Quadruplex DNA Structures Research Articles

A Review: G-Quadruplex's Applications in Biological Target Detection and Drug Delivery.

The G-quadruplex is one of the most important DNA secondary structures and has elicited widespread interest in this field of scientific research. As G-quadruplex studies have progressed, its significant roles in a variety of biological process have been revealed, including telomerase activity, genome stability, DNA replication, and oncogene expression. In addition to its biological functions, as a special DNA quadruplex structure, it also has potential for other important applications like asymmetric catalysis, probing for important biomarkers, acting as a vehicle for drug delivery, or as a structural regulator for some nanostructure designs. Here, we discuss its application to aspects of biological target detection and drug delivery. Its utility in biological target detection mainly results from its special DNAzyme activity. We would summarize some typical systems, utilizing G-quadruplex as signal readout unit, for various target detection, such as SNP, miRNAs, ions, transcription factors and so on. On the other hand, its use in drug delivery always relies on probes' structural change, as the switch of drug release. Here, we would also exhibit some typical drug delivery designs.

Calculation of the HOMO localization of Tetrahymena and Oxytricha telomeric quadruplex DNA.

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3'-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5'-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K(+) and d(TAGGGT)4T +2K(+) localized at the 5'-guanine, and that the HOMO shifted from the 5'-guanine to the 3'-guanine by the addition of a 5'-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K(+) and d(TAGGGT)4T +2K(+) matched the experimental results and suggest that d(TGGGGT)4 contains a 5'-capping cation in solution.

Selective recognition of specific G-quadruplex vs. duplex DNA by a phenanthroline derivative

A key problem in designing G-quadruplex ligand is how to discriminate quadruplex DNA specifically from other DNAs, searching ligands targeted at special G-quadruplex structure with high selectivity is a major challenge. Herein, a phenanthrolin-dicarboxylate ester (PD) is proved to exhibit selectivity toward parallel and hybrid G-quadruplex structures with propeller and edge-wise loops, over duplex DNA and antiparallel quadruplex structure with diagonal loop. Such preferred binding of PD to these special G-quadruplex structures is possibly resulted from steric hindrance of: (1) the four substituent groups in PD molecule which prevent close interaction with duplex DNA and (2) the diagonal loop above the G-tetrads in antiparallel G-quadruplex which hinder face-to-face stacking of planar phenanthrolin ring to G-tetrads. In line with its stabilizing ability of G-quadruplex, PD molecule exhibit a inhibitory ability telomerase activity, as well as the potent cytotoxic activity against several human cancer cell lines, which makes it an interesting anti-tumor drug lead.

The NEIL glycosylases remove oxidized guanine lesions from telomeric and promoter quadruplex DNA structures.

G-quadruplex is a four-stranded G-rich DNA structure that is highly susceptible to oxidation. Despite the important roles that G-quadruplexes play in telomere biology and gene transcription, neither the impact of guanine lesions on the stability of quadruplexes nor their repair are well understood. Here, we show that the oxidized guanine lesions 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) reduce the thermostability and alter the folding of telomeric quadruplexes in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases can remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplexes. Interestingly, a hydantoin lesion at the site most prone to oxidation in quadruplex DNA is not efficiently removed by NEIL1 or NEIL3. However, NEIL1, NEIL2 and NEIL3 remove hydantoins from telomeric quadruplexes formed by five TTAGGG repeats much more rapidly than the commonly studied four-repeat quadruplex structures. We also show that APE1 cleaves furan in selected positions in Na+-coordinated telomeric quadruplexes. In promoter G-quadruplex DNA, the NEIL glycosylases primarily remove Gh from Na+-coordinated antiparallel quadruplexes but not K+-coordinated parallel quadruplexes containing VEGF or c-MYC promoter sequences. Thus, the NEIL DNA glycosylases may be involved in both telomere maintenance and in gene regulation.

