3 research outputs found
Selective Binding of Zn<sup>2+</sup> Complexes to Human Telomeric G‑Quadruplex DNA
The Zn<sup>2+</sup> complex of 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-<i>N</i>,<i>N</i>-dimethylnaphthalen-1-amine, ZnÂ(DSC),
binds selectively to the biologically relevant human telomeric (H-Telo)
G-quadruplex. An increase in the ZnÂ(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
ZnÂ(DSC) with binding constants in the low micromolar range (2.5 ÎĽM).
Isothermal calorimetric titrations confirm low micromolar dissociation
constants with a 2:1 stoichiometry. The interaction between H-Telo
and ZnÂ(DSC) is highly pH-dependent, consistent with binding to the
unpaired thymines in the G-quadruplex loops. As a result, ZnÂ(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-ZnÂ(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 ZnÂ(DSC) to thymine sequences. NMR studies
were consistent with the binding of ZnÂ(DSC) to thymine-containing
oligonucleotides including CCTCC, CTTCC, and CTCTC. Studies showed
that the dansyl group of ZnÂ(DSC) interacts with thymines in CTTCC.
Fluorescence spectroscopy and ITC data indicate that ZnÂ(DSC) forms
2:1 adducts with thymines that are spaced (CTCTC) but not tandem thymines
(CTTCC). These data are consistent with one ZnÂ(DSC) complex binding
to two separate loops in the G-quadruplex. A second Zn<sup>2+</sup> complex containing an acridine pendent, ZnÂ(ACR), binds tightly to
pentanucleotides with both tandem and spaced thymines. ZnÂ(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
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 Ă— 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 rÂ(CAG) and rÂ(CUG) repeats, respectively. We report the
structures of two RNA constructs containing three copies of a rÂ(CAG)
[rÂ(3Ă—CAG)] or rÂ(CUG) [rÂ(3Ă—CUG)] motif that were modeled
with nuclear magnetic resonance spectroscopy and simulated annealing
with restrained molecular dynamics. The 1 Ă— 1 internal loops
of rÂ(3Ă—CAG) are stabilized by one-hydrogen bond (<i>cis</i> Watson–Crick/Watson–Crick) AA pairs, while those of
rÂ(3Ă—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
The zincÂ(II) complex of 1-(4-quinoylyl)Âmethyl-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 ZnÂ(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 ZnÂ(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 zincÂ(II)
complexes containing 1,4,7,10-tetraazacyclododecane (cyclen) with
aromatic pendent groups were studied for binding to DNA including
1-(2-quinolyl)Âmethyl-1,4,7,10-tetraazacyclododecane (cy2q), 1-(4-biphenyl)Âmethyl-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
ZnÂ(cybp) complex binds with moderate affinity but little selectivity
to DNA hairpins with thymine bulges and to DNA lacking bulges. Similarly,
ZnÂ(dsc) binds weakly both to thymine bulges and hairpins with fully
complementary stems. The zincÂ(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 ÎĽM) 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 ZnÂ(cy4q) bound to DNA containing a
thymine bulge is consistent with binding of the zincÂ(II) complex to
the thymine N3<sup>–</sup> and stacking of the quinoline on
top of the thymine. The thymine-bulge bound zincÂ(II) complex is pointed
into the major groove, and there are interactions with the guanine
positioned 5′ to the thymine bulge