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The helix-coil transition of DNA duplexes and hairpins observed by multiple fluorescence parameters

机译：通过多个荧光参数观察到DNA双链体和发夹的螺旋-螺旋转变

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The thermal denaturation of 8-20-bp DNA duplexes labeled with fluorescein and tetramethylrhodamine at opposing 5'-ends was investigated by monitoring the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, the fluorescence resonance energy transfer between fluorescein and rhodamine, and, for the 20-bp duplex, the UV absorption. Melting experiments with the single strands of the duplexes revealed that the single strands can form hairpins stabilized by only a few base pairs. The thermal denaturation curves of the duplexes were fitted well to an extended all-or-none model assuming that only the fully base-paired duplex, the maximally base-paired hairpin, and the random coil conformations are present simultaneously. The extent-of-melting versus temperature curves derived from the different spectroscopic parameters are nearly identical, provided that the analysis of the baselines is carried out correctly; the Delta H and Delta S of the dissociation compare well with predictions based on nearest neighbor interaction values available in the literature. Our results imply that for all the oligonucleotides other than the 34-bp oligomer, no partially melted intermediates other than hairpins are present in the reaction mixture in amounts that can be detected by our methods. The melting of the hairpins was also studied directly using single-stranded oligonucleotides. The melting of a 34-bp duplex can be accounted for by a statistical zipper model. [References: 39]
机译：通过监测染料的荧光强度，四甲基若丹明的荧光各向异性，荧光素与若丹明之间的荧光共振能量转移和，对于20 bp的双链体，吸收紫外线。用双链体的单链进行的熔解实验表明，单链可以形成仅由几个碱基对稳定的发夹。假设仅同时存在完全碱基配对的双链体，最大碱基配对的发夹和无规卷曲构象，则双链体的热变性曲线很好地适合于扩展的全无模型。如果正确地进行了基线分析，则从不同光谱参数得出的熔融程度与温度的曲线几乎相同。解离的Delta H和Delta S与基于文献中可用的最近邻相互作用值的预测相比较很好。我们的结果表明，对于除34 bp寡聚物以外的所有寡核苷酸，反应混合物中均不存在发夹以外的部分熔融中间体，其量可以通过我们的方法检测到。还使用单链寡核苷酸直接研究了发夹的解链。可以通过统计拉链模型解释34 bp双链体的熔解。 [参考：39]

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