other coding
sequences. http://www.ambion.com/techlib/misc/siRNA_finder.html
Method #1: Custom siRNA Synthesis
• Commercial synthesis of siRNA (~1-2 week turn around; getting quicker)
• Expensive, but little to no hands-on time
• Must screen siRNAs to identify an effective one
• Synthesis can easily be scaled up
• siRNAs can be labeled
Duration Results: mRNA Expression
Northern of GAPDH
Method #2: In vitro Transcription
• In vitro transcribe sense and antisense RNA strands from dsDNA template (hybridized
DNA oligonucleotides); hybridize RNA strands to create siRNAs, clean up
• Inexpensive – a fraction of the cost of chemical synthesis
• Fast turn around – synthesize and have ready for transfection in one day
• Just as effective as chemically synthesized siRNAs, and can be used at lower concentration
• Must screen siRNAs to identify an effective one
• siRNAs can be labeled
• Obtain 2 desalted DNA oligonucleotides (with 8
bases complementary to T7 promoter primer)
• Anneal oligonucleotides to T7 promoter primer
• Fill-in reaction with Klenow
• Transcribe with T7 RNA polymerase
• Hybridize, digest and clean up
Method #3: RNase III/Dicer Digestion
• Cocktail of several siRNAs generated by RNase III/Dicer digestion of long dsRNA
• RNase III/Dicer Cocktails effectively induce RNAi in mammalian systems
• RNase III/Dicer cleaves dsRNA into 12–30 bp dsRNA fragments with 2 to 3 nucleotide
3' overhangs, and 5' phosphate and 3' hydroxyl termini.
• No need to screen for effective siRNA
• Cocktail of siRNAs provides better chance for strong RNAi effect on first try; typically no
problems with nonspecific effects
• Can label siRNA cocktail
• Does not identify which siRNA sequence is effective
siRNA Cocktails Made with RNase III
- Complementary RNA strands (100-500 nt) transcribed from dsDNA template and
then hybridized to form long dsRNA.
- DNase & RNase used to remove DNA template and unhybridized RNA strands.
- RNase III digests dsRNA into population of 12-15mer dsRNAs that are functional
as siRNAs.
- Clean up reaction (removes long dsRNA) to ready it for transfection.
RNase III Generated siRNA Cocktails
Effect of GAPDH Cocktail on Other Genes
GENES EXAMINED
Method #4: siRNA Expression Vectors
• Plasmids encoding siRNA sequences for expression in vivo by Pol III promoters (U6, H1)
• Just as effective as chemically synthesized and in vitro transcribed siRNAs; same target
sequences can be used
• uses standard cloning techniques, eliminating need to synthesize or work with RNA
• Can be used for transient expression or for transient selection and longer duration
silencing when selectable markers are included
• Long term gene silencing:
- analysis of loss-of-function phenotypes within cell lines
- potential for gene therapy
Example of Expression Vector
Selectable Plasmids targeting GFP
Use of Plasmid Expression Vectors
Step 1: Identify siRNA target sequence and design siRNA encoding DNA insert
Step 2: Clone insert into siRNA Expression Vector
Step 3: Select for clones containing insert, test, grow up
Step 4: Transfect expression vector carrying insert into cells
Step 5: Assay directly for RNAi after 18-48 hrs
or
select for transient expression
or
select for stable integration
Duration Studies: Long Term Reduction with Hygromycin Selectable Plasmid
Reduction of GFP after three
weeks of hygromycin selection
Method #5: siRNA Expression
Cassettes (SECs)
• Rapid, PCR-based method for preparing siRNA
expression system
• One-day turn around; avoids labor intensive
cloning used with standard expression vector
systems
• Allows quick screening of siRNA target sequences
and siRNA sequence : promoter combinations
• SECs are easily inserted into receptor vectors (e.g.
with selectable markers for long term duration
studies)
• A precursor SEC generated by PCR using two gene specific DNA oligo
nucleotides and primers provided in the kit (using a one-step or two-step
PCR approach). The precursor SEC comprises an RNA pol III promoter and
adjacent hairpin siRNA template.
