Institute of Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-University, Max-von-Laue-Strasse 1, 60438 Frankfurt, Germany.
Chembiochem. 2010 Mar 22;11(5):664-72. doi: 10.1002/cbic.200900778.
A series of short RNA duplexes containing one or two 1-ethynylpyrene-modified adenine bases was synthesised. The melting behaviour of these duplexes was examined by monitoring temperature-dependent pyrene fluorescence. In the singly modified RNA duplexes, the bases flanking the ethynylpyrene-rA were varied to examine the sequence specificity of the fluorescence change of pyrene upon RNA hybridisation. Because an increase in pyrene fluorescence upon melting of the duplex can be correlated with intercalation of pyrene, and a decrease is usually associated with the position of pyrene outside the strand, a relationship between the flanking bases and the tendency of the dye to intercalate has been established. It was found that pyrene intercalation is less likely to take place if the modified base is flanked only by A-U base pairs. Flanking G-C base pairs, even only in the 5'-direction of the modified base, will favour intercalation. In addition, we examined a doubly modified compound that had a pyrene located on each strand. The spectra indicated that the two pyrenes were close enough for interaction. Upon melting of the strand, a fluorescence blue shift corresponding to the dissociation of the pyrene-pyrene complex could be observed in addition to the intensity effect already known from the singly modified compounds. Two melting curves based on the different properties of the fluorophore could be extracted, leading to different melting points corresponding to the global duplex melting and to the change of local pyrene environment, respectively.
合成了一系列含有一个或两个 1-乙炔基嘌呤修饰腺嘌呤碱基的短 RNA 双链体。通过监测温度依赖性的芘荧光来研究这些双链体的熔融行为。在单修饰的 RNA 双链体中，改变了围绕乙炔基嘌呤-rA 的碱基，以研究芘在 RNA 杂交时荧光变化的序列特异性。因为双链体熔融时芘荧光的增加可以与芘的嵌入相关联，而减少通常与芘在链外的位置相关联，因此已经建立了侧翼碱基与染料嵌入倾向之间的关系。发现如果修饰碱基仅由 A-U 碱基对包围，则芘嵌入不太可能发生。即使在修饰碱基的 5'方向只有 G-C 碱基对，也会有利于嵌入。此外，我们还研究了一种在每条链上都有一个芘的双修饰化合物。光谱表明，两个芘足够接近以进行相互作用。在链的熔融过程中，除了已经从单修饰化合物中知道的强度效应之外，还可以观察到与芘-芘配合物解离相对应的荧光蓝移。可以根据荧光团的不同性质提取两个熔融曲线，导致分别对应于全局双链体熔融和局部芘环境变化的不同熔点。
