Dismantling Dangerous Genetic Codes With A Faster, More Flexible RNA Mimic

July 30, 1998

OK, so maybe you can't fool Mother Nature, but that hasn't stopped a chemist at Washington University in St. Louis from imitating her.

James K. Bashkin, D. Phil., assistant professor of chemistry at Washington University, has created a molecule that mimics the behavior of a kind of naturally occurring RNA (ribonucleic acid) called a ribozyme, which acts as a catalyst. Bashkin's ribozyme mimic can dismantle the dangerous genetic codes involved in propagating viral and fungal diseases, certain cancers and even the HIV virus. The discovery shows great promise for improved viral and cancer chemotherapy and other potential drug treatments.

In 1994, Bashkin was the first scientist to make synthetic ribozymes, and the first to publish on the concept, which was conceived and patented in the mid-1980s while working with colleagues at Monsanto's Corporate Research Laboratories in St. Louis. About the same time, two other researchers suggested an alternative, organic approach to ribozyme mimics, which differed from Bashkin's metal-based ideas. Since then, several academic research groups and biotechnology companies have been hot on the trail of developing and testing Bashkin's ribozyme mimic design, or variations of it.

In the May 1998 issue of the Journal of Chemical Communications, Bashkin reported a newer, improved ribozyme mimic with the flexibility to home in on several specific regions of an HIV gene and cleave the RNA to destroy the harmful genetic message. He reported a higher level of specificity and greater dismantling speed than before. Several biotechnology companies now are planning to test the mimics in live cell cultures, moving Bashkin's concept closer to clinical trials.

"Dramatic Control"

"We've developed dramatic control over the specificity of the mimics as well as their reaction rate, or speed," said Bashkin. "Specificity is important because if you miss the targeted spot, you have the possibility of damaging normal RNA. Speed is vital to destroy the harmful target right away. The method is compatible with living cells and shows great promise for chemotherapy." Because of his status as the inventor of metal-based ribozyme mimics, Bashkin was the invited editor of the May 1998 issue of Chemical Reviews, an issue devoted entirely to DNA and RNA cleavage. In the issue, he and his students reviewed the progress made around the world in this area, and scientists worldwide reviewed additional aspects of this chemistry and biochemistry.

Ribozymes are a form of RNA that act as catalysts. DNA (deoxyribonucleic acid) is a double-stranded molecule, located in the nucleus of every human cell; it contains the genetic plans of the genes contained on 46 chromosomes. RNA (ribonucleic acid) is a single-stranded molecule that acts in tandem with DNA. It is responsible for carrying out the plans of DNA by directing the formation of proteins.

Bashkin has chemically engineered a new class of DNA molecule that recognizes specific chemical sequences of messenger RNA (mRNA) and destroys them to prevent making harmful proteins. The molecule, which he calls a "functional ribozyme mimic," uses water molecules, abundant in cells, in a chemical reaction to attack specific, targeted sequences of mRNA. Because his functional mimic is a catalyst, it is not destroyed in this action but instead remains intact to destroy many more targeted mRNA molecules.

The chemist and his Washington University co-workers prepare the molecules in a machine called a DNA synthesizer, which assembles molecules based on the chemical subunits adenine, guanine, cytosine and thymine, the four base pairs of DNA. To a strand of DNA, Bashkin attaches a 28-atom reactive arm that includes a metal-based inorganic catalyst, chosen from several families of metal-based catalysts that Bashkin and his group are investigating. His ribozyme mimics, using specific base-pair sequences, recognize the HIV mRNA and breaks the mRNA at certain base pairs. This blocks the mRNA from acting as a template for protein production.

A key recent change to Bashkin's ribozyme mimics is the incorporation of a small chemical fragment near the catalyst that replaces an actual DNA base pair. Bashkin says the replacement gives the ribozyme mimics their flexibility and also makes preparation easier, speeding up the testing of the molecules. "They're much easier to prepare because they're based on a partial DNA building block, not a complete one, so they're not as chemically complex," he says. Bashkin's achievement could lead to cheaper chemotherapy drugs that produce far fewer side effects because his ribozyme mimics recognize the chemistry of dangerous mRNA, leaving normal mRNA alone. Chemotherapy typically causes severe side effects because the method cannot completely differentiate between healthy and diseased cells.

Making Sense Of Antisense

Bashkin's advances should help make sense of the "antisense" technique for controlling gene expression. Antisense gets its name from the way complementary, or matching, DNA or RNA sequences bind to specific mRNA molecules, inactivating their genetic message. The technique goes hand-in-hand with the landmark discovery of ribozymes in the 1980s by University of Colorado researcher Thomas Cech, Ph.D., and Yale researcher Sidney Altman, Ph.D. Cech and Altman shared a Nobel Prize in 1989 for their revolutionary discovery that RNA can function as a catalyst. It had been thought that only proteins could serve as biological catalysts, or enzymes.

Bashkin's co-author of the Chemical Communications paper, Andrew T. Daniher, Ph.D., is now working for Cech in Boulder, Colo.

"None of this would have been possible without the talented group of graduate students, postdoctoral workers and undergraduates that I have had the pleasure of working with," Bashkin notes.

The work has been supported by the National Science Foundation, the Donors of the Petroleum Research Fund (administered by the American Chemical Society), Monsanto Company, the Lucille P. Markey Foundation, the American Cancer Society and the Pharmaceutical Manufacturer's Association.

Bashkin's ribozyme mimic is designed to be a catalyst that cannot destroy itself but that can destroy many copies of the targeted RNA. Naturally occurring ribozymes may be used in chemotherapy, but they are large, expensive and fragile RNA molecules. Bashkin's mimics, however, are based on stouter DNA molecules; they are smaller, simpler and lighter than naturally occurring ribozymes, and can be made to better resist enzyme degradation as well.

"We wish nothing but success for natural ribozymes as potential drugs," says Bashkin. "But we believe that it is vital to provide an alternative that may have significant advantages in drug development."

Benchmark For All Catalysts

Along with developing the mimics, Bashkin and his group have created several families of catalysts for their design and have developed a testing procedure for a wide variety of compounds that have similar catalytic potential. "We're using the assay as a benchmark for all of our catalysts and those that others have developed, so we have a way to see what really works best," he says.

"The assay also lets us study how the individual catalysts work to help us avoid being caught by any chemical surprises when we use them in a biological system. It's becoming a very popular assay."

Bashkin says the antisense community is large and growing, and his concepts can be incorporated into any of the antisense approaches.

"Most (antisense) efforts are directed toward things like getting good RNA target sequences and developing good DNA analogs with good pharmacological properties to use for humans," he says. "What we're doing can be added to improve any approach by making the best antisense compounds possible. The potential of catalytic antisense drugs is tremendous. It's the only method we have that uses the genetic code to recognize dangerous RNA and then destroys that RNA in a biocompatible manner."

Washington University in St. Louis

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