Duplex DNA Sequences Research Articles

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Mechanical Properties and Strand Invasion of Duplex Telomere DNA Probed using Magnetic Tweezers

Telomeres are specialized chromatin structures that protect chromosome ends from nucleolytic processing by DNA repair machinery. The foundation of human telomere structure consists of a long array of tandem duplex DNA sequences (TTAGGG) and terminates with a single-stranded 3’ end. To protect the chromosome end, telomeres are thought to adopt a lariat structure known as a telomere-loop (T-loop)1. T-loops are stabilized by DNA displacement loops (D-loops) generated by the invasion of a single-stranded telomeric DNA tail into an adjacent region of duplex telomere. Recent studies suggest that telomere-associated proteins promote strand invasion during telomeric D-loop formation through the application of torque to the DNA2. Although the molecular mechanism of T-loop formation has been described using biochemical approaches, the torque response and internal structural equilibrium of duplex telomeric DNA are not well characterized. To probe the mechanical properties of duplex telomeric DNA, we developed a magnetic tweezers assay to detect the response of single telomeric DNA molecules to precisely applied degrees of tension and torque. Rotation-extension curves under varying tension demonstrate that the repetitive telomere DNA sequence is more refractory to torque-induced denaturation than a non-telomeric control molecule of comparable GC content. In addition, force-extension analysis of negatively supercoiled telomeric DNA in the presence of different counter-ions (K+ vs. Li+), reveals that transient torque-induced denaturation of duplex telomeric DNA promotes a structural transition into stable DNA G-quadruplexes. Lastly, using a single molecule DNA topology-based assay, we directly monitor the torque-dependent invasion of single stranded telomere DNA primers into duplex telomeric DNA tethers. Our results provide insight into the molecular mechanisms of telomere-associated proteins and enzymes during structural remodeling of telomeres.1. Griffith JD, et al. Cell 97: 503-514.2. Amiard S, et al. Nat Struct Mol Biol 14:147-154.

An isocytidine derivative with a 2-amino-6-methylpyridine unit for selective recognition of the CG interrupting site in an antiparallel triplex DNA.

Sequence-specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non-natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N(2)-arylmethyl isodC derivatives and incorporated them into triplex-forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2-amino-6-methylpyridine moiety (AP-isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.

Sequence-specific DNA interactions with calixarene-based langmuir monolayers.

The interactions of an amphiphilic calixarene, namely p-guanidino-dodecyloxy-calix[4]arene, 1, self-assembled as Langmuir monolayers, with short double stranded DNA, were investigated by surface pressure-area (π-A) isotherms, surface ellipsometry and Brewster angle microscopy (BAM). Three DNA 30mers were used as models, poly(AT), poly(GC) and a random DNA sequence with 50% of G:C base pairs. The interactions of these model DNA duplexes with 1-based Langmuir monolayers were studied by measuring compression isotherms using increasing DNA concentrations (10(-6), 10(-5), 10(-4), and 5 × 10(-4) g L(-1)) in the aqueous subphase. The isotherms of 1 showed an expansion of the monolayer with, interestingly, significant differences depending on the duplex DNA sequence studied. Indeed, the interactions of 1-based monolayers with poly(AT) led to an expansion of the monolayer that was significantly more pronounced that for monolayers on subphases of poly(GC) and the random DNA sequence. The structure and thickness of 1-based Langmuir monolayers were investigated by BAM and surface ellipsometry that showed differences in thickness and structure between a monolayer formed on pure water or on a DNA subphase, with here again relevant dissimilarities depending on the DNA composition.

Abstract B204: Targeting key DNA/RNA heteroduplex of telomerase by small-molecule ligands.

