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) Antisense RNA UACUUUGGGCAC AUGAAACCCGUG

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 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 siRNA (small interfering RNA) http://en.wikipedia.org/wiki/Small_interfering_RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a class of 20-25 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.

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) http://en.wikipedia.org/wiki/MiRNA A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 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 20-25 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.

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.