• The precursor SEC is used in a large scale PCR to generate the final SEC.
• The SEC is column purified to remove primers, dNTPs, enzyme and salts to
ensure efficient transfection
Cautions!!!!!!
Transfecting Plasmids, SECS, siRNAs
• Transfection is critical to success of RNAi expt; many variables affect efficiency
• Efficiency of transfection agents can vary dramatically
• Most are optimized for delivery of plasmid DNA, not PCR products or RNA; those
optimized for RNA are for mRNA delivery and don’t perform well for siRNA
• Key is to use agent optimized for plasmid, PCR or siRNA delivery – transfection
efficiency must be high enough to be able to measure silencing
Plasmid & PCR Transfections
• Protocols and reagents for plasmid transfection are numerous; efficiency is highly
variable depending on cell line transfected (0-90%)
• Conditions for plasmids are different than those for PCR products (SECs)
Optimized siRNA Transfections
Two reagents optimized for siRNA delivery:
• siPORT Amine - polyamine mixture
• siPORT Lipid - mixture of cationic & neutral lipids
Silencer™ siRNA Transfection Kit
• Contains two agents optimized for siRNA delivery
• Includes well characterized GAPDH synthetic siRNA and
a scrambled GAPDH negative control
GAPDH
Scrambled
Labeled siRNAs: Applications
• Analyze ability of siRNA to attenuate target gene expression
• Determine transfection efficiency
• Track siRNA migration within a cell
• Study siRNA metabolism
• Study siRNA in living cells in real time
GAPDH siRNA in HeLa S3 Cells
siRNA = Red
Nucleus = Blue
Protein = Green
Establishment of strategies for the conditional expression of shRNA
Tet-on inducible RNAi expression
Tet
TetR
TetO
wtTBP
RNAi vector
mutTBP
pTRE/mutTBP
mutTBP
mutTATA box
Human/mouse U6 promoter
RNAi
TetR : Tet repressor
TetO : Tet operator
mutTBP : mutant TBP
wtTBP : wild type TBP
Tet
TetR
H1 promoter TO
RNAi
EMBO Rep. 2003 Jun;4(6):609-15.
Genetic Network for the Maintenance of Pluripotency in ES Cells;
Application of pSTAT3ER/4-HT system
pRNAi
pSTAT3ER
ESC
ESC/pSTAT3ER/
pRNAi
ESC/pSTAT3ER
+ LIF
+ 4-HT
+/- LIF
Synchronized
differentiation
- LIF
- 4-HT
About siRNA
RNAi (RNA interference) is a phenomenon that small double-stranded RNA (Referred as small
interference RNA or siRNA) can knock down the expression of its corresponding gene. RNAi has been
observed in plant, C.elegans and Drosophila long time ago. It was until recently that RNAi was
discovered to work in mammalian system [1].
Small interference RNA (siRNA) is 19-22 nt double-stranded RNA. It works by cleaving and destroying
its cognate RNA. siRNA first assembles into RNA-induced silencing complexes (RISCs), and it then
activates the complex by unwinding its RNA strands. The unwound RNA strands subsequently guide the
complex to the complementary RNA molecules, where the complex cleaves and destroys the cognate
RNA, which results in RNAi phenomenon.
RNAi has evolved into a powerful tool to study gene functions. Here are some of its applications:
1. A stable cell line with a specific gene knocked-out can be established, and its phenotype can be studied.
2. A knock-out mouse line can be established using trangenic siRNA method [8].
3. siRNA can be put into a vector with an inducible promoter to study its effect.
4. siRNA can be delivered by using viral vector [6,7] and used for gene therapy purpose.
5. siRNA can be mimicked by chemical molecule and used for drug development.
DNA vector-based siRNA technology
siRNA can be obtained by chemical synthesis or by DNA-vector based RNAi technology.
Using DNA vector based siRNA technology, a small DNA insert (about 70 bp) encoding a
short hairpin RNA targeting the gene of interest is cloned into a commercially available vector.
