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Georgia Institute of Technology, Emory University, Whitehead Institute for Biomedical Research

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Georgia Researchers Use ‘Molecular Beacons’ in Pharmacogenetics Work

A new class of “molecular beacons” being developed at the Georgia Institute of Technology and Emory University may have applications in pharmacogenomics, and may be used to rapidly diagnose certain cancers and viral infections.

Researchers at Georgia Tech and Emory are developing better signaling, targeting, and delivery systems for the beacons, which comprise a fluorescent dye molecule and a quencher molecule on opposite ends of an oligo engineered to match specific genetic sequences associated with disease. The research is believed to be the first technique for imaging RNA in living cells.

“The idea is to use the optical molecular beacons for cellular studies outside the body,” according to Gang Bao, associate professor in the Department of Biomedical Engineering. “You can combine that with a delivery system and additional technologies to do shallow tissue imaging. With the magnetic beacons, we could do deep-tissue imaging.”

Bao is scheduled to present results of his group’s research at the national meeting of the American Chemical Society in New Orleans this week.

Molecular beacons, developed in the mid-1990s, are used to detect sequences of nucleic acids in homogeneous solutions. Bao, together with teammates Andrew Tsourkas and Phil Santangelo, has “improved the basic system” of molecular beacons “to enhance accuracy and efficacy in living cells,” according to Georgia Tech.

Initially, the dye and quencher molecules are held closely together in a kind of “hairpin shape” as the quencher prevents the dye from emitting fluorescence. When delivered into cells, the beacons seek out matching sequences” in genetic mRNA. “If the beacons encounter and bind with their specific mRNA targets, Watson-Crick base-pairs holding the dye and quencher together break, allowing emission of a specific fluorescent signal when excited by light,” according to the statement.

Soon, the research team encountered a problem: Normal enzyme activity in living cells can separate the dye molecule from the quencher, producing a false positive signal. To address this, the researchers developed a system in which two beacons attach to the same target mRNA on adjacent binding sites.

“When that happens, fluorescence resonance energy transfer between a donor beacon and an acceptor beacon creates a red-shifted optical signal that can be distinguished from false signals produced by the enzymatic digestion of single beacons,” the team said.

“FRET is extremely sensitive to the distance between donor and acceptor molecules, so it occurs only when the donor and acceptor molecules are bound to the same mRNA target,” said Bao. “Therefore, detecting fluorescence due to FRET can significantly reduce signal contamination from beacon degradation and spontaneous opening.”


Whitehead Staff Uncover Way to Screen CML Patients for Resistance to Gleevec

Researchers at the Whitehead Institute for Biomedical Research have devised a way to identify genetic mutations linked to resistance to a certain cancer drug that exist before patients are treated. This finding may help physicians monitor patients for resistance problems before they occur, according to the Whitehead.

The scientists developed the screen to identify SNPs believed to cause patients with chronic myeloid leukemia to become resistant to Gleevec, the pioneering pharmacogenetic-based drug that targets a protein produced by BCR/ABL, the gene that causes CML.

“In the leukemia cells of a CML patient, BCR/ABL is constantly mutating,” said George Daley, leader of the research team at Whitehead that developed the screening strategy. “Cells carrying certain mutations can resist the drug and continue to grow while the remainder of leukemia cells are suppressed. We have found a way to discover those mutations experimentally.”

To arrive at their conclusions, Daley’s team used recombinant DNA methods to randomly mutate the BCR/ABL gene in order to mimic potential variations that might be found in CML patients. The mutated genes were then transferred into millions of mouse blood cells and exposed to Gleevec. While most cells succumbed to the drug, some of the cells with specific variations thrived, the researchers reported in their study.

“We looked for those cells that continued to grow, extracted them and sequenced their genes,” said Mohammad Azam, a postdoctoral researcher at Whitehead and lead author of the study, which was published in the March 21 issue of Cell. In all, that examination revealed 15 mutations that other researchers “had previously linked to Gleevec resistance in CML patients — plus 97 more.”

“With the catalogue of mutations in hand, physicians could one day examine patients who’ve relapsed because of resistance to Gleevec and pinpoint which of the 112 mutations is causing problems, or better still, detect the presence of a mutation before the patient relapses,” the Whitehead said in a statement.

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