2. Introduction
• Small interfering RNA (siRNA), sometimes known as short interfering
RNA or silencing RNA.
• It is a class of double-stranded RNA molecules, 20-25 base pairs in length
with 2-nt at 3’ overhangs.
• siRNA plays many roles, but its most notable is in the RNA
interference (RNAi) pathway, where it interferes with the expression of
specific genes with complementary nucleotide sequence.
• They are naturally produced as part of the RNA interference (RNAi)
pathway by the enzyme Dicer.
• They can also be exogenously (artificially) introduced by investigators to
bring about the knockdown of a particular gene.
3. What Is RNA Interference (RNAi)?
• RNA interference (RNAi) is a biological process in
which
RNA
molecules
inhibit
gene
expression, typically by causing the destruction of
specific mRNA molecules.
• Historically,
it
was
known
by
other
names, including co-suppression, post transcriptional
gene silencing (PTGS), and quelling.
• In 2006, Andrew Fire and Craig C. Mello shared
the Nobel Prize in Physiology or Medicine for their
work on RNA interference in the nematode worm C.
elegans, which they published in 1998.
4. • RNA interference (RNAi) is a
phenomenon in which doublestranded
RNA
(dsRNA)
suppresses expression of a target
protein by stimulating the specific
degradation of the target mRNA.
• RNAi involves
process.
a
multistep
• The RNAi pathway is found in
many eukaryotes and it is initiated
by the enzyme Dicer, which
cleaves long double-stranded
RNA(dsRNA) molecules
into
short double stranded fragments
of ~20 nucleotides that are called
siRNAs.
5. • Each siRNA is unwound into two
single-stranded (ss) ssRNAs, namely
the passenger strand and the guide
strand.
• The
passenger
strand
is
degraded, and the guide strand is
incorporated into the RNA-induced
silencing complex (RISC).
• The most well-studied outcome is
post-transcriptional
gene
silencing, which occurs when the
guide strand base pairs with a
complementary sequence in a
messenger RNA molecule and
induces cleavage by Argonaute, the
catalytic component of the RISC
6. The discovery of RNAi
Fire and
Mello., Nature 391:
806
dsRNA leads to
silencing in
C.elegans
1990
1998
2000
Elbashir et
al., Nature 411: 494
siRNAs trigger
RNAi in
mammalian
cells
2001
2002
Takeshita et
al., PNAS 102:
12177
In vivo siRNA
delivery
2004
PTGS in plants
(post
transcriptional
gene silencing)
dsRNA
processed to
siRNAs in RNAi
pathway
siRNA expressed
from vectors
Napoli et
al., Plant Cell 2:
279
Zamore et al.,
Cell 101: 25
Brmmelkamp et al.,
Science 296: 550
2009
Phase I
preclinical trail
7. siRNA synthesis
• Chemical synthesis.
• In vitro transcription.
• RNase III/DICER digestion of long dsRNA.
General Guidelines
1.
2.
3.
4.
5.
6.
7.
8.
siRNA targeted sequence is usually 21 nt in length.
Avoid regions within 50-100 bp of the start codon and the termination codon.
Avoid intron regions.
Avoid stretches of 4 or more bases such as AAAA, CCCC.
Avoid regions with GC content <30% or > 60%.
Avoid repeats and low complex sequence.
Avoid single nucleotide polymorphism (SNP) sites.
Perform BLAST homology search to avoid off-target effects on other genes or
sequences.
8. Chemical synthesis of siRNA
• Commercial synthesis.
• Expensive process.
• Must screen siRNAs to identify an effective one.
• Synthesis can easily be scaled up.
• siRNAs can be labeled for identification.
9. In vitro transcription of siRNA
• In vitro transcribe sense and
antisense RNA strands from
dsDNA template; hybridized
RNA strands to create siRNAs.
• Fast turn around.
• Lower concentration.
• Must screen siRNAs to identify
an effective one.
• siRNAs can be labeled.
10. RNase III/DICER digestion
• Cocktail of several siRNAs generated by RNase III/Dicer digestion
of long dsRNA.
• Leaves same overhang characteristics.
