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Antisense RNA technology

anti sense RNA technology is one the method for gene silencing. it found very effective making Flavr savr tomato. hope this ppt will help you.

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Antisense RNA technology

  1. 1.  1
  2. 2. -Sapate P D, Student(ABW/34/2011), Lokmangal Agril biotech college, Wadala. 2
  3. 3. INTRODUCTION   Antisense RNA is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell  Antisense RNA introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and creating barrier to the translation machinery.  E.g. hok/sok system of the E. coli R1 plasmid. 3
  4. 4.   This translational arrest causes reduced amount of protein expression.  Well-known examples of GM plants produced by this technology- The Flavr Savr tomato , Two cultivars of ring spot-resistant papaya. 4 After 45 days….
  5. 5. General outline  5
  6. 6. Diff. between antisense technology & RNAi   The intended effect in both will be same i.e. gene silencing but the processing is little but different.  Antisense technology degrades RNA by enzymes RNaseH while RNAi employed the enzyme DICER to degrade the m RNA.  RNAi are twice larger than the antisense oligonucleotide. 6
  7. 7. HISTORY   First time at “ free university of Amsterdam”, used antisense RNA technology against the gene determining flower color of petunia .  Antisense effect first demonstrated by zemencnick & Stephenson in 1970 on “Rous sarcoma virus”.  First time antisense oligonucleotides are synthesized by Eckstein and colleagues. 7
  8. 8.   In 1995 Guo and Kemp hues: injection of either antisense or sense RNAs in the germ line of C. elegans was equally effective at silencing homologous target genes. 8
  9. 9. Nature’s antisense system   There is HOK(host killing)/SOK(suppress killing) system in R1 plasmid in E.Coli.  when E. coli cell undergoes division , daughter cell inherit hok gene & sok gene from parent. But due to short life of cell, the sok gene is get degraded. So in normal cell, hok gene get over expressed & cell get die.  But when R1 plasmid is get inherited , it having the sok gene & sok promoter.  Then it transcripts sok gene & it is get overexpressed against hok gene. 9
  10. 10. HOW VIRUS REPLICATE ?  10
  11. 11. MECHANISM   In this technique, Short segments of single stranded RNA are introduced.  These oligonucleotides are complementary to the mRNA, which physically bind to the mRNA.  So , they block the expression of particular gene.  In case of viruses, antisense oligonucleotides inhibit viral replication with blocking expression of integrated proviral genes.  Usually consist of 15–20 nucleotides. 11
  12. 12.   Translation of mRNA may be blocked by two possible mechanisms , These are:- 1] by base specific hybridization – which prevents access by translation machinery i.e. “hybridization arrest”. 2] by forming RNA/DNA duplex which is recognized by nuclease RNaseH , specific for digesting RNA in an RNA/DNA duplex. 12
  13. 13.   RNaseH is a non-specific endonuclease, catalyzes the cleavage of RNA via hydrolytic mechanism.  RNaseH has ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex. 13
  14. 14.   Unique DNA sequence  Efficient cellular uptake  Minimal nonspecific binding  Target specific hybridization  Non-toxic antisense construct 14 Characteristics of antisense oligonucleotides
  15. 15.   The antisense technology can be modified in THREE modes because of chemical modifications of the oligonucleotides.  These modes are due to activation of RNaseH & internucleotides linkages which do not activate enzyme. 15 Approaches
  16. 16.   The antisense oligonucleotides binds the target sequence causing both “hybridisation arrest ” & “RNaseH activation”.  Degradation of mRNA by RNaseH results into release of oligonucleotides.  They may bind to other copies of target mRNA.  These oligonucleotides are also susceptible to other nucleases.  This a major parameter affecting catalytic mode of degradation. 16 1st approach
  17. 17.   In this, antisense oligonucleotides binds to target sequence result in translation arrest but they do not activate enzyme RNaseH.  Oligoribonucleotides & there analogues , oligodeoxyribonucleotides , various non phosphate & phosphate internucleotides linkages fall in this category.  They show resistance against nucleuses enzyme and never get degraded by them. 17 2nd approach
  18. 18.   They also show effective translational arrest .  But the major problem is that they are generally required higher molar concentrations than those which activate RNaseH. 18
  19. 19.   It combines features of both previous approaches.  They contains both internucleotides linkages which are responsible for RNaseH activation & which shows resistance against them.  Digestion of mRNA target in RNA-DNA duplex releases oligonucleotides which are resistance against nuclease enzyme, hence are more effective than oligonucleotides in 1st approach. 19 3rd approach
  20. 20.   They may form hybrids of oligodeoxyribonucleotides & Oligoribonucleotides.  The antiviral activity of an antisense oligonucleotides depends usually on specific binding to a target nucleic acid. 20
  21. 21.  Over view 21
  22. 22.   Thomas and coworkers coined the term ‘ribozymes’.  These are RNA molecules which have catalytic activity which degrade nucleotides .  Ribozyme Bind to the target RNA moiety and inactivate it by cleaving the phosphodiester backbone at a specific cutting site.  Ribozyme destroy RNA that carries the massage of disease.  These are effectively used against HIV virus. 22 Ribozymes
  23. 23.  23 Mechanism of ribozyme
  24. 24. APPLICATION  1. Flavr Savr tomato-antisense RNA used against an enzyme polygalacturonase, an softening enzyme which is responsible for ripening. 2. Transgenic ACMV-resistant cassava plants* – Used against African cassava mosaic virus (ACMV) which causes cassava mosaic disease causing major economic loss in Africa. 3. Formivirsen-is the first antiviral drug developed against CMV. 24
  25. 25.  25 Antisense as drug
  26. 26. conclusion   Antisense technology shows potential for diverse application to field of basic research & therapy.  One of the most approved approaches for inactivating a single specific gene.  But it may sometime give undesirable effect.  Generally , antisense RNA still lack effective design, biological activity, and efficient route of administration.  Antisense technologies form a very powerful weapon for studying gene function and for discovering more specific treatments of disease. 26
  27. 27.   Attempts are made to genetically engineer transgenic plants to express antisense RNA instead activate the RNAi pathway, although the processes result in “gene silencing”. 27
  28. 28. References :-   A textbook of biotechnology 2nd edition by H. D. Kumar www.youtube.com  Nature biotechnology.  www.ncbi.nlm.nih.com (PubMed ID 17173627)* www.google.com 28
  29. 29.  Queries ? (If any) 29