3 research outputs found

    Selective Binding of Zn<sup>2+</sup> Complexes to Human Telomeric G‑Quadruplex DNA

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    The Zn<sup>2+</sup> complex of 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-<i>N</i>,<i>N</i>-dimethylnaphthalen-1-amine, Zn­(DSC), binds selectively to the biologically relevant human telomeric (H-Telo) G-quadruplex. An increase in the Zn­(DSC) dansyl group fluorescence with a simultaneous shift in emission is consistent with the complex binding to H-Telo. The H-Telo G-quadruplex has two binding sites for Zn­(DSC) with binding constants in the low micromolar range (2.5 μM). Isothermal calorimetric titrations confirm low micromolar dissociation constants with a 2:1 stoichiometry. The interaction between H-Telo and Zn­(DSC) is highly pH-dependent, consistent with binding to the unpaired thymines in the G-quadruplex loops. As a result, Zn­(DSC) selectively binds to H-Telo over duplex DNA. In contrast to Zn<sup>2+</sup>, Fe<sup>2+</sup> and Co<sup>2+</sup> do not complex to the DSC macrocycle appreciably under the conditions of the experiment. The Cu<sup>2+</sup> complex of DSC does not interact measurably with the H-Telo G-quadruplex. Interestingly, the H-Telo-Zn­(DSC) adduct self-assembles from its individual components at physiological pH and 100 mM KCl. The self-assembly feature, which is specific for the Zn<sup>2+</sup> ion, suggests that this system may be viable as a Zn<sup>2+</sup> sensor. Pentanucleotides were studied in order to better describe the binding of Zn­(DSC) to thymine sequences. NMR studies were consistent with the binding of Zn­(DSC) to thymine-containing oligonucleotides including CCTCC, CTTCC, and CTCTC. Studies showed that the dansyl group of Zn­(DSC) interacts with thymines in CTTCC. Fluorescence spectroscopy and ITC data indicate that Zn­(DSC) forms 2:1 adducts with thymines that are spaced (CTCTC) but not tandem thymines (CTTCC). These data are consistent with one Zn­(DSC) complex binding to two separate loops in the G-quadruplex. A second Zn<sup>2+</sup> complex containing an acridine pendent, Zn­(ACR), binds tightly to pentanucleotides with both tandem and spaced thymines. Zn­(ACR) indiscriminately binds to both H-Telo and duplex DNA

    Structure and Dynamics of RNA Repeat Expansions That Cause Huntington’s Disease and Myotonic Dystrophy Type 1

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    RNA repeat expansions cause a host of incurable, genetically defined diseases. The most common class of RNA repeats consists of trinucleotide repeats. These long, repeating transcripts fold into hairpins containing 1 × 1 internal loops that can mediate disease via a variety of mechanism(s) in which RNA is the central player. Two of these disorders are Huntington’s disease and myotonic dystrophy type 1, which are caused by r­(CAG) and r­(CUG) repeats, respectively. We report the structures of two RNA constructs containing three copies of a r­(CAG) [r­(3×CAG)] or r­(CUG) [r­(3×CUG)] motif that were modeled with nuclear magnetic resonance spectroscopy and simulated annealing with restrained molecular dynamics. The 1 × 1 internal loops of r­(3×CAG) are stabilized by one-hydrogen bond (<i>cis</i> Watson–Crick/Watson–Crick) AA pairs, while those of r­(3×CUG) prefer one- or two-hydrogen bond (<i>cis</i> Watson–Crick/Watson–Crick) UU pairs. Assigned chemical shifts for the residues depended on the identity of neighbors or next nearest neighbors. Additional insights into the dynamics of these RNA constructs were gained by molecular dynamics simulations and a discrete path sampling method. Results indicate that the global structures of the RNA are A-form and that the loop regions are dynamic. The results will be useful for understanding the dynamic trajectory of these RNA repeats but also may aid in the development of therapeutics

    Structural Basis for Bifunctional Zinc(II) Macrocyclic Complex Recognition of Thymine Bulges in DNA

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    The zinc­(II) complex of 1-(4-quinoylyl)­methyl-1,4,7,10-tetraazacyclododecane (cy4q) binds selectively to thymine bulges in DNA and to a uracil bulge in RNA. Binding constants are in the low-micromolar range for thymine bulges in the stems of hairpins, for a thymine bulge in a DNA duplex, and for a uracil bulge in an RNA hairpin. Binding studies of Zn­(cy4q) to a series of hairpins containing thymine bulges with different flanking bases showed that the complex had a moderate selectivity for thymine bulges with neighboring purines. The dissociation constants of the most strongly bound Zn­(cy4q)–DNA thymine bulge adducts were 100-fold tighter than similar sequences with fully complementary stems or than bulges containing cytosine, guanine, or adenine. In order to probe the role of the pendent group, three additional zinc­(II) complexes containing 1,4,7,10-tetraazacyclododecane (cyclen) with aromatic pendent groups were studied for binding to DNA including 1-(2-quinolyl)­methyl-1,4,7,10-tetraazacyclododecane (cy2q), 1-(4-biphenyl)­methyl-1,4,7,10-tetraazacyclododecane (cybp), and 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-<i>N</i>,<i>N</i>-dimethylnaphthalen-1-amine (dsc). The Zn­(cybp) complex binds with moderate affinity but little selectivity to DNA hairpins with thymine bulges and to DNA lacking bulges. Similarly, Zn­(dsc) binds weakly both to thymine bulges and hairpins with fully complementary stems. The zinc­(II) complex of cy2q has the 2-quinolyl moiety bound to the Zn<sup>II</sup> center, as shown by <sup>1</sup>H NMR spectroscopy and pH–potentiometric titrations. As a consequence, only weak (500 μM) binding is observed to DNA with no appreciable selectivity. An NMR structure of a thymine-bulge-containing hairpin shows that the thymine is extrahelical but rotated toward the major groove. NMR data for Zn­(cy4q) bound to DNA containing a thymine bulge is consistent with binding of the zinc­(II) complex to the thymine N3<sup>–</sup> and stacking of the quinoline on top of the thymine. The thymine-bulge bound zinc­(II) complex is pointed into the major groove, and there are interactions with the guanine positioned 5′ to the thymine bulge
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