The flash-quench technique in protein-DNA electron transfer: reduction of the guanine radical by ferrocytochrome c

Electron transfer from a protein to oxidatively damaged DNA, specifically from ferrocytochrome c to the guanine radical, was examined using the flash-quench technique. Ru(phen)2dppz2+ (dppz = dipyridophenazine) was employed as the photosensitive intercalator, and ferricytochrome c (Fe3+ cyt c), as the oxidative quencher. Using transient absorption and time-resolved luminescence spectroscopies, we examined the electron-transfer reactions following photoexcitation of the ruthenium complex in the presence of poly(dA-dT) or poly(dG-dC). The luminescence-quenching titrations of excited Ru(phen)2dppz2+ by Fe3+ cyt c are nearly identical for the two DNA polymers. However, the spectral characteristics of the long-lived transient produced by the quenching depend strongly upon the DNA. For poly(dA-dT), the transient has a spectrum consistent with formation of a [Ru(phen)2dppz3+, Fe2+ cyt c] intermediate, indicating that the system regenerates itself via electron transfer from the protein to the Ru(III) metallointercalator for this polymer. For poly(dG-dC), however, the transient has the characteristics expected for an intermediate of Fe2+ cyt c and the neutral guanine radical. The characteristics of the transient formed with the GC polymer are consistent with rapid oxidation of guanine by the Ru(III) complex, followed by slow electron transfer from Fe2+ cyt c to the guanine radical. These experiments show that electron holes on DNA can be repaired by protein and demonstrate how the flash-quench technique can be used generally in studying electron transfer from proteins to guanine radicals in duplex DNA.     

Ru(TAP)2(dppz)]2+: a DNA intercalating complex, which luminesces strongly in water and undergoes photo-induced proton-coupled electron transfer with guanosine-5'-monophosphate

The lowest excited state of [Ru(TAP)2(dppz)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyrido[3,2-a:2',3'-c]phenazine) 1 is strongly luminescent, even in water, and very oxidizing. Therefore it is able to oxidise not only guanosine-5'-monophosphate (GMP), as demonstrated by laser flash photolysis, but also guanine-containing polynucleotides such as calf thymus DNA and [poly(dG-dC)]2. The luminescence quenching was found to be faster in H2O than in D2O, as is the back reaction, indicating that both processes probably proceed by proton-coupled electron transfer. These properties, that are controlled by the triplet MLCT state in which the charge has been transferred from the Ru to a TAP ligand, contrast with those of the well known [Ru(phen)2(dppz)]2+ 2.     

Sequence-dependent interactions of cationic naphthalimides and polynucleotides

The binding interactions of three naphthalimide derivatives with heteropoly nucleic acids have been evaluated using fluorescence, absorption and circular dichroism spectroscopies. Mono- and bifunctionalized naphthalimides exhibit sequence-dependent variations in their affinity toward DNA. The heteropoly nucleic acids, [Poly(dA-dT)]2 and [Poly(dG-dC)]2, as well as calf thymus (CT) DNA, were used to understand the factors that govern binding strength and selectivity. Sequence selectivity was addressed by determining the binding constants as a function of polynucleotide composition according to the noncooperative McGhee-von Hippel binding model. Binding affinities toward [poly(dA-dT)](2) were the largest for spermine-substituted naphthalimides (Kb = 2-6 x 10(6) M(-1)). The association constants for complex formation between the cationic naphthalimides and [poly(dG-dC)]2 or CT DNA (58% A-T content) were 2-500 times smaller, depending on the naphthalimide-polynucleotide pair. The binding modes were also assessed using a combination of induced circular dichroism and salt effects to determine whether the naphthalimides associate with DNA through intercalative, electrostatic or groove-binding. The results show that the monofunctionalized spermine and pyridinium-substituted naphthalimides associate with DNA through electrostatic interactions. In contrast, intercalative interactions are predominant in the complex formed between the bifunctionalized spermine compound and all of the polynucleotides.     