Abstract 2607: G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies

Abstract Introduction: Many cancer-related pro-oncogenes have quadruplex forming oligonucleotide sequences in their promoter regions. These genes include, amongst others, c-Myc, Bcl-2, K-ras, or HIF-1a which are individually or together over-expressed in the vast majority of neoplasms. For instance, c-Myc, a key regulator of cell cycle and proliferation, is commonly over-expressed in both hematological and solid cancers. The c-myc promoter region contains a guanine-rich sequence (PU27), capable of forming four-stranded quadruplex DNA structures, which negatively regulates c-myc transcription. Also, Bcl-2, is commonly over-expressed in a variety of hematological malignancies and the Bcl-2 quadruplex-forming sequence is located upstream of the P1 promoter of the Bcl-2 gene; it is also implicated in negative regulation of Bcl-2 transcription. Treatment of cancer cells over-expressing c-Myc and / or Bcl-2 with oligonucleotides encoding the c-Myc (PU27) and Bcl-2 (Bcl-2-GRO) guanine-rich sequences, located in their respective promoter regions, selectively inhibits c-Myc or Bcl-2 gene expressions, cancer cells proliferation and induces cell death. Methods: Four cancer cell lines obtained from ATCC were used in these studies (Raji, HL-60, U937, A549). Cell Titer-Glo© Assay was used for the cell proliferation experiments. Cell cycle analysis was determined in the HL-60 cell line using fixed doses of PU27 and Bcl-2-GRO and time dependent FACS analysis. Internalization studies were analyzed by FACS analysis using FAM labeled PU27 and DY647 labeled Bcl-2-GRO. In vivo tumor growth inhibition studies were done in females NOD/SCID mice (6 animals per group); 5x106 cells in a Matrigel mixture were implanted in the flank of the animals; tumors developed and treatments with PU27 and Bcl-2-GRO at different dose levels alone and in combination were performed while body weights were monitored during the course of the study. Results: Treating the Raji, HL-60, U937, or A549 cancer cell lines with PU27 or Bcl-2-GRO causes inhibition of proliferation with IC50 between 200 nM and 5mM after 144 hours depending on expression of the target pro-oncogene. When treating the same cell lines using a combination of these two sequences, a significant synergy is observed, leading to more than 100 fold increase in potency. The same synergistic effect was seen when PU27 was combined with paclitaxel, leading to picomolar IC50. Internalization of these oligonucleotides was very rapid and quantitative. Localization studies show that PU27 and Bcl-2-GRO are localized in the nucleus. Cell cycle results showed a significantly increase in G0-G1 cell cycle arrest for the combination. In vivo xenograft tumor growth inhibition studies confirmed the in vitro synergistic results with 60 to 80% tumor growth inhibition while treatments were well tolerated (no body weight loss). Citation Format: Gilles H. Tapolsky, Kara Sedoris, Shelia Thomas, Francine Rezzoug, Donald M. Miller. G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2014-2607

Pyrrolobenzodiazepines (PBDs) do not bind to DNA G-quadruplexes.

The pyrrolo[2,1-c][1,4] benzodiazepines (PBDs) are a family of sequence-selective, minor-groove binding DNA-interactive agents that covalently attach to guanine residues. A recent publication in this journal (Raju et al, PloS One, 2012, 7, 4, e35920) reported that two PBD molecules were observed to bind with high affinity to the telomeric quadruplex of Tetrahymena glaucoma based on Electrospray Ionisation Mass Spectrometry (ESI-MS), Circular Dichroism, UV-Visible and Fluorescence spectroscopy data. This was a surprising result given the close 3-dimensional shape match between the structure of all PBD molecules and the minor groove of duplex DNA, and the completely different 3-dimensional structure of quadruplex DNA. Therefore, we evaluated the interaction of eight PBD molecules of diverse structure with a range of parallel, antiparallel and mixed DNA quadruplexes using DNA Thermal Denaturation, Circular Dichroism and Molecular Dynamics Simulations. Those PBD molecules without large C8-substitutents had an insignificant affinity for the eight quadruplex types, although those with large π-system-containing C8-substituents (as with the compounds evaluated by Raju and co-workers) were found to interact to some extent. Our molecular dynamics simulations support the likelihood that molecules of this type, including those examined by Raju and co-workers, interact with quadruplex DNA through their C8-substituents rather than the PBD moiety itself. It is important for the literature to be clear on this matter, as the mechanism of action of these agents will be under close scrutiny in the near future due to the growing number of PBD-based agents entering the clinic as both single-agents and as components of antibody-drug conjugates (ADCs).

