Download Antisense RNA

Interfering RNA (干擾RNA)
RNA interference (RNAi)
Antisense RNA
Micro RNA (miRNA)
ribozyme
Comparision Of different Antisense stratgies
What is antisense RNA??
• Antisense RNA is a single-stranded RNA that is
complementary to a messenger RNA (mRNA) strand
transcribed within a cell.
• They are introduced in a cell to inhibit the translation
machinery by base pairing with the sense RNA and
activating the RNase H, to develop a particular novel
transgenic.
mRNA sequence(sense) AUGAAACCCGUG
Antisense RNA
UACUUUGGGCAC
Antisense RNA mechanism
 RNase H appears to be a
ubiquitous 到處都有
enzyme in eukaryotes and
bacteria.
 The antisense
oligonucleotide bind to
RNA form the
heteroduplex substrate.
Flavr-Savr
• Flavr-Savr the first FDA approved GM food developed
by Calgene in 1992.
• Licensed in may 17, 1994.
• Ripening of tomato causes production of an enzyme
Polygalactourodase in a gradual increasing level, which
is responsible for softening of the tomato and which
becomes the cause of rottening.
• So, tomato never last for few extra days in ripening
condition without rottening. Calgene introduced a gene
in plant which synthesize a complementary mRNA to
PG gene and inhibiting the synthesis of PG enzyme.
Antisense Therapy
• Antisense therapy is a mode of treatment for genetic
disorder 遺傳疾病 or infections 感染
• A complementary 互補 mRNA strand is synthesized on
the basis of the known pathogenic sequence
• Antisense drugs are being researched to treat cancers,
HIV, cytomegalovirus (CMV, 巨細胞病毒) infection
• Formivirsen is the first antisense antiviral drug
developed to treat CMV, licensed by FDA in 1998.
• Successful reducing the viral load of HIV by
developing modified T-Cell, which is antisense to the
HIV envelope protein.
RNA interference (RNAi)
is a mechanism that inhibits gene expression
at the stage of translation 轉 譯 or by
hindering the transcription of specific genes.
a system within living cells that takes part in
controlling genes activity.
Two types of small RNA molecules –small
interfering RNA (siRNA), microRNA (miRNA)
Mello and Fire named the process RNAi, were
awarded the Nobel Prize.
Mechanism of RNA interference
The Players In Interference
• RNA
– siRNA: double strand RNA 21-22 nt.
– miRNA: single strand RNA 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
RNase III-like dsRNA-specific ribonuclease
Enzyme involved in the initiation of RNAi.
digest dsRNA into uniformly sized small RNAs (siRNA)
• Drosha : cleaves base hairpin in to form pre miRNA;
which is later processed by Dicer
Mechanism of RNAi
1. dsRNA are chopped into short interfering
RNAs (siRNA) by Dicer.
• 2. The siRNA-Dicer complex recruits additional
components to form an RNA-Induced
Silencing Complex (RISC). The siRNA unwinds.
• 3. The unwound siRNA base pairs with
complementary mRNA, thus guiding the RNAi
machinery to the target mRNA.
• 4. The target mRNA is effectively cleaved and
subsequently degraded – resulting in gene
silencing.
Small interfering RNAs (siRNAs)
• Double strand RNA
• Each strand of siRNA has:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’ overhangs
miRNA
• miRNAs are genomically encoded non-coding
RNAs that help regulate gene expression, particularly
during development
• Originate from capped & polyadenylated full length
precursors (pri-miRNA)
• Hairpin precursor ~70 nt (pre-miRNA) Mature
miRNA ~22 nt (miRNA)
Difference between miRNA and 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.
Problems and solutions of RNAi
• Delivery of siRNA to tissue is a problem both because:
– The material must reach the target organ
– And must also 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
• Toxic effects: although very specific, it can still cause
unintended damage
• Solutions: Viral delivery, bacterial delivery, use of liposomes
or nanoparticles, chemical modification of siRNA to improve
stability (still only half-life several hours)
Conclusions
• The RNAi technology shows the potential for
diverse applications to therapy
• offers almost unlimited scope for the development
of new methods of drug design
• The beauty and future potential of antisense
depends on the design of multiple drugs based on
our increasing knowledge of genes and their
functions.
• However, the full commitment of this promise is
yet to be established.
Single bout exercise can change
miRNAs
• Single bout of moderate cycling ↑ and ↓several
miRNA in human muscles
– rapid, yet transient regulation with endurance
exercise, contribute to the positive adaptations of
endurance exercise.
Antisense RNA and exercise
• Slow-to-fast muscle type conversion in soleus
比目魚肌 after unloading
– Antisense RNA suppress IIa MHC gene
• Resistance exercise IIb  IIx MHC in white
gastronemius muscle 腓腸肌
– Normally, 90% MHC expression in white
gastronemius is Iib
– Normally, antisense RNA to IIx
– After resistance training, ↓antisense RNA to IIx
microRNA and its targets in venous blood
after running at 80% VO2peak for 30 min
Tonevitsky, 2013
Regulatory network of miRNA in
venous blood after exercise
Tonevitsky, 2013
Changes in
circulating
miRNA after
acute resistance
exercise
Sawada, 2013
Changes in miRNAs and their target
proteins after exercise in human muscles
Russell, 2013
Ribozymes: ribonucleic acid + enzyme
• RNA molecules which have catalytic activity
which degrade nucleotides .
• Ribozyme bind to the target RNA moiety and
inactivate it by cleaving the phosphodiester
backbone at a specific cutting site.
• Ribozyme destroy RNA that carries the
massage of disease.
• Can be effectively used against HIV virus.
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