Antisense RNA technology involves introducing short oligonucleotides that are complementary to a target gene's mRNA, interrupting normal gene expression. This can partially or fully suppress protein production from the gene. Antisense RNA works by binding to the target mRNA, preventing translation into protein via mechanisms like RNaseH degradation. It has applications in cancer treatment, fruit ripening control in agriculture, and drug development by the pharmaceutical industry. Challenges include rapid degradation of antisense oligonucleotides inside cells, but chemical modifications now help overcome this. Antisense therapy is emerging as a potential tool for gene therapy and treatment of various diseases.

1. ANTI SENSE RNA-
THEORY AND
APPLICATIONKAVYA G
18LS301010
MSC BIOTECHNOLOGY

2. INTRODUCTION
 A conventional definition of antisense refers to the laboratory modifications or manipulation of RNA
so that its components form a complementary copy of normal or “SENSE” messenger RNA.
 The binding or hybridization of antisense nucleic acid sequences to a specific mRNA target through a
number of different mechanisms, interrupt normal cellular processing of the genetic message of a
gene.
 This interruption, sometimes referred to as ‘knock down’ or ‘knock out’ depending upon whether or
not the message is either partially or completely eliminated.
 The overall goal in introducing on antisense agent into cells either invitro is to suppress or completely
block the production of the gene product.

3. THEORY

4. CONT…..
 Among several strategies for inactivating a single chosen gene, the most approved one is ‘Antisense
technology’
 This modification does not involve actual substraction but inactivating of gene or suppressing gene
activity.
 When normal gene is transcribed, the RNA produced is called as sense RNA which is Complementary
to DNA, but if the orientation of gene to be transcribed is reversed with respect to promoter, the RNA
transcribed from it would be reversed too and the RNA is said to be ‘ANTISENSE RNA’.
 Antisense RNA refers to the short oligonucleotides which are designed to be complementary to a
specific gene sequence.
 The antisense effect of a synthetic oligonucleotides sequence was first demonstrated in the late 1970s
by Zamecnik and Stephenson. Using nucleotide sequences from the 5’ and 3’ ends of the 35S RNA of
rous sarcoma virus, they found a repeated sequence of 21 nucleotides that appeared to be crucial to
viral integration. They concluded that the oligonucleotides was inhibiting viral integration by
hybridizing to the crucial sequences and blocking them.

5. PRINCIPLE
The main principle of antisense RNA technology is to prevent the
production from a targeted gene. Initially in cell the cellular
nucleases dramatically reduced the effectiveness of antisense
oligonucleotides by rapidly degrading these molecules after
administration. These obstacles are overcome by synthesizing
synthetic oligonucleodes by altering the nature of phosphate
diester bond by replacing the oxygen with sulphur such modified
oligonucleotides are termed phosphorothionates

8. APPLICATION
1. MEDICAL APPLICATION
 Antisense technology may play a major role in cancer chemotherapy. It is a tool of exceptional
value in the fictionalization of genes and their validation as potential targets for cancer therapy.
 Additionally, there is now substantial voidance that antisense drugs are safe, and a growing body
of data showing activity to animal models of human diseases including cancer and suggesting
efficacy in patients with cancer.
 Antisense technology is arguably the most advanced genomically based drug discovery
technology. It has been shown to be capable of generating very specific inhibitors, and a significant
number of antisense drugs are in development as anticancer agents

10. Cont….
 A number of potential mechanisms by which antisense drugs may work have been identified and they
have been partitioned into two broad groups: occupancy only and occupancy activated destabilization.
The occupancy is the only mechanism, translation arrest and inhibition of splicing have been most
extensively characterized. Occupancy-induced destabilization, as thetarget RNA, is epitomized by
RNaseH degradation of the target RNA, and this mechanism is the most often used and least
characterized antisense mechanism.
 There are now a number of reports of antisense inhibition of human tumour zenograft and other animal
models of human cancers.
 For many of these studies, appropriate controls, including a variety of mismatched oligonucleotides were
used and there is evidence that the effect of the antisense agents were indeed due to an antisense
mechanism.
 Human tumor zenografts have shown that it is feasible to reduce target RNA and protein levels and to
induce antiproliferative effects in some of these models.