Circular dichroism spectroscopic studies reveal pH dependent binding of curcumin in the minor groove of natural and synthetic nucleic acids

For the first time, an interaction between the non-toxic, cancer chemopreventive agent curcumin and both natural and synthetic DNA duplexes has been demonstrated by using circular dichroism (CD) and absorption spectroscopy techniques. Upon addition of curcumin to calf thymus DNA, poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) solutions, an intense positive induced CD band centered around 460-470 nm was observed depending on the actual pH and Na+ ion concentration of the medium; no CD signal was obtained, however, with single stranded poly(dC). Interaction of curcumin with calf thymus DNA was observed already at pH 6.5 in contrast with poly(dG-dC).poly(dG-dC) which induces no extrinsic Cotton effect above a pH value of 5. The protonated, Hoogsteen base-paired structure of poly(dG-dC).poly(dG-dC) is necessary for curcumin binding while the alternating AT-rich polymer formed complexes with curcumin only at certain Na+ concentrations. Evaluation of the spectral data and molecular modeling calculations suggested that curcumin, this dietary polyphenolic compound binds in the minor groove of the double helix. The mechanism of the induced CD activity, the effects of the pH and Na+ ions on the ligand binding and conformation of the double helix are discussed in detail. As well as being an essentially new phenolic minor groove binder agent curcumin is also a promising molecular probe to study biologically important, pH and cation induced conformational polymorphisms of nucleic acids.     

Dual molecular light switches for pH and DNA based on a novel Ru(II) complex. A non-intercalating Ru(II) complex for DNA molecular light switch

A new Ru(II) complex of [Ru(phen)(2)(Hcdpq)](ClO(4))(2) {phen = 1,10-phenanthroline, Hcdpq = 2-carboxyldipyrido[3,2-f:2',3'-h]quinoxaline} was synthesized and characterized. The spectrophotometric pH and calf thymus DNA (ct-DNA) titrations showed that the complex acted as a dual molecular light switch for pH and ct-DNA with emission enhancement factors of 17 and 26, respectively. It was shown to be capable of distinguishing ct-DNA from yeast RNA with this binding selectivity being superior to two well-known DNA molecular light switches of [Ru(bpy)(2)(dppz)](2+) {bpy =2,2'-bipyridine, and dppz = dipyrido-[3,2-a:2',3'-c]phenazine}and ethidium bromide. The complex bond to ct-DNA probably in groove mode with a binding constant of (4.67 ± 0.06) × 10(3) M(-1) in 5 mM Tris-HCl, 50 mM NaCl (pH = 7.10) buffer solution, as evidenced by UV-visible absorption and luminescence titrations, the dependence of DNA binding constants on NaCl concentrations, DNA competitive binding with ethidium bromide, and emission lifetime and viscosity measurements. To get insight into the light-switch mechanism, theoretical calculations were also performed by applying density functional theory (DFT) and time-dependent DFT.     

The mode of binding ACMA-DNA relies on the base-pair nature

A thermodynamic and kinetic study on the mode of binding of 9-amino-6-chloro-2-methoxi-acridine (ACMA) to poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) has been undertaken at pH = 7.0 and I = 0.1 M. The spectrophotometric, kinetic (T-jump), circular dichroism, viscometric and calorimetric information gathered point to formation of a fully intercalated ACMA complex with poly(dA-dT)·poly(dA-dT) and another one only partially intercalated (7%) with poly(dG-dC)·poly(dG-dC). The ACMA affinity with the A-T bases was higher than with the G-C bases. The two polynucleotide sequences give rise to external complexes when the ACMA concentration is raised, namely, the electrostatic complex poly(dA-dT)·poly(dA-dT)-ACMA and the major groove binding complex poly(dG-dC)·poly(dG-dC)-ACMA. A considerable quenching effect of the ACMA fluorescence is observed with poly(dA-dT)·poly(dA-dT), ascribable to face-to-face location in the intercalated A-T-ACMA base-pairs. The even stronger effect observed in the presence of poly(dG-dC)·poly(dG-dC) is related to the guanine residue from on- and off-slot ACMA positions.     

