Gene Targeting: Antisense RNA technology and RNA i

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Gene Targeting: Antisense RNA technology and RNA interference technology Gene Targeting: Antisense RNA technology and RNA interference technology by Call Me Gene

Gene Targeting: Antisense RNA technology and RNA interference technology
By N. L. Eskeland, Ph.D. and N. C. Bailey, Ph.D.

Two methods that pharmaceutical companies are using to make drugs that can block the expression of disease-causing proteins are: antisense RNA, and the dsRNAi (double strand RNA interference) technologies.

Antisense RNA ? Antisense RNA technology emerged in the 1980s. It is based on short DNA oligonucleotides that are designed to pair with a piece of mRNA to block the translation of mRNA into protein.

The antisense molecules are usually short, ~19-25 nucleotides(nt) long, chemically-modified complementary nucleotide chains that hybridize to a specific complementary area of mRNA, degrading the mRNA and thus preventing its translation by the ribosome to a functional protein.

RNAi (RNA interference) ? Small interfering RNAs were discovered in the late 1990s when experiments on the nematode Caenorhabditis elegans revealed that a 21-23 nucleotide dsRNA (double strand RNA) mediated post-transcriptional gene silencing. Small interfering RNAs (siRNA) are produced in vivo when the ribonuclease enzyme Dicer cleaves long dsRNA small synthetic dsRNA that are either directly introduced into the organism, or through a transgene or virus. RNA-induced silencing complex (RISC) gets activated by siRNA, which is incorporated into the complex, and cleaves the target mRNA. Pharmaceutical founders achieved their success in mammalian cell gene silencing with siRNAs at concentrations tens-to-hundreds of times lower than those typically used in antisense experiments. The remarkable effectiveness of siRNAs lies in the fact that target mRNA destruction is achieved by eliciting a natural cellular process.

Mechanism of the dsRNA mediated gene silencing process
Long double-stranded RNAs (dsRNAs; usually greater than 200 nt) are introduced in a cell that contains the gene that needs to be silenced. First, the dsRNAs get processed into 20-25 nt small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer (initiation step). Second, the siRNAs assemble into complexes that contain endoribonuclease. The complexes are known as RNA-induced silencing complexes (RISCs). The siRNA strands then guide the RISCs to complementary target RNA molecules, where they cleave and destroy the RNA.

In mammalian cells, introduction of long dsRNA (>30 nt) initiates a potent antiviral response, which results in nonspecific inhibition of protein synthesis and RNA degradation. The mammalian antiviral response can be avoided by introducing or expressing siRNAs in the cells.

Several biotechnology companies are using antisense RNA and RNAi technologies to target cancer and cardiovascular illnesses. In addition, scientists are using both methods in functional genomics to elucidate the functions of proteins.

It is worth noting that Andrew Z. Fire and Craig C. Mello were awarded the 2006 Nobel Prize in Physiology or Medicine for their discovery of RNA interference.
Questions:

1. When introduced into a mammalian system, which RNA is more stable-the antisense single strand RNA (ssRNA) or the siRNA? Why?

Answer: si RNA is more stable than ssRNA since it is double-stranded. It is not subject to degradation by RNAse enzymes.

2. Check the internet for companies using gene targeting technology and determine the type of diseases being studied with either antisense RNA, or the RNA interference technology.

References: The above article is adapted from the high school workbook 'Know Your Gene', found at http://www.science2discover.com http://www.ambion.com/techlib/append/RNAi_mechanism.html http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01suschultz/homepage.html

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