Pb EXAFS Studies on DNA Quadruplexes:  Identification of Metal Ion Binding Site

Abstract

Nucleic acid quadruplexes are composed of guanine quartets stabilized by specific metal ions. X-ray diffraction can provide high-resolution information on the structure and metal binding properties of quadruplexes, but only if they can be crystallized. NMR can provide detailed information on the solution structure of such quadruplexes but little quantitative data concerning the metal binding site. Here we apply extended X-ray absorption fine structure (EXAFS) measurements to characterize the metal ion binding site, in frozen solution, of the unimolecular quadruplex formed by the thrombin binding aptamer, d(G 2 T 2 G 2 TGTG 2 T 2 G 2 ) (TBA), in the presence of Pb 2+ ions. The Pb L III -edge X-ray absorption spectrum of this metal-DNA complex is very similar to that we obtain for a Pb 2+ -stabilized quartet system of known structure constructed from a modified guanine nucleoside (G1). The Fourier transforms of the Pb 2+ complexes with both TBA and G1 show a first-shell interaction at about 2.6 Å, and a weaker, broader shell at 3.5-4.0 Å. Quantitative analysis of the EXAFS data reveals the following: (i) very close agreement between interatomic distances at the metal coordination site for the Pb 2+ -G1 complex determined by EXAFS and by X-ray crystallography; (ii) similarly close agreement between interatomic distances measured by EXAFS for the Pb 2+ -G1 and Pb 2+ -TBA complexes. These results provide strong evidence for binding of the Pb 2+ ion in the region between the two quartets in the Pb 2+ -TBA complex, coordinated to the eight surrounding guanine O6 atoms. The specific binding of Pb 2+ to DNA examined here may be relevant to the genotoxic effects of this environmentally important heavy metal. Furthermore, these results demonstrate the utility of EXAFS as a method for quantitative characterization of specific metal binding sites in nucleic acids in solution.Metal ion-DNA interactions are important in nature, often impacting the genetic material's structure and function. To better understand the molecular details of these interactions, it is essential that we have a reliable picture of the metal ion coordination site. This is not always so straightforward. Thus, X-ray diffraction can provide high-resolution information on the structure and metal binding properties of nucleic acids, but only if diffraction-quality crystals can be obtained. NMR can provide detailed information on solution structure, but usually little quantitative data concerning the metal binding site. This paper describes how extended X-ray absorption fine structure (EXAFS) 1 measurements can be used to gain detailed information about a Pb 2+ binding site within a folded DNA oligonucleotide.DNA and RNA can form quadruplex structures in the presence of certain metal ions, based on the guanine quartet, which is shown in Figure 1A. Only a small number of X-ray crystal structure determinations on quadruplexes have been reported, and these have located the metal binding site in the region between two quartet stacks, or coplanar with one...

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

“…1B, the CD spectrum of T7-TMR-TA15 is of relatively low amplitude in the absence of Pb 2+ , which suggests that the aptamer probe possesses little or no G-quadruplex structure in the absence of Pb 2+ . When the probe incubated with Pb 2+ , the spectrum displayed a positive peak at longer wavelength near 310 nm and a negative peak near 268 nm, which is consistent with the Pb 2+ -stabilized anti-parallel G-quadruplex structure [64,72]. To further confirm that the large reduction in FA resulted from Pb 2+ -inducing G-quadruplex formation, we examined a mutant aptamer probe (T7-TMR-TA15-G1T) obtained by only substitution of the first G with T. The CD spectrum of this mutant aptamer probe showed no conformation change and no characteristics of G-quadruplex structure in the presence of Pb 2+ (see Fig.…”

“…Because the internally labeled TMR can intramolecularly interact with the adjacent guanine bases by photo-induced electron transfer (PET) mechanism due to its good electron donating property [67][68][69][70][71], the rotation of TMR is firmly restricted, which results in the aptamer probe having large FA value. In the presence of Pb 2+ , because Pb 2+ can interact with aptamer probe and induce conformational change from a random-coil structure into a highly-ordered G-quadruplex [65,72], this would weaken or eliminate the intramolecular interaction between the TMR and the adjacent guanine bases involved in formation of the G-quadruplex. Thus, the fluorophore TMR has more freedom and rotates faster.…”

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

“…XAS provides structural information regarding both the first coordination sphere and the environment beyond the immediate coordination metal ligands. Though XAS has previously been used to study metal coordination to DNA (25,26), we believe this work represents a unique application of XAS techniques to the study of metals bound to RNA.…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…Nonetheless, higher-affinity binding sites, like the ones in the G-quadruplex channel [171] or in a short RNase P helix P4 model [172] can be characterized in remarkable detail, not to forget complexes of kinetically more inert metal ions [173,174].…”