DNA-binding geometry dependent energy transfer from 4′,6-diamidino-2-phenylindole to cationic porphyrins

The circular and linear dichroism (CD and LD) spectral properties of the meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP)–DNA complex at a [porphyrin]/[DNA] ratio below 0.015 showed that TMPyP intercalates between DNA base pairs. Contrarily, when cis–bis(N-methylpyridinium-4-yl)porphyrin (BMPyP) is associated with DNA, no CD spectrum was induced and a bisignate LD spectrum was observed. These spectral properties of both the TMPyP and BMPyP were essentially retained when the minor groove of the DNA was saturated with 4′,6-diamidino-2-phenylindole (DAPI). The fluorescence of the DNA-bound DAPI was effectively quenched by BMPyP and TMPyP. The quenching by BMPyP can be described through a pure static mechanism while TMPyP quenching produced an upward bending curve in the Stern–Volmer plot. Quenching efficiency was by far greater than predicted by the “sphere of action model”, suggesting that the DNA provides some additional processes for an effective energy transfer.     

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72

Background

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures.

Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.

The effects of macromolecular crowding were tested on several reactions catalyzed by T4 RNA ligase. The rate of cyclization of oligoriboadenylates was stimulated up to 10-fold by relatively high concentrations of several polymers (polyethylene glycol (PEG) 8000 or 20,000; bovine plasma albumin; Ficoll 70). In addition, higher concentrations of PEG 8000 or PEG 20,000 allowed the novel formation of large linear products from the oligoriboadenylates. Also stimulated by high concentrations of PEG 8000 were the rate at which T4 RNA ligase joined p(dT)10 to oligoriboadenylates and the rate at which the enzyme activated p(dT)n by transfer of an adenylyl moiety from ATP to the oligonucleotides. These results with T4 RNA ligase are compared to earlier studies on the effects of crowding on DNA ligases.     

Conformational change of a G-quadruplex under molecular crowding conditions

Abstract

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K⁺ and Na⁺). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na⁺ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K⁺ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.

Communicated by Ramaswamy H. Sarma     

What is the true enzyme kinetics in the biological system? An investigation of macromolecular crowding effect upon enzyme kinetics of glucose-6-phosphate dehydrogenase

Enzyme kinetic parameters for rate equations are vital in metabolic network simulation, a major part of systems biology research efforts. Measurements of Michaelis–Menten kinetic parameters K_m and K_cat have been performed for enzymes glucose-6-phosphate dehydrogenase (G6P DH) under crowded conditions using molecular crowding agents bovine serum albumin (BSA) and polyethylene glycol (PEG) of 8000 Da molecular weight. An increase in K_cat was observed at very low concentrations of crowding agent, and also at high crowder concentrations when the experiment was performed at 45 °C with PEG. The observed pattern in K_cat for G6P DH at high crowder concentrations has been explained via modelling using excluded volume theory. An increase in rate was observed at 45 °C for G6P DH versus 30 °C; this has been modelled via the Arrhenius equation.     

Temperature-induced changes of the packing of double-stranded linear DNA molecules in particles of liquid-crystalline dispersions

The circular dichroism spectra of liquid-crystalline dispersions obtained by phase exclusion of linear double-stranded DNA molecules from aqueous saline solutions of polyethylene glycol (120 ≤ C_PEG ≤ 300 mg/mL) have been investigated. The formation of liquid-crystalline dispersions at polyethylene glycol concentrations ranging from 120 to 200 mg/mL was accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism; this is indicative of cholesteric packing of the double stranded DNA molecules in the particles of the dispersion. Liquid-crystalline dispersions formed at PEG concentrations higher than 220 mg/mL and room temperature did not show any abnormal bands in the circular dichroism spectra; this is indicative of hexagonal packing of double-stranded DNA molecules in the particles of the dispersions. Heating of optically inactive liquid crystal dispersions induced a transition of the dispersions into a different state accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism. This transition is considered within the concept of the transformation of a hexagonal packing of DNA molecules into a cholesteric packing. A qualitative mechanism of such a transition is proposed that is formulated in the terms of the “quasinematic” layers of double-stranded DNA molecules that change their spatial orientation under the competing influences of the osmotic pressure of the solvent, orientational elasticity of the cholesteric packing, and thermal fluctuations.     

Flow dichroism of deoxyribonucleic acid solutions

The flow dichroism of dilute DNA solutions (A₂₆₀ ≈ 0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5–160 sec.^?1 were studied. The DNA samples were whole, “half,” and “quarter” molecules of T4 bacteriophage DNA, and linear and circular λb₂b₅c, DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear λ DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.     

