Effect of periphery cationic substituents of porphyrin on the B-Z transition of poly[d(A-T)2], poly[d(G-C)2] and their binding modes.

The binding mode of cationic porphyrin (trans-BMPyP) with poly[d(G-C)2] and poly[d(A-T)2] was examined according to the site of the periphery cationic methyl pyridine ion of the cationic porphyrin (o-, m-, p-) as well as the possibility of a B-Z transition depending on the binding modes by measuring the absorption spectrum and circular dichroism (CD). The negative band found in the soret region showed the intercalation mode of m- and p-trans-BMPyP-poly[d(G-C)2] to the DNA base pairs, but no B-Z transition was induced. On the other hand, the distinctive bisignate band found in the soret region of the CD spectrum for m- and p-trans-BMPyP-poly[d(A-T)2] suggests that m- and p-trans-BMPyP have an effective extensive stacking-based binding mode along with the skeleton of poly[d(A-T)2], wherein the B-Z transition was induced through extensive stacking. The difference in binding mode was attributed to the difference in the molecular structure depending on the site of the periphery cationic methyl pyridine ion in the cationic porphyrin. In other words, o-trans-BMPyP is nonplanar because of the steric hindrance of the cationic methyl pyridine ion at the o-site. In contrast, m- and p-trans-BMPyP are planar, but not all porphyrins with a planar structure undergo the B-Z transition. In conclusion, a B-Z transition is induced if the structure of a porphyrin is planar and the binding mode allows the porphyrins to be stacked effectively along the DNA skeleton, not in a binding mode where the porphyrin is intercalated to the DNA.Communicated by Ramaswamy H. Sarma.

Binding Properties of Various Cationic Porphyrins to DNA in the Molecular Crowding Condition Induced by Poly(ethylene glycol).

The binding modes of various cationic porphyrins to DNA in an aqueous solution and under the molecular crowding condition induced by poly(ethylene glycol) (PEG) were compared by normal absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy techniques. Large negative CD and LD signals in the Soret absorption regions of the meta- and para-TMPyP [meso-tetrakis (n-N-methylpyridiniumyl) porphyrin (meta, n = 3) and (para, n = 4)] were apparent in the aqueous solution, indicating an intercalative-binding mode, while a positive CD spectrum and a less intense negative LD spectrum for the ortho-TMPyP (n = 2)-complexed DNA suggested a major-groove-binding mode. These binding modes are retained under a molecular crowding condition, suggesting that the PEG cluster cannot access the TMPyPs that are intercalated between the DNA base pairs or that bind at the major groove. The spectral properties of the ortho-, meta-, and para-trans-BMPyP [trans-bis(N-methylpyrodinium-n-yl)diphenyl porphyrin, n = 2,3,4]-bound DNA in an aqueous solution correspond to neither the intercalative-binding nor the groove-binding mode, which is in contrast with the TMPyP cases. The spectral properties under the molecular crowding condition are altered considerably for all of the three trans-BMPyPs compared to those in an aqueous solution, suggesting that the matted PEG cluster is in contact with the cationic trans-BMPyPs, causing a change in the polarity of the porphyrin environment. Consequently, trans-BMPyPs bind to the external side of the DNA.

Conformational change of a G-quadruplex under molecular crowding conditions.

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.

Neighboring base sequence effect on DNA damage.

Guanine is the most strongly oxidized base in DNA; generation of a guanine radical cation as an intermediate in an oxidation reaction leads to migration through a resulting cationic hole in the DNA π-stack until it is trapped by irreversible reaction with water or other free radicals. In the case of normal sequences, the primary position of Guanine oxidations by one-electron oxidants such as carbonate radical anions, BPT(7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene), and riboflavin are 5'-G in GG doublets and the central G in a GGG triplet. According to results, the properties of guanine oxidation on abasic site containing sequences are independent from the position of AP(apurinic/apyrimidinic) site in the presence of carbonate radical anions under a short irradiation time, although this radical is exposed to solvent by the existence of an abasic site. The lack of abasic site effect on guanine oxidative damage by the carbonate radical may be due to a sequence-independent property of the initial electron transfer rate in the hole injection step, or may relate to an electron transfer mechanism with large reorganization energy dependency. Consequently, the carbonate radical anions may easily migrate to another single G in the charge re-distribution step. Meanwhile, there is a strong dependency on the presence of an AP(apurinic/apyrimidinic) site in the cleavage patterns of guanine oxidations by physically large oxidizing agents, such as BPT(7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene) and riboflavin. These radicals show strong AP(apurinic/apyrimidinic) site dependency and clear G-site selectivity.Communicated by Ramaswamy H. Sarma.