Photooxidation of guanine by a ruthenium dipyridophenazine complex intercalated in a double-stranded polynucleotide monitored directly by picosecond visible and infrared transient absorption spectroscopy

Transient species formed by photoexcitation (400 nm) of [Ru(dppz)(tap)2]2+ (1) (dppz = dipyrido[3,2-a:2',3'-c]phenazine; tap=1,4,5,8-tetraazaphenanthrene) in aqueous solution and when intercalated into a double-stranded synthetic polynucleotide, [poly(dG-dC)]2, have been observed on a picosecond timescale by both visible transient absorption (allowing monitoring of the metal complex intermediates) and transient infrared (IR) absorption spectroscopy (allowing direct study of the DNA nucleobases). By contrast with its behavior when free in aqueous solution, excitation of 1 when bound to [poly(dG-dC)]2 causes a strong increase in absorbance at 515 nm due to formation of the reduced complex [Ru(dppz)(tap)2]+ (rate constant=(2.0+/-0.2) x 10(9) s(-1)). The subsequent reformation of 1 proceeds with a rate constant of (1.1+/-0.2) x 10(8) s(-1). When the process is carried out in D2O, the rates of formation and removal of [Ru(dppz)(tap)2]+ are reduced (rate constants (1.5+/-0.3) x 10(9) and (0.7+/-0.2) x 10(8) s(-1) respectively) consistent with proton-coupled electron transfer processes. Picosecond transient IR measurements in the 1540-1720 cm(-1) region in D2O solution confirm that the reduction of 1 intercalated into [poly(dG-dC)]2 is accompanied by bleaching of IR ground-state bands of guanine (1690 cm(-1)) and cytosine (1656 cm(-1)), each with similar rate constants.     

Spectroscopic study of the interaction of pazelliptine with nucleic acids

The antitumor drug pazelliptine (PZE) binds to natural and synthetic DNA sequences at 100 mM NaCl, pH 7.0, as deduced from the absorption and fluorescence data. Scatchard plots constructed from the results obtained with poly(dG-dC)-poly(dG-dC) give binding constants of base pairs in the range (2–6) × 10⁵ M⁻¹. The modifications in the absorption and fluorescence spectra observed when PZE binds to various polynucleotides, namely poly(dA-dT)-poly(dA-dT), poly(dA)-poly(dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA. reveal a change in the protonation state of the drug upon binding, increasing the apparent pK_a of its 9-N nitrogen atom. The PZE excited state properties serve as a sensitive probe to distinguish between homo and hetero A-T sites as well as between AT and GC sites. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to the bound PZE chromophore, a result consistent with an intercalative mode of binding of the drug to DNA. The emission is enhanced when PZE is bound to A-T base pairs ( 30% increase of φ_F) whereas it is quenched in the vicinity of G-C base pairs ( 90% decrease of φ_F). Furthermore, the fluorescence spectrum obtained with calf thymus DNA is hardly distinguishable from that obtained with poly(dG-dC)-polu(dG-dC), suggesting a binding of PZE to G-C rich regions.     

Ruthenium(II) complexes of redox-related, modified dipyridophenazine ligands: synthesis, characterization, and DNA interaction

The synthesis, spectral characterization, and electrochemical properties of [Ru(phen)2(qdppz)]2+, which incorporates a quinone-fused dipyridophenazine ligand (naphtho[2,3-a]dipyrido[3,2-h:2',3'-f]phenazine-5,18-dione, qdppz), are described in detail. Chemical or electrochemical reduction of [Ru(phen)2(qdppz)]2+ leads to the generation of [Ru(phen)2(hqdppz)](2+)--a complex containing the hydroquinone form (hqdppz = 5,18-dihydroxynaphtho[2,3-a]-dipyrido[3,2-h:2',3'-f]phenazine) of qdppz. Absorption and viscometric titration, thermal denaturation, topoisomerase assay, and differential-pulse voltammetric studies reveal that [Ru(phen)2(qdppz)]2+ is an avid binder of calf-thymus DNA due to a strong intercalation by the ruthenium-bound qdppz, while [Ru(phen)2(hqdppz)]2+ binds to DNA less strongly than the parent "quinone"-containing complex. DNA-photocleavage efficiencies of these complexes also follow a similar trend in that the MLCT-excited state of [Ru(phen)2(qdppz)]2+ is more effective than that of [Ru(phen)2(hqdppz)]2+ in cleaving the supercoiled plasmid pBR 322 DNA (lambda exc = 440 +/- 5 nm), as revealed by the results of agarose gel electrophoresis experiments. The photochemical behaviors of both the quinone- and hydroquinone-appended ruthenium(II) complexes in the presence of DNA not only provide valuable insights into their modes of binding with the duplex but also lead to detailed investigations of their luminescence properties in nonaqueous, aqueous, and aqueous micellar media. On the basis of the results obtained, (i) a photoinduced electron transfer from the MLCT state to the quinone acceptor in Ru(phen)2(qdppz)]2+ and (ii) quenching of the excited states due to proton transfer from water to the dipyridophenazine ligand in both complexes are invoked to rationalize the apparent lack of emission of these redox-related complexes in the DNA medium.     

