RESUMO
Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na(+) than for Li(+). Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance.
Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Telômero/química , Telômero/genética , Sequência de Bases , Dicroísmo Circular , Ligação de Hidrogênio , Oligonucleotídeos/química , TermodinâmicaRESUMO
Thrombin-binding aptamer (TBA) is a 15-nt DNA oligomer that efficiently inhibits thrombin. It has been shown that TBA folds into an anti-parallel unimolecular G-quadruplex. Its three-dimensional chair-like structure consists of two G-tetrads connected by TT and TGT loops. TBA undergoes fast degradation by nucleases in vivo. To improve the nuclease resistance of TBA, a number of modified analogs have been proposed. Here, we describe anomeric modifications of TBA. Non-natural α anomers were used to replace selected nucleotides in the loops and core. Significant stabilization of the quadruplex was observed for the anomeric modification of TT loops at T4 and T13. Replacement of the core guanines either prevents quadruplex assembly or induces rearrangement in G-tetrads. It was found that the anticoagulant properties of chimeric aptamers could be retained only with intact TT loops. On the contrary, modification of the TGT loop was shown to substantially increase nuclease resistance of the chimeric aptamer without a notable disturbance of its anticoagulant activity.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Anticoagulantes/química , Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Sequência de Bases , Dicroísmo Circular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Termodinâmica , Tempo de TrombinaRESUMO
The porphyrin-based photosensitizers capable of binding to DNA are perspective drug candidates. Here we report the interactions with calf thymus DNA of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (P1) and its derivatives containing Zn(II) or Ni(II) in the coordination sphere. These interactions were studied with absorption and circular dichroism spectroscopy. NiP1 and ZnP1 formed different types of complexes with DNA. NiP1 intercalated into the double helix, whereas ZnP1 bound the DNA groove. Compound P1 displayed both binding modes. The ZnP1-DNA binding constant was approximately three times smaller than the respective values for P1-DNA and NiP1-DNA complexes. Light induced degradation of the reactive oxygen species (ROS) trap 1,3-diphenylisobenzofuran in the presence of P1 and its metal derivatives revealed that NiP1 was a weaker photooxidative agent, whereas P1 and ZnP1 generated ROS to similar extents. Nevertheless, the DNA photodamaging effect of ZnP1 was the most pronounced. Illumination of the supercoiled plasmid caused single-strand DNA photocleavage in the presence of P1 and ZnP1; double strand breaks were detectable with micromolar concentrations of ZnP1. The concentration of ZnP1 required for plasmid photonicking was two times smaller than that of P1 and ~20 times lower than that for NiP1. Thus, the modes of P1, NiP1 and ZnP1 binding to DNA determine the differential photodamaging potency of these porphyrins. A greater accessibility to the solvent of the groove binder ZnP1, compared to the shielded intercalator NiP1 and the intercalated P1 molecules, allows for an efficient local generation of ROS followed by DNA photocleavage.
Assuntos
DNA de Cadeia Simples/química , Metaloporfirinas/química , Níquel/química , Fármacos Fotossensibilizantes/química , Compostos de Piridínio/química , Zinco/química , Sequência de Aminoácidos , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/efeitos da radiação , Metaloporfirinas/síntese química , Metaloporfirinas/farmacologia , Metaloporfirinas/efeitos da radiação , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/efeitos da radiação , Compostos de Piridínio/síntese química , Raios UltravioletaRESUMO
Novel generations of antitumor anthraquinones are expected to be advantageous over the conventional chemotherapeutic agents. Previous structure-activity relationship studies demonstrated an importance of the positively charged side chains conjugated to anthra[2,3-b]thiophene-5,10-dione scaffolds. Exploring a role of individual side chain moieties in binding to the duplex and G-quadruplex DNA, modulation of telomerase and topoisomerase I activities, intracellular accumulation and cytostatic potency, we herein analyzed a series of reported and newly synthesized guanidine-containing derivatives of anthra[2,3-b]thiophene-5,10-dione. We found that the number of cationic side chains (namely, two) is critical for a tight interaction with human telomeric G-quadruplex (TelQ). Along with a larger drug-TelQ association constant, the telomerase attenuation by anthrathiophenediones with two basic groups in the side chains was more pronounced than by the analogs bearing one basic group. For mono-guanidinated compounds the substituent with the amino group in the side chain provided better TelQ affinity than the methylamine residue. The intracellular uptake of the mono-guanidino derivative with two side chains was >2-fold higher than the respective value for the bis(guanidino) derivative. This difference can explain a lower antiproliferative potency of bis(guanidine) containing compounds. Thus, the modifications of side chains of anthra[2,3-b]thiophene-5,10-dione differently modulated drug-target interactions and cellular effects. Nevertheless, the selected compound 11-(3-aminopropylamino)-4-(2-guanidinoethylamino)anthra[2,3-b]thiophene-5,10-dione 13 demonstrated a high affinity to TelQ and the ability to stabilize the quadruplex structure. These properties were paralleled by reasonable potency of 13 as a telomerase/topoisomerase I inhibitor and an antiproliferative agent. These results indicate that the structural elements of anthra[2,3-b]thiophene-5,10-dione derivatives can be balanced to yield a candidate for further preclinical study.
