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Antisense Nucleotides

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.

see also Gene Therapy; Nucleases; Nucleotide; RNA Interference; Transgenic Plants.

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>.

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Antisense Nucleotides

Antisense Nucleotides

Antisense nucleotides are either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecules that are complementary to a messenger RNA (mRNA) molecule. Because these molecules are complementary to given mRNA, they will bind to the RNA and form a free double-stranded molecule or double-stranded region of a chromosome . The double-stranded molecules are not able to interact with ribosomes and, as a result, a particular protein is unable to be made. Inhibiting the production of a given protein may be important in the control and treatment of many diseases such as cancer.

Two approaches to antisense nucleotides have been tried: (1) direct introduction of antisense nucleotides into cells and (2) synthesis of antisense nucleotides within the cell. In the first approach, short antisense oligonucleotides are introduced directly into cells in hopes that they will interact with the appropriate mRNA. Scientists are using different nucleotides that are complementary to different regions of the mRNAbeginning, middle, or endin an attempt to determine the most effective sequence. Unfortunately, enzymes within cells often degrade these short oligonucleotides before they can interact with the target mRNA. Replacing the phosphate linkages in the nucleotides with sulfur or other linkages seems to prevent degradation.

The second approach involves using a vector (a vehicle for transferring genetic material) containing the entire gene to transfer DNA into the cells. This DNA will theoretically integrate into the chromosome, duplicate at each cell division, and remain within the cells. These vectors are constructed so that the control sequences for transcription are on the DNA strand opposite to the one that is usually used for transcription. Therefore, when inducers are added, the cells make the antisense RNA, which then binds to mRNA from the normal gene. In many cases, the amount of an undesirable protein is reduced.

The use of antisense nucleotides is in its infancy, but the results have been promising in reducing certain types of cancer in animals. The procedure has the potential of becoming widely used in the future to treat a variety of diseases, provided that it has low risks associated with it.

see also DNA; Gene; Hybridization; RNA

William R. Wellnitz

Bibliography

Campbell, Neil A., Jane B. Reece, and Lawrence G. Mitchell. Biology, 5th ed. Menlo Park, CA: Benjamin Cummings, 1999.

Pasternak, Jack J. An Introduction to Human Molecular Genetics. Bethesda, MD: Fitzgerald Science Press, Inc., 1999.

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antisense RNA

antisense RNA An RNA molecule whose base sequence is complementary to that of the RNA transcript of a gene, i.e. the ‘sense’ RNA, such as a messenger RNA (mRNA). Hence, an antisense RNA can undergo base pairing with its complementary mRNA sequence. This blocks gene expression, either by preventing access for ribosomes to translate the mRNA or by triggering degradation of the double-stranded RNA by ribonuclease enzymes. Like antisense DNA, antisense RNA has therapeutic potential for modifying the activity of disease-causing genes. Also, genes encoding antisense RNAs can be used in genetic engineering to alter the makeup of organisms. For example, the FlavrSavr tomato was engineered with an artificial gene for antisense RNA that prevented expression of a gene for an enzyme involved in ripening, in order to retard spoilage. In the 1980s it was discovered that double-stranded RNA molecules have a much greater ability to suppress their corresponding genes than single-stranded RNAs, due to a phenomenon called RNA interference. This is now the focus of much research.

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antisense

antisense, DNA or RNA manipulated in a laboratory so that its components (nucleotides) form a complementary copy of normal, or "sense," messenger RNA (mRNA; see nucleic acid). Antisense techniques are used to deactivate disease-causing or undesirable genes so that they cannot produce harmful or unwanted proteins. (Conventional drugs bind directly with disease-causing protein molecules, but their imperfect specificity may lead them to bind with other protein molecules, resulting in unwanted side effects. Antisense molecules are extremely specific.) In some applications of this technique, the antisense nucleic acid segment is inserted into an inactivated or nonvirulent virus, then introduced into the cell. The antisense segment pairs with the mRNA, preventing the synthesis of protein by the mRNA. Antisense has applications in agricultural biotechnology, where it has been used to deactivate the gene that causes softening in tomatoes, and in medicine, especially in cancer and antiviral therapy. See also gene therapy.