Synthesis and DNA binding of novel water-soluble cationic methylcobalt porphyrins

Organocobalt derivatives of tetracationic water-soluble porphyrins are difficult to prepare via the typical reductive alkylation of the Co(II)(por) (porH(2) = porphyrin ligand). None have been reported. The problem may arise because the porphyrin core is made relatively electron poor by the positively charged peripheral groups. We have circumvented this problem by using the [Co(III)(NH(3))(5)CH(3)](2+) reagent, which inserts the Co(III)-CH(3) moiety directly into porH(2) in water under basic conditions. The method afforded two new [CH(3)Co(por)](4+) derivatives, [CH(3)CoTMpyP(4)](4+) and [CH(3)CoTMAP](4+), where [TMpyP(4)](4+) and [TMAP](4+) are the coordinated, NH-deprotonated forms of meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin and meso-tetrakis(N,N,N-trimethylaniliniumyl)porphyrin, respectively. The binding of the two new [CH(3)Co(por)](4+) cations to DNA and to the synthetic DNA polymers [poly(dA-dT)](2) and [poly(dG-dC)](2) was studied. Using published criteria by which changes in DNA viscosity and in the visible and CD spectra in the Soret region can be used to assess DNA binding, we conclude that both are outside binders. A large hypochromicity of the Soret bands of the [CH(3)Co(por)](4+) cations observed upon outside binding to DNA may indicate a high degree of self-stacking. The visible absorption and CD spectra of the [CH(3)Co(por)](4+) cations in the presence of 1:1 mixtures of [poly(dA-dT)](2) and [poly(dG-dC)](2) are nearly identical to those with [poly(dA-dT)](2) alone and are very different from those of [poly(dG-dC)](2) alone. Thus, both cations show a high preference for outside binding at AT-rich over GC-rich DNA sites. Upon binding of each of the [CH(3)Co(por)](4+) cations to all of the DNA polymers, the Soret bands exhibit blue shifts, whereas the Soret bands of the corresponding [(H(2)O)(2)Co(por)](5+) cations exhibit red shifts. The blue shifts strongly suggest that the [CH(3)Co(por)](4+) cations, particularly [CH(3)CoTMAP](4+), become five-coordinate forms to some extent on DNA binding; this result is the first good evidence for the presence at equilibrium of five-coordinate CH(3)Co(III)(N(4)) forms in water.     

DNA-binding and physical studies of Pt(4'-NR2-trpy)CN+ systems (trpy = 2,2':6',2'-terpyridine)

This paper focuses on DNA-binding interactions exhibited by Pt(dma-T)CN(+), where dma-T denotes 4'-dimethylamino-2,2':6',2'-terpyridine, and includes complementary studies of the corresponding pyrr-T complex, where pyrr-T denotes 4'-(N-pyrrolidinyl)-2,2':6',2'-terpyridine. The chromophores are useful for understanding the interesting and rather intricate DNA-binding interactions exhibited by these and related systems. One reason is that the terpyridine ligands employed provide intense visible absorption and enhanced photoluminescence signals. Incorporating cyanide as a coligand further aids analysis by suppressing covalent binding. Physical methods utilized include X-ray crystallography for structures of the individual inorganic complexes. Viscometry as well as spectral studies of the absorbance, emission, and circular dichroism (CD) yield information about interactions with a variety of DNA hosts. Although there is no sign of covalent binding under the conditions used, most hosts exhibit two phases of uptake. Under conditions of high loading (low base-pair-to-platinum ratios), the dma-T complex preferentially binds externally and aggregates on the surface of the host, except for the comparatively rigid host [poly(dG-dC)]2. Characteristic signs of the aggregated form include a bisignate CD signal in the charge-transfer region of the spectrum and strongly bathochromically shifted emission. When excess DNA is present, however, the complex shifts to intercalative binding, preferentially next to G[triple bond]C base pairs if available. Once the complex internalizes into DNA it becomes virtually immune to quenching by O2 or solvent, and the emission lifetime extends to 11 micros when [poly(dI-dC)]2 is the host. On the other hand, the host itself becomes a potent quenching agent when G[triple bond]C base pairs are present because of the reducing strength of guanine residues.     

