1. Interfering RNA (干擾RNA)
RNA interference (RNAi)
Antisense RNA
Micro RNA (miRNA)
ribozyme

2. Comparision Of different Antisense stratgies

3. 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)
Antisense RNA UACUUUGGGCAC
AUGAAACCCGUG

4. 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.

5. 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.

6. 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.

7. 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.

8. Mechanism of RNA interference

9. The Players In Interference
RNA
siRNA: double strand RNA nt.
miRNA: single strand RNA 19-25nt. Encoded by non protein coding genome
RISC:
RNA induced Silencing Complex, that cleaves mRNA
Enzymes
Dicer : produces 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

10. Mechanism of RNAi
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.

11. 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
siRNA (small interfering RNA)
Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a class
of nucleotide-long RNA molecules that interfere with the expression of genes.
They are naturally produced as part of the RNA interference (RNAi) pathway by the enzyme Dicer.
They can also be exogenously (artificially) introduced by investigators to bring about th
knockdown of a particular gene.
siRNA's have a well defined structure.
Briefly, this is a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt overhangs
on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group.
Transfection of an exogenous siRNA is problematic, since it is only transient, and the
dsRNA structure cannot easily be permanently maintained.
One way of overcoming these problems is to modify the siRNA in such a way as to allow it
to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction
of a loop between the two strands, thus producing a single transcript, which can be processed
into a functional siRNA.
This transcription cassette usually uses an RNA polymerase III promoter, which direct the
transcription of small nuclear RNA's, such as U6 or H1. It is assumed (although not known
for certain) that the resulting short hairpin RNA (shRNA) transcript is processed by Dicer.
Introduction of too much siRNA can result in non-specific events due to activation of the interferon
pathway. Most papers suggest that this is probably due to activation of the dsRNA sensor PKR,
although retinoic acid inducible Gene I (RIG-I may also be involved
One method of reducing the non-specific effects is by turning the shRNA into a micro RNA.
Micro RNA's are naturally occurring, and, as such, tolerated better by the cell.
By engineering an siRNA sequence into an miRNA structure, non-specific effects can
potentially be eliminated.

12. 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)
miRNA (micro-RNA)
A miRNA (micro-RNA) is a form of single-stranded RNA which is typically nucleotide long.
It is thought to regulate the expression of other genes.
miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence
includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is
transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base
pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA).
In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA.
The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5,
a carrier protein. The Dicer enzyme then cuts nucleotides from the base of the hairpin to release
the mature miRNA.
In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes
place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.

13. 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.

14. 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)

15. 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.

16. 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.

17. 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

18. microRNA and its targets in venous blood after running at 80% VO2peak for 30 min
Tonevitsky, 2013

19. Regulatory network of miRNA in venous blood after exercise
Tonevitsky, 2013

20. Changes in circulating miRNA after acute resistance exercise
Sawada, 2013

21. Changes in miRNAs and their target proteins after exercise in human muscles
Russell, 2013

22. 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.