Antisence RNA and RNA Interference Technology

RNA Interference (RNAi) and Antisense Technology: A Comprehensive Overview
RNA interference (RNAi) and antisense technology are powerful molecular biology techniques that play critical roles in gene regulation and therapeutic applications. Both methods are designed to silence or knock down gene expression, but they operate through distinct mechanisms. Here, we explore the fundamental principles, mechanisms, and applications of RNAi and antisense technology.

RNA Interference (RNAi)
1. Introduction to RNAi:
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression by neutralizing targeted mRNA molecules. It is a natural mechanism that protects cells from viruses and transposable elements, and it also regulates gene expression.

2. Mechanism of RNAi:
The RNAi pathway involves several steps:

Double-stranded RNA (dsRNA) introduction: The process begins when double-stranded RNA (dsRNA) is introduced into the cell. This dsRNA can come from various sources, including viruses, transposons, or experimentally introduced synthetic RNA.
Dicer enzyme action: The dsRNA is recognized and cleaved by an enzyme called Dicer, producing small interfering RNA (siRNA) fragments, typically 21-23 nucleotides in length.
RISC complex formation: The siRNA is then incorporated into the RNA-induced silencing complex (RISC). Within RISC, the siRNA is unwound to form a single-stranded RNA (ssRNA).
Target recognition and degradation: The ssRNA within RISC guides the complex to the complementary mRNA target. Upon binding, the target mRNA is cleaved and degraded, preventing its translation into protein.
3. Applications of RNAi:

Gene function studies: RNAi is widely used in functional genomics to study gene function by knocking down specific genes.
Therapeutic applications: RNAi has potential therapeutic applications in treating diseases caused by overexpression or aberrant expression of genes, such as cancer, viral infections, and genetic disorders.
Agricultural biotechnology: RNAi is used to develop genetically modified crops with desirable traits, such as pest resistance and improved nutritional content.
Antisense Technology
1. Introduction to Antisense Technology:
Antisense technology involves the use of antisense oligonucleotides (ASOs) to bind to complementary mRNA sequences, thereby inhibiting their translation into proteins. This technology can modulate gene expression at the mRNA level.

2. Mechanism of Antisense Technology:
The mechanism of antisense technology includes several key steps:

Design and synthesis of ASOs: Antisense oligonucleotides are designed to be complementary to specific mRNA sequences. These synthetic ASOs can be modified to increase their stability and binding affinity.
Binding to target mRNA: The ASO binds to the complementary mRNA sequence through Watson-Crick base pairing.

1. 1

2. DNA mRNA Protein
transcriptiontranslation
Central Dogma
2

3. Single-strandedRNAthatiscomplementarytoa
messengerRNA(mRNA)strandtranscribedwithina
cell.
Maybeintroducedintoacelltoinhibittranslationof
acomplementarymRNAbybasepairingtoit.
RNAregulators–controlgeneexpressionbybinding
tosequencesonmRNA,inhibitingtranslation.
Antisense RNA

4. INTRODUCTION
What is antisense RNA Technology?
Antisense RNA Technology >tool used for
inhibition of gene expression.
In this technique Short segments of single
stranded DNA called oligodeoxynucleotidesare
introduced.
These oligonucleotides are complementary to
the mRNA, which physically bind to the
mRNA.
This technique prevents the synthesis of
specific protein.
Powerful weapon for Studying gene function and
fordiscovering more specific treatments of
diseases.

5. History
1990-Richard Jorgenson gave the phenomenon of ‘cosupression
of gene expression’.
1992-Romano and Macino gave the phenomenon of as Virus
Induced Gene Silencing , set of such phenomenon was termed as
post transcriptional gene silencing.
1995-RNA silencing was first documented in animals by Guo and
Kemphues, and for this phenomenon they coined the term antisense
mediated silencing.
1998-Andrew Fire and Craig C.Mello coined the term RNA
interference (RNAi) .

6. 2001-Thomas Tuschl, discovered with his colleagues that
RNAi could be prompted through the use of shorter pieces
of RNA known as small interfering RNAs (siRNAs).
2001-Gregory Hannon identified, described, and named
the "Dicer" enzyme, which chops dsRNA into siRNAs, as
well as the RNA-induced silencing complex (RISC), which
mediates the silencing process by degrading the homologous
mRNA.
2002-Effect named "short hairpin-activated gene silencing"
or SHAGging was introduced.
2004 Morris et al.Observed that siRNA silences genes at
the transcriptional level.
History
Contd….

7. Levels of gene silencing
1. Transcriptional gene silencing (TGS)
It Causes gene silencing by:
• DNA methylation
• Heterochromatin formation.
• Programmed DNA elimination.
2. Post transcriptional gene silencing
(PTGS)
It is known commonly as RNA
interference
(RNAi). It causes silencing by
destruction of
the mRNA of the gene to which the
siRNA
shows perfect complementarity.

