When Yale biologist Ronald R. Breaker and a colleague at Scripps Research Institute created the first DNA enzyme in 1994, they wondered whether such enzymes existed eons ago only to be abandoned during evolution as a failed experiment, or whether nature has yet to explore their unique biological benefits.
"This question strikes at the very heart of biology -- whether DNA was meant to do more than just store genetic code," says Mr. Breaker, an assistant biology professor who joined the Yale faculty in 1995. "We can readily generate chemically active DNA enzymes in a test tube, but we have no evidence that they exist in nature.
"DNA enzymes may have existed in primitive organisms to provide both a genetic blueprint and a means of triggering cell division in one compact package, only to be thrown in the trash heap by natural selection when more efficient protein enzymes evolved," he says.
In the December issue of the journal Chemistry and Biology, Professor Breaker describes how he created a new class of self- cleaving DNA enzymes that can fold into chemically active molecules and cut themselves into segments. Collaborating on the research were Yale postdoctoral fellow Nir Carmi and former undergraduate Lisa A. Shultz.
The next step, he notes, is to genetically engineer a DNA enzyme that can cut apart the genetic code of a harmful organism like the HIV virus, rendering it harmless.
DNA enzymes also could make effective biosensors for detecting toxic chemicals in the environment or for medical diagnostics -- an idea for which Yale and Professor Breaker have jointly filed a provisional patent application. Specific enzymes would be tailor-made to break down only in the presence of target molecules.
Professor Breaker's discovery builds upon the Nobel Prize- winning research of Yale biochemist Sidney Altman, who shared the 1989 prize in chemistry for isolating and identifying the first RNA enzymes, or ribozymes. That discovery in the late 1970s and early 1980s, which exploded the myth that RNA is merely a passive carrier of genetic code incapable of triggering cell activity, eventually spawned a whole new branch of genetic engineering and spurred the search for similar DNA enzymes.
"My best guess is that DNA enzymes will be able to do most things that ribozymes can do. If you can get a ribozyme to work as a therapeutic agent, you should be able to make an analogous DNA enzyme to do the same thing," says Professor Breaker, adding that four other research groups in the United States and Germany have replicated his earlier success by creating additional DNA enzymes.
Some ribozymes already have been developed to function as precision scissors that can snip out flawed gene segments and splice in corrected versions. The method has potential for treating diseases ranging from cystic fibrosis to muscular dystrophy and sickle cell anemia.
Because DNA is a million times more stable than RNA, DNA enzymes could enjoy a much longer shelf life than ribozymes, although it is not clear whether they would better withstand the intense chemical onslaught of the body. It is likely, however, that DNA enzymes can be developed that will cut and splice both RNA and the sturdier DNA, opening a new avenue for treating genetic diseases and fighting viruses, says Professor Breaker.
Because DNA also lends itself well to test-tube evolution techniques, it can be synthesized readily in the laboratory, and different strains of enzymes can be genetically engineered for specific purposes, Professor Breaker says.
To find the self-cleaving DNA, he and his colleagues began by synthesizing more than 10, trillion-trillion random DNA sequences using a computerized DNA synthesizer. Then they washed a grid containing the sequences with a compound of copper and ascorbic acid -- vitamin C -- thereby breaking down some of the DNA sequences. By cloning the DNA sequences that are washed away by the copper compound and then repeating the process eight times, the Yale biochemists isolated the desired enzymes.
"The DNA is perfectly stable until you throw in a metal ion -- copper in our latest experiments -- and then it falls apart. While a normal, double-stranded, or double helix, version of DNA is too rigid to fold itself into a biological catalyst, the single-stranded DNA that is washed off apparently is flexible enough to fold itself into an enzyme," he says.
The experimental design is classic test-tube evolution -- Professor Breaker sets up a system of natural selection to produce DNA sequences with the characteristics he desires through evolution. "Our next step is to take these enzymes and force nature to use them," he says. "For example, we are on the verge of forcing an engineered bacterium to rely on the function of a self-cleaving DNA enzyme."
The Yale scientists also hope to use DNA enzymes to attack and destroy single-stranded DNA inside cells, which would be an important advance. The HIV virus, for example, goes through a single-stranded DNA stage while reproducing.