Believed to be the first technique for imaging RNA in living cells, a new class of beacons under development at the Georgia Institute of Technology and Emory University also has potential applications in the rapid diagnosis of viral infections, as well as drug discovery and pharmacogenomics. Their ability to rapidly detect viruses makes the beacons potentially valuable in the battle against bio-terrorism.
Georgia Tech and Emory researchers are developing improved signaling, targeting and delivery systems for the beacons, which consist of a fluorescent dye molecule and a quencher molecule on opposite ends of an oligonucleotide engineered to match specific genetic sequences associated with disease.
Initially, the dye and quencher molecules are held close together in a hairpin shape, the quencher preventing fluorescent emission from the dye. When delivered into cells, the beacons seek out matching sequences in genetic material known as messenger RNA (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.
Details of the research, sponsored by the Wallace H. Coulter Foundation and the National Science Foundation, will be presented March 26 at the 225th national meeting of the American Chemical Society in New Orleans, LA.
Researchers led by Gang Bao, associate professor in the Department of Biomedical Engineering operated jointly by Georgia Tech and Emory, are improving earlier beacon systems to overcome problems specific to their use in living cells. They have also made progress developing magnetic beacons suitable for use in body tissues too deep for optical imaging to work.
"We want to cover the whole spectrum," Bao explained. "The idea is to use the optical molecular beacons for cellular studies outside the body. 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."
Developed in the mid-1990s, molecular beacons are used today by researchers to detect sequences of nucleic acids in homogeneous solutions. Bao and collaborators Andrew Tsourkas and Phil Santangelo have improved the basic system to enhance accuracy and efficacy in living cells.
Normal enzyme activity within living cells can separate the dye molecule from the quencher, producing a false positive signal. To address that problem, Bao and his collaborators developed a system in which two beacons attach to the same target mRNA on adjacent binding sites. When that happens, fluorescence resonance energy transfer (FRET) 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.
"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," Bao explained. "Therefore, detecting fluorescence due to FRET can significantly reduce signal contamination from beacon degradation and spontaneous opening."
In addition to these innovations, as part of studies to optimize the beacons, researchers have learned how molecular beacon design and other factors affect binding time, specificity and accuracy.
Optical signals can be measured in laboratory tests and in living tissues near the skin, but the light cannot penetrate into deep tissues. To address that need, the researchers are developing beacons containing magnetic nanoparticles. These magnetic beacons take advantage of the fact that when two magnetic nanoparticles attach to adjacent sites of a target mRNA, the disturbance they create in water molecules can be detected with magnetic resonance imaging (MRI).
So far, the researchers have shown that the clustering of magnetic nanoparticles can be detected with MRI, and they have combined the nanoparticles with oligonucleotides necessary for recognizing and binding to target mRNA.
Bao and colleagues are pursuing other improvements, including an ability to target specific organ systems, more rapidly disperse the beacons into cells, and recognize genetic sequences that signify the presence of viruses. The latter work, in which the researchers have shown their ability to detect viral mRNA, could be the basis for tests able to identify viruses within a few hours – instead of days.
With Dr. Karim Godamunne, who holds both a medical degree and an MBA, Bao has formed a start-up company – Vivonetics – to commercialize the patent-pending technology. The company recently received a $50,000 commercialization grant from the Georgia Research Alliance. Many challenges lie ahead.
In early and more curable stages of cancer, the amount of marker RNA is low in bodily fluids such as blood or pancreatic fluid. That means the researchers must develop a detection system sensitive enough to pick up very faint signals.
A definitive cancer diagnosis requires recognizing several markers, so they will also have to use several beacons systems together, each targeting different markers and producing different signals. For in-vivo use, researchers will have to show that the beacons don't harm healthy cells.
For cancer, Bao envisions a comprehensive system in which molecular beacons detect cancerous cells in lab-tested bodily fluids. When appropriate fluids cannot be obtained, other beacons could be introduced into the body to detect the cancerous cells. Beacons could then be used to monitor the success of cancer therapy. And because they specifically attach to mRNA, beacons could perhaps also be used to slow down or halt the growth of cancer cells.
Technical contacts:
1. Gang Bao (404-385-0373); E-mail: (gang.bao@bme.gatech.edu);
2. Dr. Karim Godamunne (404-385-4064); E-mail: (karim.godamunne@venturelab.gatech.edu).