Depletion interactions and modulation of DNA‐intercalators binding: Opposite behavior of the “neutral” polymer poly(ethylene‐glycol)

In this work we have investigated the role of high molecular weight poly(ethylene‐glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single‐molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an “inert polymer” with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non‐neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA‐ligand interactions. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 227–233, 2016.     

5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin destabilizes the antiparallel telomeric quadruplex d(TTAGGG)<Subscript>4</Subscript>

5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin (TMPyP3) is a DNA-binding derivative of porphyrins. A comparative study of the binding of this ligand to biologically significant DNA structures was performed. For this purpose, the interactions of TMPyP3 with the antiparallel telomeric G-quadruplex d(TTAGGG)₄, oligonucleotide dTTAGGGTTAGAG(TTAGGG)₂ (not forming a quadruplex structure), double-stranded d(AC)₈ · d(GT)₈, and single-stranded d(AC)₈ and d(GT)₈ DNA molecules have been studied. Analysis of absorption isotherms has demonstrated that the binding constants and the number of binding sites for the complexes TMPyP3: DNA increase in the following order: d(AC)₈ < d(GT)₈ < d(AC)₈ · d(GT)₈ = d(TTAGGG)₄ < dTTAGGGTTAGAG(TTAGGG)₂. It has been for the first time demonstrated that the constant for TMPyP3 binding to unfolded dTTAGGGTTAGAG(TTAGGG)₂ strand (1.3 × 10⁷ M⁻¹) is approximately threefold higher than for the G-quadruplex d(TTAGGG)₄ (4.7 × 10⁶ M⁻¹). Binding of two TMPyP3 molecules to d(TTAGGG)₄ decreases the thermostability of G-quadruplex (ΔT_m = −8°C). Circular dichroism spectra of the TMPyP3 complexes with d(TTAGGG)₄ suggest that the ligand partially unfolds the G-quadruplex structure. Structural destabilization of the telomeric G-quadruplex by TMPyP3 can explain the relatively low activity of this ligand as a telomerase inhibitor and a low cytotoxicity for cultured tumor cells.     

Liquid-crystalline dispersions formed by the complexes of linear double-stranded DNA molecules with dendrimers

The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.     

Human telomeric G-quadruplex formed from duplex under near physiological conditions: Spectroscopic evidence and kinetics

The structural interconversion between the G-quadruplex and duplex in vivo is an important subject. In the present study, we used human telomeric DNA duplex composed of GGG(TTAGGG)₃/CCC(TAACCC)₃ as a model system to investigate its properties under near physiological conditions by spectroscopic methods. Circular dichroism and fluorescence spectra demonstrated that G-quadruplex structure can be formed from duplex at near physiological pH (pH 7.4), salt concentration (150 mM K⁺), and temperature (37 °C) in the presence of molecular crowding agent PEG (400 g/l), whereas the G-quadruplex structure cannot be formed at 25 °C in buffer containing 150 mM K⁺ in the presence of PEG. It is found that the formation rate of G-quadruplex structure depends on the temperature and the concentrations of both PEG and K⁺. This work suggests that human telomeric G-quadruplex structure may be potentially formed from Watson–Crick duplex in vivo.     

5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin destabilizes the anti-parallel telomeric quadruplex d(TTAGGG)4

We studied the parameters of binding of 5,10,15,20-tetra-(N-methyl-3-pyridyl)porphyrin (TMPyP3) to the anti-parallel human telomeric G-quadruplex d(TTAGGG)4, the oligonucleotide dTTAGGGTTAGAG(TTAGGG)2 that does not form a quadruplex structure, as well as to the double stranded d(AC)8 x d(GT) and single stranded d(AC)8 and d(GT)8 DNAs. The analysis of absorption revealed that the binding constants and the number of DNA binding sites for TMPyP3 were d(AC)8 < d(GT)8 < d(AC)8 x d(GT)8 = d(TTAGGG)4 < dTTAGGGTTAGAG(TTAGGG)2. We demonstrated for the first time that the binding constant of TMPyP3 with the non-quadruplex chain dTTAGGGTTAGAG(TTAGGG)2 (1.3 x 10(7) M(-1)) is approximately 3 times bigger than the binding constant with the quadruplex d(TTAGGG)4 (4.6 x 10(6) M(-1)). Binding of two TMPyP3 molecules to d(TTAGGG)4 led to a decrease of thermostability of the G-quadruplex (deltaT(m) = -8 degrees C). Circular dichroism spectra of TMPyP3:d(TTAGGG)4 complexes revealed a shift of DNA structure from the G-quadruplex to an irregular chain. We hypothesize that partial destabilization of the telomeric G-quadruplex by TMPyP3 might be a reason for relatively low potency of this ligand as a telomerase inhibitor, as well as its marginal cytotoxicity for cultured tumor cells.     