Computational study of the interaction of proflavine with d(ATATATATAT)2 and d(GCGCGCGCGC)2

The interaction of proflavine (PR) with two B-DNA decamers of alternating AT and GC sequence, called [deca(dG-dC)]₂ and [deca(dA-dT)]₂, respectively, was computationally investigated by the ONIOM method, exploiting a three-layer QM/QM/MM hybrid approach. The highest level QM method was applied to the model system, which comprises the intercalation site (5th and 6th base pairs) and the inserted PR molecule. The connecting sugar phosphate backbone was added in the medium layer region. Both higher and medium level layers, differing in the size of the basis set used, were treated by the DFT MPWB1K functional. The full system in the lower layer was described by the empirical AMBER force field. The calculated values of the interaction energy of PR with [deca(dG-dC)]₂ and [deca(dA-dT)]₂, as well as with the dinucleotides d(GpC)₂, and d(ApT)₂, the latter considered either in vacuo and in the mimicked water solvent, support for a static higher affinity toward G-C compared to the A-T DNA base sequences, in agreement with structural results from crystallographic studies. Furthermore, the different structural characteristics of the [deca(dG-dC)]₂/PR complex compared to those of the [deca(dA-dT)]₂/PR, furnish a possible interpretation of apparently controversial experimental thermodynamic data, explained in terms of two possible modes of non-covalent binding of ligands with DNA, namely intercalation and external binding, respectively.     

DNA Sequence and Ancillary Ligand Modulate the Biexponential Emission Decay of Intercalated [Ru(L)2dppz]2+ Enantiomers

The bi‐exponential emission decay of [Ru(L)₂dppz]²⁺ (L=N,N′‐diimine ligand) bound to DNA has been studied as a function of polynucleotide sequence, enantiomer, and nature of L (phenanthroline vs. bipyridine). The lifetimes (τ_i) and pre‐exponential factors (α_i) depend on all three parameters. With [poly(dA‐dT)]₂, the variation of α_i with [Nu]/[Ru] has little dependence on L for Λ‐[Ru(L)₂dppz]²⁺ but a substantial dependence for Δ‐[Ru(L)₂dppz]²⁺. With [poly(dG‐dC)]₂, by contrast, the Λ‐enantiomer α_i values depend strongly on the nature of L, whereas those of the Δ‐enantiomer are relatively unaffected. DNA‐bound linked dimers show similar photophysical behaviour. The lifetimes are identified with two geometries of minor‐groove intercalated [Ru(L)₂dppz]²⁺, resulting in differential water access to the phenazine nitrogen atoms. Interplay of cooperative and anti‐cooperative binding resulting from complex–complex and complex–DNA interactions is responsible for the observed variations of α_i with binding ratio. [Ru(phen)₂dppz]²⁺ emission is quenched by guanosine in DMF, which may further rationalise the shorter lifetimes observed with guanine‐rich DNA.     

Synthesis of [Ru(phen)(2)dppz](2+)-tethered oligo-DNA and studies on the metallointercalation mode into the DNA duplex