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

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.

Sequence selective photoinduced electron transfer of a pyrene-porphyrin dyad to DNA.

The binding modes of a pyrene-porphyrin dyad, (1-pyrenyl)-tris(N-methyl-p-pyridino)porphyrin (PyTMpyP), to various DNAs (calf thymus DNA (Ct-DNA), poly[d(G-C)2], and poly[d(A-T)2]) have been investigated using circular dichroism and linear dichroism measurements. Based on the polarization spectroscopic results, it can be shown that the pyrenyl and porphryin planes are skewed to a large extent for PyTMPyP in an aqueous environment and in the binding site of poly[d(G-C)2]. In this complex, a photoinduced electron transfer (PET) process between the pyrenyl and porphyrin moieties occurs. On the other hand, PET was not observed in the PyTMPyP-poly[d(A-T)2] complex, whereas the fluorescence intensity of TMPyP was enhanced. The molecular planes of the pyrene and porphyrin moieties are almost parallel in the poly[d(A-T)2] and Ct-DNA adducts. Moreover, the generation of 1O2 species occurs only for the PyTMPyP-Ct-DNA and PyTMPyP-poly[d(A-T)2] complexes. We discuss the photophysical properties of PyTMPyP which are attributed to the binding patterns and the sequence of DNA bases.

RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro.

Mg2+ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg2+ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg2+ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg2+ and similar to those of full-length RecA in the presence of saturating Mg2+. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg2+ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.

Binding Mode of Cationic Porphyrin with CT-DNA: Importance of the Location and the Number of Positively Charged of Periphery Cationic Ions of Porphyrin.

The binding modes of o-, m-, and p-trans-BMPyP with DNA were studied using their spectroscopic properties. Also, the binding modes were compared based on the location and number of periphery cationic methylpyridine ions of the cationic porphyrins. The optical absorption spectra of the o-, m-, and p-trans-BMPyP when bound to DNA presented red shifts and hypochromicity compared to the optical absorption spectrum of DNA-free cationic porphyrins. m-trans-BMPyP-DNA presented the largest red shifts and hypochromicity. The results of the circular dichroism spectral analysis indicated positive and negative bisignate absorption bands in the Soret band of the porphyrins in the case of all concentration ratios of o- and p-trans-BMPyP-DNA, and two negative absorption bands were observed in m-trans-BMPyP-DNA. Compared to the size of the absorption band of the DNA optical absorption spectrum, the results of the reduced linear (LDr) spectral analysis indicated mainly small sizes of Soret absorption bands (the absorption spectrum of porphyrins) and positive LDr values for o- and p-trans-BMPyP-DNA. In consideration of several of such spectroscopic properties, the binding of o- and p-trans-BMPyP with DNA can be said to be distant to insertion modes. Although the case of m-trans-BMPyP to DNA is an insertion mode, the m-trans-BMPyP molecular surface presented much tilt within the intercalation pocket. The results of comparing the binding modes of TMPyP having four periphery cationic methylpyridine ions of cationic porphyrin indicated that regardless of the number of periphery cationic methylpyridine ions of cationic porphyrin, in the case of the ortho-position, nonplanarity due to steric hindrance of the periphery cationic methylpyridine ions presented outside or groove-binding modes indicative of interaction with DNA phosphates. Unlike the ortho-position, the para-position presented different binding modes based on the number of periphery cationic methylpyridine ions. Only cationic porphyrins having four periphery cationic methylpyridine ions were inserted into the DNA. Lastly, regardless of the number of periphery cationic methylpyridine ions, all meta-positions were inserted into the DNA. This indicated that at the least the location and the number of periphery cationic methylpyridine ions of the porphyrins used in this experiment were important elements that determine insertion into DNA base pairs.

