Antisense Nucleotides

Antisense nucleotides are strings of RNA or DNA that are complementary to "sense" strands of nucleotides. They bind to and inactivate these sense strands. They have been used in research, and may become useful for therapy of certain diseases.

Antisense RNA

Messenger RNA (mRNA) is a single-stranded molecule used for protein production at the ribosome. Because its sequence is used for translation, mRNA is called a "sense" strand or sense sequence. A complementary sequence to that mRNA is an "antisense" sequence. For instance, if the mRNA sequence was AUGAAACCCGUG, the antisense strand would be UACUUUGGGCAC. Complementary sequences will pair up in RNA just as they do in DNA. When this happens to an mRNA, however, it can no longer be translated at the ribosome, no protein synthesis occurs, and the "duplex" RNA is degraded.

This phenomenon has been used experimentally and commercially to block the synthesis of specific proteins in transgenic organisms (ones to which a foreign gene has been added). The strategy is to add a synthetic gene that, when transcribed, will make the antisense RNA sequence for the target protein's mRNA.

This technique was first used commercially in 1988 for the FlavrSavr tomato. The gene chosen for inactivation was polygalacturonase (PG), whose enzyme unlinks pectins in the plant cell wall, thereby softening it. The intent was to increase the time the fruit could be left to ripen without softening, thus increasing flavor of commercial tomatoes. The Calgene company created a transgenic tomato plant expressing the antisense RNA for PG mRNA, and reduced PG production by up to 90 percent. Although the tomato was not a commercial success, it demonstrated the potential for this strategy.

Antisense RNA is currently being investigated as a human therapy for certain forms of cancer. The goal is to use gene therapy techniques to insert an antisense gene into tumor cells. Many cancers are due to overexpression of the genes that promote cell proliferation, called tumor suppressor genes. Antisense RNA might be able to inhibit this overexpression. Another target is the BCL-2 gene, whose protein prevents apoptosis, or programmed cell death. In certain cancers, the BCL-2 gene is overactive, preventing death of cells and leading to their proliferation. Antisense therapy against BCL-2 is currently being tested under the trade name Genazyme.

Antisense DNA

Antisense DNA strands can also be made (note that in the double helix, the side of the DNA that is transcribed is itself antisense). Short antisense strands of DNA can be introduced into cells, which then bind with target mRNA. Antisense DNA is currently an approved therapy for cytomegalovirus infections of the eye, under the trade name Vitravene. Vitravene targets two different viral proteins. Antisense DNA is also being explored for therapy of HIV, some cancers, and other diseases.

One advantage of using antisense therapy in treating infectious diseases such as virus infections is that it can be tailored to the particular strain in circulation, and then modified as the virus mutates. One difficulty in applying this therapy is successfully delivering the antisense DNA or RNA to all target tissues (for instance, making sure the antisense strands reach infected blood cells for HIV). Another problem is maintaining prolonged suppression of target protein expression, since the antisense molecule will eventually be degraded by the cell's nuclease enzymes. One strategy to prevent degradation is to chemically modify the DNA to interfere with nuclease action.

RNA Interference

Investigation of the mechanism of action of antisense RNA led to the surprising discovery that naturally occurring double-stranded RNA molecules (dsRNA) suppress gene expression as well as or better than antisense sequences. This suppression by dsRNA of expression of the related gene is called RNA interference. dsRNA molecules are cut into short segments by nucleases; the antisense strand of such a segment then peels off and binds with its complementary mRNA. This new, double-stranded RNA is then subject to further nuclease attack. RNA interference is believed to be an ancient means of protecting against double-stranded RNA viruses. Further understanding of RNA interference may lead to improvements in or replacement of antisense therapies.

Richard Robinson

Bibliography

Smith C. J. S., et al. "Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes." Nature 334 (1988): 724-726.

Tamm I., B. Dorken, and G. Hartmann. "Antisense Therapy in Oncology: New Hope for an Old Idea?" Lancet 358, no. 9280 (2001): 489-497.

Internet Resource

"Antisense DNA." Michigan State University. <http://www.cem.msu.edu/~cem181h/projects/97/antisense/dia1.gif>.