A UV Resonant Raman Spectroscopic Study of the Interaction of Metallic Derivatives of Tetrakis(4-N-methylpyridyl)porphine with Polynucleotides

Abstract

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)₂, poly(dA)poly(dT), poly(dG.dC)₂ and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC)₂. Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.     

Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ was studied by UV–vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV–vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]₂ and poly(dC)·poly(dG)·poly(dC)⁺ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)⁺ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Enhanced Cellular Uptake of G-Rich Oligonucleotides

Abstract

Sequence-dependency of cellular uptake of oligonucleotides into Vero cells has been studied. Cellular uptake of 5′-[³⁵S]-labelled homopolymers decreased in the order (dG)₁₆ >> (dT)₁₆> (dA)₁₆ > (dC)₁₆. The change of two base-pairs (dG → dA) in a dG-rich antisense oligonucleotide with good antiviral activity dramatically decreased cellular uptake and abolished antiviral activity.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.     

Studies on the processivity of highly purified calf thymus 8S and 7.3S DNA polymerase alpha

Template-challenge experiments indicate no gross difference in processivity of the calf thymus DNA polymerase α A and C enzymes. Both enzymes appear to be distributive. Results showing the apparent processive nature of both enzymes on poly (dC). oligo (dG)₁₀ when challenged with poly (dA). oligo (dT)₁₀ are explicable by the failure of both enzymes to bind to the challenging template rather than by the presence of an initiation factor which preferentially binds to certain templates.     

Mechanistic study of E. coli DNA topoisomerase I: cleavage of oligonucleotides.

E. coli DNA topoisomerase I catalyzes DNA topoisomerization by transiently breaking and rejoining single DNA strands (1). When an enzyme-DNA incubation mixture is treated with alkaline or detergent, DNA strand cleavage occurs, and the enzyme becomes covalently linked to the 5'-phosphoryl end of the cleaved DNA (2). Using oligonucleotides of defined length and sequence composition, this cleavage reaction is utilized to study the mechanism of E. coli DNA topoisomerase I. dA7 is the shortest oligonucleotide tested that can be cleaved by the enzyme. dT8 is the shortest oligo(dT) that can be cleaved. The site of cleavage in both cases is four nucleotides from the 3' end of the oligonucleotide. No cleavage can be observed for oligo(dC) and oligo(dG) of length up to eleven bases long. dC15 and dC16 are cleaved at one tenth or less the efficiency of oligo(dA) and oligo(dT) of comparable length.     

Binding of 5S estradiol receptor to poly-deoxynucleotides

Calf uterus cytosol was incubated with (³H)estradiol and fractionated on Sephadex G-200. Two (³H)estradiol-binding protein fractions were obtained with sedimentation coefficients of 5.1 S and 3.5 S, respectively. The 5.1 S fraction bound to poly dT, poly dA:dT and poly dG:dC to a higher extent than to calf thymus DNA. The 3.5 S fraction did not bind to DNA.     

Berberine–DNA complexation: New insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA–dT).poly(dA–dT) and poly(dG–dC).poly(dG–dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K_a versus [Na⁺] for poly(dA).poly(dT) and poly(dA–dT).poly(dA–dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (ΔCp^o) of berberine binding to poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), Clostridium perfringens and calf thymus DNA were − 98, − 140, − 120 and − 110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins.

During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.     

Sequence Specific Binding of Beta Carboline Alkaloid Harmalol with Deoxyribonucleotides: Binding Heterogeneity,Conformational, Thermodynamic and Cytotoxic Aspects

Background

Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.

Methodology/Principal Findings

Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.

Conclusions/Significance

Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC₅₀ value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA).poly(dT) and poly(dG).poly(dC), and with triple helical poly(dA).[poly(dT)](2) and poly(dC).poly(dG).poly(dC)(+) were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA).poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG).poly(dC) and -poly(dC).poly(dG).poly(dC)(+) complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.     