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

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

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

The interaction of Δ- and Λ-[Ru(phen)2DPPZ]2+ (DPPZ = dipyrido[3,2-a:2', 3'-c]phenazine, phen = phenanthroline) with a G-quadruplex formed from 5'-G2T2G2TGTG2T2G2-3'(15-mer) was investigated. The well-known enhancement of luminescence intensity (the 'light-switch' effect) was observed for the [Ru(phen)2DPPZ]2+ complexes upon formation of an adduct with the G-quadruplex. The emission intensity of the G-quadruplex-bound Λ-isomer was 3-fold larger than that of the Δ-isomer when bound to the G-quadruplex, which is opposite of the result observed in the case of double stranded DNA (dsDNA); the light switch effect is larger for the dsDNA-bound Δ-isomer. In the job plot of the G-quadruplex with Δ- and Λ-[Ru(phen)2DPPZ]2+, a major inflection point for the two isomers was observed at x ≈ .65, which suggests a binding stoichiometry of 2:1 for both enantiomers. When the G base at the 8th position was replaced with 6-methyl isoxanthopterin (6MI), a fluorescent guanine analog, the excited energy of 6-MI transferred to bound Δ- or Λ-[Ru(phen)2DPPZ]2+, which suggests that at least a part of both Ru(II) enantiomers is close to or in contact with the diagonal loop of the G-quadruplex. A luminescence quenching experiment using [Fe(CN)6]4- for the G-quadruplex-bound Ru(II) complex revealed downward bending curves for both enantiomers in the Stern-Volmer plot, which suggests the presence of Ru(II) complexes that are both accessible and inaccessible to the quencher and may be related to the 2:1 binding stoichiometry.

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

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

Relationship between the photoinduced electron transfer and binding modes of a pyrene-porphyrin dyad to DNA.

The binding modes of a pyrene-porphyrin dyad, (1-pyrenyl)-tris(N-methyl-p-pyridino)porphyrin (PyTMpyP), to DNA and its photophysical properties have been investigated using various spectroscopic techniques. The circular dichroism (CD) spectrum of PyTMpyP bound to DNA (PyTMpyP-DNA) showed one negative and two positive bands in the Soret region. The CD signal in the pyrene absorption region was positive. The shape of the CD spectrum does not support an intercalative binding mode of TMpyP, which would typically afford a negative CD band in the absence of the pyrene moiety. Linear dichroism (LD) experiments revealed a very small signal in the Soret region, which also challenges the intercalation of TMpyP into DNA. Upon excitation of the pyrene moiety, the emission intensity of porphyrin in aqueous solution was quenched due to a photoinduced electron transfer (PET) process between the pyrenyl and porphyrin moieties. On the other hand, the emission of porphyrin was markedly enhanced upon binding to DNA, as the PET process from the excited pyrene moiety to TMpyP was suppressed when bound to DNA. The PET process occurs in the timescale of 65 ps, and could be detected by femtosecond transient absorption spectroscopic methods. Two fluorescence decay times were observed for PyTMpyP in aqueous solution (0.78 and 4.8 ns). Both decay times increased upon binding to DNA owing to environment and/or conformational changes in PyTMpyP. The driving force (ΔG) of the PET process was evaluated under conditions of minor and major groove binding. The PET process and photophysical properties of the PyTMpyP dyad were concluded to be influenced by the binding mode.

Chiral Selective Stacking of a Cationic Porphyrin along Z-Form Poly[d(A-T)2].

In this study, the binding mode of porphyrin-free single-stranded poly[d(AT)] and trans-BMPyP was observed in the Z-form trans-BMPyP-poly[d(A-T)2] complex induced by extensive stacking depending on the temperature and concentration through circular dichroism (CD). The Z-form trans-BMPyP-poly[d(A-T)2] complex (R = 0.30) retained the Z-form DNA structure at a low temperature (20 °C) by the trans-BMPyP molecules. When the temperature was increased to 60 °C, the DNA was almost unfolded as a single-stranded poly[d(AT)], but the extensive stacking binding mode of trans-BMPyP was maintained and the shape of the porphyrin Soret band was symmetrically changed in comparison with the shape of the Z-form DNA. However, when the temperature was raised to 80 °C, the extensive stacking binding mode of trans-BMPyP was also unfolded almost completely. The binding mode of the trans-BMPyP-single-stranded poly[d(AT)] complex was very similar to the already known binding mode of porphyrins and a double-stranded DNA. The binding mode was dependent on the concentration ratio ([porphyrin]/[DNA]): a monomeric binding mode at a concentration ratio of 0.04, a moderate groove binding mode at a concentration ratio between 0.08 and 0.16, and extensive stacking at a concentration ratio between 0.20 and 0.30. The same result was obtained when the temperature of the Z-form DNA (R = 0.30) was increased to 60 °C. However, those binding modes were not found in cis-BMPyP, which was because, in the extensive stacking of trans-BMPyP along the DNA skeleton, the distance between the two positive methylpyridine ions at the trans site and thymine, one of the DNA bases, is decreased, creating a much more hydrophobic environment. In addition, the poly AT sequences found from the CD spectra for the binding of trans-BMPyP-poly[d(A-T)2] and trans-BMPyP-poly[d(AT)] (R = 0.30) showed that both of them underwent effective extensive stacking and that the chirality of extensive stacking was dependent on the form of DNA.