To explore the binding properties of [Ru(phen)(2)dppz](2+) complex (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) in a sequence-specific manner in DNA duplex, it was tethered through the dppz ligand to a central position as well as both at the 3'- and 5'-ends of oligodeoxyribonucleotide (ODN). The middle [Ru(phen)(2)dppz](2+)-ODN tethered was resolved and isolated as four pure diastereomers, while the 3'- or 5'-[Ru(phen)(2)dppz](2+)-ODNs were inseparable on RP-HPLC. Thermal stability of the (Ru(2+)-ODN).DNA duplexes is found to increase considerably (DeltaT(m) = 12.8-23.4 degrees C), depending upon the site of the covalent attachment of the tethered [Ru(phen)(2)dppz](2+) complex, or the chirality of the [Ru(phen)(2)dppz](2+)-linker tethered at the middle of the ODN, compared to the unlabeled counterpart. Gross differences in CD between the [Ru(phen)(2)dppz](2+)-tethered and the native DNA duplexes showed that the global duplex conformation of the former has considerably altered from the B-type, but is still recognized by DNase I. The thermal melting studies, CD measurements, as well as DNase I digestion data, are interpreted as a result of intercalation of the dppz moiety, which is realized by threading of the Ru(phen)(2) complex part through the DNA duplex core. DNase I footprinting with four diastereomerically pure middle ([Ru(phen)(2)dppz](2+)-ODN).DNA duplexes furthermore showed that the tethered [Ru(phen)(2)dppz](2+)-linker chirality dictates the stereochemical accessibility of various phosphodiester moieties (around the intercalation site) toward the cleavage reaction by the enzyme. The diastereomerically pure ruthenium-modified duplexes, with the well-defined pi-stack, will be useful to explore stereochemistry-dependent energy- and electron-transfer chemistry to understand oxidative damage to the DNA double helix as well as the long-range energy- and electron-transfer processes with DNA as a reactant.     

A STUDY OF SOME POLYPYRIDYLRUTHENIUM(II) COMPLEXES AS DNA BINDERS AND PHOTOCLEAVAGE REAGENTS

The nature of the binding of several ruthenium polypyridyl complexes containing 2,2'-bipyridine (bipy), 4,4'-dimethyl-2,2'-bipyridine (DMB), 1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (DPP), 2,2',2"-terpyridine (terpy), 2,2'-biquinoline (biq), 1,4,5,8-tetraazaphenanthrene (TAP) and 1,4,5,8,9,12-hexaazatriphenylene (HAT), with calf thymus DNA, poly[d(A-T)] and poly[d(G-C)] were studied by absorption and emission spectroscopy, DNA melting techniques, and emission lifetime measurements. In low ionic strength phosphate buffer, spectroscopic changes and DNA stabilization depended on the polypyridyl ligands present, and indicated binding that varied from substantially electrostatic to intercalative. Ru(bipy)2(HAT)2+ and Ru(phen)3(2+), which bind by partial intercalation, also show a strong preference for poly[d(A-T)]. The emission quantum yields for most complexes were increased in the presence of DNA. An exception was Ru(TAP)3(2+) which has a markedly reduced emission quantum yield and lifetime in the presence of poly[d(G-C)] or CT-DNA, due to photoredox interaction with quanines. Emission decays of the complexes generally showed multiexponential behaviour. The ability of the ruthenium complexes to sensitise DNA cleavage was determined using pBR322 plasmid DNA. Ru(TAP)3(2+) is the most efficient sensitiser while uncharged complexes and complexes with very short-lived excited states do not cleave DNA.     

Luminescent Ir(III) complex exclusively made of polypyridine ligands capable of intercalating into calf-thymus DNA

Efficient intercalation of a luminescent Ir(III) complex exclusively made of polypyridine ligands in natural and synthetic biopolymers is reported for the first time. The emission of the complex is largely enhanced in the presence of [poly(dA-dT)(2)] and strongly quenched in the presence of [poly(dG-dC)(2)]. By comparing the emission decays in DNA and in synthetic polynucleotides, it is proposed that the emission quenching of the title compound by guanine residues in DNA is no longer effective over a distance of four dA-dT base pairs.     

Time-dependent binding mode of a cationic porphyrin dimer to poly[d(G-C)2] and Poly[d(A-T)2

The time-dependent binding mode of a porphyrin dimer to poly[d(G-C)2] and poly[d(A-T)2] was investigated by spectroscopic methods including absorption and circular and linear dichroism (CD and LD) spectroscopy. Immediately after mixing with poly[d(G-C)2], the porphyrin dimer exhibited red-shift and hypochromism in the absorption spectrum and negative CD and LD spectra. With further red-shift in absorption, the CD and LD magnitude in the Soret region became increasingly negative over time. After it was stabilized, the magnitude of the reduced LD (LDr) in the Soret region was larger than that in the DNA absorption region, indicating that the second porphyrin was also intercalated. Following the rapid intercalation of the first porphyrin, the very slow intercalation of the second followed with first-order kinetics. In the poly[d(A-T)2] case, a bisignate CD spectrum was observed in the Soret region suggesting stacking of the porphyrins. The small alteration in the CD spectrum and increased absorbance, which followed the initial rapid spectral change, was of the second order. This alteration in the spectral properties was attributed to the conformational change of poly[d(A-T)2] near the binding site because the overall shape of the CD spectrum was conserved in spite of the changes in the absorption spectrum.     