8. Transcriptional gene silencing
Transcriptional gene silencing -Result of modifications
of either the histones or DNA.
Gene silencing by modification of Histones and DNA
Modification of nucleosomes alter the accessibility of the
gene to the transcriptional machinery and regulatory
proteins
Heterochromatin is commonly involved in gene silencing,
and affects large sections of DNA.
Methylation of particular DNA sequences can also silence
transcription in many eukaryotes.

9. Post transcriptional gene
silencing
RNA interference (RNAi) is a molecular mechanism
in
which fragments of double stranded nucleic acid
(dsRNA)
interfere with the expression of a particular gene .
The dsRNA can be either, MicroRNA (miRNA) or
Small
interference RNA (siRNA)
https://lh6.ggpht.com/unnamed.jpg

12. Theantisenseeffectofa
oligonucleotidesequencewas
firstdemonstratedin1970sby
ZamecnikandStephenson,in
Roussarcomavirus.
AS-ONsusuallyconsistof15–
20nucleotides,whichare
complementarytotheirtarget
mRNA.

13. WhentheseAS-ONcombined
withtargetmRNA,aDNA/RNA
hybridform,whichdegradedby
theenzymeRNaseH.
RNase H

14. RNaseHisanon-specific
endonuclease,catalyzesthe
cleavageofRNAviahydrolytic
mechanism.
RNaseHhasribonuclease
activitycleavesthe3’-O-Pbond
ofRNAinaDNA/RNAduplex.

15. Mechanism of antisense activity

16. Craig Mello Andrew Fire
(2006 Nobel Prize in Physiology & Medicine)
dsRNA was the suppressing agent!!!
Did simple mathematics
Sense + (-sense) 0 (interference)
RNA interference (RNAi)-first noticed during RNA injection
experiments into the nematode Caenorhabditis elegans.

17. RNAinterference(RNAi)representsanevolutionary
conservedcellulardefensemechanismfor
controllingtheexpressionofaliengenesin
filamentousfungi,microbes,plants,andanimals.
MostwidelyheldviewisthatRNAievolvedtoprotect
genomefromviruses,andperhapstransposonsor
mobileDNAs.
Natural,biologicalmechanisminplants,insectsand
mammals

19. Double-stranded RNA
inject
C. elegans
sense
antisense
•InjectionofdsRNAforspecificgenesintoC.
eleganscausedaspecificdisappearanceofthe
correspondinggeneproductfromboththesomatic
cellsandtheF1progeny.
•dsRNAwasabletoinhibitgenefunctionata
distancefromsiteofinjection
•Appearedtocrosscellboundaries,suggestingthata
smalldiffusiblemoleculemayberesponsibleforthe
repressingeffect.
•VerysmallamountsofdsRNA,requiredtorepress
proteinproduction,suggestingacatalyticor
amplificationprocesswasoccurring.
19

20. Unc-22 (Uncoordinated 22) –Codes for a non essential myofilament
•Injection of either the senseor the antisense RNA strands of a
particular gene into the organism caused little reduction in the
expression of the gene.
•Co-injection of both the sense and antisense RNA strands caused a
massive reduction in the expression of the gene

21. RNA silencing
Steps:
dsRNA or stem-loop containing RNA
Processing & assembly
Silencing complex
Methods of silencing
•mRNA cleavage / RNAi
•Translational repression
•Transcriptional gene silencing
•DNA elimination

22. Definition:RNAinterference(RNAi)isasequencespecificgene
silencingphenomenoncausedbythepresenceofdoublestranded
RNA.
ItiscalledasPost-TranscriptionalGeneSilencing(PTGS)
InPTGS,thetranscriptofthesilencedgeneissynthesizedbutdoesnot
accumulatebecauseitisrapidlydegraded.Thisisamoregeneralterm
thanRNAi,sinceitcanbetriggeredbyseveraldifferentmeans.
Thisisaprocessbywhichdouble-strandedRNA(dsRNA)directs
sequence-specificdegradationofmRNA
dsRNAprocessedtoshortinterferingRNA(siRNA)
RNA Interference (RNAi)

23. Remarkable Properties of RNAi
RNAiis highly gene-specific
dsRNA appears to move freely across cell boundaries
RNAiworks in flies, microbes, fungi, mice, animals
and plants

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Small RNAs that provide target specificity to the silencing machinery
Short interfering RNAs (siRNAs),
Repeat-associated siRNAs(rasiRNAs)
MicroRNAs(miRNAs)
siRNAs
Processed from dsRNA precursors made up of two distinct
strands of perfectly base-paired RNA
siRNAs -21–25bp dsRNA with dinucleotide 3' overhangs
that are processed from longer dsRNA by Dicer in the
RNA interference pathway.
Introduction of synthetic siRNAs can induce RNA
interference in mammalian cells.
siRNAs can also originate from endogenous dsRNA
precursors.