Assuntos
Quadruplex G , Guanidina/química , Telomerase/antagonistas & inibidores , Tiofenos/metabolismo , Tiofenos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Espaço Intracelular/metabolismo , Camundongos , Tiofenos/química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologiaRESUMO
A series of DNA aptamers bearing triazole internucleotide linkages that bind to thrombin was synthesized. The novel aptamers are structurally analogous to the well-known thrombin-inhibiting G-quadruplexes TBA15 and TBA31. The secondary structure stability, binding affinity for thrombin and anticoagulant effects of the triazole-modified aptamers were measured. A modification in the central loop of the aptamer quadruplex resulted in increased nuclease resistance and an inhibition efficiency similar to that of TBA15. The likely aptamer-thrombin binding mode was determined by molecular dynamics simulations. Due to their relatively high activity and the increased resistance to nuclease digestion imparted by the triazole internucleotide linkages, the novel aptamers are a promising alternative to known DNA-based anticoagulant agents.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Nucleotídeos/química , Inibidores de Serina Proteinase/farmacologia , Trombina/antagonistas & inibidores , Trombina/metabolismo , Triazóis/química , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , Sítios de Ligação , Relação Dose-Resposta a Droga , Quadruplex G , Conformação Molecular , Simulação de Dinâmica Molecular , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Relação Estrutura-Atividade , Trombina/química , Triazóis/farmacologiaRESUMO
Cationic porphyrin-based compounds capable of interacting with DNA are currently under extensive investigation as prospective anticancer and anti-infective drugs. One of the approaches to enhancing the DNA-binding affinity of these ligands is chemical modification of functional groups of the porphyrin macrocycle. We analyzed the interaction with DNA of novel derivatives containing carboxymethyl and ethoxycarbonylmethyl substituents at quaternary nitrogen atoms of pyridinium groups at the periphery of the porphyrin macrocycle. The parameters of binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (P1) and 5,10,15,20-tetrakis(N-ethoxycarbonylmethyl-4-pyridinium)porphyrin (P2) to double-stranded DNA sequences of different nucleotide content were determined using optical spectroscopy. The association constant of P1 interaction with calf thymus DNA (K = 3.4 × 10(6) M(-1)) was greater than that of P2 (K = 2.8 × 10(5) M(-1)). Preferential binding of P1 to GC- rather than AT-rich oligonucleotides was detected. In contrast, P2 showed no preference for particular nucleotide content. Modes of binding of P1 and P2 to GC and AT duplexes were verified using the induced circular dichroism spectra. Molecular modeling confirmed an intercalative mode of interaction of P1 and P2 with CpG islands. The carboxyl groups of the peripheral substituent in P1 determine the specific interactions with GC-rich DNA regions, whereas ethoxycarbonylmethyl substituents disfavor binding to DNA. This study contributes to the understanding of the impact of peripheral substituents on the DNA-binding affinity of cationic porphyrins, which is important for the design of DNA-targeting drugs.
Assuntos
DNA/química , Simulação de Acoplamento Molecular , Porfirinas/química , Compostos de Piridínio/química , Porfirinas/síntese química , Compostos de Piridínio/síntese químicaRESUMO
The indolocarbazole derivative 12-(α-L-arabinopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (AIC) has demonstrated a high potency (at nanomolar to submicromolar concentrations) towards the NCI panel of human tumor cell lines and transplanted tumors. Intercalation into the DNA double helix has been identified as an important prerequisite for AIC cytotoxicity. In this study, we provide evidence for preferential binding to the G-quadruplex derived from the c-Myc oncogene promoter (Pu18 d(AG(3)TG(4))(2); G-c-Myc). The association constant for AIC:G-c-Myc complex was ~100 times and 10 times greater than the respective values for the complexes AIC:c-Myc duplex and AIC:telomeric d(TTAGGG)(4) G-quadruplex. The concentrations at which AIC formed complexes with G-c-Myc were close to those that attenuated the steady-state level of the c-Myc mRNA in the human HCT116 colon carcinoma cell line. We suggest that preferential binding of AIC to G-c-Myc rather than to the c-Myc duplex might favor the quadruplex formation in the cells, thereby contributing to downregulation of the c-Myc expression by AIC.
