ANTISENSE ANTISENSE TECHNOLOGYTECHNOLOGY
Presented ByDesh Bandhu Gangwar
M.Tech Biotech (2 year)
Concerned FacultyDr. Gunjan Garg
Assistant ProfessorSchool of Biotechnology
INTRODUCTIONINTRODUCTION
What is Antisense Technology ?
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.
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.
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Antisense Antisense OligonucleotidesOligonucleotides
What are Antisense Oligonucleotides?
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.
When these AS-ON combined with target mRNA, a DNA/RNA hybrid form,which degraded by the enzyme RNase H.
RNase H
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.
Mechanism of antisense Mechanism of antisense activityactivity
Types Of Types Of AS-AS-ONON
First generation AS-ON
Second generation AS-ON
Third generation AS-ON
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
First generation First generation AS-ON 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.
Sites of chemical modification
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.
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 Characterstics of first generation AS-ONAS-ON
Second generation Second generation AS-ONAS-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.
Characterstics of second Characterstics of second generation AS-ONgeneration AS-ON
Best stability to nucleases.
Increased affinity to target mRNA.
Less toxic than first generation
AS-ON.
Can not activate R Nase.
Third generation AS-Third generation AS-ONON
Newest and most promising.Enhance binding affinity and
biostability.
Peptide nucleic acids (PNAs)Locked nucleic acid (LNA)Tricyclo-DNA (tcDNA)Cyclohexene nucleic acids (CeNA)
Peptide nucleic acidsPeptide 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
Locked nucleic acidLocked 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
RibozymesRibozymesThomas 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.
Mechanism of RibozymesMechanism of Ribozymes
Types Of Types Of RibozymesRibozymes
Tetrahymena group I intron
RNase P
Hammer head ribozyme
Hairpin ribozyme
Hepatitis delta virus ribozyme
Cycle of RNA cleavage by hammerhead ribozyme
Ribozymes in clinical Ribozymes in clinical trialstrials
ANGIOZYME - VEGF-receptor1
HERZYME - HER-2
HEPTAZYM
RNA interferenceRNA interferenceRNA 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.
Mechanism of RNA interferenceMechanism of RNA interference
Comparision Of different Antisense Comparision Of different Antisense stratgiesstratgies
Applications Of Antisense Applications Of Antisense technologiestechnologies
Story of Flavr Savr…
Antisense therapy
ß-thalassemiaCytomegalovirus retinitisHemorrhagic fever virusesDuchenne muscular dystrophyCancerHIV/AIDSHigh cholesterol
Antisense Drug Therapy
REFERENCEREFERENCE 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
QUERIES?QUERIES?
THANK YOUTHANK YOU
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