EFFICIENCY OF PHOTOAFFINITY LABELING DNA HOMOPOLYMERS AND COPOLYMERS WITH ETHIDIUM MONOAZIDE *

Abstract— Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently-linked following photoactivation at the same drug/DNA input ratio. There were significant sequence-related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent-adduct formation decreased in the order poly(dG-dC)^. poly(dG-dC)> poly-(dG). poly(dC)poly(dA-dT). poly(dA-dT)poly(dA). poly(dT). Ethidium monoazide was about 2-fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G-C pairs than the A-T base counterparts. In low ionic buffers (0.015 M Na⁺), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40-fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity of the drug for that site.     

MONOADDITION OF DICTAMNINE TO SYNTHETIC DOUBLE-STRANDED POLYDEOXYRIBONUCLEOTIDES IN UVA AND THE EFFECT OF PHOTOMODIFIED DNA ON TEMPLATE ACTIVITY

Abstract— The photoreactivity of dictamnine, a furoquinoline alkaloid, towards different synthetic DNAs has been studied. The ratio of the photobinding of [₃H]-dictamnine to poly(dA-dT) poly(dA-dT): poly(dG-dC) poly(dG-dC): poly(dA-dU) poly(dA-dU): poly(dA) poly(dT), in relation to that of calf thymus DNA, is 18:1:0.5:0.3. Prior treatment of calf thymus DNA with dictamnine in light inhibits the subsequent incorporation of 8-methoxypsoralen (8-MOP). These results suggest that the sites in DNA for the photobinding of dictamnine are probably identical with those for monoad-ducts of 8-MOP. Furthermore, the template activity of photomodified DNA in the RNA polymerase reaction is considerably inhibited for poly(dA-dT)poly(dA-dT), to a lesser extent for calf thymus DNA, but almost not affected for the linear copolymer, poly(dA)-poly(dT).     

Steric protection of a photosensitizer in a N,N-bis[2-(2-pyridyl)ethyl]-2-phenylethylamine-copper(II) bowl that enhances red light-induced DNA cleavage activity

Ternary copper(II) complexes [Cu(py2phe)B](ClO4)2 (1-3), where py2phe is a tripodal ligand N,N-bis[2-(2-pyridyl)ethyl]-2-phenylethylamine and B is a heterocyclic base (viz., 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2), or dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3)), are prepared and their DNA-binding and photoinduced DNA-cleavage activities are studied. Complex 1 has been structurally characterized by single crystal X-ray crystallography. The molecular structure shows an axially elongated square-pyramidal (4 + 1) coordination geometry in which the phen ligand binds at the basal plane. The tripodal ligand py2phe displays an axial-equatorial binding mode with the amine nitrogen bonded at the axial site. A chemically significant CH-pi interaction involving the CH moiety of the phenyl group of the tripodal ligand and the aromatic ring of phen is observed. The complexes display good binding propensity to calf thymus DNA giving a relative order of 3 (dppz) > 2 (dpq) > 1 (phen). The DNA binding constants (K(b)) for 1-3, determined from absorption spectral studies, are 6.2 x 10(3), 1.0 x 10(4), and 5.7 x 10(4) M(-1), respectively. The complexes show chemical nuclease activity in the presence of 3-mercaptopropionic acid as a reducing agent forming hydroxyl radicals as the cleavage active species. The photoinduced DNA-cleavage activity of the complexes has been studied using UV radiation of 365 nm and red light of 632.8 and 694 nm. The phen complex in absence of any photosensitizing moiety does not show any DNA cleavage upon photoirradiation. The dpq and dppz ligands with their photoactive quinoxaline and phenazine moieties display significant photoinduced DNA-cleavage activity. The dppz complex is more active than its dpq analogue because of the better steric protection of the DNA-bound photosensitizing dppz ligand from the solvent molecules. Control experiments reveal the formation of singlet oxygen in the light-induced DNA-cleavage reactions. The observed efficient photoinduced DNA-cleavage activity of 2 and 3 is akin to the "light switch" effect known for the tris-chelates of ruthenium(II).