Pyrene excimer signaling molecular beacons for probing nucleic acids
Pyrene excimer signaling molecular beacons for probing nucleic acids
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DOI：
10.1021/ja076411y
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发表时间：
2008-01-09
影响因子：
15
通讯作者：
Tan, Weihong
中科院分区：
文献类型：
--
作者：
Conlon, Patrick;Yang, Chaoyong James;Tan, Weihong
设计了分子信标DNA探针，5‘端含有1-4个芘单体，3’端含有猝灭剂DABCyL，用于实时探测DNA序列。在没有靶序列的情况下，多个芘标记的分子信标(MBS)呈现茎闭合的构象，导致芘准分子的荧光猝灭。在靶标存在的情况下，信标转变为茎开构象，从而将芘标记从猝灭剂分子中分离出来，并产生与靶标浓度成正比的准分子发射信号。稳态荧光分析在缓冲液中的检测极限为亚纳摩尔，而时间分辨信号使细胞生长介质中的低纳摩尔目标检测成为可能。研究发现，通过增加连接在5‘端的Py单体的数量可以改变准分子的发射强度。每增加一种Py单体，都会显著增加杂化的分子轨道的准分子发射强度、量子产额和激发态寿命。长的荧光寿命(接近40 ns)，大的斯托克斯位移(130 Nm)，以及可调的强度使得这种多芘部分成为核酸探针中传统荧光团标记的有效替代。
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 (similar to 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.