Fluorescent nucleotide base analogs as probes of nucleic acid structure, dynamics and interactions

Highly fluorescent nucleotide base analogs provide sensitive probes for studying the structure, dynamics and interactions of nucleic acids. These analogs can be incorporated site-specifically into oligonucleotides through standard automated synthetic methods, allowing them to serve as sensitive probes of changes in the microenvironment of bases that may result from variation in buffer condition, ion concentration, temperature or molecular interactions. Global conformational changes in nucleic acids and nucleic acid complexes can also be detected using fluorescent base analogs. A significant strength of fluorescent base analogs is their similarity in molecular constitution and chemical properties to natural bases. In contrast to bulkier chromophores, incorporation of nucleotide base analogs into oligonucleotides can normally be accomplished without introducing significant structural or chemical changes that might alter the measurement. Here, we review the characteristics of currently available fluorescent nucleotide base analogs that have made them useful probes in fluorescence studies of nucleic acids and nucleic acid complexes. A range of applications, that include measurement of fluorescence emission quenching, spectral shifting, depolarization (anisotropy), fluorescence lifetime and fluorescence resonance energy transfer, will be presented to demonstrate the broad utility of fluorescence base analog probes in characterizing the steady-state equilibrium properties and real-time kinetics in nucleic acid systems.