Abstract Small molecules can be designed to target the DNA/RNA hybrid (DRH) duplex that forms during the catalytic cycle of telomerase. Formation of this unique heteroduplex, which is typically between 4-11 base pairs long, is a key step in the mechanism of telomerase extension. Small molecules that bind to this duplex may inhibit telomerase by either stabilizing the structure thereby preventing strand dissociation (a key step in the catalytic cycle of telomerase), or by sufficiently distorting the substrate duplex causing misalignment of key catalytic groups. As telomerase is frequently over-expressed in cancer cells, the DRH duplex is a good therapeutic target. Thus we initiated a screening programme initially based on the National Cancer Institute (NCI) compound libraries which provide a high degree of structural diversity. Three novel scaffolds were identified in a Fluorescent Resonance Energy Transfer (FRET) based assay that showed at least a 5°C selective stabilization of the DNA/RNA hybrid duplex at 5μM ligand concentration. Medicinal chemistry modifications were then employed on these scaffolds to generate focused libraries of analogues with drug like properties. In total, 30 molecules were synthesized based on the identified scaffolds and subsequently screened against seven different DNA sequences including telomeric and promoter G-quadruplexes, and DRH sequences. A number of compounds showed selective DNA/RNA hybrid stabilization potential without significant affinity for G-quadruplex or duplex DNA sequences. These ligands provided hybrid DNA/RNA stabilization in the range of 5.2-9.5°C at 1μM ligand concentration. A second generation molecules where amides have been replaced by secondary amines are currently being synthesized to obtain SAR. Selected analogues from both compounds set are currently being evaluated in a variety of biological assays to confirm selective telomerase inhibition, and in cell based experiments to assess their potential as antitumour agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B204. Citation Format: Mohammad K. Islam, David E. Thurston, Khondaker M. Rahman. Targeting key DNA/RNA heteroduplex of telomerase by small-molecule ligands. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B204.

DNA triplex folding: Moderate versus high salt conditions

Some specific sequences in duplex DNA can give rise to local formation of a triple helical DNA called triplex together with a separate strand. Recent single-molecule FRET experiments, performed on DNA strands designed to fold into a triplex, allow us to measure the folding and unfolding time distributions under neutral p H conditions. The average times of both processes are of the order of 1 s. The folding time is moderately sensitive to salt concentration. The average unfolding time is found to be nearly constant. Interestingly, the distributions of the unfolding time revealed heterogeneous kinetics at moderate salt concentration (∼ 10 mM), but not at high salt (∼ 100 mM). We relate this salt dependence to different folding paths and folded states, which are governed by the (salt-dependent) stiffness of the third single-stranded donor sequence. Finally we comment on the formation of intramolecular triplex named H-DNA in a torsionally constrained duplex under physiological salt conditions, which mimics the in vivo situation of triplex folding.

On the Formation and Properties of Interstrand DNA–DNA Cross-Links Forged by Reaction of an Abasic Site with the Opposing Guanine Residue of 5′-CAp Sequences in Duplex DNA

We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5'-CAp sequence under conditions of reductive amination (NaCNBH(3), pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions, and the cross-link, once formed, was stable to a variety of workup conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LC-MS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N(2)-amino group of the opposing guanine residue in 5'-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH(3)/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N(2)-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH(3).

Templated chemistry for monitoring damage and repair directly in duplex DNA

We report the fluorogenic detection of the product of base excision repair (an abasic site) in a specific sequence of duplex DNA. This is achieved by DNA-templated chemistry, employing triple helix-forming probes that contain unnatural nucleobases designed to selectively recognize the site of a missing base. Light-up signals of up to 36-fold were documented, and probes could be used to monitor enzymatic removal of a damaged base.

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

Templated chemistry for sequence-specific fluorogenic detection of duplex DNA.

We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple-helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an α-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double-stranded DNA, at least for homopurine-homopyrimidine target sites, under native and nondenaturing conditions.

Structural contributions to the intracellular targeting strategies of antimicrobial peptides

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide–DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic α-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an α-helix structure is not required for the DNA binding and condensation activity of buforin II amide.

