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Scripps research scientists develop general-purpose method for detecting trace chemicals

Described in an advance, online publication of the journal Nature Biotechnology on February 22, 2009, the team's general method could be adapted for detecting a wide variety of compounds, including many that are relevant to diagnostic medicine and environmental work.

"This technology could be used to measure drugs and metabolites in the body or to measure toxic compounds in soil or groundwater," says Professor Gerald Joyce, M.D., Ph.D., who authored with paper with a postdoctoral fellow in his lab, Bianca Lam, Ph.D. Joyce is the dean of the faculty at Scripps Research, where he is also a professor in the Department of Molecular Biology, the Department of Chemistry, and The Skaggs Institute for Chemical Biology.

Joyce's new method is based on a class of special RNA replicator molecules that he and his colleagues reported earlier this year. In the presence of specific chemicals, these RNA molecules will replicate exponentially, detectably amplifying their concentration. And the more of the target chemical that is present, the faster the RNA molecules will replicate.

"The development of these RNA replicators provides researchers with a valuable new tool for detecting the presence of specific molecules and measuring their levels," says Richard Ikeda, Ph.D., who oversees enzymology grants at the National Institute of General Medical Sciences of the National Institute's of Health, which partially funded the research. "There is tremendous potential for application of this technology in diagnostic, environmental, and chemical testing."

Similar to DNA, RNA is a basic component of cells and plays many roles in the body, including helping to transfer genetic information from DNA into active protein enzymes, which carry out many of the body's vital functions. Scientists have known for many years that some types of RNA molecules are themselves enzymes. More than 40 years ago, Nobel laureate Francis Crick proposed that RNA, and in particular self-replicating RNA, may have once been the basis of life on the early Earth more than four billion of years ago.

In the few decades since this "RNA world" was first proposed, research on RNA has blossomed. But it was not until recently that Joyce and colleagues provided the first example of self-replicating RNA enzymes, a discovery that they reported in the journal Science last month. The goal of the Scripps Research scientists is both to better understand the origins of life and to come up with designer molecules with useful properties that can be exploited for medicine and other applications.

The basis of the new chemical detection method is a system of paired RNA enzymes that can "cross-replicate" each other. Each enzyme in the pair is composed of two pieces, and the enzymes only can function when the two pieces are joined together. When they are active, each enzyme joins together the pieces that form the other enzyme. Each makes the other, and continues to do so repeatedly, requiring only a small starting amount of the two enzymes and a steady supply of the subunits.

In this latest research, Joyce and his colleagues extended this basic system by adding "aptamers" to the RNA enzymes. This modifies the enzymes such that they will only replicate if they first bind a completely separate chemical. The chemical must be present for cross replication to occur at all, and the higher the concentration of this target chemical, the faster the system will replicate.

In their paper, Joyce and his colleagues showed that they could use cross-replicating RNA enzymes containing aptamers to detect and quantify two different chemicals. One of these was the drug theophylline, which is used to treat asthma and chronic obstructive pulmonary disease. People who are taking this drug often have to have their theophylline blood levels closely monitored. Complications arise because existing diagnostic technologies lack specificity they cannot, for instance, distinguish between theophylline and similar chemicals, such as caffeine.

The cross-replicating RNA molecules do not have this limitation. In the study, if theophylline was present, the molecules cross-replicated. If caffeine was present, they did not. The concentration of theophylline was proportional to the speed of replication, which could be determined through a routine laboratory assay.

Because RNA aptamers can be made to recognize nearly any protein and many other molecules, says Joyce, this method should be general enough to detect a variety of chemicals significant in human health and the environment. Another potential application is in the area of molecular computing. At their heart, computers perform operations by moving and storing electronic charges in logical circuits. These same basic operations can also be accomplished biologically, using molecules instead of electrons as the basis of the logical circuitry. Self-replicating RNAs that are triggered by target chemicals could provide the functional basis for some of these operations, says Joyce.


Contact: Mika Ono
Scripps Research Institute

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