Nucleic acid structure and sequence probing using fluorescent base analogue tCO

The fluorescent cytosine analog tCO is on average the brightest probe of its kind and, moreover, it introduces minimal perturbations to the normal secondary structure of DNA. Here several ways of how tCO, with an advantage, can be used as a local fluorescent probe in nucleic acid systems are presented. Most importantly, we show that tCO is an excellent probe for the detection of individual melting processes of complex nucleic acid structures containing a large number of separate secondary structure motifs. Since conventional UV-melting investigations merely monitor the global melting process of the whole nucleic acid structure, e.g. multi-hairpin systems in RNA/DNA, and thus is incapable of estimating individual melting transitions of such systems, tCO represents a new method of characterization. Furthermore, we find that tCO may be used to detect bulges and loops in nucleic acids as well as to distinguish a matched base-pair from several of the mismatched.