RESUMO
The modes of binding of 5'-[4-(aminoiminomethyl)phenyl]-[2,2'-Bifuran]-5-carboximidamide (DB832) to multi-stranded DNAs: human telomere quadruplex, monomolecular R-triplex, pyr/pur/pyr triplex consisting of 12 T*(T x A) triplets, and DNA double helical hairpin were studied. The optical adsorption of the ligand was used for monitoring the binding and for determination of the association constants and the numbers of binding sites. CD spectra of DB832 complexes with the oligonucleotides and the data on the energy transfer from DNA bases to the bound DB832 assisted in elucidating the binding modes. The affinity of DB832 to the studied multi-stranded DNAs was found to be greater (K(ass) approximately 10(7)M(-1)) than to the duplex DNA (K(ass) approximately 2 x 10(5)M(-1)). A considerable stabilizing effect of DB832 binding on R-triplex conformation was detected. The nature of the ligand tight binding differed for the studied multi-stranded DNA depending on their specific conformational features: recombination-type R-triplex demonstrated the highest affinity for DB832 groove binding, while pyr/pur/pyr TTA triplex favored DB832 intercalation at the end stacking contacts and the human telomere quadruplex d[AG(3)(T(2)AG(3))(3)] accommodated the ligand in a capping mode. Additionally, the pyr/pur/pyr TTA triplex and d[AG(3)(T(2)AG(3))(3)] quadruplex bound DB832 into their grooves, though with a markedly lesser affinity. DB832 may be useful for discrimination of the multi-sranded DNA conformations and for R-triplex stabilization.
Assuntos
DNA/química , Furanos/química , Sequência de Bases , Dicroísmo Circular , DNA/metabolismo , Furanos/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Telômero/química , Telômero/metabolismoRESUMO
Novel indolocarbazole derivative 12-(alpha-L-arabinopyranosyl)indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7-dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)(20), d(GC)(20) and calf thymus DNA (binding constants (1.6x10(6)) M(-1)< or =K(a)< or =(3.3x10(6)) M(-1)). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C< or =1 microM) were identical to the concentrations that triggered p53-dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I-mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.
Assuntos
Antineoplásicos/farmacologia , Arabinose/análogos & derivados , Carbazóis/farmacologia , DNA/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose , Arabinose/química , Arabinose/farmacologia , Carbazóis/química , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Feminino , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/metabolismo , Camundongos , Camundongos Endogâmicos , Espectrometria de Fluorescência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5'-GT-2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3' (R(2AP)CW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3' (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R(2AP)CW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19 degrees C to 33 degrees C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R(2AP)CW triplex and up to six YOYO molecules to CW hairpin.
Assuntos
Benzoxazóis/química , DNA/química , Corantes Fluorescentes/química , Substâncias Intercalantes/química , Compostos de Quinolínio/química , Sequência de Bases , Estrutura Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/química , TermodinâmicaRESUMO
The parallel (recombination) 'R-triplex' can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T.A) and 6MI*(C.G) triplets were monitored by their fluorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. The fluorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from fluorescence of the single base analog. The similarity suggests that fluorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative 'branch migration' and 'strand exchange' structures and discuss why these are less likely than the R-type triplex.
Assuntos
2-Aminopurina/análise , DNA/química , Conformação de Ácido Nucleico , Xantopterina/análise , 2-Aminopurina/química , Pareamento de Bases , Sequência de Bases , DNA/genética , Etídio/análise , Fluorescência , Polarização de Fluorescência , Conformação de Ácido Nucleico/efeitos da radiação , Desnaturação de Ácido Nucleico/efeitos da radiação , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Temperatura , Termodinâmica , Raios Ultravioleta , Xantopterina/análogos & derivados , Xantopterina/químicaRESUMO
Oligonucleotides 3'-d(GT)(5)-(CH(2)CH(2)O)(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)(4)G-(CH(2)CH(2)O)(3)-dG(GT)(4)G-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. Dyn. 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 (tau) of Et:qGT (q = 0.15 +/- 0.01 and tau = 24 +/- 1 ns) and Et:hp-GT (q = 0.10 +/- 0.01 and tau = 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 omega = 3.9 +/- 0.1 and the binding constant Kappa = (6.4 +/- 0.1).10(4) M(-1). The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of innerspaces 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 Kappa = (1.5 +/- 0.1).10(5) M(-1). Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.