Effect of tetramethylammonium ions on conformational changes of DNA in the premelting temperature range

The reversible conformational change of DNAs and polydeoxyribonucleotides occurring before melting was followed by circular dichroism. Δθ/δT, the rate of change of ellipticity θ with temperature, was used mainly as a measure of this premelting phenomenon. If sodium ions were replaced by tetramethylammonium ions Δθ/δT decreased for poly (dA) poly (dT) and poly (dA.dT) poly (dT.dA), but increased for poly (dG.dC) poly (dC.dG). DNAs of different base composition showed no more premelting (Δθ/ΔT ~ 0) even at low molarities of TMACl provided the Na/TMA ratio was very small. For all cases studied the θ values at 0°C and at a given ionic strength were smaller in NaCl than in TMACl. When studying the series of ammonium ions from NH⁺₄ to (C₂H₅)₄,N⁺ the Δθ/ΔT values first decreased, going through zero with TMA⁺ io and then increased again. A tentative and qualitative explanation of our results can be given: (a) Hydration of the polymers increases in presence of TMA ions and their average stability decreases; locally, however, (AT) pairs are preferentially stabilized by TMA ions owing to a specific interaction at the level of O₂ of thymine. (b) In order to explain the different behaviour of (AT) polymers and DNA, it is assumed that only the B structure is able to accommodate TMA ions in the small groove of the double stranded helix.     

Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin.

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.     

Conformational Peculiarities of Polynucleotides with a Nonrandom Base Sequence According to the 1H→ 3H Exchange Rate in C8H Groups of Purinic Residues

Abstract

We have determined the ¹H→³H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT)·poly(dA-dT), poly(dG-dC)·poly(dG- dC) and poly(dA-dC)·poly(dG-dT) as well as homopolynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4–6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25°C. The rate constants for adenine (k_A) and guanine (k_G) of polynucleotides were compared with corresponding constants for E.coli DNA, dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution.

Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT)·poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating “wrinkled” DNA model. The conformations of poly(dG-dC)·poly(dG-dC) and poly(dA-dC)·poly(dG-dT), according to the exchange data obtained, are within the B form. For homopolynucleotides in 0.15 M NaCl, the k_A value for poly(dA)·poly(dT) is nearly the same as k_A for B-DNA, which indicates the similarity of their conformations, whereas the k_G value for poly(dG)·poly(dC) is 1.7-fold lower in comparison with the k_G value in B-DNA. This seems to be connected with the existence of B? A conformation equilibrium for poly(dG)·poly(dC) in solution.

The increase of NaCl concentration to 3 M results in a B→Z transition in the case of poly(dG-dC)·poly(dG-dC) and in the shift of B?A equilibrium towards the A-form in the case of poly(dG)·poly(dC), as is evidenced by alterations of their K_G values. Poly(dA-dT)·poly(dA-dT) in 6 M CsF and poly(dA-dC)·poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the “X-type” CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA)·poly(dT) in 6 M CsF corresponds to the “heteronomous” DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.     

Receptor properties of corticosteroid binder IB

Corticosteroid binder IB, present in liver and kidney, is pronounced in liver cytosol after injection of [³H]triamcinolone acetonide. Following injection of the radioactive ligand, livers homogenized in the presence of 20 mm molybdate, 2 mm leupeptin hemisulfate, 2 mm antipain, or 2 mm phenylmethylsufonyl fluoride produce cytosols with Chromatographie profiles of binders II and IB identical to controls, as determined by DEAE-Sephadex chromatography, suggesting that IB is a cellular constituent rather than a product of protease action (sensitive to the above inhibitors) after cell breakage. Generation of IB in kidney cytosols in vitro appears to be unrelated to protease activity. Liver binder IB has an S value of 5–6 and a Stokes radius of about 26 Å producing a calculated range of molecular weight from 40,000 to 50,000 with frictional coefficient and axial ratio close to spherical values. As expected of a steroid receptor, IB, like II, binds to DNA and to liver cell nuclei but IB binds more tightly as evidenced by the fact that KCl is more effective in eluting II than IB from nuclei. Because recovery of bound radioactivity from acceptors is sometimes difficult to achieve, indirect experiments have been used frequently to determine the binding. Pyridoxal phosphate extracts liver IB and II equally from nuclei but spermidine is ineffective. While IB and II can be extracted partially from nuclei by pancreatic DNase I, more binder II is extracted by this method than IB. Micrococcal nuclease is poorly effective in either case. Binder II is extracted to a greater degree from DNA-cellulose than is IB by spermidine, MgCl₂, pyridoxal phosphate, and NaCl. IB binds more extensively to homodeoxypolymers than II. The extent of binding of liver IB to homodeoxypolymers is in the order: poly(dC) ≥ poly(dG) > poly(dA) ? poly(dT), whereas the order for liver binder II is: poly(dG) ≥ poly(dT) > poly(dC) ? poly(dA). Binders IB and II may be separate gene products or IB may arise in the cell from post-translational action. In the latter case, the activity of a protease cannot be ruled out.