Fluorescence of terbium ion-nucleic acid complexes: a sensitive specific probe for unpaired residues in nucleic acids

The interaction of the lanthanide cation Tb3+ with the phosphate moieties of non-hydrogen-bonded residues of nucleic acids has been shown to result in substantial enhancement of the fluorescence of this cation. The excitation spectrum for this fluorescence is characteristic of the base moiety of the residue to which the Tb3+ is bound, while the emission spectrum is characteristic of the cation itself. The intensity of the fluorescence enhancement, however, is dependent upon the base of the ligand moiety, with G inducing the strongest enhancement, C and T rather less, and A very little. Base-paired residues of nucleic acids induce no such fluorescence enhancement, even though the cation is more tightly bound to double helical regions than to residues in single strands. The enhancement of Tb3+ fluorescence upon binding to non-hydrogen-bonded residues therefore provides a highly specific conformational probe for such residues. This probe has been exploited successfully for the purpose of analyzing the kinetics of reassociation of DNAs (C0t analysis) and as a specific stain for single-strand DNA bands on polyacrylamide gels.