Antisense technology uses short segments of DNA called oligonucleotides that are complementary to messenger RNA (mRNA) to prevent specific proteins from being synthesized. When introduced into cells, these oligonucleotides physically bind to the target mRNA, preventing translation. There are several generations of antisense oligonucleotides that differ in their chemical modifications to improve nuclease resistance and binding affinity. Ribozymes and RNA interference are related techniques that use catalytic RNA or small interfering RNA respectively to inactivate target RNA through cleavage. Antisense technology has potential applications in treating diseases like cancer, viral infections, and genetic disorders.

1. ANTISENSE TECHNOLOGY Presented By Desh Bandhu Gangwar M.Tech Biotech (2 year) Concerned Faculty Dr. Gunjan Garg Assistant Professor School of Biotechnology

2. INTRODUCTION What is Antisense Technology ?

4. 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 .

5. 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. .

6. Antisense Oligonucleotides What are Antisense Oligonucleotides?

7. 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.

8. When these AS-ON combined with target mRNA, a DNA/RNA hybrid form,which degraded by the enzyme RNase H . RNase H

9. RNase H is a non-specific endonuclease , catalyzes the cleavage of RNA via hydrolytic mechanism. RNase H has ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex.

10. Mechanism of antisense activity

11. Types Of AS-ON First generation AS-ON Second generation AS-ON Third generation AS-ON

12. A successful AS-ON depends on the following characteristics : Unique DNA sequence Efficient cellular uptake Minimal nonspecific binding Target specific hybridization Non-toxic antisense construct Nuclease resistant to protect AS-ON

13. First generation AS-ON First synthesized by Eckstein and colleagues . Phosphorothioate - oligo deoxy nucleotides are the major representatives of first generation DNA analogs that are the best known.

14. Sites of chemical modification

15. Phosphorothioate linkages in Ons primarily used to enhance their nuclease resistance . In this 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.

16. Better stability to nucleases but still degrades. Decreased affinity to target mRNA. Enhanced specificity of hybridization. Toxic in nature. Can activate R Nase H. Characterstics of first generation AS-ON

17. 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.

18. 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.

19. 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)

20. 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

21. 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

22. 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.

23. Mechanism of Ribozymes

24. Types Of Ribozymes Tetrahymena group I intron RNase P Hammer head ribozyme Hairpin ribozyme Hepatitis delta virus ribozyme

25. Cycle of RNA cleavage by hammerhead ribozyme

26. Ribozymes in clinical trials ANGIOZYME - VEGF-receptor1 HERZYME - HER-2 HEPTAZYM

27. RNA interference RNA interference (RNAi) is a system within living cells that takes part in controlling genes activity. Two types of small RNA molecules – (miRNA) and (siRNA) are central to RNA interference. Mello and Fire named the process RNAi, were awarded the Nobel Prize.

28. Mechanism of RNA interference

29. Comparision Of different Antisense stratgies

30. Applications Of Antisense technologies Story of Flavr Savr…

31. Antisense therapy ß -thalassemia Cytomegalovirus retinitis Hemorrhagic fever viruses Duchenne muscular dystrophy Cancer HIV/AIDS High cholesterol

32. Antisense Drug Therapy

33. 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

34. QUERIES?

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