Pyrene Excimer Signaling Molecular Beacons for Probing Nucleic Acids

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• 作者：Patrick Conlon ; Chaoyong James Yang ; Yanrong Wu ; Yan Chen ; Karen Martinez ; Youngmi Kim ; Nathan Stevens ; Angel A. Marti ; Steffen Jockusch ; Nicholas J. Turro ; Weihong Tan
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：January 9, 2008
• 年：2008
• 卷：130
• 期：1
• 页码：336 - 342
• 全文大小：277K
• 年卷期：v.130,no.1(January 9, 2008)
• ISSN：1520-5126

文摘

Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencherDABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In theabsence 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 quenchermolecule and generated an excimer emission signal proportional to the target concentration. Steady-statefluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signalingenabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensitycould be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additionalpyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields,and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime (~40 ns), large Stokesshift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternativeto traditional fluorophore labeling in nucleic acid probes.