Transcript SiRNA and Epigenetic

siRNA and Epigenetic
Asma Siddique
Saloom Aslam
Syeda Zainab Ali
Topics under discussion
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Brief History
Intro to different terms
What is RNAi?
What is siRNA?
siRNA formation
Difference between miRNA and SiRNA
siRNA design
Therapies
Challenges
Clinical trials
History
 SiRNA was first discovered by David
Baulcombe’s group as part of posttranscriptional gene silencing in plants,
in 1993.
Different terms
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RNAi
siRNA
shRNA
MiRNA
RISC…
DICER, ARGONAUTE FAMILY PROTEINS and OTHER
PROTEINS
 Off- target effects
What is RNA interference?
 Gene silencing mechanism...siRNA and miRNA
 Known as the RNA interference machinery.
Once it finds a double-stranded RNA (Dicer),
separates the two molecule, cuts it up.
 Way to silence genes by preventing the
formation of the proteins that they code for.
Transitive RNAi
 Organisms have RNA dependant RNA
polymerase that uses the mRNA
targeted by the initial anti-sense SIRNA
as a template for the synthesis of more
siRNA.
 These secondary siRNA also target other
parts of mRNA.
 When mRNA forms a duplex with a
complementary antisense RNA
sequence, translation is blocked:
1. The ribosomes cannot gain access to
nucleotides in mRNA
2. Duplex RNA is quickly degraded by
ribonucleases
 Double stranded RNA corresponding to a
particular gene is a powerful
suppressant of that gene.
 The suppressive effect of anti sense RNA
probably depends on its ability to form
dsRNA.
siRNA
 siRNA known as short\small interfering RNA,
are a class of 20-25 nucleotide-long RNA
molecules that interfere with the expression of
genes. It has 2-nt overhangs on either end,
including a 5' phosphate group and a 3'
hydroxy (-OH) group.
 They are 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.
Sources of siRNA
 Plant cells make these from the
double stranded RNA of invading
viruses.
 Scientists make these as agents to
turn off the expression of specific
genes.
SiRNA formation
Delivery of trigger dsRNA
Generation of siRNA pool
Capture, unwinding of SiRNA by RISC
Binding of SiRNA associated RISC with
target mRNA… ATP dependant
 Destruction of target mRNA
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 SiRNA can also inhibit the transcription
of genes:
1. Perhaps by binding to complementary
sequences on DNA
2. Perhaps by binding to the nascent RNA
transcript as it is formed.
 How these SiRNAs synthesized in the
cytosol –gain access to the DNA in the
nucleus is unknown.
miRNA
 A miRNA (micro-RNA) is a form of singlestranded RNA which is typically 20-25
nucleotide long.
 It is thought to regulate the expression
of other genes.
 They act by either destroying or
inhibiting translation of several mRNA
(by
binding to a region of complimentary sequences in the
3’UTR of mRNA)
Studies have shown that miRNAs play a role in
the most critical biological events including
development, proliferation, differentiation, cell
fate determination, apoptosis, signal
transduction, organ development,
hematopoietic lineage differntiation, host viral
interactions and carcinogenesis.
Effective SiRNA design?
 Select the target region from the open reading
frame of a given DNA sequence…50-100 nt
down stream of the start codon.
 Search for sequences 5’AA(N19)UU, in the
mRNA sequence and choose those with approx
50% GC content.
 BLAST search
 Strand incorporation depends upon weaker
base pairing…more AT content more
incorporation.
Therapies
 Synthetic siRNA molecules that bind to gene
promoters can repress transcription of that
gene. Repression is mediated by methylation of
the DNA in the promoter ; methylation of
histones in the vicinity.
 Rnai can use as a weapon to counter infections
by RNA viruses by destroying their mRNA’s.
 Screening genes for their effect on drug
sensitivity.
 Why RNA triggers and DNA does not?
 More tightly packed
 More stable
 RNA is easily hydrolysed
Challenges of RNAi
 Finding a vector or delivery system
 At what age, a patient should receive
treatment
 RNAi therapy is long term or only
temporary?
 Long dsRNA fragments reduce gene
expression in mammals
Clinical trials underway
 “wet” macular degeneration (targeting VEGF
which encodes vascular endothelial growth
factors)
 AIDS (targeting an exon used by the HIV
envelope protein)
 Hepatitis B (targeting four different sequences
in the viral genome)
 Some cancers
Any questions???