RNAi technology

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RNAi TECHNOLOGY

• BY,
• NARENDRA

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RNA
DNA
Protein
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CONTENTS

• RNAi
• Mechanism
• Application
• Conclusion
• References

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non coding, but rather control proteins that do
code
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Discovery of RNAi Nature (1998) 391806-811
Andrew Fire
Craig Mello
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So, what is RNAi ?

• RNA interference (RNAi) The phenomenon of gene
  silencing using dsRNA was termed RNA
  interference.
• It is sequence specific
• Involves the degradation of dsRNA ssRNA
  molecule (usually mRNA).

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RNAi MECHANISM
DICER - RNAse III, ds-specific endonuclease
- Dimer, 2 catalytic domains. - produce 2-3 nt
3 overhangs. - ATP-dependent ribonuclease
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RNAi MECHANISM

• RISC
• - RNA-induced silencing complex
• - RISC contains siRNA
• - precursor activated by ATP
• - find and destroy mRNA of complementary
  sequence
• contains endo- and exonuclease, cleaves the
  hybrid in the middle followed by degradation

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technology
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STAGES IN RNAi TECHNOLOGY

• Target selection
• Target site selection
• Target RNA molecule design
• Modification
• Delivery into the site.

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RNAi technology in plants

• Slow fruit softening in tomato
• Pectin major component of fruit cell wall in
  tomato.
• Enzyme Polygalacturonase (PG) degrades pectin.
• RESULTS, Softening of fruit
  deterioration in fruit.
• Solution Tg Tomato with anti-PG is introduced.
• Showed reduced expression of PG.
• Tomatoes have about 2 weeks longer shelf life.

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A NOVEL STRATEGY IN INSECT PEST MANAGEMENT

• In 2007, researchers demonstrated the potential
  of RNAi in managing the highly destructive pest
  H. armigera.
• They identified gene P450 monooxygenase in the
  cotton plant.
• This gene detoxifies the toxic compound gossypol.
• Gossypol is toxic to insects.
• Detoxification leads to the larval tolerance.

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• So, to this problem researchers transferred hpRNA
  construct directed against CYP6AE14(P450
  monooxygenase) into plant fed the cotton ball
  worm.
• This demonstrated that Tg plant provided
  sufficient levels of dsRNA to suppress gene
  expression in insect midgut stunt in growth.

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TARGET GENE TARGET PLANT PARASITE HOST PLANT Tg PHENOTYPE
MSP H. glycines Soybean Reduction in eggs
Mi- tnc M. Incognita Tobacco Reduction of transcripts in the progeny.
Splicing factor M. Incognita Tobacco Reduction in number of galls females.
Hs4F01 H. Schachtii Arabidopsis Reduction in number of females, down regulation of transcript level.
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ON GOING CLINICAL TRIALS IN HUMANS
COMPANY DISEASE TARGET CLINICAL TRIAL
ALNYLAM PHARMACEUTICALS RESPIRATORY SYNCYTIAL VIRUS (RSV) VIRAL N GENE PHASE 2
OPKO HEALTH AGE RELATED MACULAR DEGENERATION (AMD) VGEF PHASE 3
MERCK CO. AMD VEGF-R1 PHASE 2
QUARK PHARMACEUTICALS AMD RTP 801 PHASE 1
QUARK PHARMACEUTICALS ACUTE RENAL FAILURE P53 PHASE 1
CALANDO CANCER POLYRIBONUCLEOTIDE REDUCTASE PHASE 1
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RNAi technology

• Disadvantages
• It does not knockout a gene for 100
• siRNA tends to activate unwanted pathways
• Expensive
• Ethical problems
• Advantages
• Highly gene specific
• High gene silencing efficiency
• Screening targeted plants takes less time
• Highly inducible
• Fast

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CONCLUSION

• RNAi is still developing field of biology, which
  could prove to be of enormous important for
  strategies.
• Summarizing, the development of new RNAi based
  drugs is feasible, but it will take many more
  years

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COMPANIES
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References

• Mauro Ferrari, Experimental therapies Vectoring
  siRNA therapeutics into the clinic, Nature
  Reviews Clinical Oncology 7, 2010 485-486.
• Mike may, Technology review, Biotechniques,
  2007,27-9,16.  
• Daniel Cejka, Doris Losert and Volker Wacheck.
  Short interfering RNA (siRNA) tool or
  Therapeutic? Clinical Science, 2006 110, 4758.
• Jun-Hal Niu, Heng Jian , Jian-mei Xu, Yang-Dong
  Guo Qian Liu, RNAi technology extends its
  reach Engineering plant resistance against
  harmful eukaryotes , African journal of
  biotechnology, 2010, vol 9(45), 7573-7582.
•  

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References

• Guiliang Tang, Gad Galili, Xun Zhuang. RNAi and
  microRNA breakthrough technologies for the
  improvement of plant nutritional value and
  metabolic engineering. Metabolomics 2007,
  3357369.
•  David P. Clark, Regulation at RNA level, In
  Text book of Molecular biology Elsevier
  academic press, 2005, Pg 291-294.
• Anne Dallas, Alexander V. Vlassov, RNAi A novel
  antisense technology and its therapeutic
  potential, Med Sci Monit, 2006 12(4) 67-74.

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small but mighty.the RNA world!
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