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Calixarene tool kit can read epigenetic codes A group of researchers at the University of Victoria has demonstrated that calixarene molecules can be used to read information encoded on DNA-packaging proteins called histones. The discovery provides a new tool for the emerging field of epigenetics, the study of heritable information stored in molecules other than DNA and RNA. In the past, histones were thought of as spools around which DNA was wound. More recently, post-translational modifications to the histones — for example, acetylation or methylation of certain amino acids — have been shown to play a role in determining which genes get expressed at which times. This epigenetic ‘histone code’ can be probed by antibodies in enzyme-linked immunosorbent assays (ELISAs). But such assays have shortcomings. “Some code elements are really similar and difficult to distinguish,” says Fraser Hof, professor of chemistry at the University of Victoria, noting that the failure rate with antibodies is over 20 per cent. Hof’s group has been working on an alternative approach based on calixarenes. These cup-shaped macromolecules bind preferentially to certain histone code elements. In a paper recently published in the Journal of the American Chemical Society, Hof’s group described a new assay in which various calixarenes, each paired with a fluorescent dye, were exposed to peptides bearing the modifications of the histone code. The dyes were quenched by binding to the calixarenes, but histone code elements compete for the binding site. Since each calixarene has a different affinity for a given code element, a pattern of fluorescent responses results. Taken together, the signals lead to a unique ‘fingerprint’ for each code element.
Cup-shaped calixarene molecules can bind to the post-translational modifications that are added to the amino acids of proteins called histones. here, a monobrominated p-sulfonatocalix[4]arene (spheres) binds to a trimethyl group (stick figures) which is attached to a lysine residue. Such a system could assist researchers probing the epigenetic code, which regulates how genes are turned on and off in complex organisms.
A set of only three calixarenes was sufficient to distinguish histone code elements with a high degree of reproducibility. “We really didn't expect this to work so well; I thought we were going to need up to 10 different sensors,” says Hof. Even better, the system works in real time, unlike ELISA. The team hopes it can be used to study the activity of the enzymes that add and remove histone code elements.
the change in charge is often small compared with the background, such sensors are not sensitive enough to detect analytes at low — but still physiologically relevant — concentrations. The new assay developed by Das and his colleagues relies on a neutralizer made of peptide nucleic acid (PNA). The charge of this synthetic DNA analogue can be tuned by adding cationic amino acids to the end, while its affinity for the DNA probe can be controlled by introducing mismatches to its sequence. A properly designed PNA sequence will neutralize the probe but will be dislodged when the molecule of interest binds to the probe instead. This results in a bigger charge difference than with DNA alone and allows for the detection of neutral molecules, even at low concentrations. In a paper published in Nature Chemistry, the team shows that the new system works effectively with probes designed for DNA, RNA, ATP and even cocaine. Best of all, the electrodes can be miniaturized and embedded on chips, allowing for fast and portable systems capable of detecting hundreds of analytes simultaneously. A spin-off company founded by Kelley, Xagenic Inc., is working toward developing commercial systems. The technology could have applications in medicine, forensics and many other fields.
September 2012 CAnAdiAn ChemiCAl news
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