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In Living Color
A four-color probe for messenger RNA biomarkers consists of a gold nanoparticle (yellow circle) decorated with short loops of DNA (blue), each with a fluorescent dye on the end (colored circles). When the DNA loops are closed, and the fluorophores are near the gold nanoparticles, the gold quenches the fluorescence (left). But when the DNA sequences bind to their target mRNAs, they open, and the dyes can glow (right).
Credit: Anal. Chem.
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Medical Diagnostics: Gold nanoparticles armed with fluorescent molecular beacons can recognize four cancer biomarkers at the same time, reducing the chance of a false
Cancer researchers have long looked for ways to detect cancerous cells at early stages of the disease. Tools that help doctors diagnose cancer early could increase the success of treatments. One approach has been to search for specific biomarkers, such as RNA molecules, that cancer cells produce large amounts of. But because healthy cells also produce these molecules, testing for just one or two such biomarkers runs the risk of getting a false positive result. Now, researchers in China have developed a cancer probe designed to reduce the chance of falsely identifying cancer cells. They have synthesized a four-color fluorescent probe that can detect the presence of four RNA biomarkers at the same time (Anal. Chem. 2013, DOI:10.1021/ac402700s).
In 2007, researchers at Northwestern University, led by Chad A. Mirkin, developed a strategy to spot these biomarkers by modifying gold nanoparticles to release “nanoflares”—short strands of DNA with a fluorescent dye attached—in the presence of a target messenger RNA sequence (J. Am. Chem. Soc. 2007, DOI: 10.1021/ja0776529). Last year, a team led by Bo Tang ofShandong Normal University, in China, built on that idea, creating a fluorescent three-color probe that used nanoflares to detect three different mRNA biomarkers simultaneously in live cells (Angew. Chem. Int. Ed. 2012, DOI:10.1002/anie.201203767).
But three colors were not enough—an extra color would help to further prevent false positive results, says Shandong Normal University’s Na Li, co-lead author on the new research. Also, Tang and colleagues wanted to cut down the time it took to spot the biomarkers. To meet these two goals, Tang’s team developed a four-color probe with a different signaling mechanism: Instead of using nanoflares, they used so-called molecular beacons that glowed in the presence of mRNA biomarkers.
To make the molecular beacons, the researchers started with snippets of single-stranded DNA that bind to one of four mRNA biomarkers from breast and liver cancer cells. They attached a different fluorescent dye to the end of each of the four DNA strands and then coated 13-nm-diameter gold nanoparticles with the labeled DNA. The strands fold onto themselves to form loops anchored at one end to the nanoparticle. When each fluorescently labeled loop is wrapped up tight, the fluorescent dye rests close to the gold nanoparticle, and the gold quenches the fluorescence. But when the DNA loops bind to their target mRNA sequences, they unfold. That opening moves the fluorescent dyes perched at the loose ends of the DNA strands away from the gold nanoparticle, allowing them to shine.
To test their nanoprobes, the team incubated the armored gold nanoparticles with breast and liver cells, both cancerous and normal ones. The cells take up the particles, and the probes bump into mRNAs inside the cytoplasm. The researchers then measured the fluorescence produced in the cells using a confocal microscope. Both types of cancer cells lit up with all four colors, indicating that all the tested biomarkers were being overexpressed. The healthy breast cells remained dark. But the healthy liver cells glowed in two colors, showing the importance of looking for multiple mRNAs to distinguish between healthy and cancer cells, Li says.
The process of unfolding the DNA loops is faster than sending out a nanoflare, so the new probe gets answers from living cells within three hours, instead of 12 hours with the nanoflare probes, Li says.
The new work “represents a major advancement in the design of gold-nanoparticle-based nanobeacons,” says Chunhai Fanof the Shanghai Institute of Applied Physics, whose group also has made three-color fluorescent nanoprobes. He explains that the surface chemistry necessary for the team to add a fourth color to the nanoprobe is tricky: Adding a new color means determining how to deposit the proper ratio of the probes and picking the dyes to prevent an overlap of colors from each beacon.
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