Abstract
The interaction of terbium(III) (Tb3+) with synthetic and native nucleic acids, including circular supercoiled DNA, has been studied. Fluorescence spectroscopy has revealed that: (a) the intrinsic fluorescence of Tb3+ is greatly enhanced upon binding either xanthine or guanine residues; (b) slight chemical modification of the nucleoside bases abrogates energy transfer; (c) binding affinity increases with chain length; (d) protonation of the N-7 site of xanthine or guanine obviates energy transfer in Tb3+-homopolynucleotide complexes; and (e) unimpaired base residues in supercoiled DNA are capable of measurably enhancing Tb3+ fluorescence. In addition, equilibrium dialysis experiments with 160Tb3+ show that double helical polynucleotides (which do not enhance Tb3+ emission) bind more cation/molar nucleotide residue than do single-stranded polynucleotides. Melting profiles demonstrate that at low Tb3+: base ratios, the lanthanide destabilizes helical DNA and prevents reannealing of strands following thermal denaturation. These data suggest the existence of two Tb3+ binding sites on the unpaired residues of polynucleotide chains: the phosphate moiety and electron donor groups on the nucleoside bases.
Highlights
In confirmation of earlier reports [9, 14,15,16], we fiid that the greater the secondary structure of a nucleic acid, the less is its capability of enhancing intrinsic Tb3+fluorescence as a function of (Tb3+)fluorescence
Type I pBR322 DNA was purified from a crude Hirt extract [21]of a lysate of pBR322-containbium(II1) and europium(III),as their resonance energy levels ing Escherichia coli by banding DNA on a 52.5% (w/w) CsCl, 0.4 fortuitously overlap with the triplet energy states of protein mg/ml of ethidium bromide density gradient for 64 h a t 40,000 rpm and nucleic acid ligands irradiated with ultraviolet light [19]
Quantitative removal of ethidium ions extremely valuable as fluorescent probes for alkaline was assayed by fluorescence spectroscopy ( L = 545 nm; X, = 590 metal binding sites in proteins [1,2,3], tRNA [4,5,6,7], and rRNA
Summary
Thermal Denaturation of DNA-Heating and cooling curves of DNA were constructed as described previously [29]. Titration curves manifest a progression in shape from sigmoidal (monomer)to hyperbolic (polymer), with a concomitant decrease in (Tb3+)required to same buffer to a final concentration of p~ and subjected to an saturate a constantamount of nucleicacid. At saturating increase in temperature at a rate of 1 "C/min in the presence of 17.5 levels of Tb3+,optimal energy transfer is effected bya deoxyp~ TbCL (higher concentrations induced microprecipitation upon heating); absorbance a t 260 nm was monitored on a single-beam. DNA samples were cooled at the same rate (1"C/min). As a guanylic acid chain length of four (oligo(dG)& chain lengths longer than the tetramer generate progressively lower levels of enhancement, apparently reflecting their greater tendency control, 25 and 100 p~ CuC12 (Mallinckrodt) was added in place of
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