Ethidium Probing of the Parallel Double- and Four-stranded Structures Formed by the Telomeric DNA Sequences dG(GT)4G and d(GT)5

Abstract

Oligonucleotides 3′-d(GT)₅-(CH₂CH₂O)₃-d(GT)₅-3′ (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140–142 (1992)]. Four d(GT)₅ strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3′-dG(TG)₄G-(CH₂CH₂O)₃- dG(GT)₄G-3′ (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G·G and T·T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dynam. 18, 493–503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (τ) of Et:qGT (q = 0.15 ±0.01 and τ = 24 ±1 ns) and Et:hp-GT (q = 0.10 ± 0.01 and τ = 16.5 ± 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient ω = 3.9 ± 0.1 and the binding constant K= (6.4 ± 0.1)·10M^?1. The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of inerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732–739 (2001)]. The anticooperative type of Et binding was observed in case of hp- GT ps-DNA, with the maximum number of bound Et molecules, N = 4 ÷ 5, and the association constant K = (1.5 ± 0.1)·10⁵ M^?1. Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.     

The telomeric dG(GT)4G sequence can adopt a parallel-stranded double helical conformation

Oligonucleotides 3'-d(GTGTGTGTGG)-L-d(GGTGTGTGTG)-3' (hp-GT) and 3'-d(G4STG4TG4STG4STGG)-L-d(GGTGTGTGTG)-3' (hp-SGT), (L=(CH2CH2O)3), were shown by use of several optical techniques to form a novel parallel-stranded (ps) intramolecular double helix with purine-purine and pyrimidine-pyrimidine base pairing. The rotational relaxation time of hp-GT was similar to that of a 10-bp reference duplex, and the fraction of unpaired bases was determined to be approximately 7%, testifying to the formation of an intramolecular double helical hairpin by the sequence under the given experimental conditions. A quasi-two-state mode of ps-double helix formation was validated, yielding a helix-coil transition enthalpy of -135 +/- 5 kJ/mol. The G x G and T x T (or 4ST x T) base pair configurations and conformational parameters of the double helix were derived with molecular modeling by force field techniques. Repetitive d(GT) sequences are abundant in telomers of different genomes and in the regulatory regions of genes. Thus, the observed conformational potential of the repetitive d(GT) sequence may be of importance in the regulation of cell processes.     

Conformational polymorphism and extensibility of DNA quadruplexes formed from d(GT)n repeats

We showed earlier that oligonucleotides 3'-d(GT)5-pO(CH2CH2O)3p-d(GT)5-3' form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K = (3.3 +/- 0.1) x 10(4) M-1, cooperativity coefficient omega = 2.5 +/- 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N = 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.     

Intramolecular G-quadruplexes from microsatellite d(GT)12 sequence in the presence of K+

Polymorphic d(GT)n microsatellite sequences are known to drastically affect genes expression. By use of CD spectroscopy, UV melting, fluorescence polarization of EtBr probe and FRET, we detected formation of a new fold with three G-quartets by d(GT)12 oligonucleotide in 0.01 M Na phosphate buffer, pH 8.0, in the presence 0.1 M KCl. Monomolecular type of the structure was verified with measurements of rotational relaxation time (p = 28 +/- 0.5 ns) of EtBr:d(GT)12 complex. CD spectra supported G-quartets formation. A distance between FITC, covalently attached to 5'-end of d(GT)12, and intercalated EtBr molecule was estimated using FRET (R < or =17 A). These data are in agreement with the proposed self folding of d(GT)12.     

The Telomeric dG(GT)4G Sequence Can Adopt a Parallel-Stranded Double Helical Conformation

Abstract

Oligonucleotides 3′-d(GTGTGTGTGG)-L-d(GGTGTGTGTG)-3′ (hp-GT) and 3′- d(G^4STG^4STG^4STG^4STGG)-L-d(GGTGTGTGTG)-3′ (hp-SGT), (L=(CH₂CH₂O)₃), were shown by use of several optical techniques to form a novel parallel-stranded (ps) intramolecular double helix with purine-purine and pyrimidine-pyrimidine base pairing. The rotational relaxation time of hp-GT was similar to that of a 10-bp reference duplex, and the fraction of unpaired bases was determined to be ～ 7%, testifying to the formation of an intramolecular double helical hairpin by the sequence under the given experimental conditions. A quasi-two- state mode of ps-double helix formation was validated, yielding a helix-coil transition enthalpy of ?135±5 kJ/mol. The G-G and T·T (or ^4ST·T) base pair configurations and conformational parameters of the double helix were derived with molecular modeling by force field techniques. Repetitive d(GT) sequences are abundant in telomers of different genomes and in the regulatory regions of genes. Thus, the observed conformational potential of the repetitive d(GT) sequence may be of importance in the regulation of cell processes.     

