RNA Interference(RNAi) is a conserved biological response of eukaryotes against double-stranded RNA causing silencing of the gene expression. This mechanism has an important role in defending cells against viral genes and transposons. RNAi technology has become the latest "next big thing," progressing from a barely understood colour silencing mechanism found in flowers to a powerful tool that is going to become a new therapeutic tool for treating illnesses ranging from AIDS to cancer to Huntington’s disease. Even more exciting is the potential of RNAi in agriculture where it has provided a way to control pests and diseases as well as increase nutritional value of food.

1. MINOR CREDIT SEMINAR ON
by
DR. PRANATEE BEHERA
M5651
Division of Animal Biotechnology

2. • Post-transcriptional biological process
• Eukaryotes
• Suppresses gene expression
• Target - dsRNA
• mRNA degradation/ inhibition of translation
• microRNA (miRNA) and small interfering RNA
(siRNA)

3. Why RNAi!!!
• 30% of human genome regulated
• Defense against
viruses
transposons
• Invaluable tool in therapeutics
• “breakthrough of the year”- 2002 science

4. Discovery : an accident, a puzzle !!
• Napoli and Jorgensen -
first to report an RNAi type
of phenomenon (1990).
• Goal – to get deep violet
coloration in petunias.
• overexpressed chalcone
synthase in petunias.

5. expected
observed
Cossack dancer

6. Quelling: In fungal system
• Romano and Macino (1992)
• Fungus Neurospora crassa
• al-1 gene- carotenoid synthesis
• Expected:
orange pigment
• Observed:
albino

7. Antisense RNA technology

8. Andrew Fire And Craig Mellow
1998
“trigger for gene silencing was not single-
stranded RNA (ssRNA) but double-
stranded RNA (dsRNA)”

9. Experiment that led to Discovery of RNAi
Injected dsRNA
corresponding
to unc22 gene
ssRNA was effective only when sense strand was injected
followed by the antisense or vice versa
C. elegans

10. Happy ending………
noble prize
in 2006

12. RNAi
RNA interference
MECHANISM OF
RNAi

13. siRNA gene silencing

14. miRNA gene silencing
Along with DGCR8
form
microprocessor
complex

15. piRNA
• Piwi-Interacting RNA
• Most recently discovered class of longer small
RNAs(25-30nt)
• Dicer independent
• Direct cleavage of transposon mRNA

18. RNAi and HBV
• Nucleonics
• NUC B1000 (plasmid DNA
producing 4 different
shRNA)
• Phase 1 clinical trial

19. Other diseases
• Huntington disease – huntingtin protein
(chromosome 4)
• HIV AIDS- CD4 protein of human, p24 viral protein
• Cancer
• Hepatitis C

20. RNAi and agriculture
Pest resistance
L. Gu, D.C. Knipple / Crop Protection 45 (2013) 36-40
Root worm is
destructive
pest of corn
RNAi targets
genes essential
to the
rootworm
survival
Rootworms that feed
on RNAi-corn plants
are eliminated

21. Crop improvement
Pathogen resistance
• Fungal-resistant bananas
• Nematode-resistant soybeans
• Golden mosaic virus resistant
bean
• Africa researchers are
employing RNAi to combat viral
diseases cassava (Manihot
esculenta)
• Ring spot virus resistant
papaya
• More cost-effective and
environmental friendly
Ring spot in papaya

22. Nutritional improvement
• Tomatoes and potatoes with increased carotenoids
“Hey!!!! I am with more
carotenoids…. Eat me”
RNAi target is
b-carotene
hydroxylase gene

23. Reduced- lignin alfalfa
Alfalfa a key
component of hay but
contains lignin which
cattle can’t digest
RNAi targets the gene
that control lignin
Reduced lignin
can be easily
digested

24. Others…
• Nicotine-free tobacco
• Non-allergenic peanuts
• Decaffeinated coffee
• Nutrient fortified maize

25. Limitations in delivery of si-or miRNA
• Nuclease enzyme
• Unsafe delivery of mi- or siRNAs
• Off-target effects or Unspecific silencing (Saxena et
al., 2003)
• Activation of intracellular PKR and immune pathways
that are linked to toll-like receptor activation.
(Williams, 1997; Alexopoulou et al., 2001)

26. Chemical modifications
Liposome mediated transfer
Scientists are really smart!!
LNA

27. • Specificity of silencing effects and efficacy in
vitro and in vivo.
• But clinical trials are nowhere complete
• With the increase in technological advancements and
novel breakthroughs though it can only be but
anticipated that the future of RNAi therapeutics is
going to be a bright one.
Conclusion

28. References :
• Fire, A. et al (1998), Potent and specific genetic interference by double-
stranded RNA in Caenorhabditis elegans, Nature 391:806-811
• Elbashir, Sayda M. et al (2001), RNA interference is mediated by 21- and
22-nucleotide RNAs, Genes & Dev. 15: 188-200
• Patrick Y. Lu. et al(2005), In Vivo Application of RNA Interference: From
Functional Genomics to Therapeutics, Advances in Genetics.54: 115–142
• Gregory J. Hannon(2002), review article RNA interference, Nature. 418:
244-251
• Sabine Branti(2002),Antisense-RNA regulation and RNA interference,
Biochimica et Biophysica Acta (BBA) - Gene Structure and
Expression.1575(1-3):15-25
• Napoli, C., Lemieux, C., Jorgensen, R. (1990) Introduction of a chimeric
chalcone synthase gene into petunia results in reversible co-suppression
of homologous genes in trans. Plant Cell 2,279-289
• Judge Adam D et al(2005) , Sequence-dependent stimulation of the
mammalian innate immune response by synthetic siRNA, Nature
Biotechnology 23, 457 - 462
• Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C.
(1998) Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans. Nature 391,806-811

29. “A small but mighty world ,
that is……RNA world”
Thank you
for
your kind attention