This document provides an overview of RNA interference (RNAi), including its definition, mechanism, generation of small interfering RNA, delivery methods, and applications. RNAi is a conserved mechanism in eukaryotes where double-stranded RNA is cut into small interfering RNA by the enzyme Dicer. These siRNAs then bind to complementary mRNA and induce its degradation via the RNA-induced silencing complex. The document discusses the key components of RNAi, differences between siRNAs and microRNAs, generation of siRNAs, challenges with siRNA delivery, and applications of RNAi technology in areas like cancer treatment, stem cell research, and infectious disease.

1. RNA interference (RNAi)
From:-
Dr. Abhishek Kumar Mishra
Assistant Professor
Department of Biosciences
Swami Rama Himalayan University
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2. Learningobjectives
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At the end of the discussion you will be able to learn:-
 Definition of RNA interference
 Mechanismof RNAinterference
 Generation of smallinterfering RNA
 Smallinterfering RNAdeliverymethods
 Applicationsof RNAinterference

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RNAsilencing&RNAinterference
Several terms are used to described RNA silencing; usually there are
three phenotypically different but mechanistically similar phenomena:
1. Cosuppression or post-trascriptional gene silencing (PTGS) in plants
2. Quelling in fungi
3. RNA interference in animal kingdom

4. RNAi is a form of RNA based immunity, targeting viruses and
genomic repeated sequences
RNAi is viewed as a conserved ancient mechanism protecting
genomes from nucleic acid invaders
The technique has proven effective in Drosophila,
Caenorhabditis elegans, plants and in mammalian cell cultures
RNAi evolved as a mechanism most likely to protect organisms
from viruses modulate transposon activity, eliminate aberrant
transcription products
RNA interference (RNAi)

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Needfor interference
• DefenseMechanism
-Defense against Infection by viruses, etc
-As a defense mechanism to protect against transposons
and other insertional elements
• GenomeWide Regulation
-RNAi plays a role in regulating development and genome
maintenance
-30% of human genome regulated

6. PTGSin plants: Discoveryof Jorgensenand Napoli(1990)
• They were trying to make petunias more purple
• Overexpression of petunia gene
• Entered homologous RNA
Expected: more pigments
Observed:white sectors
Cosuppression:
Loss of mRNAs of both endo-and transgene
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7. Discovery
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Inject worms with dsRNA
corresponding to a gene
(important for muscle function)
involved in wiggling (unc-22)

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RNAiOverview
• During RNAi Double-stranded RNAs cut into short double-
stranded RNAs, s(small) i(interfering) RNA's, by an enzyme
called Dicer
• These then base pair to an mRNA through a dsRNA-enzyme
complex. This will either lead to degradation of the mRNA
strand
• Highly specific process
• Very potent activity
• So far only been seen in eukaryotes
• Evidence 30% of genome is regulated by RNAi

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ThecomponentsIn Interference
• RNA
-siRNA: dsRNA 21-22 nt.
-miRNA: ssRNA 19-25nt. Encoded by non protein coding
genome
• RISC:
-RNA induced Silencing Complex, that cleaves mRNA
• Enzymes
-Dicer : produces 20-21 nt cleavages that initiate RNAi
-Drosha : cleaves base hairpin in to form pre miRNA; which is
later processed by Dicer

10. Small interfering RNAs (siRNA)
• Small interfering RNAs that have an integral role in the phenomenon
of RNA interference (RNAi), a form of post-transcriptional gene
silencing
• RNAi: 21-25 nt fragments, which bind to the complementary portion
of the target mRNA and tag it for degradation
• A single base pair difference between the siRNA template and the
target mRNA is enough to
block the process.
• Each strand of siRNA has:
a.5’-phosphate termini
b.3’-hydroxyl termini
c.2/3-nucleotide 3’ overhangs 16

11. MicroRNA(miRNA)
• Originate from capped & polyadenylated full length precursors(pri-
miRNA)
• Hairpin precursor~70 nt (pre-miRNA) Mature miRNA~22 nt
(miRNA)
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12. DifferencebetweenmiRNAand siRNA
• Function of both species is regulation of gene expression.
• Difference is in where they originate. siRNA originates with dsRNA.
• siRNA is most commonly a response to foreign RNA (usually viral)
and is often 100% complementary to the target.
• miRNA originates with ssRNA that forms a hairpin secondary
structure. miRNA regulates post-transcriptional gene
expression and is often not 100% complementary to the
target.
• Also miRNA help to regulate gene expression, particularly during
induction of heterochromatin formation serves to downregulate
genes pre- transcriptionally (RNA induced transcriptional silencing
or RITS)
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13. DICER
RNaseIII-like dsRNA-specificRibonuclease
Enzymeinvolved in the initiation of RNAi.
It is able to digest dsRNA into uniformly
sizedsmallRNAs (siRNA).
Dicer family proteins are ATP-dependent
nucleases.
RNAseIII enzymeactsasadimer
Loss of dicer→loss of silencing processing in
vitro
Dicer homologs exist in many organisms
including C.elegans, Drosphila, yeast and
humans (Dicer isa conserved protein)
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14. Dicer’sdomains
1 4 2 2
Dicer is a Ribonuclease (RNAse III family) with 4 distinct domains:
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Composed of 4 different domains:-
1.Amino-terminal helicase domain
2.Dual RNAse III motifs in the carboxy terminal segment
3.dsRNA binding domain
4.PAZ domain (110-130 amino-acid domain present in protein like
Argo, Piwi..);it is thought to be
important for protein-protein interaction
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15. • RISC is a large (~500-kDa)
RNA-multiprotein complex,
which triggers mRNA
degradation in response to
siRNA
• Unwinding of double-
stranded siRNA by ATP
independent helicase
• The active components of an
RISC are endonucleases called
argonauteproteins which cleave
the target mRNA strand
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RNA induced silencing complex (RISC)