Structure of d(GT)n repeating sequences with parallel and antiparallel chains]

The ability of oligonucleotides 3'-d(GT)5pO(CH2)5Opd(GT)5-5' (anti[d(GT)]) and 3'-d(GT)5pO(CH2)6Opd(GT)5-3' (par[d(GT)]) to form hairpins and higher associates is studied. Optical methods of thermal denaturation and circular dichroism as well as the fluorescence of ethidium bromide and acridine orange bound to oligonucleotides were used. At room temperatures the formation of hairpin structure with parallel and antiparallel strands is possible. Thermodynamic parameters of par[d(GT)] and anti[d(GT)] are similar and equal to delta H = -15 kcal/mol, delta S = -50 cal/mol. deg. In the temperature range 3-10 degrees C par[d(GT)] and anti[d(GT)] form four-stranded structures with parallel chains, in which layers of four G-residues alternate with unpaired T-residues being bulged out easily. On comparison of occurrence of alternating (GT)n, (GC)n and (G)n sequences in genome it can be stated that (GT)n biological functions could be connected with conformational possibilities of the four-stranded parallel structures with unpaired T-residues.     

Lipari-Szabo approach as a tool for the analysis of macromolecular gadolinium(III)-based MRI contrast agents illustrated by the [Gd(EGTA-BA-(CH2)12)] n n + polymer

The parameters governing the water proton relaxivity of the [Gd(EGTA-BA-(CH2)12)]nn+ polymeric complex were determined through global analysis of 17O NMR, EPR and nuclear magnetic relaxation dispersion (NMRD) data [EGTA-BA2- = 3,12-bis(carbamoylmethyl)- 6,9-dioxa-3,12-diazatetradecanedioate(2-)]. The Lipari-Szabo approach that distinguishes the global motion of the polymer (tau g) from the local motion of the Gd(III)-water vector (tau l) was necessary to describe the 1H and 17O longitudinal relaxation rates; therefore for the first time it was included in the global simultaneous analysis of the EPR, 17O NMR and NMRD data. The polymer consists on average of only five monomeric units, which limits the intramolecular hydrophobic interactions operating between the (CH2)12 groups. Hence the global rotational correlation time is not very high (tau g298 = 3880 +/- 750 ps) compared to the corresponding DTPA-BA-based polymer (about 15 monomeric units), where tau g298 = 6500 ps. As a consequence, the relaxivity is limited by the rotation, which precludes the advantage obtained from the fast exchanging chelating unit (kex298 = 2.2 +/- 0.1 x 10(6) s-1).     

Synthetic analogs for oxovanadium(IV/V)-glutathione interaction: an NMR, EPR, synthetic and structural study of oxovanadium(IV/V) compounds with sulfhydryl-containing pseudopeptides and dipeptides

The reaction of [VO(CH3COO)2(phen)] (phen = 1,10-phenanthroline) with the sulfhydryl-containing pseudopeptides (scp), N-(2-mercaptopropionyl)glycine (H3mpg), N-(2-mercaptopropionyl)cysteine (H4m2pc), N-(3-mercaptopropionyl)cysteine (H4m3pc) and the dipeptides glycylglycine (H2glygly) and glycyl-L-alanine (H2glyala), in the presence of triethylamine, results in the formation of the compounds Et3NH[VO(mpg)(phen)] (1), (Et3NH)2[VO(m2pc)] (4), [(Et3NH)2[VO(m3pc) (5), [VO(glygly)(phen)] x 2CH3OH (2 x 2CH3OH) and [VO(glyala)(phen)] x CH3OH (3 x CH3OH). Evidence for the molecular connectivity in 2 x CH3OH was established by X-ray crystallography, showing the vanadium(IV) atom ligated to a tridentate glygly2- ligand at the N(amine), N(peptide) and O(carboxylato) atoms. Combination of the correlation plot of the EPR parameters gz versus Az, together with the additivity relationship supported the prediction of the equatorial donor atom sets of the V(IV)O2+ center at various pH values for the V(IV)O2+-glutathione system considered in this study. Model NMR studies (interaction of vanadium(V) with the scp H3mpg) showed that there is a possibility of vanadium(V) ligation to glutathione.     