Selective Binding of Distamycin A Derivative to G‐Quadruplex Structure [d(TGGGGT)]4

Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are found in many regions of human genome and in the telomeres of all eukaryotic organisms. Since small molecules that target G-quadruplexes have been found to be effective telomerase inhibitors, the identification of new specific ligands for G-quadruplexes is emerging as a promising approach to develop new anticancer drugs. Distamycin A is known to bind to AT-rich sequences of duplex DNA, but it has recently been shown to interact also with G-quadruplexes. Here, isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound 1) and the [d(TGGGGT)]4 quadruplex. Additionally, to compare the binding behaviour of netropsin and compound 1 to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)]4 has been performed. Experiments show that netropsin and compound 1 are able to bind to [d(TGGGGT)]4 with good affinity and comparable thermodynamic profiles. In both cases the interactions are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity.

Synthesis, Structural Characterization, and Quadruplex DNA Binding Studies of Platinum(II)-Terpyridine Complexes

Three new substituted terpyridine ligands and their corresponding platinum(II) complexes have been prepared and fully characterized (including the X-ray crystal structures of two of the complexes). These compounds have shown to stack via pi-pi interactions both in the solid state and in solution. The interactions of these compounds with quadruplex DNA (both HTelo and c-myc sequences) have been studied by fluorescent indicator displacement (FID) assays, surface plasmon resonance (SPR), and in one case by circular dichroism (CD). The complexes have shown to interact strongly with quadruplex DNA (with binding constants of ca. 10(6) and DC(50) values between 2.24 and 0.26, as determined by SPR and FID, respectively). We have also investigated the interaction between the complexes and a sequence of duplex DNA to gain some insight into the selectivity of the compounds for quadruplex structures. FID and SPR studies have shown that the complexes have a modest selectivity (1 order of magnitude) for quadruplex, particularly c-myc, versus duplex DNA.

Empirical relationship between intra-purine and intra-pyrimidine differences in conserved gene sequences.

DNA sequences seen in the normal character-based representation appear to have a formidable mixing of the four nucleotides without any apparent order. Nucleotide frequencies and distributions in the sequences have been studied extensively, since the simple rule given by Chargaff almost a century ago that equates the total number of purines to the pyrimidines in a duplex DNA sequence. While it is difficult to trace any relationship between the bases from studies in the character representation of a DNA sequence, graphical representations may provide a clue. These novel representations of DNA sequences have been useful in providing an overview of base distribution and composition of the sequences and providing insights into many hidden structures. We report here our observation based on a graphical representation that the intra-purine and intra-pyrimidine differences in sequences of conserved genes generally follow a quadratic distribution relationship and show that this may have arisen from mutations in the sequences over evolutionary time scales. From this hitherto undescribed relationship for the gene sequences considered in this report we hypothesize that such relationships may be characteristic of these sequences and therefore could become a barrier to large scale sequence alterations that override such characteristics, perhaps through some monitoring process inbuilt in the DNA sequences. Such relationship also raises the possibility of intron sequences playing an important role in maintaining the characteristics and could be indicative of possible intron-late phenomena.

Cross-Link Dimer Formation of the Acetaldehyde-Derived Cyclic 1,N2-Propano-2'-deoxyguanosine Adduct Using Electrochemical Oxidation

The electrochemically oxidative lesion of the acetaldehyde-derived cyclic propano adduct 2 of 2'-deoxyguanosine 1 was identified as the cross-linked dimer 4 of adduct 2. Cross-link formation is explained by the nucleophilic preference of the exocyclic amino group in 2 to the carbocation 3 electrogenerated by 1-proton and 2-electron transfers. Dimer formation was also detected in duplex DNA during exposure to acetaldehyde followed by electrochemical oxidation. The dimer has been deduced to be an intrastrand cross-link generated specifically in the G-G sequence in duplex DNA, which is expected to contribute to acetaldehyde-mediated genotoxicity.

