Sequence dependence of fluorescence emission and quenching of doubly thiazole orange labeled DNA: Effective design of a hybridization-sensitive probe
Sequence dependence of fluorescence emission and quenching of doubly thiazole orange labeled DNA: Effective design of a hybridization-sensitive probe
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DOI：
10.1021/bc800201m
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发表时间：
2008-08-01
影响因子：
4.7
通讯作者：
Okamoto, Akimitsu
中科院分区：
文献类型：
--
作者：
Ikeda, Shuji;Kubota, Takeshi;Okamoto, Akimitsu
我们设计了一种双噻唑橙标记的核苷，与目标 DNA 杂交时显示出高荧光强度，并能有效猝灭单链状态。了解该探针的荧光发射和猝灭在多大程度上取决于探针序列以及为什么存在这种序列依赖性对于有效的探针设计非常重要，我们合成了 30 多个探针序列并测量了它们的荧光强度。当探针与目标DNA链杂交时，有较强的发射，而杂交前发射强度要弱得多；然而，探针的自二聚化抑制了荧光猝灭。特别是，自二聚体结构中与标记核苷酸相邻的G/C碱基对导致杂交前探针的低猝灭能力。另一方面，标记位点周围形成的错配碱基对降低了荧光强度，因为邻近序列是束缚的噻唑橙色染料的结合位点。即使标记的核苷酸位于探针链的末端，杂交也会增强探针的荧光；然而，部分缺乏双链体结构导致杂交体的荧光强度降低。
We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.