Solution, solid state and biological characterization of ruthenium(III)-DMSO complexes with purine base derivatives

Two new complexes of Ru(III) with purine base derivatives, [mer-RuCl(3)(acv)(DMSO-S)(C(2)H(5)OH)].C(2)H(5)OH (1) (acv=acyclovir, DMSO=dimethyl sulfoxide) and [trans-RuCl(4)(guaH)(DMSO-S)].2H(2)O (2) (guaH=protonated molecule of guanine), were prepared from the same Ru(III) precursor, [trans-RuCl(4)(DMSO-S)(2)](-), by substitution of one DMSO-S. Coordination of acv induced also replacement of one chloride by an ethanol molecule. This reactivity difference was explained by striking contrasts in the hydrogen bonding schemes of the two complexes, evidenced in their X-ray crystal structures. In 1 the guanine derivative acyclovir is coordinated to ruthenium through the N(7) atom, while in 2 the protonated guanine molecule is bound through the N(9) atom. Both complexes were also characterized by various physico-chemical methods in the solid state and in the solution. In vitro, the biological activity of 2 and of the previously described complexes [mer-RuCl(3)(acv)(DMSO-S)(CH(3)OH)].0.5CH(3)OH (3) and [mer-RuCl(3)(acv)(DMSO-S)(H(2)O)].H(2)O (4) on tumour cells appear to be very similar to that of NAMI-A (NAMI-A=[ImH][trans-RuCl(4)(DMSO-S)Im]). All compounds are only weakly active on tumour cell proliferation but show an interesting proadhesive effect that suggest possible activity on tumour malignancy.     

Crystal packing and hydrogen bonding in platinum(II) nucleotide complexes: X-ray crystal structure of [Pt(MeSCH(2)CH(2)SMe)(5'-GMP-N7)(2)].6H(2)O

We have synthesised the complex [Pt(CH(3)SCH(2)CH(2)SCH(3))(5'-GMP-N7)(2)].6H(2)O (1), where 5'-GMP is 5'-guanosine monophosphate, and determined its X-ray crystal structure. Pt(II) adopts a square-planar geometry in which the bases are coordinated head-to-tail (HT) in the Delta configuration. The nucleotide conformation in this complex is almost identical to that in the previously reported complex [Pt(en)(5'-GMP-N7)(2)].9H(2)O (2), in which there is outer sphere macrochelation via intramolecular H-bonding between the monoanionic phosphate groups and the coordinated ethylenediamine (en) NH. It is therefore apparent that intermolecular interactions rather than intramolecular H-bonding determines the orientation of the sugar-phosphate side-chain in these Pt(II) bisnucleotide complexes in the solid state.     

Conformational Polymorphism and Extensibility of DNA Quadruplexes Formed by d(GT)nRepeats

We showed earlier that oligonucleotides 3"-d(GT)₅-pO(CH₂CH₂O)₃p-d(GT)₅-3" form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K= (3.3 ± 0.1)·10⁴M^–1, cooperativity coefficient = 2.5 ± 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N= 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.     

Synthesis, structure and luminescence studies of heterometallic gold(I)-copper(I) and -silver(I) alkynyl clusters/aggregates.

A series of pentanuclear gold(I)-copper(I) and -silver(I) mixed-metal alkynyl complexes, [(n)Bu(4)N][Au(3)M(2)(C triple bond CC(6)H(4)R-p)(6)] [M = Cu, R = OMe, O(n)Bu, O(n)Hex, Me, Et; M = Ag, R = Et, O(n)Hex] have been synthesized. The complexes were found to be emissive both in the solid state and in fluid solutions. DFT calculations at the B3LYP level of theory were performed on [Au(3)M(2)(C triple bond CC(6)H(4)Me-p)(6)](-) (M = Cu, Ag) to provide an understanding on the electronic structure of the complexes.     

Evidence for the tetraplex structure of the d(GT)n repetitive sequences in solution.

