Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Chemistry. 2009 Nov 2;15(43):11551-8. doi: 10.1002/chem.200901607.
We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynucleoside fluorophores (ODFs)--a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3' end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl-labeled complementary strand resulted in strong quenching of fluorescence in 85% of the twenty ODF sequences. The high efficiency of quenching was also established by their large Stern-Volmer constants (K(SV)) of between 2.1 x 10(4) and 4.3 x 10(5) M(-1), measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a K(SV) that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the pi stack of fluorophores in the amplified quenching of fluorescence.
我们描述了在 DNA 环境中进行的研究，以测试常见的荧光猝灭剂 dabcyl 如何与寡脱氧核苷酸荧光团（ODF）相互作用——这是一个基于 DNA 支架构建的堆叠、电子相互作用的荧光团系统。我们测试了二十种不同的四聚体 ODF 序列，其中包含不同组合和顺序的芘（Y）、苯并芘（B）、苝（E）、二甲氨基芪（D）和间隔物（S）单体连接到 DNA 寡聚物的 3'端。该探针序列与 dabcyl 标记的互补链杂交导致在二十种 ODF 序列中的 85%中荧光强烈猝灭。猝灭效率也通过它们的大 Stern-Volmer 常数（K(SV)）来证明，范围在 2.1 x 10(4) 和 4.3 x 10(5) M(-1)之间，使用游离的 dabcyl 猝灭剂进行测量。有趣的是，ODF 的猝灭显示出强烈的序列依赖性。这在 ODF 序列的倒排中尤为明显；例如，序列 BYDS 的 K(SV)大约比具有相同染料组成的 BSDY 高两个数量级。其他倒排，例如 EDSY 和 ESYD，在被 dabcyl 猝灭时也显示出不同的响应。光谱分析表明，与单体发射带相比，明显的激基缔合物和激基复合物发射带的猝灭效率至少高出一个数量级。这表明荧光的放大猝灭中，荧光团的 pi 堆叠的离域激发态发挥了重要作用。
