Targeting messenger RNA

The antisense approach is use of nucleic acids to reduce the expression of a specific target gene. As shown in Figure 58.2, a small piece of DNA, an oligodeoxynu-cleotide that is in the reverse orientation (antisense) to a portion of a target messenger RNA (mRNA) species, is introduced into a cell and a DNA-RNA duplex is formed by complementary Watson-Crick base pairing. Cessation of protein synthesis then may result from the rapid... 

Small interfering RNAs (siRNA) are short double-stranded RNAs that bind to target messenger RNAs (mRNA) and interfere with translation of encoded proteins. This method for gene silencing is under investigation for therapy of diseases including cancer and Carbon nanotubes have been used... 

Discovered in the 1980s, ribozymes are RNA molecules with a very distinct hammerhead structure. They function as enzymes and directly cut RNA. Synthetic ribozymes targeted to specific messenger RNAs have been used in many laboratory studies with cells. Efforts to develop ribozyme treatments targeted to specific cancer genes and for other uses have not succeeded in clinical trials, partly because the structure of the ribozyme that is needed to allow it to work as an enzyme was destroyed when it was injected. 

RNA is only a small fraction of total RNA, but it is expressed in high concentrations in tissues that express the target protein. Therefore messenger RNA isolated from enriched sources is likely to contain RNA sequences that encode for the target protein. 

During this process some key questions are (1) Does the DNA sequence inserted into the expression vector contain all the sequences essential for the target protein (2) Is the messenger RNA expressed at the predicted length and produced in high enough levels (3) Does the recombinant protein react with antibody (4) Is there evidence of functional activity of the recombinant protein Methods for answering these questions are listed in Table 4.4. 

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