2-(1-Ethynylpyrene)-adenosine as a folding probe for RNA - pyrene in or out

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.