Structural transformation induced by locked nucleic acid or 2′–O-methyl nucleic acid site-specific modifications on thrombin binding aptamer

BackgroundLocked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades. However, how they affect DNA quadruplex structures remains largely unknown. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect G-quadruplex structure formation using a thrombin binding aptamer (TBA), the most studied extracorporal G-quadruplex-forming DNA sequence, which is frequently modified to increase its analytical performance.ResultsThe experimental results showed that when two or more nucleotides were substituted with LNA or OMeNA, the anti-parallel TBA structure was transformed into an unstructured random conformation in a 50 mM K+ environment; OMeNA appeared to have greater power to induce this transformation. However, the native TBA was unstructured in a 50 mM Ca2+ environment, whereas four or more LNA- or OMeNA- substitutions could convert this unstructured TBA into a parallel quadruplex structure. PAGE mobility measurements suggested that these TBAs might be a dimeric form.ConclusionLNA or 2'-OMeNA site-specific modifications induced G-quadruplex structural transformation of TBA, which enriched our understanding of the intrinsic G-quadrupex forming property and affinity of LNA and OMeNA modifications. This study demonstrates possible applications in the regulation of gene expression (i.e. manual intervention of gene therapy), genetic analyses, molecular diagnosis and the construction of nano-scale biostructures.

Exploring non-covalent interactions in guanine- and xanthine-based model DNA quadruplex structures: a comprehensive quantum chemical approach

The study aimed to cast light on the structure and internal energetics of guanine- and xanthine-based model DNA quadruplexes and the physico-chemical nature of the non-covalent interactions involved. Several independent approaches were used for this purpose: DFT-D3 calculations, Quantum Theory of Atoms in Molecules, Natural Bond Orbital Analysis, Energy Decomposition Analysis, Compliance Constant Theory, and Non-Covalent Interaction Analysis. The results point to an excellent degree of structural and energetic compatibility between the two types of model quadruplexes. This fact stems from both the structural features (close values of van der Waals volumes, pore radii, geometrical parameters of the H-bonds) and the energetic characteristics (comparable values of the energies of formation). It was established that hydrogen bonding makes the greatest (∼50%) contribution to the internal stability of the DNA quadruplexes, whereas the aromatic base stacking and ion coordination terms are commensurable and account for the rest. Energy decomposition analysis performed for guanine (Gua) and xanthine (Xan) quartets B4 and higher-order structures consisting of two or three stacked quartets indicates that whereas Gua structures benefit from a high degree of H-bond cooperativity, Xan models are characterized by a more favorable and cooperative π-π stacking. The results of electron density topological analysis show that Na(+)/K(+) ion coordination deeply affects the network of non-covalent interactions in Gua models due to the change in the twist angle between the stacked tetrads. For Xan models, ion coordination makes tetrads in stacks more planar without changing the twist angle. Therefore, the presence of the ion seems to be essential for the formation of planar stacks in Xan-based DNA quadruplexes. Detailed study of the nature of ion-base coordination suggests that this interaction has a partially covalent character and cannot be considered as purely electrostatic. Investigation of the H-bond and ion-base coordination strengths by various independent approaches agrees well with the results of QTAIM analysis.

