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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.
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Uploaded byPravin Sapate
Antisense RNA technology
The document discusses antisense RNA technology, which involves the introduction of single-stranded RNA to inhibit the translation of complementary mRNA, reducing protein expression in genetically modified organisms like the Flavr Savr tomato. It compares antisense technology with RNA interference (RNAi) regarding their mechanisms of gene silencing and highlights the historical and practical applications of antisense RNA and ribozymes in biotechnological advancements. Antisense technology shows promise but faces challenges in effective design, biological activity, and administration routes.
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Antisense RNA technology
- 1.
- 2. -Sapate P D, Student(ABW/34/2011), Lokmangal Agril biotech college, Wadala. 2
- 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. Thistranslational 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.
- 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. 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. In1995 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. 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. HOW VIRUS REPLICATE? 10
- 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. Translationof 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. RNaseHis 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. UniqueDNA sequence Efficient cellular uptake Minimal nonspecific binding Target specific hybridization Non-toxic antisense construct 14 Characteristics of antisense oligonucleotides
- 15. Theantisense 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. Theantisense 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. Inthis, 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. Theyalso show effective translational arrest . But the major problem is that they are generally required higher molar concentrations than those which activate RNaseH. 18
- 19. Itcombines 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. Theymay form hybrids of oligodeoxyribonucleotides & Oligoribonucleotides. The antiviral activity of an antisense oligonucleotides depends usually on specific binding to a target nucleic acid. 20
- 21. Over view 21
- 22. Thomasand 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 Mechanismof ribozyme
- 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 Antisenseas drug
- 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. Attemptsare made to genetically engineer transgenic plants to express antisense RNA instead activate the RNAi pathway, although the processes result in “gene silencing”. 27
- 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. Queries ? (If any) 29
- 30. THANKs FORYOUR KIND ATTENSION 30