The ability of oligonucleotides 3'-d(GT)5pO(CH2)6Opd(GT)5-5' (anti[d(GT)]) and 3'-d(GT)5pO(CH2)6Opd(GT)5-3' (par[par[d(GT)]) to form tertiary structures has been studied. Circular dichroism (CD) as well as the fluorescence of the ethidium bromide (EtBr) complexes with oligonucleotides and hydrodynamic volume measurements in solutions containing 0.01 M phosphate buffer, pH 7 and NaCl in concentrations from 0.1 M to 1 M, have been used. The data obtained in the temperature interval from 3 degrees C to 10 degrees C are in good agreement with the structure suggested earlier where the par[d(GT)] and anti[d(GT)] form structures with four parallel strands in which layers of four G-residues alternate with unpaired bulged-out T-residues. Ethidium bromide interacts with the structure in a cooperative manner. Two ethidium bromide molecules intercalate between two layers of four G-residues.     

Low-temperature (180 K) crystal structures of tetrakis-mu-(niflumato)di(aqua)dicopper(II) N,N-dimethylformamide and N,N-dimethylacetamide solvates, their EPR properties, and anticonvulsant activities of these and other ternary binuclear copper(II)niflumate complexes

Two pseudopolymorphs, solvates, of [Cu(2)(II)(niflumate)(4)(H(2)O)(2)] of unknown structure were obtained following solution of [Cu(2)(II)(niflumate)(4)(H(2)O)(2)] in N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF). Low-temperature crystal structures obtained for these solvates revealed that they were ternary aqua DMA and DMF solvates: [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMA and [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMF. Intermolecular hydrogen bonding interactions account for the formation of these stable DMA and DMF solvates. These pseudopolymorphs contain a centrosymmetric binuclear center with Cu-Cu bond distances ranging from 2.6439(7) to 2.6452(9) A; the coordination sphere of Cu(II) is characterized by one long Cu-O (water) bond length of 2.128(3)-2.135(3) A and four short Cu-O (carboxylate) bonds of 1.949(3)-1.977(3) A. Crystal parameters for the DMA pseudopolymorph: a=10.372(1), b=19.625(2), c=17.967(2) A, beta=97.40(1) degrees , V=3626.8(6) A(3); monoclinic system; space group: P2(1)/a and for the DMF pseudopolymorph: a=10.125(2), b=18.647(3), c=19.616(4) A, alpha=74.38(2)(o), beta=88.18(2)(o), gamma=79.28(2)(o), V=3504(1) A(3); triclinic system; space group: P1. EPR spectra of these solids are identical and show strong antiferromagnetic coupling between the copper atoms, similar to the spectrum obtained for [Cu(2)(II)(niflumate)(4)(DMSO)(2)]. The [Cu(2)(II)(niflumate)(4)(H(2)O)(2)], [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMA, [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMF, [Cu(2)(II)(niflumate)(4)(DMF)(2)], and[Cu(2)(II)(niflumate)(4)(DMSO)(2)] evidenced protection against maximal electroshock-induced seizures and Psychomotor seizures at various times after treatment, consistent with the well known antiinflammatory activities of Cu chelates, but failed to protect against Metrazol-induced seizures while evidencing some Rotorod Toxicity consistent with a mechanism of action involving sedative activity.     

Mixed ligand complexes of platinum(II) and palladium(II) with cytokinin-derived compounds Bohemine and Olomoucine: X-ray structure of [Pt(BohH+-N7)Cl(3)].9/5H2O [Boh=6-(benzylamino)-2-[(3-(hydroxypropyl)-amino]-9-isopropylpurine,Bohemine

