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RESEARCH PROFILE RNA enzyme reactions on a chip
COURTESY OF TRACY PAXON
bozyme cleavage reaction] Researchers at Pennsylvania along faster in order to get State University have found good kinetic data,” says a way to simplify the kinetic Ewing. analysis of RNA enzymes, or In the system’s current ribozymes, by using capillary configuration of only five array electrophoresis on a michannels, a lag time of ~1 min crochip with automated samoccurs between the start of ple introduction. The new the reaction and when the approach, which is described first sample hits the channel. in this issue of Analytical The cleavage reaction is iniChemistry (pp 6921– 6927), tiated offline. The sample is could provide a better underthen put into a chamber and standing of ribozyme cleavpressurized, says Paxon. “We age reactions for use in cliniare in the process of incorcal settings. porating another valve such Ribozymes show great that we can pressurize our promise for treating various chamber faster,” she adds. illnesses from infectious disThe researchers are also tryeases to cancer. But their ap- Penn State researchers have developed an automated capillary sample ing to increase the number plication has been limited introduction system for monitoring RNA enzyme kinetics on a microchip. of channels. It takes ~14 s because of a lack of fundaautomatically, says Paxon. between the first and second injections, mental knowledge about their cleavage To demonstrate their new technique, says Paxon. “We would like to decrease mechanisms, structure, and stability. the researchers studied a model ribozyme that to ~2 s so that we would be able to Kinetic studies could help provide this called leadzyme, which undergoes selfget 7 data points in the same amount of information. cleavage in the presence of Pb2+ ions. time it is taking to get 2 right now,” she Traditionally, polyacrylamide gel elecadds. With only 5 channels, by the time trophoresis has been used to study riThe leadzyme makes an ideal model beof the second injection, the reaction has bozyme kinetics. A cleavage reaction is cause it is small, reacts at room temperaproceeded beyond 50% cleavage. “For initiated, and at various time intervals, a ture, and is easy to label with a fluoressample is removed, quenched, and sepacent tag. In addition, the rate of cleavage any kinetic reaction, in order to get reliable data, you need to have more than rated in parallel on a gel. The process is can be manipulated simply by adjusting one data point before 50% cleavage,” time-consuming, labor-intensive, and tethe concentration of Pb2+. she explains. dious. To automate and simplify the pro“The system that our collaborators One alternative is to actually perform cedure, the Penn State researchers creat[coauthors Trevor Brown and Phil the reaction on a chip, says Ewing. That ed a microchip-based device with a novel Bevilacqua at Penn State] eventually want is a completely different approach, howsample introduction system for continu- to get at is actually the hepatitis delta ever, and the researchers are still working ous analysis of the reaction. virus [HDV],” says Ewing. “We would In a previous version of the device, like to know more about the cleavage re- on ways to accomplish that. Interest in ribozymes appears to be “Everything was done manually,” says action and to understand the mechanism on the rise. “Ribozymes were fairly oblead author Tracy Paxon, a doctoral stuof [the HDV] ribozyme,” he adds. If a scure in the early 1980s because they dent in Andy Ewing’s lab at Penn State. person is infected with the hepatitis B were just discovered,” says Bevilacqua. “Every time an injection needed to be virus and has this HDV ribozyme, proThey gained more notoriety after the made, the user would toggle a manual gression of the disease is much faster, Nobel Prize was awarded in 1989 for valve,” she explains. To improve upon says Paxon. “The long-term goal is to their discovery. “I think now people are this earlier version, the researchers intebe able to control the activity of these using them a little bit more because of grated a six-port valve that could be pro- ribozymes,” she adds. grammed with their software. The valve The HDV ribozyme reacts much faster the possible therapeutic uses. Analytical was coupled to a manipulator, so that than the model leadzyme, making it diffi- chemists are in general doing more with RNA, not just with ribozymes, but also every time the manipulator moves a cult to study its kinetics using the current with aptamers,” he adds. a capillary into the channel, the six-port configuration of the system. “We need —Britt E. Erickson valve closes and an injection is made to get more channels and move [the ri426 A
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