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.
Antisense OligonucleotidesWhat are AntisenseOligonucleotides?
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 combinedwith target mRNA, a DNA/RNAhybrid form,which degraded bythe enzyme RNase H. RNase H
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.
Types Of AS-ONFirst generation AS-ONSecond generation AS-ONThird generation AS-ON
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
First generation AS-ONFirstsynthesized by Eckstein and colleagues.Phosphorothioate - oligo deoxy nucleotides are the major representatives of first generation DNA analogs that are the best known.
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.
Characterstics of first generation AS-ONBetter stability to nucleases but still degrades.Decreased affinity to target mRNA.Enhanced specificity of hybridization.Toxic in nature.Can activate R Nase H.
Second generation AS-ONSecond 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 generation AS-ONBest stability to nucleases.Increased affinity to target mRNA.Less toxic than first generation AS-ON.Can not activate R Nase.
Third generation AS-ONNewest 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 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 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
RibozymesThomas 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.
Ribozymes in clinical trialsANGIOZYME - VEGF-receptor1HERZYME - HER-2HEPTAZYM
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.
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