Pyrene excimer signaling molecular beacons for probing nucleic acids.
Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.
Conlon
Patrick
P
Center for Research at Bio/Nano Interface, Department of Chemistry and Shands Cancer Center, University of Florida Genetics Institute, University of Florida, Gainesville, Florida 32611-7200, USA.
Yang
Chaoyong James
CJ
Wu
Yanrong
Y
Chen
Yan
Y
Martinez
Karen
K
Kim
Youngmi
Y
Stevens
Nathan
N
Marti
Angel A
AA
Jockusch
Steffen
S
Turro
Nicholas J
NJ
Tan
Weihong
W
D001483:Base Sequence; D015342:DNA Probes / Q000737:chemistry*; D005453:Fluorescence; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis*; D011721:Pyrenes; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D015342:DNA Probes; D009696:Nucleic Acids; D011721:Pyrenes; C063127:4-(4-dimethylaminophenylazo)benzoic acid; C030984:pyrene; D004124:p-Dimethylaminoazobenzene