Unambiguous Detection of Target DNAs by Excimer−Monomer Switching Molecular Beacons

Abstract

A new class of molecular beacons were developed in which pyrene fluorophores were connected both at 3' and 5' ends of a single-stranded oligonucleotide. The two pyrene-based fluorophores were synthesized from the same starting material, so that the preparation of the beacons was simplified. The detection strategy of the beacons for target DNAs is based on "excimer-monomer emission switching" of the pyrene fluorophores: excimer emission of the pyrene moieties changed to monomer one when the beacons hybridized with the targets. This type of two-state mode of fluorescence allows unambiguous detection of the target DNAs because strict 1:1 correlation between the nonhybridized and the hybridized beacons can be monitored by the presence of isoemissive points of the fluorescence changes. The beacons can detect target 19-mer DNAs and can discriminate the targets from their single-nucleotide mismatches at 1 nM concentration. Advantages of the excimer-monomer switching molecular beacons were discussed in comparison with conventional ones.

Light-switching excimer probes for rapid protein monitoring in complex biological fluids

“…Some spatially sensitive fluorescent dyes, such as pyrene (13)(14)(15)(16) and BODIPY Fl (17,18), can form excimers upon close encounter of an excited-state molecule with another ground-state molecule. The excimer emits at a longer wavelength than a monomer does.…”

“…For labeling oligonucleotide with pyrene, several methods were reported (13,24). Unfortunately, for dually labeling pyrene on both nucleic acid termini, the reported procedures proceeded in low yields partially because of incompatible solubility of the pyrene derivatives and nucleic acids.…”

“…This strategy is a combined approach of wavelength switching and time-resolved measurement to solve the significant problems for protein monitoring in its native environment. Our approach is to label molecular aptamer with pyrene, similar to what has been reported in using pyrene for molecular beacons (13). The aptamer sequence that binds with high affinity to the target protein platelet-derived growth factor (PDGF)-BB is labeled with pyrene molecules at both ends.…”

Rapid fluorescence detection of immunoglobulin E using an aptamer switch based on a binding-induced pyrene excimer

“…[29][30][31][32][33][34][35] The pyrene molecules in proximity generate uorescence peaks different from those of separated pyrenes. The combination of the aptamer and pyrene excimer provides an aptamer excimer probe for rapid detection of targets based on target-binding induced structure switching of the aptamer.…”

Development of a DNA sensor using a molecular logic gate

“…Fujimoto et al reported the detection of target DNAs by excimer-monomer switching of pyrene using the fluorescence resonance energy transfer (FRET) process [26]. A DNAbased nanomachine was reported by Liu et al using the FRET phenomenon [20].…”