• No need to screen for effective siRNA.
• siRNA cocktail can be labeled.
• Does not identify single effective siRNA sequence.
• Non-specific effects.
11. siRNA cocktails made with RNase III
• Complementary RNA strands
(100-500nt) transcribed from
dsDNA template and then
hybridized to long dsRNA
• DNase and RNase used to remove
DNA template and unhybridized
RNA strands
• RNase III digests dsRNA into
population of 12-15mer dsRNA
that fuction as siRNAs
• Clean up.
12. siRNA Delivery
• Chemical-mediated transfection
- adherent cell lines and some primary cell types
• Electroporation
- primary cells, suspension cells, and many difficult to transfected
cell lines
• Viral vector
- most all type of cells, in vivo application
- long-term silencing
- adenovirus, lentivirus, retrovirus
13. Significance of the RNAi
1. RNAi protects against viral
infection.
2. RNAi secures genome stability
by keeping mobile elements
silent.
3. RNAi-like mechnisms repress
protein synthesis and regulate the
development of organisms.
4. RNAi-like mechanisms keep
chromatin condensed and
suppress transcription.
5. RNAi offers a new experimental
tool to repress genes specifically.
6. RNAi might be a useful
approach in future gene therapy.
14. Applications……..
• RNA interference (RNAi) has become an almost-standard method for
in vitro knockdown of any target gene of interest. From the
mechanism, it becomes clear that small interfering RNAs (siRNAs)
play a pivotal role in triggering RNAi.
• The use of RNAi for therapeutic purposes holds a great deal of
potential for the treatment of viral and genetic diseases, and cancer.
• The specific aim of siRNA therapies is to control gene expression by
gene silencing. Many methods are being developed to deliver siRNA
to a spe-cific target site in the cell. The delivery methods that hold the
most
potential
include
lipo-somes,
polymers,
chemical
modifications, nanoparticles, and aptamers. These delivery me-thods
are being used to facilitate therapeutic applications employing
siRNA. siRNA can be applied to the treatment of viral and genetic
diseases and cancer.
15. • RNA interference is a vital part of the immune response to viruses and
other foreign genetic material, especially in plants where it may also
prevent the self-propagation of transposons. Plants such as Arabidopsis
thaliana express multiple dicer homologs that are specialized to react
differently when the plant is exposed to different types of viruses.
• Although animals generally express fewer variants of the dicer enzyme
than plants, RNAi in some animals has also been shown to produce an
antiviral response.
• In both juvenile and adult Drosophila, RNA interference is important
in antiviral innate immunity and is active against pathogens such
as Drosophila X virus.
• A similar role in immunity may operate in C. elegans, as argonaute
proteins are upregulated in response to viruses and worms that
overexpress components of the RNAi pathway are resistant to viral
infection.
16. • The role of RNA interference in mammalian innate immunity is
poorly understood, and relatively little data is available. Alternative
functions for RNAi in mammalian viruses also exist, such as miRNAs
expressed by the herpes virus that may act as heterochromatin
organization triggers to mediate viral latency.
• Other proposed clinical uses center on antiviral therapies, including
topical microbicide treatments that use RNAi to treat infection
by herpes simplex virus type 2 and the inhibition of viral gene
expression in cancerous cells, knockdown of host receptors and
coreceptors for HIV, the silencing of hepatitis A and hepatitis
B genes, silencing of influenza gene expression, and inhibition
of measles viral replication. Potential treatments for neurodegenerative
diseases have also been proposed, with particular attention being paid
to the polyglutamine diseases such as Huntington's disease.
17. • The natural bicolor floral traits of the horticultural petunia (Petunia
hybrida) cultivars Picotee and Star are caused by the spatial
repression of the chalcone synthase A (CHS-A) gene, which encodes
an anthocyanin biosynthetic enzyme.
• Here the Picotee and Star petunias carry the same short interfering
RNA (siRNA)-producing locus, consisting of two intact CHS-A
copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail
orientation.
• The precursor CHS mRNAs are transcribed from the two CHS-A
copies throughout the bicolored petals, but the mature CHS mRNAs
are not found in the white tissues.
