Probing metal binding sites in DNA by using europium(III) luminescence spectroscopy
Switala, Brooke A.
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Metal ion binding sites in nucleic acids are difficult to locate and characterize; NMR spectroscopy and X-ray crystallography are the two most widely used methods. Lanthanide luminescence is a third promising technique that can give insight into metal ion binding sites in DNA, which this work focuses on. Nine different hairpin DNA sequences were studied with 30 μM EuCl 3 at pH=6.5, MES=0.020 M, I =0.100 M NaCl. These DNA hairpin structures were determined to be very stable both with and without EuCl 3 . Eu(III) excitation spectra, luminescence lifetimes and melting temperatures were performed for each sequence and then compared to the others. Eu(III) bound to the hairpins of Bev3 (GGAC GCA GTCC) and Bev3 L1 (GGAC GCAT GTCC) showed the longest luminescence lifetimes and luminescence intensity increases in the excitation spectrum. These DNA hairpins with Eu(III) also had the smallest dissociation constants. Each variation in the hairpin sequence showed a decrease in luminescence lifetime, a decrease in intensity in the excitation spectrum and a reduction in the dissociation constant of Eu(III) and the hairpin. From the thermal melting experiments it was determined that out of all nine hairpins, the Bev 3 is the only one with a melting temperature influenced by EuCl 3 . This means there is a destabilization in the folded hairpin structure and the metal ion perturbs the structure. From the luminescence results of these two DNA hairpins and the mutated hairpins, it was determined that there could be two different Eu(III) binding sites in the DNA, possibly one in the hairpin loop and one in the stem. The G-A sheared mismatch which is contained in the structures is necessary for binding and it may be a key structural component. Also the cytosine located in the hairpin loop is necessary for binding; its involvement may be structural or part of the metal ion binding site. The luminescence lifetimes of Bev 3 in D 2 O and H 2 O were used to calculate that 6.4 +/- 0.5 waters remain bound to the Eu(III). Assuming a coordination number of nine, this suggests that 2-3 waters are displaced by direct DNA coordination.