Length and sequence effect on the B-Z transition of [d(A-T)n]2 oligonucleotide induced by a cationic porphyrin.

trans-BMPyP induced the B-Z transition for alternated AT oligonucleotides as it was evident by inversed CD spectrum. The transition occurred simultaneously with appearance of the extensive stacking of porphyrin. Complete B-Z transition required at least 14 base-pairs long. Insertion of one or two GC base pairs prevented the B-Z transition.

Oxidative DNA cleavage by Cu(II) complexes: Effect of periphery substituent groups.

A series of structurally-related [Cu(R-benzyl-dipicolylamine)(NO3)2] complexes, where R=methoxy- (1), methyl- (2), H- (3), fluoro- (4), and nitro-group (5), were synthesized, and their activity on DNA cleavage was investigated by linear dichroism (LD) and electrophoresis. The addition of a benzyl group to the dipicolylamine ligand of the [Cu(dipicolylamine)(NO3)2] complex (A), i.e., the [Cu(benzyl-dipicolylamine)(NO3)2] complex (3), caused significant enhancement in the efficiency of oxidative cleavage of both super-coiled (sc) and double stranded (ds) DNA, as evidenced by the electrophoresis pattern and faster decrease in the LD intensity at 260nm. The efficiency in DNA cleavage was also altered with further modifications of the benzyl group by the introduction of various substituents at the para-position. The cleavage efficiency appeared to be the largest when the methyl group was attached. The order of efficiency in DNA cleavage was methyl>methoxy≈H>fluoro≈nitro group. When an electron-withdrawing group was introduced, the cleavage efficiency decreased remarkably. The reactive oxygen species involved in the cleavage process were the superoxide radical and singlet oxygen. A possible mechanism for this variation in the DNA cleavage efficiency was proposed.

Effects of guanine bases at the central loop on stabilization of the quadruplex DNAs and their interactions with Meso-tetrakis(N-methylpyridium-4-yl)porphyrin.

The thermal stability of the G-quadruplex formed from the thrombin-binding aptamer, 5'G2T2G2TGTG2T2G2, in which the guanine (G) base at the central loop was replaced with an adenine (A) or inosine (I) base, was examined to determine the role of the central G base in stabilizing the quadruplex. Replacement of the central G base by the I base resulted in a slight decrease in thermal stability. On the other hand, the stability of the G-quadruplex decreased to a significant extent when it was replaced with the A base. The optimized structure of the G-quadruplex, which was obtained by a molecular dynamic simulation, showed that the carbonyl group of the C5 position of the central G base could form hydrogen bonds with the G1 amine group at the C7 position on the upper G-quartet. This formation of a hydrogen bond contributes to the stability of the G-quadruplex. The spectral property of meso-tetrakis(N-methylpyridium-4yl)porphyrin (TMPyP) associated with the G-quadruplex was characterized by a moderate red shift and hypochromism in the absorption spectrum, a positive CD signal, and two emission maxima in the fluorescence emission spectrum, suggesting that TMPyP binds at the exterior of the G-quadruplex. Spectral properties were slightly altered when the G base at the central loop was replaced with A or I, while the fluorescence decay times of TMPyP associated with the G-quadruplex were identical. Observed spectral properties removes the possibility of intercalation binding mode for TMPyP. TMPyP binds at the exterior of the quadruplex. Whether it stacks on the central loop or binds at the side of the quadruplex is unclear at this stage.

Formation of Poly[d(A-T)2] Specific Z-DNA by a Cationic Porphyrin.

Typical CD spectrum of the right-handed poly[d(A-T)2] was reversed when trans-bis(N-methylpyrimidium-4-yl)diphenyl porphyrin (trans-BMPyP) was bound, suggesting that the helicity of the polynucleotide was reversed to the left-handed form. The formation of the left-handed Z-form poly[d(A-T)2] was confirmed by (31)P NMR, in which a single (31)P peak of B-form poly[d(A-T)2] was split into two peaks, which is similar to the conventional B-Z transition of poly[d(G-C)2] induced by the high ionic strength. The observed B-Z transition is unique for poly[d(A-T)2]. The other polynucleotides, including poly[d(G-C)2], poly(dG)·poly(dC) and poly(dA)·poly(dT) remained as the right-handed form in the presence of the same porphyrin. This observation suggests that the porphyrin array that was formed along the poly[d(A-T)2] provides a template to which left-handed poly[d(A-T)2] is associated with an electrostatic interaction.

Conformations of adducts formed between the genotoxic benzo[a]pyrene-7,8-dione and 2'-deoxycytidine.

