VOLUME 12, NUMBER 20 • APRIL 20, 2001

Gold Quenches Fluorescence Better than Dabcyl in Molecular Beacons, Rockefeller Physicists Determine

Researchers at The Rockefeller University’s Center for Studies in Physics and Biology are designing tools that will help molecular biologists achieve greater accuracy in some of their experimental work. Benoit Dubertret, a post-doctoral fellow in the Laboratory for Experimental Condensed Matter Physics, and his colleagues have designed a probe that is eight times more powerful than conventional molecular beacons at detecting single nucleotide mismatches out of a mixture of random sequences. Their findings appear in the April issue of Nature Biotechnology.

Called a probe, or a molecular beacon, the material created by the researchers joins a family of molecular tools that have been in use for about five years. For the first time gold is being used as a quencher in a molecular beacon.

Working in the laboratory of Detlev W. Bronk Professor Albert Libchaber, Dubertret has created a hybrid probe composed of a single strand of DNA, a 1.4-nanometer diameter gold particle and a fluorophore.

Molecular beacons can be made in many different colors using a broad range of fluorescent dyes called fluorophores. The ends of the single-stranded DNA forming the beacon can self-hybridize, forming a stem loop structure. In this state, the fluorophore and the quencher are held in close proximity, resulting in quenching of fluorescence. Fluorescence is restored when the stem loop structure is altered, as is the case when a complementary single-stranded DNA sequence hybridizes to the loop of the beacon.

The recognition of targets by beacons is so specific that single-nucleotide differences can be readily detected. The hybrid molecules are useful for detecting RNAs within living cells, for monitoring the synthesis of specific nucleic acids in sealed reaction vessels, for homogenous one-tube assays for genotyping single-nucleotide variations of DNA, and in PCR reactions for the detection of multiple pathogenic agents.

One of the innovations of Dubertret and Libchaber’s work is that they have determined that a very small gold colloid can quench fluorescence very well. Dubertret explains, "Although this fact had been predicted in the 1980s, it had never before been demonstrated experimentally." And part of the challenge was determining just how small to make the gold colloid.

In a comparison test, the scientists replaced the 1.4-nanometer gold cluster with a 0.8 nanometer gold cluster to explore the dynamic quenching potential of the metal. The smaller particle did not quench fluorescence even though it has the same surface ligands and active group.

In the future, researchers should be able to use the metal to an even greater quantitative advantage than formerly possible using dabcyl, the non-fluorescent chromophore used on all earlier molecular beacons. It should be possible to optimize gold quenchers for each dye and use the differently colored gold-quenched beacons for simultaneous quantitation of multiple sequences.

The great advantage to using gold-quenched beacons is their greater sensitivity to single nucleotide mismatches of DNA sequence. This strength results from gold’s ability to quench a wider range of colors than dabcyl, and to quench all colors better.

In comparison tests between dabcyl and gold-quenched beacons, the gold-quenched beacon fared significantly better than the dabcyl-quenched beacon, especially in a low-salt buffer, which tends to impair detection rates of dabcyl-quenched beacons in all colors. A better quencher also would widen the range of current applications of the beacon, such as detection of smaller amounts of DNA or RNA.

While the gold-quenched biomolecules created by Dubertret and Libchaber are likely to increase the accuracy of molecular beacons as they currently are used, other applications also are possible.

"We created the new beacon because we wanted a better-quenching molecule to attach to an optical fiber, for use as a biosensor," says Dubertret. A biosensor is a device that can read and transmit responses to physical or chemical changes. The devices can be implanted in the human body to monitor various physiological states or conditions.

Support for this research was provided by the Mathers Foundation.