Nanocluster Beacon (NCB): A DNA-Silver Nanocluster Probe that Fluoresces upon Hybridization

Abstract

Oligonucleotide-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that have seen applications in cellular imaging and chemical/biological detection. Recently we discovered the red fluorescence emission of DNA/Ag NCs could be enhanced more than 500 fold when brought into close proximity to a guanine-rich DNA sequence (Yeh et al., Nano Letters, 10 (8): 3106-3110, 2010). Based on this finding, we developed a new type of molecular probe (termed NanoCluster Beacon, NCB) that fluoresces upon target DNA binding. Compared to molecular beacons, NCBs require only a single labeling step and do not rely on F [[Unable to Display Character: ő]]rster energy transfer as fluorescence switching mechanism. Moreover, there is no need to remove the silver nanocluster precursors (Ag+ and BH4-) used during nanocluster formation, as these are essentially non-fluorescent. Our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers. It is also the first demonstration that a turn-on probe can be made based on fluorescent noble metal nanoclusters.Our initial investigation demonstrated NCB detection of an influenza target with a signal-to-background (S/B) ratio five times better than that of a conventional molecular beacon. Here, we expand upon this work to demonstrate a method of using NCBs to differentiate single-nucleotide variations. Our method discriminates single-nucleotide variants by colorimetric change of the NCB probes rather than fluorescence intensity change. This added dimensionality enables the increase in fluorescence intensity (on/off switching) to quantify the amount of target, whereas the fluorescence color identifies single-nucleotide variants. Samples with single-nucleotide variations can be unambiguously identified on a common gel imager with naked eyes, making this method a reliable and low-cost assay with simple readout format.Ref: H.-C. Yeh et al., “A DNA-silver nanocluster probe that fluoresces upon hybridization,” Nano Letters 10 (8): 3106-3110, 2010.