A Family Of G-Rich Genomic Sequences Iinteract Specifically With The Pu27 Silencer Element Of The c-Myc Promoter

IntroductionA substantial proportion (∼80%) of c-Myc expression is regulated by the NHEIII1 element in the c-myc P1 promoter. The NHEIII1 element contains a G/C rich negative regulatory sequence (Pu27), which can form an intramolecular DNA quadruplex structure which results in c-myc silencing. Quadruplex DNA is a four-stranded structure which is quite stable and can form either “intramolecular” (one DNA strand) quadruplex, or “intermolecular” (two or four DNA strands) quadruplex. These quadruplex-forming sequences are very commonly present in the promoters of eukaryotic genes and generally are negative regulators of transcription. The equilibrium between quadruplex (transcriptionally inactive) and duplex (transcriptionally active) DNA structure is influenced by protein binding, salt concentration and supercoiling torsion (as well as other factors). Constitutive overexpression of the c-myc P0-noncoding transcript, containing the Pu27 sequence, inhibits tumorigenesis and growth of tumor cells. Oligonucleotides encoding the Pu27 sequence bind specifically to the Pu27 sequence in the c-myc promoter, inhibit c-myc expression and result in decreased cellular proliferation and cell death. This most likely reflects the formation of intermolecular quadruplex between the oligonucleotide and target sequence. In addition to the Pu27 sequence in the c-myc promoter, we have recently identified a family of 13 nearly identical Pu27 sequences in the noncoding human genome. MethodsPu27 family members were identified by a BLAT search from the UCSC Genome Browser. Circular dichroism was used to document quadruplex formation by each of the 14 Pu27 family members. Specific interaction of each Pu27 “family member” with the “parent” Pu27 sequence was documented by electrophoretic mobility shift (EMS) studies. Growth inhibition was measured for the U937, Raji, K562 and HL60 leukemic cell lines. C-Myc expression was measured by RT-PCR and Western blot analysis. ResultsWe have identified fourteen nearly identical copies of the Pu27 sequence in the noncoding genome. They are located on Chromosomes 8 (c-Myc promoter), 1, 3 (2), 5, 7, 9, 10 (2), 11, 16, 17, 20, and X. Other than the c-Myc Pu27 sequence, these sequences do not appear to be located in promoter regions. Using gel shift analysis, we demonstrated that each of the Pu27 family member sequences binds in a sequence specific manner to the “parent” sequence in the c-myc promoter. Other genomic quadruplex-forming sequences (from the K-ras, Bcl2 and VEGF promoters) do not bind to the c-myc Pu27 promoter sequence. We have previously shown that treatment of leukemic cell lines with an oligonucleotide containing the Pu27 sequence results in downregulation of c-myc transcription and inhibition of cell proliferation with subsequent cell death. The Pu27 oligonucleotide does not affect the growth of nontransformed cell lines. Each of the Pu27 family members also inhibits the growth of all of the leukemic cell lines tested, with varying efficacy. This occurs with an IC50 as low as <200 nM in all of tumor cell lines tested. Analyzing data from the Human Cancer Genome project we have shown that one of the Pu27 family members (located on chromosome 5) is frequently deleted in leukemic cells of patients with AML (data from Human Cancer Genome Project). ConclusionsThis novel family of genomic sequences interacts specifically with the Pu27 c-Myc silencer sequence and down regulates c-myc expression and cellular proliferation. This suggests that they likely play a role in regulating c-Myc expression either through direct DNA-DNA interaction or sequence-specific interaction with transcripts of one or more of the Pu27 sequences. We hypothesize that DNA-DNA interactions between these sequences may also stabilize quadruplex formation by the c-myc promoter sequence, locking it in the “off” (transcriptionally inactive) position. Disruption of this interaction may play an important role in leukemia pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.

Carbohydrate–DNA Interactions at G‐Quadruplexes: Folding and Stability Changes by Attaching Sugars at the 5′‐End

Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate-DNA conjugates containing the oligonucleotide sequences of G-quadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose-TBA conjugate shows stacking interactions between the carbohydrate and the DNA G-tetrad in addition to hydrogen bonding and hydrophobic contacts. We have also shown that attaching carbohydrates at the 5'-end of a quadruplex telomeric sequence can alter its folding topology. These results suggest the possibility of modulating the folding of the G-quadruplex by linking carbohydrates and have clear implications in molecular recognition and the design of new G-quadruplex ligands.