11. CONT…..
2. AGRICUTULARAL APPLIACATION (Genetic manipulations of fruit ripening)
a. Genes related to ethylene biosynthesis:
 Small multi-gene families code for the ACS and ACO enzyme involved in the ethylene biosynthesis.
 It was observed that tomato fruits of transgenic plant carrying ACS in antisense orientation completely
blocked the ripening by decreasing ethylene production to 99%, and these fruits did not ripen without
exogenous ethylene.
 Similarly, anti-ACS apple showed reduced autocatalytic ethylene production and increased shelf life.
 Tomato fruits from transgenic tomato plants carrying tomato ACO in antisense orientation and melon
fruits from transgenic melon plants carrying ACO from melon under the regulation of CaMV35S
promoter showed a reduction in ethylene production and delayed ripening

12. CONT…
 b. Genes related to cell wall softening:
 Several enzymes like polygalacturonase (PG), PME, expansin, cellulose, xyloglucanase etc. are implicated in
wall softening during ripening.
 Transgenic homozygous tomato plants carrying PG gene in antisense orientation showed a reduced PG
activity 1% of the normal value. It has been shown that in fruits with antisense PG, the degradation of cellular
wall pectins was inhibited but other aspects of maturation, such as ethylene production and lycopene
accumulation were not affected.
 Ripened fruits from transgenic strawberry plants carrying pectate lyase from strawberry in antisense
orientation under the expression of CaMV35S were significantly firmer than control fruits and this gene
could be an excellent candidate for biotechnological improvement of fruit softening in strawberry.
 Suppression of Exp1 in tomato lead to firmer fruits as compared to wild type and also substantially inhibited
polyuronide depolymerization late in ripening, but did not prevent the breakdown of structurally important
hemicelluloses, a major contributor to softening.
 Antisense suppression of deoxyhypusine synthase in tomato delays fruit softening and alters growth and
development.
 Transgenic strawberry plants carrying antisense pectate lyase cDNA from strawberry under the control of the
35S promoter showed significantly firmer ripened fruits than controls.

13. CONT….
 Highest reduction of softening in these fruits occurred during the transition from the white to the red
stage. The postharvest softening of apple fruit was also diminished.

14. CONT…
c. Genes related to lycopene production:
 Lycopene possesses beneficial antioxidant properties due to which the researchers aimed to increase its
content.
 Transgenic approaches were employed to increase tomato fruit lycopene content. Suppression of
lycopene cyclase (Lcy) gene resulted in increased lycopene content in the transgenic tomato fruits.
 Suppression of TDET1 (Tomato DE-ETIOLATED1) mRNA accumulation by RNA interference
(RNAi) specifically in fruits using TDET1-derived inverted-repeat constructs driven by three different
fruit-specific promoters, P119, 2A11 and TFM7 was shown to enhance the carotenoid and flavonoid
content in mature fruit.
OTHER APPLICATIONS
 Modification of flower colour in decorative plants
 Antisense SAHH gene for resistance against a broad spectrum of viruses in plants
 Mutant gene for a protein necessary for cell to cell movement of virus in the host plant

15. CONT…..
3. INDUSTRIALAPPLICATION
 Antisense is an important technology for drug discovery and development . It is broadly used by the
pharmaceutical industry as a tool for functional genomics and as highly specific drugs for a wide range
of diseases.
 Cumulative clinical and preclinical data suggest that antisense technology has the potential to create a
new sector of the pharmaceutical industry.
 Several antisense drugs are now in late stage clinical development with one antisense drug developed
by Isis, Vitravene®, marketed in the US and Europe.
 Antisense inhibitors can target specific aspects of ocular disease processes and have ideal properties as
therapies for the eye.
 Antisense drugs have already been shown to be effective in treating ocular diseases with the
commercialization of Vitravene®, approved for the treatment of CMV Retinitis.

17. CONCLUSION
 Antisense technologies have been hailed as one of the best options for gene manipulation to be
used in studying gene function and for discovering new and more specific treatments for a wide
range of diseases in humans, animals, and plants.
 However, this success has been mainly restricted to laboratory and research organizations. There
has been only one antisense drug in the market, even after almost three decades of research on
antisense technologies.
 Its clinical applications thus far were barred by numerous problems and untrammeled challenges.
Despite all the challenges, antisense therapy has held its ground for more than two decades and
now it is ready to emerge as a potential tool for gene therapy.
 With more than a dozen molecules in the clinical development phases, the stage is set for
antisense therapy to emerge as a potential option for treatment of a wide range of diseases.
 The antisense strategy of inhibiting gene expression has tremendous potential in the fields of
functional genomics, drug discovery and therapy

18. REFERENCES
 https://www.researchgate.net/publication/318084598_Antisense_Technology
 Agrawal S., Goodchild J., Civeira M.P., Thornton A.H., Sarin P.S. and Zamecnik P.C. (1988)
Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human
immunodeficiency virus. Proc. Natl. Acad. Sci. USA. 85 (19): 7079-7083.
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117510/