title: A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters.
abstract: DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag(+) ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.
authors.affiliation: Department of Mechanical Engineering, University of California Santa Barbara, USA.
authors.forename: Jackson Travis
authors.initials: JT
authors.lastname: Del Bonis-O'Donnell
authors.forename: Daniel
authors.initials: D
authors.lastname: Vong
authors.forename: Sumita
authors.initials: S
authors.lastname: Pennathur
authors.forename: Deborah Kuchnir
authors.initials: DK
authors.lastname: Fygenson
mesh_terms: D015342:DNA Probes / Q000737:chemistry*; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D053768:Metal Nanoparticles; D012834:Silver; D013050:Spectrometry, Fluorescence
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D012834:Silver