The insert-containing vector can be transfected into the cell, and it expresses the short hairpin
RNA. The hairpin RNA is rapidly processed by the cellular machinery into 19-22 nt double
stranded RNA (siRNA).
The following is a list of GenScript siRNA expression vectors:
1. U6 like promoter:
pRNA-U6.1/Neo
pRNA-U6.1/Hygro
H1 like promoter
pRNA-H1.1/Neo
pRNA-H1.1/Hygro
Advantages of DNA vector-based siRNA technology
Comparing to chemically synthesized siRNA, DNA-vector based technology has a lot of
advantages:
1.Vector based siRNA is more effective than synthetic siRNA for inhibition of gene expression [2].
2.Very stable and easy to handle:
Synthetic siRNA is not stable, which has to be protected during shipping and de-protected before use. Unlike
synthetic siRNA, vector based siRNA is the same as DNA, and it is very stable and can be easily tranfected into
cell using routine DNA transfection reagents, such as Lipofectamine.
3.Stable cell line can be established: vector based siRNA allows you to obtain a stable cell line, and observe
long-term effects of RNAi.
4.Inducible system can be established: vector based siRNA allows you to establish an inducible system by using
a vector with an inducible promoter.
5.A knock-out mouse line can be established using trangenic siRNA method [8].
6.Unlimited supply: once a DNA construct is made, you will have unlimited supply of siRNA.
7.Cost-effective: synthetic siRNA has to be re-ordered once it is used up whereas vector-based siRNA only need
to be ordered one time.
One big obstacle for vector-based siRNA technology is that it takes a lot of time and trouble to make the DNA
constructs. GenScript can provide you custom siRNA construct! Follow this link to Learn more about how
Genscript siRNA technology works for you.
siRNA Target Site Selection
We have developed some software tools to facilitate your design process. It is recommended that at
least 3 vector-based siRNA should be prepared for each gene in order to find a potent and specific
siRNA. Here are the reasons:
1.Not all siRNA target sequences are equally potent: Because of secondary structure and other factors, some target
sequences are more potent than others. It is better to test at least three vector-siRNA constructs to find the most potent
one.
2.Not all siRNA silencing effects are gene-specific: It has been reported that some siRNA silencing effects are not
gene-specific because of various of reasons. It is better to validate your experiments results using three vector-based
siRNA constructs.
3.Results from synthetic siRNA or siRNA cassette cannot be completely transferred to vector-based siRNA construct:
Vector-based siRNA is different from synthetic siRNA oligos or siRNA cassette. Although the results from synthetic
siRNA oligos or siRNA cassette can suggest the most potent siRNA targets, the results cannot be completely duplicated
in vector-based siRNA for unknown reasons.
4.The experiment is still the gold test stone: Although we are pride of our vector-based siRNA design program, the best
design is still not as good as what the experiments can tell you.
References
1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells. Nature 411: 494-498.
2. Yu JY, DeRuiter SL, Turner DL. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in
mammalian cells. Proc Natl Acad Sci U S A. 99(9):6047-6052.
3. Brummelkamp, T.R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in
mammalian cells. Science 296: 550-553.
4. Jacque, J.-M., Triques, K., and Stevenson, M. (2002) Modulation of HIV-1 replication by RNA interference. Nature 418: 435438.
5. Sui, G., Soohoo, C., Affar, E.B., Gay, F., Shi, Y., Forrester, W.C., and Shi, Y. (2002) A DNA vector-based RNAi technology to
suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(8): 5515-5520.
6. Shen C, Buck AK, Liu X, Winkler M, and Reske SN. (2003) Gene silencing by adenovirus-delivered siRNA. FEBS Lett 539(13):111-114.
7. Barton GM, and Medzhitov R. (2002) Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A
99(23):14943-14945.
8.Kunach T, Gish G, Lickert H, Jones N, Pawson T, and Rossant J. (2003) Transgenic RNA interference in ES cell-derived
embryos recapitulates a genetic null phenotype. Nature Biotechnology 21:559-561.