Intramolecular dimers: A new strategy to fluorescence quenching in dual-labeled oligonucleotide probes
Intramolecular dimers: A new strategy to fluorescence quenching in dual-labeled oligonucleotide probes
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DOI：
10.1021/ja025678o
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发表时间：
2002-06-19
影响因子：
15
通讯作者：
Cook, RM
中科院分区：
文献类型：
--
作者：
Johansson, MK;Fidder, H;Cook, RM
许多基因组分析使用前荧光寡核苷酸探针，在5‘端用荧光团共价标记，在3’端用猝灭剂标记。人们普遍认为，在这种没有茎结构的探针中，猝灭是通过Forster共振能量转移(FRET或FET)发生的，应该选择荧光团和猝灭剂来最大化它们的光谱重叠。我们研究了两个具有相同序列和猝灭剂的不同荧光团的双标记探针：5‘Cy3.5-β-肌动蛋白-3’BHQ1和5‘FAM-β-肌动蛋白-3’BHQ1。对它们的吸收光谱、相对荧光量子产率和荧光寿命的分析表明，这两种双标记探针都发生了静态猝灭，这是Cy3.5-BHQ1探针的主要猝灭机制。吸收光谱与激子二聚体的形成是一致的，激子二聚体是位于Cy3.5荧光团和BHQ1猝灭剂之间的分子内杂二聚体。
Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through Forster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.