25. miRNAs
Originate from a single, long transcript that forms
imperfectly base-paired hairpin structures
miRNAs -19–23 nt single-stranded RNAs,
Endogenous in origin
miRNAs were discovered through their critical roles in
development and cellular regulation
Represent a large class of evolutionarily conserved
RNAs.
repeat-associated siRNAs, or rasiRNAs.
Endogenous siRNAs derived from repetitive sequences
within the genome

26. How does RNAi work?
1.Initiation step
dsRNA digested into 21-25 ntsiRNAs (guide
RNAs) with the help of Dicer
2.Effector step
siRNA assemble into RISC
siRNA
•guide the RISC
•cleave
•destroy complementary RNA
Mechanism of RNAi

27. Major Actors in RNAi
1.Dicer:ResponsiblefortheprocessingofdsRNAinto
siRNAs.InitiatesRNAi,progressivelycleavesdsRNA21-
25bpintervalstogeneratesiRNAswith2-3’overhangsat3’
ends&phosphorylated5’ends
2.SmallinterferingRNA(siRNA):Producedinvivoby
cleavageofdsRNA.AmplificationbyanRNA-dependent
RNApolymerase(RdRP)occur.IncorporatedintotheRISC
guidingittomRNA
3.RNA-InducedSilencingComplex(RISC):Nuclease
complexcomposedofproteinsandsiRNA.Targetsand
destroysendogenousmRNAscomplementarytothesiRNA

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•Mature RISC, fully
assembled with guide strand
of siRNA –siRISC,
analogous complex
assembled with miRNA,
miRISC
•Perfect complimentarity of
an siRNA or miRNA with its
target results in mRNA
cleavage, partial base
pairing leads to
transcriptional repression
miRNA biogenesis

30. Players in miRNA biogenesis
Drosha
Nuclear RNase III enzyme.
Initiates miRNA biogenesis by cleaving pri-miRNA into
pre-miRNA
Pasha
Partner of drosha is a dsRNA binding protein. Human
DGCR8
Exportin-5
Nuclear transmembrane protein that transports pre-
miRNA form nucleus to cytoplasm. Works in conjunction
with GTP-Ran

31. siRNAs can be produced by:
• Chemical synthesis
• Enzymatic synthesis
• RNase III/Dicer cleavage of long dsRNA
• Plasmid based in vivo expression
• siRNA Expression Cassettes (SECs)

32. Applications

33. Case study-
Flavr SavrTomato
The first FDA approved genetically modified
food
Licensed in 1994
will not soften while ripening on the vine.
Increased shelf life , tomatoes can be shipped
safely, keep their color, and have their natural
flavors.
BEFORE THE FLAVR SAVR
Picked before ripe and gassed with ethylene to
give red color –keeps them from becoming
spoiled
Tomatoes lose their taste and taste more like
“cardboard.”

34. Making of Flavr Savr
Enzyme
Polygalacturonase breaks
down structural
polysaccharide pectin in
wall of a plant.
This is part of the
natural decay process in
a plant
Flavr savr tomatoes have
been constructed that
express an antisense
mRNA complementary to
mRNA for an enzyme
involved in ethylene
production
These tomatoes make only
10% of normal amount of
enzyme thus delaying
ethylene production.

35. GM vs. traditional tomato
Flavr Savr Tomato Traditional Tomato
The Flavr Savr
tomato ripens on the
vine –resulting in
fuller flavor. It is
modified so that it
remains firm after
harvesting
The traditional tomato
must be harvested
while it is still green
and firm so that it is
not crushed on the
way to the
supermarket.
The traditional tomato
is sprayed with
ethylene after
shipping to induce
ripening.
Ripe and
Increased
Flavor.
Ripe but
decreased
flavor.
Supermarket

36. Modification of Brassicaseed oil by antisense expression of a
stearoyl-acylcarrier protein desaturasegene
Moleculargenetransfertechniqueshavebeenusedtoengineerthe
fattyacidcompositionofBrassicarapaandBrassicanapus(canola)
oil.
Stearoyl-acylcarrierprotein(stearoyl-ACP)desaturase(EC
1.14.99.6)catalyzesthefirstdesaturationstepinseedoil
biosynthesis,convertingstearoyl-ACPtooleoyl-ACP.
Seed-specificantisensegeneconstructsofB.rapastearoyl-ACP
desaturasewereusedtoreducetheproteinconcentrationandenzyme
activityofstearoyl-ACPdesaturaseindevelopingrapeseedembryos
duringstoragelipidbiosynthesis.

37. Changed fatty acid composition of brassica oil
Theresultingtransgenicplantsshoweddramaticallyincreasedstearate
levelsintheseeds.
Acontinuousdistributionofstearatelevelsfrom2%to40%wasobservedin
seedsofatransgenicB.napusplant,illustratingthepotentialtoengineer
specializedseedoilcompositions.

38. outcomes:
Basically, the gene in the tomato stops the
tomato from softening during ripening so
that it is easier to ship but keeps its
natural flavors too.
The tomato also has a much longer shelf
life and keeps from spoiling quickly.
PROBLEMS WITH FLAVR SAVR :
Safety-health risks, some environmental
risks
Possible monopolies for businesses
Ethical concerns
Only rich countries can afford it

39. CONCLUSION…
The Antisense RNA technology shows the
potential for
diverse applications to basic research and
therapy.
Antisense technology offers almost unlimited
scope for
the development of new methods of drug design
and one
of the most approved approaches among several
others,
for inactivating a single chosen gene.

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Thank You