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antisense DNA

antisense DNA A single-stranded DNA molecule that can bind to a complementary base sequence in a particular messenger RNA (mRNA) molecule and so prevent synthesis of the protein encoded by the mRNA. Antisense DNA thus has the potential to block the expression of a particular gene, hence its potential as a therapeutic weapon to combat certain diseases. A short DNA strand, called an antisense oligodeoxynucleotide (ODN), is constructed consisting of 15–20 deoxynucleotides complementary to a segment of the target mRNA. By binding to the mRNA, the ODN may either prevent translation of the mRNA by ribosomes or trigger degradation of the mRNA by cellular enzymes. To be effective drugs, ODNs have to be chemically modified to resist degradation by DNase enzymes and must be attached to some kind of vector that will deliver them to the target cells. An antisense ODN product has already been approved in the USA for the treatment of cytomegalovirus infections of the eye. See also antisense RNA.

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complementary DNA

complementary DNA (cDNA) A form of DNA prepared in the laboratory using messenger RNA (mRNA) as template, i.e. the reverse of the usual process of transcription in cells; the synthesis is catalysed by reverse transcriptase. cDNA thus has a base sequence that is complementary to that of the mRNA template; unlike genomic DNA, it contains no noncoding sequences (introns). cDNA is used in gene cloning for the expression of eukaryote genes in prokaryote host cells, or as a gene probe to locate particular base sequences in genomic DNA.

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Complementary DNA

Complementary DNA

Resources

Complementary deoxyribonucleic acid (DNA) is DNA in which the sequence of the constituent molecules on one strand of the double-stranded structure chemically matches the sequence on the other strand.

A useful analog is to picture a key and a lock. While there are many different types of keys, only one design matches the contours of the lock and so will fit into the lock. The different chemical molecules that make up DNA also do not pair up nonspecifically. A lock-in-key fit operates at the molecular level.

The chemical molecules that make up DNA are known as nucleotide bases. There are four common types of bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). In the chemical lock-and-key fit, an A on one strand is able to form a bond only with a T; for the two strands of DNA to intertwine together, the As on one strand of DNA must pair with Ts on the other strand. Similarly, C on one strand always pairs with a G on the other strand. Thus, the sequence of one DNA strand determined the sequence of the other strand, which is described as being complementary.

Complementary DNA (cDNA) is a copy of a region of a strand of DNA. For example, if the original DNA strand had a sequence of ATT, the complementary sequence will be TAA. The cDNA will bind to the complementary site on the DNA strand.

Complementary DNA is important naturally, in the manufacture of new copies of DNA, and has become an important experimental tool. In DNA replication, the two strands are unwound from one another. A molecule called DNA polymerase runs the length of each strand, making a complementary copy of each strand. In other words, each strand acts as a blueprint to produce a complementary strand. The two new strands are complementary to one another, and so can join together in a process called annealing. The old strands also anneal. The result is two complete copies of DNA.

Complementary DNA has been exploited to develop research techniques and to produce genetically altered commercial products. A classic example of cDNA is the technique of polymerase chain reaction (PCR). PCR mimics the process of DNA manufacture in a test tube. In a series of reactions, a target stretch of DNA is copied, and the copies themselves serve as templates for more copies. The original DNA sequence is amplified to make a billion copies within minutes.

Because ribonucleic acid (RNA) is made using DNA as the blueprint, the phenomenon of complementary strands also extends to RNA. Like DNA, RNA is made of four bases; three of the bases are the same as found in DNA (A, C, and G). But, instead of T, RNA contains a base called uracil (U). In RNA, A pairs with U and C pairs with G. Complementary RNA (cRNA)is a copy of a strand of RNA that will bind to the appropriate region of the original molecule. If the original RNA stand had a base sequence of AUU, for example, the sequence of the cRNA strand would be UAA.

The association of a DNA or RNA strand to its complement is one of the basic research tools of the molecular biologist. Binding of a complement can identify target regions of DNA or RNA, and can be used to disrupt the process of DNA manufacture. If the complementary DNA is labeled with a compound that fluoresces, then the binding of the fluorescent probe can actually be visualized using a microscope. This permits the so-called real time examination of DNA synthesis.

Resources

Books

Drlica, Karl. Understanding DNA and Gene Cloning: A Guide for the Curious. New York: Wiley, 2003.