The new square-planar Pt(II) and Pd(II) complexes with cytokinin-derived compounds Bohemine and Olomoucine, having the formulae [Pt(BohH(+))Cl(3)].H(2)O (1), [Pt(Boh)(2)Cl(2)].3H(2)O (2), [Pt(Boh-H)Cl(H(2)O)(2)].H(2)O (3), [Pt(OloH(+))Cl(3)].H(2)O (4), [Pd(BohH(+))Cl(3)].H(2)O (5), [Pd(Boh)Cl(2)(H(2)O)] (6), [Pd(Boh-H)Cl(H(2)O)].EtOH (7) and [Pd(OloH(+))Cl(3)].H(2)O (8), where Boh=6-(benzylamino)-2-[(3-(hydroxypropyl)amino]-9-isopropylpurine and Olo=6-(benzylamino)-2-[(2-(hydroxyethyl)amino]-9-methylpurine, have been synthesized. The complexes have been characterized by elemental analyses, IR, FAB+ mass, 1H, 13C and 195Pt NMR spectra, and conductivity data. The molecular structure of the complex [Pt(BohH(+)-N7)Cl(3)].9/5H(2)O has been determined by an X-ray diffraction study. Results from physical studies show that both Bohemine and Olomoucine are coordinated to transition metals through the N(7) atom of purine ring in all the complexes. The prepared compounds have been tested in vitro for their possible cytotoxic activity against G-361 (human malignant melanoma), HOS (human osteogenic sarcoma), K-562 (human chronic myelogenous leukemia) and MCF-7 (human breast adenocarcinoma) cell lines and IC(50) values have been also determined for all the complexes. IC(50) values estimated for the Pt(II)-Bohemine complexes (2.1-16 microM) allow us to conclude that they could find utilization in antineoplastic therapy. Thus, from a pharmacological point of view, Pt(II) complexes of Bohemine may represent compounds for a new class of antitumor drugs.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.     

Synthesis, structure and biomimetic properties of Cu(II)-Cu(II) and Cu(II)-Zn(II) binuclear complexes: possible models for the chemistry of Cu-Zn superoxide dismutase

Four imidazolate-bridged binuclear copper(II)-copper(II) and copper(II)-zinc(II) complexes viz., [(Bipy)(2)Cu-Im-Cu(Bipy)(2)](ClO(4))(3).CH(3)OH, [(Phen)(2)Cu-Im-Cu(Phen)(2)](BF(4))(3).2CH(3)OH, [(Bipy)(2)Cu-Im-Zn(Bipy)(2)](BF(4))(3), and [(Phen)(2)Cu-Im-Zn(Phen)(2)](BF(4))(3), (Bipy=2,2'-Bipyridyl, Phen=1-10-Phenanthroline and Im=imidazolate ion) were synthesized as a possible models for superoxide dismutase (SOD). Complex [(Bipy)(2)Cu-Im-Cu(Bipy)(2)](ClO(4))(3).CH(3)OH has been structurally characterized. This complex crystallizes in the triclinic space group P1, with the unit parameters a=8.88(5) A, b=13.79(17) A, c=20.18(18) A, alpha=76.424(8)(o), beta=85.888(6)(o), gamma=82.213(7). The metal-nitrogen bond length from 1.972-2.273 A and the distance Cu-Cu is 5.92 A. The five-coordinate geometry about the copper(II) ion is square pyramidal. Magnetic moment and electron paramagnetic resonance (e.p.r.) spectral measurements of the homobinuclear complexes have shown an antiferromagnetic exchange interaction. From the e.p.r. and UV-Vis spectral measurement studies, these complexes have been found to be stable (pH 8.5-10.5 for 1, 10.5 for 2,3 and 8.5 for 4). These complexes catalyse the dismutation of superoxide radical (O(2)(-)) at biological pH. All the observations indicate that these complexes act as good possible models for superoxide dismutase.     

Synthesis,structure and nuclease properties of several ternary copper(II) peptide complexes with 1,10-phenanthroline

Three new ternary peptide-Cu(II)-1,10-phenanthroline (phen) complexes, [Cu(L-ala-gly)(phen)].3.5H(2)O 1, [Cu(L-val-gly)(phen)] 2 and [Cu(gly-L-trp)(phen)].2H(2)O 3, have been prepared and structurally characterised. These compounds exist as distorted square pyramidal complexes with the five co-ordination sites occupied by the tridentate peptide dianion and the two heterocyclic nitrogens of the phenanthroline ligand. The bulk of the lateral chain in the peptide moiety determines the relative disposition of the phen ligand. Thus, in [Cu(L-val-gly)(phen)] 2, the phenanthroline plane is deviated towards the opposite side of the isopropyl group of the L-valine moiety. On the other hand, in [Cu(gly-L-trp)(phen)].2H(2)O 3 the absence of stacking interactions between phen and indole rings and the presence of an intramolecular CH...pi interaction should be pointed out. These complexes exhibit significant differences in their nuclease activity which depends on the nature of the peptidic moiety, the complex [Cu(gly-L-trp) (phen)].2H(2)O 3 being the most active.     

Conformational changes induced in the human protein translin and in the single-stranded oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5) upon binding of these oligodeoxynucleotides by translin

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.