Benzo[a]pyrene-7,8-dione (BPQ) is formed by the activation of benzo[a]pyrene(B[a]P), which is an environmental toxic substance that is easily exposed in daily life, due to P450/epoxide hydrolase, and is a substance that induces DNA deformation by forming adducts with DNA. In this study, to investigate the form of bonding between BPQ and DNA, the structures of adducts between BPQ and 2'-deoxycytidine were examined. To examine BPQ-dC adduct conformation, geometry optimization of a total of 16 structural isomers was performed using the density functional theory method. In the structures of BPQ-dC adducts, for the cis-form, the angle between BPQ and dC is nearly perpendicular; but for the trans-form, the bending angle is small. The trans-form had a larger energy gap between ground state and excited state than the cis-form, and had a smaller HOMO-LUMO gap than the cis-form. Therefore, it was found that the trans-form absorbs stronger light and has higher reactivity than the cis-form. Molecular electrostatic potential was calculated and analyzed. The calculated ESP contour map shows the electrophilic and nucleophilic regions of the molecule.

Effects of deficient of the Hoogsteen base-pairs on the G-quadruplex stabilization and binding mode of a cationic porphyrin.

BACKGROUND: In stabilization of the G-quadruplex, formation of a Hoogsteen base-pair between the guanine (G) bases is essential. However, the contribution of each Hoogsteen base-pair at different positions to whole stability of the G-quadruplex has not been known. In this study, the effect of a deficiency of the Hoogsteen type hydrogen bond in the G-quadruplex stability was investigated. Spectral properties of meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) associated with various G-quadruplexes were also examined. METHODS: The thermal stability of the thrombin-binding DNA aptamer 5'G1G2TTG5G6TG8TG10G11TTG14G15 G-quadruplex, in which the guanine (G) base at 1, 2, 5, 6 and 8th positions was replaced with an inosine (I) base, one at a time, was investigated by circular dichroism (CD). The absorption, CD and fluorescence decay curve for the G-quadruplex associated TMPyP were also measured. RESULTS: The transition from the G-quadruplex to a single stranded form was endothermic and induced by an increase in entropy. The order in stability was 0>8>6>2>5>1, where the numbers denote the position of the replacement and 0 represents no replacements of the G base, suggesting the significant contribution of the G1 base in the stability of the G-quadruplex. Alteration in the spectral property of TMPyP briefly followed the order in thermal stability. CONCLUSIONS: Replacement of a G base with an I base resulted in destabilization of the G-quadruplex. The missing hydrogen bond at position 1 destabilized the G-quadruplex most efficiently. TMPyP binds near the I base-replaced location namely, the side of the G-quadruplex. GENERAL SIGNIFICANCE: The Hoogsteen base-pairing is confirmed to be essential in stabilization of G-quadruplex. When G is replaced with I, the latter base is mobile to interact with cationic porphyrin.

Ligand effect and cooperative role of metal ions on the DNA cleavage efficiency of mono and binuclear Cu(II) macrocyclic ligands complexes.

Two binuclear Cu(II) complexes of N-functionalized macrocycle ligands, namely 1,3-bis(1,4,7-triaza-1-cyclonomyl)propane and 1-(3-(1,4,7-triazonan-1-yl)propyl)-1,4,7,10-tetraazacyclo-dodecane, were synthesized and their ability to hydrolyze the cleavage of supercoiled plasmid DNA (pBR322) was compared with that of structurally related non-functionalized mononuclear Cu(II) complexes. The former, binuclear Cu(II) complex with the symmetrical ligand exhibited enhanced double-strand cleavage activity compared to the other three complexes at the same [Cu(2+)] concentration. In contrast, the latter binuclear complex with unsymmetrical macrocylic ligand did not give rise to double-strand DNA cleavage. The linear DNA formation induced by the mononuclear Cu(II) 1,4,7,10-tetraazacyclo-dodecane complex was realized via a non-random double-stranded scission process. The differential cleavage activity is discussed in relation to dimer formation, effective cooperation and coordination environment of the metal center. The hydrolytic cleavage by the copper complexes without H2O2 is supported by evidence from an anaerobic reaction, free radical quenching, and nitro blue tetrazolium assay. In contrast, both the binuclear complexes cleaved supercoiled DNA efficiently to Form III (linearized DNA) in the presence of H2O2, indicating that nuclearity is a crucial parameter in oxidative cleavage. The radical scavenger inhibition study and nitro blue tetrazolium assay suggested the involvement of H2O2 and superoxide ions in the oxidative cleavage of DNA by the binuclear complexes.