Role of solvent properties of aqueous media in macromolecular crowding effects

Analysis of the macromolecular crowding effects in polymer solutions show that the excluded volume effect is not the only factor affecting the behavior of biomolecules in a crowded environment. The observed inconsistencies are commonly explained by the so-called soft interactions, such as electrostatic, hydrophobic, and van der Waals interactions, between the crowding agent and the protein, in addition to the hard nonspecific steric interactions. We suggest that the changes in the solvent properties of aqueous media induced by the crowding agents may be the root of these “soft” interactions. To check this hypothesis, the solvatochromic comparison method was used to determine the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity of aqueous solutions of different polymers (dextran, poly(ethylene glycol), Ficoll, Ucon, and polyvinylpyrrolidone) with the polymer concentration up to 40% typically used as crowding agents. Polymer-induced changes in these features were found to be polymer type and concentration specific, and, in case of polyethylene glycol (PEG), molecular mass specific. Similarly sized polymers PEG and Ucon producing different changes in the solvent properties of water in their solutions induced morphologically different α-synuclein aggregates. It is shown that the crowding effects of some polymers on protein refolding and stability reported in the literature can be quantitatively described in terms of the established solvent features of the media in these polymers solutions. These results indicate that the crowding agents do induce changes in solvent properties of aqueous media in crowded environment. Therefore, these changes should be taken into account for crowding effect analysis.     

Induced chirality of binary aggregates of oppositely charged water-soluble porphyrins on DNA matrix

The induced chirality of achiral binary aggregates of meso-tetrakis(4-N-methylpyridyl)porphyrine (TMPyP) and meso-tetrakis(4-sulfonatophenyl)porphyrine (TPPS) on a deoxyribonucleic acid (DNA) matrix was investigated. Although the negatively charged TPPS did not show induced chirality in DNA solution due to the electrostatic repulsion, induced chirality was obtained through the addition of a positively charged TMPyP. It was confirmed that the induced chirality was due to the binary complex formation between TPPS and TMPyP on the DNA matrix. Moreover, the induced chirality depended on the relative molar ratio of TPPS to TMPyP (r) and the binding modes of the complex to DNA. When r<1, induced circular dichroism (CD) spectrum of the ternary complex was similar to that of intercalated TMPyP into DNA. For r=1, the induced CD spectrum showed a reversed biphasic signal due to the complex of TMPyP and TPPS stacking along the DNA surface. At a higher r value (>1), there was an induced CD signal at 482 nm attributed to a lateral shifted arrangement of heteroaggregate of TPPS and TMPyP on DNA matrix where TMPyP acted as a spacer to mediate the growth of heteroaggregates. Increasing the concentration of sodium chloride in the solution would favor the formation of the lateral shifted arrangement of heteroaggregate of TPPS and TMPyP. The resonance light scattering (RLS) spectra confirmed the above results. Analysis of the CD spectral changes in DNA conformation showed that during the binary complex formation of TPPS and TMPyP, the intercalated TMPyP could be 'pulled out' from the base pairs of DNA, which might be useful in gene therapy. A model was proposed to account for these observations.     

Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

Abstract

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G₃T₂G₃TGTG₃T₂G₃) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I₂ to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction.

Communicated by Ramaswamy H. Sarma     

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)₁/DNA complex (K₁ = 3.7 × 10⁶) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)₁/DNA complex, (K₁ = 7.0 × 10⁴). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)₃/DNA and (TMPyP4)₄/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.     

Binding of cationic porphyrin to isolated and encapsidated viral DNA analyzed by comprehensive spectroscopic methods

The complexation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with free and encapsidated DNA of T7 bacteriophage was investigated. To identify binding modes and relative concentrations of bound TMPyP forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. Spectral decomposition, fluorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of TMPyP, e.g., external binding and intercalation both in free and in encapsidated DNA. Optical melting studies revealed that TMPyP increases the strand separation temperature of both free and native phage DNA and does not change the phase transition temperature of phage capsid proteins. From these findings we concluded that TMPyP binding does not influence the protein structure and/or the protein-DNA interaction. A combined analysis of absorption spectra and fluorescence decay curves made possible the determination of concentrations of free, externally bound, and intercalated porphyrin. As a perspective, our results facilitate a qualitative analysis of the TMPyP binding process at various experimental conditions.     

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background

G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging.

Methods

Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA–drug interactions.

Results

We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines.

Conclusions

Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed.

General significance

The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties.