Comparison of the Binding Geometry of Free-Base and Hexacoordinated Cationic Porphyrins to A- and B‑Form DNA

Although the transition from B-DNA to the A-form is essential for many biological concerns, the properties of this transition have not been resolved. The B to A equilibrium can be analyzed conveniently because of the significant changes in circular dichroism (CD) and absorption spectrum. CD and linear dichroism (LD) methods were used to examine the binding of water-soluble <i>meso</i>-tetrakis­(<i>N</i>-methylpyridinium-4-yl)­porphyrin (TMPyP) and its derivatives, Co-TMPyP, with B- and A-calf thymus DNA. B- to A-transitions occurred when the physiological buffer was replaced with a water-ethanol mixture (∼80 v/v %), and the fluorescence emission spectra of TMPyP bound to DNA showed a different pattern under ethanol–water conditions and water alone. The featureless broad emission bands of TMPyP were split into two peaks near at 658 and 715 nm in the presence of DNA under an aqueous solution. In the case of an ethanol–water system, however, the emission bands are split in two peaks near at 648 and 708 nm and 656 and 715 nm with and without DNA, respectively. This may be due to a change in the solution polarity. On the basis of the CD and LD data, TMPyP interacts with B-DNA via intercalation at a low ratio under a low ionic strength, 1 mM sodium phosphate. On the other hand, the interaction with A-DNA (80 v/v % ethanol–water system) occurs in a nonintercalating manner. This difference might be because the structural conformations, such as the groove of A-DNA, are not as deep as in B-DNA and the bases are much more tilted. In the case of Co-TMPyP, porphyrin binds preferably via an outside self-stacking mode with B- and A-DNA

Retained binding mode of various DNA-binding molecules under molecular crowding condition

<p><i>Meso</i>-tetrakis(<i>N</i>-methyl pyridinium-4-yl)porphyrin (TMPyP) intercalates between the base-pairs of DNA at a low [TMPyP]/[DNA base] ratio in aqueous solutions and molecular crowding conditions, which is induced by the addition of Poly(ethylene glycol) (PEG). Studied DNA-binding drugs, including TMPyP, 9-aminoacridine, ethidium bromide, and DAPI (4′,6-diamidino-2-phenylindole) showed similar binding properties in the presence or absence of PEG molecules which is examined by circular and linear dichroism. According to the LD^<i>r</i> (reduced linear dichroism) results of the binding drugs examined in this work, PEG molecules induced no significant change compared to their binding properties in aqueous buffering systems. These results suggest that the transition moments are not expected to be perturbed significantly by PEG molecules. In this study, the experimental conditions of PEG 8000 were maintained at 35% (<i>v/v</i>) of total reaction volume, which is equal to the optimal molar concentration (0.0536 M as final concentration for PEG 8000) to maintain suitable cell-like conditions. Therefore, there was no need to focus on the conformational changes of the DNA helical structure, such as forming irregular aggregate structures, induced by large quantities of molecular crowding media itself at this stage.</p

Enantioselective light switch effect of Δ- and Λ-[Ru(phenanthroline)₂ dipyrido[3,2-a:2′, 3′-c]phenazine]²⁺ bound to G-quadruplex DNA

<p>The interaction of Δ- and Λ-[Ru(phen)₂DPPZ]²⁺ (DPPZ = dipyrido[3,2-a:2′, 3′-c]phenazine, phen = phenanthroline) with a G-quadruplex formed from 5′-G₂T₂G₂TGTG₂T₂G_2–3′(15-mer) was investigated. The well-known enhancement of luminescence intensity (the ‘light-switch’ effect) was observed for the [Ru(phen)₂DPPZ]²⁺ complexes upon formation of an adduct with the G-quadruplex. The emission intensity of the G-quadruplex-bound Λ-isomer was 3-fold larger than that of the Δ-isomer when bound to the G-quadruplex, which is opposite of the result observed in the case of double stranded DNA (dsDNA); the light switch effect is larger for the dsDNA-bound Δ-isomer. In the job plot of the G-quadruplex with Δ- and Λ-[Ru(phen)₂DPPZ]²⁺, a major inflection point for the two isomers was observed at <i>x</i> ≈ .65, which suggests a binding stoichiometry of 2:1 for both enantiomers. When the G base at the 8th position was replaced with 6-methyl isoxanthopterin (6MI), a fluorescent guanine analog, the excited energy of 6-MI transferred to bound Δ- or Λ-[Ru(phen)₂DPPZ]²⁺, which suggests that at least a part of both Ru(II) enantiomers is close to or in contact with the diagonal loop of the G-quadruplex. A luminescence quenching experiment using [Fe(CN)₆]^4- for the G-quadruplex-bound Ru(II) complex revealed downward bending curves for both enantiomers in the Stern–Volmer plot, which suggests the presence of Ru(II) complexes that are both accessible and inaccessible to the quencher and may be related to the 2:1 binding stoichiometry.</p

<i>POMC</i> CpG methylation pattern of CpG island 1 and 2.

<p>Analysis example of the DNA methylation pattern of human peripheral blood cells (PBC) from a normal weight individual after sequencing of 10 different clones of the PCR amplification product. Filled cycles represent methylated CpGs, open cycles non-methylated CpG positions, which are numbered according to their relative position to the start of the next exon. Alu element positions and P300 binding site are marked.</p

<i>POMC</i> DNA methylation pattern of CpG island 1 and 2 in normal-weight adolescents (n = 12), laser-microdissected MSH positive cells of the human arcuate nucleus (n = 5) and human newborn samples (n = 8).

<p>CpG positions are numbered according to their relative position to the next exon start.</p

<i>POMC</i> intron2 exon3 genomic sequence, P300 ChIP assay, real-time PCR analysis, and real-time <i>POMC</i> PCR.

<p>(A) <i>POMC</i> intron2 exon3 genomic sequence with annotated ChIP primer localization and P300 binding site. (B) P300 ChIP assay was performed at three independent occasions within human peripheral blood cells. <i>POMC</i> fragment was amplified with two different primer pairs (fragment 1 and 2). Confirmation of a published P300 binding site within the insulin gene promoter in β-TC3 cells was used as a positive control <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002543#pgen.1002543-Chakrabarti1" target="_blank">[34]</a> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002543#pgen.1002543.s005" target="_blank">Figure S5</a>). (C) Real time PCR analysis (PCR fragment 2) of the P300 ChIP results of 4 obese patients with a hypermethylation variant and 6 individuals with hypomethylated intron 2-exon 3 intersection at <i>POMC</i> CpG island 2. (D) Real-time <i>POMC</i> PCR (<i>POMC</i> exon 2–3 and <i>POMC</i> exon3) of cDNA extracted from PBC of hypomethylated normal weight individuals (n = 20), hypermethylated obese patients (n = 20), and obese individuals without the hypermethylated variant (n = 20) reveal reduced POMC gene expression in the hypermethylated samples.</p

The DNA methylation was analysed with two different bisulphite based protocols in two independent cohorts.

<p>A, B, C agarose-embedded DNA was analysed by bisulphite sequencing. In D, E, F DNA was bisulphite treated in a non-agarose-bead protocol. A/D Diagram of the DNA methylation score (%) of <i>POMC</i> CpG island 2 within PBC of normal weight (red) and obese individuals (blue) (p<0,001). CpG positions are numbered according to their relative position to the next exon start. B/E Box plots analysis represents the statistic differences from the mean CpG methylation score (%) of CpG position −4 to +6 in normal weight individuals versus obese patients. C/F Percent of normal weight (red) and obese (blue) individuals, who showed DNA methylation at the annotated single CpG position (−1 to +5) in relation to the analysed number of samples.</p

Longitudinal analysis of <i>POMC</i> DNA methylation score (%) (CpG position −4 to +6) before and after the development of obesity in altogether 21 participants of the MAS birth cohort.

<p>Each line represents one individual course of DNA methylation score prior to and after weight gain.</p

Effects of the VEGF signaling on the trans-differentiation of NSCs.

<p>(A) GFP-positive NSCs and GFP-negative human umbilical venous endothelial cells were co-cultured on the matrigel in the differentiation condition of endothelial cells. Effects of Flk-1 inhibition on the trans-differentiation of NSCs were analyzed by the treatment of ZD6474. (B) Relative GFP⁺ tube length (%) and percent of GFP⁺ tube of total tube length were measured and compared * P<0.05. n = 3 for each group.</p

Trans-differentiation and migration of NSCs in the animal models with focal brain irradiation and effects of the VEGF signaling on the migration of NSCs.

<p>(A) GFP⁺ NSCs were injected into the right brain hemisphere stereotactically at 24 hours after focused brain irradiation to the left hemisphere using a GKS apparatus (50% isodose = 3 Gy). The dosimetry for GKS was calculated and presented. The irradiation focus could be within 0.5 mm of the planned target. (B) Immunohistochemistry against GFP was performed. Injected GFP⁺ NSCs migrated into the contralateral irradiated brain hemisphere and made GFP⁺ vessels. (C) The migration route and velocity were analyzed by immunohistochemistry against GFP (green) at various time points after the injection. NSC  =  NSC supplementation without GKS (n = 5 for each time point), IR + NSC  =  NSC supplementation at 24 hours after GKS (n = 5 for each time point). Arrowheads  =  injection tract. Arrows  =  GFP⁺ NSCs migrating in the subventricular zone. (D) Systemic treatment with a specific Flk-1 inhibitor (ZD6474, 50 mg/kg, oral administration 12 hours after the irradiation) inhibited the migration of injected GFP⁺ NSCs in the focally irradiated brain (n = 5 for each time point). Arrows  =  GFP⁺ NSCs migrating in the subventricular zone.</p