FRET Nanoflares Ferret Out False Positives

Scientists from Hunan University have improved intracellular mRNA sensors, making them less prone to false positives.

AsianScientist (Aug. 7, 2015) – By combining gold nanoparticles coated with DNA and fluorescently labelled donor and acceptor molecules, researchers have developed a new class of nanoprobes that can detect mRNA in living cells more accurately. Their research has been published in the Journal of the American Chemical Society.

False positive signals occur frequently in biosensors. Over the past decade, many different gold nanoparticle-based fluorescent DNA probes such as molecular beacons and nanoflares have been developed to tackle this issue.

Traditionally, these nanoprobes use single-dye labelled DNA coupled with gold nanoparticles to design a detection mechanism. In close proximity between the fluorophore and gold nanoparticles, fluorescence is quenched. In the presence of target DNA, hybridization between DNA occurs, separating the fluorophore and gold nanoparticles. This process will result in the recovery of fluorescence signals.

While these conventional probes have been developed with varying degrees of success, these probes have a greater propensity to create false positive signals that are generated by thermodynamic fluctuations, nuclease degradation, protein binding or even glutathione (GSH) competition in cells.

Large background signals generated from these conventional nanoprobes reduce the selectivity and sensitivity of biosensors. In addition, another challenging problem has also been to accurately determine the concentration of targets such as metal ions, DNA, mRNA in each sensing techniques.

In the present study, a team of researchers led by Professor Wang Kemin from Hunan University has found a way to reduce false positive signals.

Beginning with gold nanoparticles, they used gold-thiol bond formation to coat the nanoparticle surface with DNA. The DNA-gold nanoparticles were then linked to acceptor and donor fluorophores at their 5′ and 3′ ends respectively, forming what the researchers call fluorescence resonance energy transfer (FRET) nanoflares.

In the absence of target DNA, fluorescence is observed as the donor and acceptor fluorophores are separated. As soon as the target DNA binds, however, the fluorophores are displaced. This causes the formation of the hairpin structure, bringing the donor and acceptor fluorophores into close proximity and quenching the fluorescence.

The researchers showed that the FRET nanoflares could selectively detect mRNA, responding with a seven fold decrease in fluorescent signals in the presence of a single base mismatch. In intracellular sensing, the FRET nanoflares are exposed to Dnase I and glutathione (GSH) which are capable of hydrolyzing DNA and destroying gold thiol bonds. Despite the potential interference, the FRET nanoflares demonstrated low levels of background changes as compared to large signal changes in conventional nanoprobes.

Furthermore, the FRET nanoflares also demonstrated excellent abilities in detecting mRNA in two types of cancer cell lines.

While their selectivity and sensitivity have not been validated with clinical samples, the FRET nanoflares offer researchers a new possibility of reducing and minimizing false positive signals. In addition, these probes can potentially be further developed into ratiometric sensing probes that are more accurate in vivo.

The article can be found at: Yang et al. (2015) FRET Nanoflares for Intracellular mRNA Detection: Avoiding False Positive Signals and Minimizing Effects of System Fluctuations.

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Copyright: Asian Scientist Magazine; Photo: Journal of the American Chemical Society.
Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff.

Kang Yong is pursuing chemistry at the University of Illinois at Urbana-Champaign. With more than three years of experience on nanomaterials research, he is keen on making an impact on the biomedical field with these tiny materials. Outside the lab, he enjoys running, travelling and composing. As a hobby, he is setting up his own recording studio to make his own renditions of songs in his cosy apartment in Urbana, IL.

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