Bulk and single-molecule characterization of an improved molecular beacon utilizing H-dimer excitonic behavior.
Pairs of fluorophores in close proximity often show self-quenching of fluorescence by the well-known H-dimer mechanism. We use a pair of fluorophores in the new dicyanomethylenedihydrofuran (DCDHF) dye family in the design and characterization of a new fluorescent probe for nucleic acid detection, which we refer to as a self-quenched intramolecular dimer (SQuID) molecular beacon (MB). We obtain a quenching efficiency of 97.2%, higher than the only other reported value for a MB employing fluorophore self-quenching by H-dimer formation. Furthermore, the excellent single-molecule (SM) emitter characteristics of the DCDHF dyes allow observation of individual SQuID MB-target complexes immobilized on a surface, where the doubled SM emission intensity of our target-bound beacon ensures a higher signal-to-background ratio than conventional fluorophore-quencher MBs. Additional advantages of the SQuID MB are single-pot labeling, visible colorimetric detection of the target, and intrinsic single-molecule two-step photobleaching behavior, which offers a specific means of discriminating between functional MBs and spurious fluorescence.
Conley
Nicholas R
NR
Pomerantz
Andrea Kurtz
AK
Wang
Hui
H
Twieg
Robert J
RJ
Moerner
W E
WE
D001483:Base Sequence; D019281:Dimerization; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans / Q000737:chemistry*; D008969:Molecular Sequence Data; D009570:Nitriles / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry*
D005456:Fluorescent Dyes; D005663:Furans; D009570:Nitriles; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; C521333:dicyanomethylenedihydrofuran