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4-bis(4-dimethylaminostyryl)-1-methylpyridinium (1a), its derivative with a quaternary aminoalkyl chain (1b), and the symmetric 2,6-bis(4-dimethylaminostyryl)-1-methylpyridinium (2a), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA-binding and DNA-probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4-disubstituted) dyes 1a and 1b bind to quadruplex DNA structures with a near-micromolar affinity and a fair selectivity with respect to double-stranded (ds) DNA [K(a)(G4)/K(a)(ds)=2.5-8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80-100-fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double-stranded DNA. This optical selectivity allows these dyes to be used as quadruplex-DNA-selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2a displays a strong binding preference for double-stranded DNA [K(a) (ds)/K(a) (G4)=40-100), presumably due to binding in the minor groove. In addition, 2a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20-50-fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA-recognition properties and fluorimetric response of organic dyes.

The critical effect of polarization on the dynamical structure of guanine quadruplex DNA

Guanine quadruplex DNA (G-DNA), found in eukaryotic telomeres and recently in non-telomeric genomic DNA, plays important biological roles and their structures are being explored as potential targets for therapeutic intervention. Since the quadruplex structure of G-DNA is stabilized by cations, electrostatic interaction is expected to play important roles in the dynamical structure of G-DNA. In current work, MD simulation was carried out to study the dynamical structure of a special G-DNA (with sequence d(G(4)T(4)G(4))) complexed with five K(+) ions. In order to properly include polarization in MD simulation, a new set of polarized nucleic acid specific charge based on fragment quantum chemistry calculation was developed for G-DNA. Our study showed that polarization of the nucleobases by K(+) enhanced electrostatic attraction between the base and ions. This increased attractive interaction is critical to stabilizing the stem-loop junction ions in G-DNA. Without this polarization effect, as in MD simulation using a standard (nonpolarizable) force field, the top and bottom cations escaped into the solvent within just a few nanoseconds. Furthermore, an incorrect bifurcated bonding geometry of G-DNA, found in previous MD simulation study under a standard force field but not observed in experiments, disappeared in the present stimulation using the new polarized force field. The current study bridged an important gap between the simulation study and experimental observation on the dynamical structure of G-DNA.

Total Synthesis of Taspine and a Symmetrical Analogue: Study of Binding to G‐Quadruplex DNA by ESI‐MS

AbstractTaspine is an alkaloid found in the latex of certain trees belonging to the family of Euphorbiaceae, commonly called “Sangre de Drago” (Dragon's blood). Its structure and antineoplastic properties are well known. Here we report the total synthesis of taspine, starting from ferulic acid and isovanillin. The successful synthetic strategy was then applied to the synthesis of a symmetrical analogue TAS2C. Both the synthesised compounds were tested by ESI‐MS (electrospray ionisation mass spectrometry) for their ability to bind different quadruplex and duplex DNA structures.

Cationic Pentaheteroaryls as Selective G‐Quadruplex Ligands by Solvent‐Free Microwave‐Assisted Synthesis

We report herein a solvent-free and microwaved-assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4-oxadiazole moieties (1-7). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4-FID) and CD spectroscopy. Among the G-quadruplexes considered, attention was focused on telomeric repeats together with the proto-oncogenic c-kit sequences and the c-myc oncogene promoter. Compound 1, and to a lesser extent 2 and 5, preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole (TOxaPy), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties.

Structural Investigations on the Anti‐HIV G‐Quadruplex‐Forming Oligonucleotide TGGGAG and Its Analogues: Evidence for the Presence of an A‐Tetrad

Several anti-HIV aptamers adopt DNA quadruplex structures. Among these, "Hotoda's aptamer" (base sequence TGGGAG) was one of the first to be discovered. Although it has been the topic of some recent research, no detailed structural investigations have been reported. Here we report structural investigations on this aptamer and analogues with related sequences, by using UV, CD, and NMR spectroscopy as well as electrophoretic techniques. The addition of a 3'-end thymine has allowed us to obtain a single, investigable quadruplex structure. Data clearly point to the presence of an A-tetrad. Furthermore, the effects of the incorporation of an 8-methyl-2'-deoxyguanosine at the 5'-end of the G-run were investigated.

