An Investigation of G‑Quadruplex Structural Polymorphism in the Human Telomere Using a Combined Approach of Hydrodynamic Bead Modeling and Molecular Dynamics Simulation

Guanine-rich oligonucleotides can adopt noncanonical tertiary structures known as G-quadruplexes, which can exist in different forms depending on experimental conditions. High-resolution structural methods, such as X-ray crystallography and NMR spectroscopy, have been of limited usefulness in resolving the inherent structural polymorphism associated with G-quadruplex formation. The lack of, or the ambiguous nature of, currently available high-resolution structural data, in turn, has severely hindered investigations into the nature of these structures and their interactions with small-molecule inhibitors. We have used molecular dynamics in conjunction with hydrodynamic bead modeling to study the structures of the human telomeric G-quadruplex-forming sequences at the atomic level. We demonstrated that molecular dynamics can reproduce experimental hydrodynamic measurements and thus can be a powerful tool in the structural study of existing G-quadruplex sequences or in the prediction of new G-quadruplex structures

An Improved Model for the hTERT Promoter Quadruplex

<div><p>Mutations occur at four specific sites in the hTERT promoter in >75% of glioblastomas and melanomas, but the mechanism by which the mutations affect gene expression remains unexplained. We report biophysical computational studies that show that the hTERT promoter sequence forms a novel G-quadruplex structure consisting of three contiguous, stacked parallel quadruplexes. The reported hTERT mutations map to the central quadruplex within this structure, and lead to an alteration of its hydrodynamic properties and stability.</p></div

The hTERT promoter sequence and proposed structure.

<p><i>Top</i>: Sequence of the hTERT core promoter with the positions of mutations observed in melanomas shown in red. <i>Bottom</i>: Previously proposed structure of the hTERT core promoter (5). Adapted with permission from (5).</p

Results of sedimentation velocity experiments of hTERT promoter sequences in solution.

<p>The distribution of sedimentation coefficients, corrected for viscosity and temperature, are shown, revealing a major species with S_20,w of 4.05+/−0.04 for the folded wild-type sequence. S_20,w values for folded mutant sequences are slightly but significantly reduced to 3.5 - 3.6 S_20,w.</p

Oligodeoxynucleotides used in this study.

<p>Oligodeoxynucleotides used in this study.</p

Preparation of PLGA microspheres.

<p>(A) Schematic for preparation of TPCA-1/PLGA microspheres. (B) Microscopic images of microparticles loaded with TPCA-1. Scale bar, 10 μm. (C) Size distribution of TPCA-1<i>/PLGA microparticles</i>. The mean diameter is 2.4 μm.</p

Circular dichroism studies of hTERT and mutant sequences.

<p>(A) Molar circular dichroism (Δε) of the hTERT promoter sequence in solution (black). The spectrum predicted for the structure shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115580#pone-0115580-g001" target="_blank">Fig. 1</a> is shown in red. The experimentally observed spectrum for the parallel quadruplex structure formed by the c-myc promoter sequence variant 1XAV is shown in blue. (B). Molar circular dichroism of the folded wild-type and mutant sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115580#pone-0115580-g001" target="_blank">Fig. 1</a>.</p

Effects of retrobulbar-injected TPCA-1-loaded PLGA microparticles on laser-induced CNV formation.

<p>(A-C) Representative images of fluorescein angiogram were taken from the eyes receiving no injection (A), PLGA (10 mg PLGA in 100 μL PBS) injection (B), and PLGA/TPCA-1 (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) injection (C) at 7 days post drug treatment and laser injury. Laser burn was performed in the eyes immediately after retrobulbar injection. (D) Quantification of the fluorescein-leakage score was measured as described in Materials and Methods. (E-G) Representative images of laser-induced CNV stained by isolectin B4. The eyes were injected with PLGA (F) or PLGA/TPCA-1 (G) in comparison with eyes receiving no injection (E). The images were taken and calculated at 7 days after laser injury. (H) Area of CNV in each eye under the indicated conditions, measured from the isolectin B4 staining. The images in E-G were taken and calculated at 7 days after laser injury. “Untreated” represents the results from the eyes without any injection. Data is expressed as mean±SEM.; n = the number of mice used for each condition. The statistical analysis performed for all comparisons was one-way anova followed by <i>post hoc</i> Bonferroni <i>t</i>-tests using ProStat Ver5.5. **<i>p</i> < 0.01, ***<i>p</i> < 0.005.</p

Histological and flow cytometric analyses of liver, spleen, and T cell populations.

<p>(A) H&E-stained sections of liver (A, C, and E) and spleen (B, D, and F) tissues on day 7 after PLGA (10 mg PLGA in 100 μL PBS) (A and B) or PLGA/TPCA-1 (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) (C and D) retrobulbar injection. The eyes without any injection were used for control (E and F). (G-I) Flow cytometry analysis of CD4⁺ and CD8⁺ single-positive and CD4⁺/CD8⁺ double-positive T cell populations in thymus (G) and spleen (H). Apoptotic assessment of the TCR⁺ lymphocytes was performed on splenocytes using flow cytometry for annexin V exposure on the cell surface (I). Data expressed as mean±SD, n = 3 for each group.</p

Assessment of retinal histology and visual function.

<p>(A-C) H&E-stained images of the retinas from untreated mice (A), mice injected with sham-loaded PLGA microparticles (10 mg PLGA in 100 μL PBS) (B), and mice injected with TPCA-1-loaded PLGA (PLGA/TPCA-1) microparticles (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) (C). The tissues were collected at 7 days after treatment. (D) The quantitative comparison of the cell layers in the outer nuclear layer (ONL) among untreated mice, mice treated with PLGA or PLGA/TPCA-1 at 7 days post treatment. Data expressed as mean±SD, n = 3 (individual experimental mice). (E) Visual acuity measured by OKR from eyes treated with PLGA or PLGA/TPCA-1 in comparison with untreated control eyes. Data expressed as mean±SD. N = the number of eyes tested.</p