siRNA and Epigenetic

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siRNA and Epigenetic

• Asma Siddique
• Saloom Aslam
• Syeda Zainab Ali

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Topics under discussion

• 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

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History

• SiRNA was first discovered by David Baulcombes
  group as part of post-transcriptional gene
  silencing in plants, in 1993.

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Different terms

• RNAi
• siRNA
• shRNA
• MiRNA
• RISC DICER, ARGONAUTE FAMILY PROTEINS and OTHER
  PROTEINS
• Off- target effects

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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.

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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.

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• When mRNA forms a duplex with a complementary
  antisense RNA sequence, translation is blocked
• The ribosomes cannot gain access to nucleotides
  in mRNA
• Duplex RNA is quickly degraded by ribonucleases
  

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• 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.

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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.

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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.

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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
• Perhaps by binding to complementary sequences on
  DNA
• Perhaps by binding to the nascent RNA transcript
  as it is formed.

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• How these SiRNAs synthesized in the cytosol gain
  access to the DNA in the nucleus is unknown.

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miRNA

• A miRNA (micro-RNA) is a form of single-stranded
  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 3UTR of
  mRNA)

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• 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.

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Effective SiRNA design?

• Select the target region from the open reading
  frame of a given DNA sequence50-100 nt down
  stream of the start codon.
• Search for sequences 5AA(N19)UU, in the mRNA
  sequence and choose those with approx 50 GC
  content.
• BLAST search
• Strand incorporation depends upon weaker base
  pairingmore AT content more incorporation.

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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 mRNAs.
• Screening genes for their effect on drug
  sensitivity.

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• Why RNA triggers and DNA does not?

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• More tightly packed
• More stable
• RNA is easily hydrolysed

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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

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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

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• Any questions???