16. Mechanisms of RNA silencing

17. Illustrationof miRNAprocessing
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18. Generation of smallinterference RNA
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• Effective methods for the delivery of small RNA to allow a
sufficient silencing effect in the target organ(s) and/or cells are yet
to be developed
• In particular, toxicity and side effects of RNAi must be well
characterized and limited
• Therefore, careful design and selection of target sequence and
quantification of the effect on the expression of target protein and
mRNA are essential for success of gene interfering approaches
siRNA: Delivery

20. High-pressure injection
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• “High-pressure injection” was the first strategy to demonstrate
successful delivery of siRNA in vivo
• A large volume (1–2mL) of saline containing unmodified siRNA
is injected intravenously into the tail vein of mice within very
short time (in less than 7 sec), which presumably results in the
siRNA molecules being forced into several organs mainly the
liver, kidney and to a lesser degree the lung
• Certainly, such an approach seems to be impossible in human
subjects (1000 mL saline solution containing siRNA per 10 kg of
weight)

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• Delivery of siRNA to tissue is a problem both because:
– The material must reach the target organ
– And it must enter the cytoplasm of target cells
• RNA cannot penetrate cellular membranes, so systemic
delivery of siRNA is unlikely to be successful
• RNA is quickly degraded by RNAse activity in serum and even
siRNA chemically modified to be more stable has a half-life of
only a few hours at most
DeliveryProblems

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• For these reasons, other mechanisms to deliver siRNA to
target cells has been devised
These methods include:
• Viral delivery
• Theuseof liposomesor nanoparticles
• Bacterial delivery
• Chemicalmodification of siRNAto improve stability
Solution

23. Viraldelivery
• Viral delivery has been used extensively
in gene therapy to deliver DNA to target
cells
• There are 5 main classes of viruses used
in the delivery of nucleotides to cells:
• Retrovirus
• Adenovirus
• Lentivirus
• Baculovirus
• Adeno-associated-virus (AAV).
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24. Liposome'sandnanoparticles
• Liposomes and nanoparticles have been known
as an alternative to viral delivery systems.
• Unmodified siRNA has a half-life of less
than 1 hour in human plasma and siRNA is
rapidly excreted by the kidneys.
• Liposomes and nanoparticles can act as
envelopes to protect the siRNA from
metabolism and excretion, but can also carry
specific molecules designed to target the
siRNA to specific tissue types
• Liposomes such as Lipofectamine have been
used to carry siRNA into cells
• Nanoparticles such as the cationic polymer,
polyethyleneimine (PEI) have also been used
to successfully deliver siRNA to target cells 37

25. Bacterialdelivery
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• Bacterial delivery using nonpathogenic bacteria has been used to
silence genes in a process known as transkingdom RNA interference
(tkRNAi)
• Generally, the shRNA is produced in bacteria that invade and release
the RNA into eukaryotic cells (hence the term transkingdom)
• The bacteria can also be engineered to carry shRNA encoding DNA
plasmids
• The advantages of this system include:
-Safety
-Abilityto controlthe vectorusingantibiotics

26. Chemicalmodification
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• Finally, chemical modification of siRNA has been used to
improve stability and prevent degradation by serum RNAase
• Importantly, these modifications must obviously not affect the
RNA interference activity of the siRNA
• One of the most common modifications is the use of locked nucleic
acidresidues (LNA).
• A methylene bridge connects the 4.C with the 2.O in LNA residues.
This modification increases the stability of oligonucleotides in
serum, without reducing the gene silencing effect

27. ApplicationsofRNAi
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RNAi in treatment of hematologic disorders result from defective
genes
RNAi in treatment of Cancers by targeting oncogenes
• Improvement efficacy of chemotherapy and radiotherapy
• Tumor regression through creation of potentially new mode of
chemotherapy
RNAi in Stem cell research
• Knockout tumor-suppressiongenein mouseembryonicstem cell
• Observetumor phenotype
• Positive correlation between extent of Trp 53 (suppression gene) inhibition
andseverityof disease
RNAi in treatment of infectious diseases
• RNAi targetstranscriptase&inhibitsviralreplication

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RNAinterferencecharacteristics
• dsRNA needs to be directed against an exon, not an intron in
order to be effective
• Homology of the dsRNA and the target gene/mRNA is required
• Targeted mRNA is lost (degraded) after RNAi
• ssRNA does not work as well as dsRNA

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Advantageof RNAi
• Down regulationof geneexpressionsimplifies"knockout" analysis.
• Easierthan useof antisenseoligonucleotides.siRNAmoreeffective
and sensitive at lowerconcentration.
• Costeffective
• HighSpecificity
• Middle region9-14 are mostsensitive
• With siRNA,the researchercansimultaneously perform
experimentsin anycell type of interest
• Canbelabelled
• Easeof transfectionbyuseof vector

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ImportanceofRNAi
• Powerful for analyzingunknown genesin sequenced genomes.
• More efforts are beingundertaken to target every human gene via
siRNAs
• Fasteridentification of gene function
• Genetherapy: down-regulation of certain genes/ mutated alleles
• Cancertreatments
– knock-out of genesrequired for cell proliferation
– knock-out of genesencoding keystructural proteins
• Agriculture

32. THANK YOU
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