Free-probe fluorescence of light-up probes.
Free-probe fluorescence of light-up probes.
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DOI：
10.1021/ja002294u
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发表时间：
2001-01
影响因子：
15
通讯作者：
Nicke Svanvik;J. Nygren;Gunnar Westman;Mikael Kubista
中科院分区：
文献类型：
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作者：
Nicke Svanvik;J. Nygren;Gunnar Westman;Mikael Kubista
发光探针（噻唑橙子（TO）缀合的肽核酸（PNA））在与靶核酸杂交时的荧光增强取决于探针序列，主要是由于游离探针荧光的大的变化。在这里，我们研究了三种探针，其中在自由状态下的荧光变化超过50倍。我们发现，这种变化是由于一个馏分，有TO分子内“回绑定”的PNA基地。这种单分子相互作用的分子内亲和常数通过温度滴定使用吸收光谱法测定，并计算在背结合构象的探针的荧光量子产率。在30 ℃和60 ℃时，反向结合构象的探针的摩尔比分别为0.70-0.96和0.40-0.73，并且反向结合构象的荧光量子产率在30 ℃和60 ℃时分别在0.0020和0.077之间和0.00065-0.029之间变化。这些数据表明，在自由探针荧光的变化主要取决于在反向结合构象的探针的荧光量子产率，并在较小程度上的探针的倾向，采用反向结合构象。随着温度的升高，自由探针的荧光降低，由于两个减少的程度的背结合和背结合构象的荧光量子产率的降低。
The fluorescence enhancement of light-up probes (thiazole orange (TO) conjugated peptide nucleic acids (PNAs)) upon hybridization to target nucleic acid depends on the probe sequence, mainly due to large variations in free-probe fluorescence. Here we study three probes where the fluorescence in free state varies more than 50-fold. We find that this variation is due to a fraction that has TO intramolecularly "back-bound" to the PNA bases. The intramolecular affinity constant for this unimolecular interaction was determined by temperature titrations using absorption spectroscopy, and the fluorescence quantum yields of the probes in back-bound conformation were calculated. The molar ratio of probes in back-bound conformation was 0.70-0.96 at 30 degrees C and 0.40-0.73 at 60 degrees C, and the fluorescence quantum yield in back-bound conformation varied between 0.0020 and 0.077 at 30 degrees C, and 0.00065-0.029 at 60 degrees C. These data show that the variation in free-probe fluorescence depends mainly on the fluorescence quantum yield of the probe in back-bound conformation and to a much lesser extent on the tendency of the probe to adopt the back-bound conformation. With increasing temperature the free-probe fluorescence decreases owing to both reduced degree of back-binding and a decrease of the fluorescence quantum yield in back-bound conformation.