antisense technology


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antisense technology

  1. 1. ANTISENSE TECHNOLOGY Presented By Desh Bandhu Gangwar M.Tech Biotech (2 year) Concerned Faculty Dr. Gunjan Garg Assistant Professor School of Biotechnology
  2. 2. INTRODUCTIONWhat is AntisenseTechnology ?
  3. 3. In this technique Short segments of single stranded DNA called oligo de oxy nucleotides are introduced.These oligonucleotides are complementary to the mRNA, which physically bind to the mRNA.
  4. 4. Antisense technology prevent the synthesis of specific protein.Antisense technologies are a suite of techniques that, together form a very powerful weapon for studying gene function and for discovering more specific treatments of disease.
  5. 5. Antisense OligonucleotidesWhat are AntisenseOligonucleotides?
  6. 6. The antisense effect of a oligonucleotide sequence was first demonstrated in 1970s by Zamecnik and Stephenson, in Rous sarcoma virus.AS-ONs usually consist of 15– 20 nucleotides, which are complementary to their target mRNA.
  7. 7. When these AS-ON combinedwith target mRNA, a DNA/RNAhybrid form,which degraded bythe enzyme RNase H. RNase H
  8. 8. RNase H is a non-specificendonuclease, catalyzes thecleavage of RNA via hydrolyticmechanism.RNase H has ribonucleaseactivity cleaves the 3’-O-P bondof RNA in a DNA/RNA duplex.
  9. 9. Mechanism of antisense activity
  10. 10. Types Of AS-ONFirst generation AS-ONSecond generation AS-ONThird generation AS-ON
  11. 11. A successful AS-ON depends on the followingcharacteristics: Unique DNA sequence Efficient cellular uptake Minimal nonspecific binding Target specific hybridization Non-toxic antisense construct Nuclease resistant to protect AS-ON
  12. 12. First generation AS-ONFirstsynthesized by Eckstein and colleagues.Phosphorothioate - oligo deoxy nucleotides are the major representatives of first generation DNA analogs that are the best known.
  13. 13. Sites of chemical modification
  14. 14. Phosphorothioate linkages in Ons primarily used to enhance their nuclease resistance.Inthis class of ONs, non bridging oxygen atoms in phopho-diester bond is replaced by sulfur.They first used as AS-ONs for the inhibition of HIV.
  15. 15. Characterstics of first generation AS-ONBetter stability to nucleases but still degrades.Decreased affinity to target mRNA.Enhanced specificity of hybridization.Toxic in nature.Can activate R Nase H.
  16. 16. Second generation AS-ONSecond generation ONs containing nucleotides with alkyl modifications at the 2’ position of the ribose. 2’-O-methyl and 2’-O-methoxy- ethyl RNA are the most important member of this class.
  17. 17. Characterstics of second generation AS-ONBest stability to nucleases.Increased affinity to target mRNA.Less toxic than first generation AS-ON.Can not activate R Nase.
  18. 18. Third generation AS-ONNewest and most promising.Enhance binding affinity and biostability.Peptide nucleic acids (PNAs)Locked nucleic acid (LNA)Tricyclo-DNA (tcDNA)Cyclohexene nucleic acids (CeNA)
  19. 19. Peptide nucleic acids In PNAs the deoxyribose phosphate backbone is replaced by polyamide linkages, which is composed of repeating N-(2-aminoethyl)-glycine units, linked by peptide bonds PNA was first introduced by Nielsen and coworkers in 1991. They are electrostatically neutral molecules
  20. 20. Locked nucleic acid LNA was synthesized by Jesper Wengel in 1998. The ribose moiety of LNA nucleotide is modified with an extra bridge connecting the 2 oxygen and 4 carbon
  21. 21. RibozymesThomas and coworkers coined the term ‘ribozymes.Ribozymes are RNA molecules that have catalytic activity.Ribozyme Bind to the target RNA moiety and inactivate it by cleaving the phosphodiester backbone at a specific cutting site.
  22. 22. Mechanism of Ribozymes
  23. 23. Types Of RibozymesTetrahymena group I intron RNase PHammer head ribozymeHairpin ribozymeHepatitis delta virus ribozyme
  24. 24. Cycle of RNA cleavage by hammerhead ribozyme
  25. 25. Ribozymes in clinical trialsANGIOZYME - VEGF-receptor1HERZYME - HER-2HEPTAZYM
  26. 26. RNA interference RNA interference (RNAi) is a system within living cells that takes part in controlling genes activity. Twotypes of small RNA molecules – (miRNA) and (siRNA) are central to RNA interference. Melloand Fire named the process RNAi, were awarded the Nobel Prize.
  27. 27. Mechanism of RNA interference
  28. 28. Comparision Of different Antisense stratgies
  29. 29. Applications Of Antisense technologies Story of Flavr Savr…
  30. 30. Antisense therapyß-thalassemiaCytomegalovirus retinitisHemorrhagic fever virusesDuchenne muscular dystrophyCancerHIV/AIDSHigh cholesterol
  31. 31. Antisense Drug Therapy
  32. 32. REFERENCE Gene cloning and DNA analysis, Fifth edition By T.A Brown Page no. 235 Walton, S. P., Roth, C. M., Yarmush, M. L. “Antisense Technology.”The Biomedical Engineering Handbook: Second Edition. Indian journal of chemistry vol. 48 B December 2009, pp. 1721-1726 Indian journal of biotechnology vol 4,JUL 2005,pp. 316 -322 Eur. J. Biochem. 270,1628–1644 Clinical and Experimental Pharmacology and Physiology (2006) 33, 533–540
  33. 33. QUERIES?
  34. 34. THANK YOU