Gerlach, Neil. The Genetic Imaginary: DNA in the Canadian Criminal Justice System. Toronto: University of Toronto Press, 2004.

Synder, L., and W. Champness. Molecular Genetics of Bacteria, 2nd ed. Washington, DC: American Society for Microbiology Press, 2002.

Periodicals

Volchkov, V.E., V.A. Volchkova, E. Möhlberger, et al.Recovery of Infectious Ebola Virus From Complementary DNA: RNA Editing of the GP Gene and Viral Cytotoxicity. Science 229 (March 2001): 19651969.

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Complementary DNA

Complementary DNA

Complementary deoxyribonucleic acid (DNA) is DNA in which the sequence of the constituent molecules on one strand of the double stranded structure chemically matches the sequence on the other strand.

A useful analog is to picture a key and a lock. While there are many different types of keys, only one design matches the contours of the lock and so will fit into the lock. The different chemical molecules that make up DNA also do not pair up nonspecifically. A "lock in key" fit operates at the molecular level.

The chemical molecules that make up DNA are known as nucleotide bases. There are four common types of bases: adenine (A), cytosine (C), guanine (G), and thymine (T). In the chemical "lock and key" fit, an A on one strand always pairs with a T on the other strand. As well, a C on one strand always pairs with a G on the other strand. The two strands are described as complementary to one another.

Complementary DNA (cDNA) is a copy of a region of a strand of DNA. For example, if the original DNA stand had a sequence of ATT, the complementary sequence will be TAA. The cDNA will bind to the complementary site on the DNA strand.

Complementary DNA is important naturally, in the manufacture of new copies of DNA, and has become an important experimental tool. In DNA replication , the two strands are unwound from one another. A molecule called DNA polymerase runs the length of each strand, making a complementary copy of each strand. In other words, each strand acts as a blueprint to produce a complementary strand. The two new strands are complementary to one another, and so can join together in a process called annealing. The old strands also anneal. The result is two complete copies of DNA.

Complementary DNA has been exploited to develop research techniques and to produce genetically altered commercial products. A classic example of cDNA is the technique of polymerase chain reaction (PCR ). PCR mimics the process of DNA manufacture in a test tube. In a series of reactions, a target stretch of DNA is copied, and the copies themselves serve as templates for more copies. The original DNA sequence is amplified to make a billion copies within minutes.

Because ribonucleic acid (RNA) is made using DNA as the blueprint, the phenomenon of complementary strands also extends to RNA. RNA is made of four bases; adenine (A), cytosine (C), guanine (G), and uracil (U; instead of the thymine found in DNA). In the lock in key scenario, an A pairs with the U) on the other strand, and a C always pairs with a G. Complementary RNA (cRNA) is a copy of a strand of RNA that will bind to the appropriate region of the original molecule. If the original RNA stand had a base sequence of AUU, for example, the sequence of the cRNA strand would be UAA.

The association of a DNA or RNA strand to its complement is one of the basic research tools of the molecular biologist. Binding of a compliment can identify target regions of DNA or RNA, and can be used to disrupt the process of DNA manufacture. If the complementary DNA is labeled with a compound that fluoresces, then the binding of the fluorescent probe can actually be visualized using a microscope . This permits the "real time" examination of DNA synthesis .

See also DNA technology; Microorganisms.


Resources

books

Synder, L., and W. Champness. Molecular Genetics of Bacteria. 2nd ed. Washington, DC: American Society for Microbiology Press, 2002.

periodicals

Aizaki, H., Y. Aoki, T. Harada, et al., "Full-length Complementary DNA of Hepatitis C Virus From an Infectious Blood Sample." Hepatology 27 (February 1998): 621–627.

Chien, Y.-C., Y.-J. Zhu, and C.-M. Chuen. "Complementary DNA Cloning and Analysis of Gene Structure of Pyruvate Kinase From Drosophila melanogaster." Zoological Studies 38 (February 1999): 322–332.

Volchkov, V.E., V.A. Volchkova, E. Mühlberger, et al. "Recovery of Infectious Ebola Virus From Complementary DNA: RNA Editing of the GP Gene and Viral Cytotoxicity." Science 229 (March 2001): 1965–1969.

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"Complementary DNA." The Gale Encyclopedia of Science. . Retrieved September 18, 2018 from Encyclopedia.com: http://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/complementary-dna-0

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