The interactions of phenanthroline compounds with DNAs: Preferential binding to telomeric quadruplex over duplex

Compounds that bind and stabilize selectively human telomeric quadruplex DNA structures are of significant interest due to their potential to inhibit telomerase and to halt tumor cell proliferation. In our previous study, we found that three novel phenanthroline derivatives could induce and stabilize significantly the formation of an antiparallel structure of human telomeric G-quadruplex DNA (G3(T2AG3)3), moreover, these compounds could bind selectively to G-quadruplex over duplex. In order to understand their binding nature, in this work we investigated the interactions of compounds 1–3 with human telomeric G-quadruplex and i-motif (C3(A2TC3)3) DNAs together with calf thymus DNAs (ct DNA) by means of absorption, fluorescence and CD spectroscopies and competition dialysis assay. Results showed that all three compounds showed the highest affinity for G-quadruplex structure although with least affinity for ct DNA. Compounds 1–3 could also stabilize the structure of i-motif with an increase in melting temperature (ΔTm) of 7.6, 7.2 and 10.1°C, respectively, in the presence of 10 times excess of compounds. Their binding stoichiometric ratios were 2:1 and 1:1 for G-quadruplex and i-motif DNAs, respectively. The thermodynamic parameters results indicated that the binding of compound to either quadruplex or duplex DNAs was entropically driven and hydrophobic force played a major role in the reaction.

Structure–activity relationships of novel substituted naphthalene diimides as anticancer agents

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure–activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3–22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.

Spontaneous DNA Lesions Modulate DNA Structural Transitions Occurring at Nuclease Hypersensitive Element III1 of the Human c-myc Proto-Oncogene

G quadruplex (G4) DNA is a noncanonical four-stranded DNA structure that can form in G repeats by stacking of planar arrays of four hydrogen-bonded guanines called G quartets, in the presence of potassium ions. In addition to a presumed function in the regulation of gene expression, G4 DNA also localizes to regions often characterized by genomic instability. This suggests that formation of this structure may interfere with DNA transactions, including processing of DNA damage at these sites. Here we have studied the effect of two spontaneous DNA lesions, the abasic site and 8-oxoguanine, on the transition from duplex to quadruplex DNA structure occurring at nuclease hypersensitive element III(1) (NHEIII(1)) of the human c-myc promoter. We show by dimethyl sulfate footprinting and RNA polymerase arrest assays that at physiological concentrations of potassium ions NHEIII(1) folds into two coexisting G4 DNA structures, myc-1245 and myc-2345, depending on which G runs are utilized for G quartet formation. We found that a single substitution of G12 of NHEIII(1) with a single abasic site or a single 8-oxoguanine prevented formation of G4 structure myc-2345 in favor of structure myc-1245, where the lesion was accommodated in a DNA loop formed by G11-AP12/(or 8-oxoG12)-G13-G14. Surprisingly, when an additional G to A base substitution was introduced at position 3 of NHEIII(1), we observed formation of myc-2345. The extent of this structural transition was modulated by the location and type of lesion within the G11-G14 repeat. Our data indicate that spontaneous lesions formed in the G4-forming sequence of c-myc NHEIII(1) affect the structural transitions occurring at this regulatory site, potentially altering transcription factor binding and DNA repair of lesions formed in this highly regulated sequence.

UV Spectroscopy of DNA Duplex and Quadruplex Structures in the Gas Phase

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in solution. Mass spectrometry, on the other hand, is able to separate constituents of the solution based on their mass, but methods to probe the structure of each constituent are needed. Here, we explored whether gas-phase UV spectroscopy can give an indication of DNA folding in ions isolated by electrospray mass spectrometry. Model DNA single strands, duplexes, and G-quadruplexes were extracted from solution by electrospray; the anions were stored in a quadrupole ion trap and irradiated by a tunable laser to obtain the UV action spectra of each complex. We found that the duplex and quadruplex spectra are significantly different from the spectra of single strands, thereby suggesting that electronic spectroscopy can be used to probe the DNA gas-phase structure and obtain information about the intrinsic properties of high-order DNA structure.