Y-DNA melting: a short tale of three scales

We address some aspects of the thermal denaturation of Y-DNA—the three-wayjunction—on three different length scales: the effect of chain concentration onthe extrinsic melting behaviour within a simple kinetic approach, the exponentof the loop entropy in intrinsic melting as it appears in statistical mechanicsmodels, and the microscopics of stacking within the junction from moleculardynamics simulations. Our results suggest that a multiscale approach is neededto properly describe the denaturation properties of these systems. We proposeexperiments which can also shed light on the melting of short and long duplex DNAsequences.

Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase

Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-beta T and WNA-beta C, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-beta 3AP, having 3-aminopyrazole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-beta 3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-beta C did not show the stabilizing effect.

Molecular modeling and biophysical analysis of the c-MYC NHE-III1 silencer element

G-Quadruplex and i-Motif-forming sequences in the promoter regions of several oncogenes show promise as targets for the regulation of oncogenes. In this study, molecular models were created for the c-MYC NHE-III(1) (nuclease hypersensitivity element III(1)) from two 39-base complementary sequences. The NHE modeled here consists of single folded conformers of the polypurine intramolecular G-Quadruplex and the polypyrimidine intramolecular i-Motif structures, flanked by short duplex DNA sequences. The G-Quadruplex was based on published NMR structural data for the c-MYC 1:2:1 loop isomer. The i-Motif structure is theoretical (with five cytosine-cytosine pairs), where the central intercalated cytosine core interactions are based on NMR structural data obtained for a tetramolecular [d(A(2)C(4))(4)] model i-Motif. The loop structures are in silico predictions of the c-MYC i-motif loops. The porphyrin meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4), as well as the ortho and meta analogs TMPyP2 and TMPyP3, were docked to six different locations in the complete c-MYC NHE. Comparisons are made for drug binding to the NHE and the isolated G-Quadruplex and i-Motif structures. NHE models both with and without bound cationic porphyrin were simulated for 100 ps using molecular dynamics techniques, and the non-bonded interaction energies between the DNA and porphyrins calculated for all of the docking interactions.

Photoreaction of iodouracil in DNA duplex; C–I bond is cleaved via two different pathways ‘homolysis and heterolysis’

We recently found that a selective photoreaction of 5-iodouracil ( IU) occurs in 5′-(G/C)AA IU IU-3′ and 5′-(G/C)A IU IU-3′ sequences in IU-substituted duplex DNA. In this study, the photoreactivity of the 5′-G(A) n IUT-3′ sequence was examined using various IU-containing oligonucleotides. HPLC analysis revealed that their photoreactivity largely depends on the number of As between G and IU. The most efficient reactivity was observed when the number of As was two and this decreased with increasing numbers from three to five, as observed for the 5′-G(A) n IUT-3′ sequence. These results indicate that the G located 5′ from IU acts as an electron donor for IU, as in the photoreaction of BrU. In sharp contrast to the BrU photoreaction, IU was photoreactive when the number of As was zero or more than five. These results indicate that both homolytic and heterolytic pathways operate in the formation of the uracil-5-yl radical in the photoreaction of IU in duplex DNA. In addition, the ratio of these pathways is highly dependent on DNA sequence.

Theoretical prediction of binding modes and hot sequences for allopsoralen–DNA interaction

Molecular docking studies of two duplex DNA sequences as target fragments and allopsoralen as ligand were performed. The predicted sequence selectivity for allopsoralen is that intercalation is favored in 5′-TpA sites in poly-TA sequences. Molecular docking studies of two duplex DNA sequences as target fragments and allopsoralen as ligand were performed. The calculated interaction energies showed that the ligand can be docked into the minor groove as well as become intercalated. However, unlike psoralen, allopsoralen preferred binding mode for non-poly-TA sequences is minor groove binding. Calculated energies for intercalation between different base pairs suggest that the predicted sequence selectivity for allopsoralen is analogous to that observed for psoralen. Intercalation is favored in 5′-TpA sites in poly-TA sequences.