Formation of (3+1) G-quadruplexes with a long loop by human telomeric DNA spanning five or more repeats

Structural studies of human telomeric repeats represent an active field of research with potential applications toward the development of specific telomeric quadruplex-targeting drugs for anticancer treatment. To date, high-definition structures were limited to DNA sequences containing up to four GGGTTA repeats. Here we investigate the formation of G-quadruplexes in sequences spanning five to seven human telomeric repeats using NMR, UV, and CD spectroscopy. A (3+1) G-quadruplex with a long propeller loop was isolated from a five-repeat sequence utilizing a guanine-to-inosine substitution. A simple approach of selective site-specific labeling of guanine residues was devised to rigorously determine the folding topology of the oligonucleotide. The same scaffold could be extrapolated to six- and seven-repeat sequences. Our results suggest that long human telomeric sequences consisting of five or more GGGTTA repeats could adopt (3+1) G-quadruplex structures harboring one or more repeat(s) within a single loop. We report on the formation of a Watson-Crick duplex within the long propeller loop upon addition of the complementary strand, demonstrating that the long loop could serve as a new recognition motif.     

Tandem trimer pyrrole–imidazole polyamide probes targeting 18 base pairs in human telomere sequences

The binding of molecules to specific DNA sequences is important for imaging genome DNA and for studying gene expression. Increasing the number of base pairs targeted by these molecules would provide greater specificity. N-Methylpyrrole–N-methylimidazole (Py–Im) polyamides are one type of such molecules and can bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. Our recent work has demonstrated that tandem hairpin Py–Im polyamides conjugated with a fluorescent dye can be synthesized easily and can serve as new probes for studying human telomeres under mild conditions. Herein, to improve their selectivities to telomeres by targeting longer sequences, we designed and synthesized a fluorescent tandem trimer Py–Im polyamide probe, comprising three hairpins and two connecting regions (hinges). The new motif bound to 18 bp dsDNA in human telomeric repeats (TTAGGG)_n, the longest sequence for specific binding reported for Py–Im polyamides. We compared the binding affinities and the abilities to discriminate mismatch, the UV-visible absorption and fluorescence spectra, and telomere staining in human cells between the tandem trimer and a previously developed tandem hairpin. We found that the tandem trimer Py–Im polyamide probe has higher ability to recognize telomeric repeats and stains telomeres in chemically fixed cells with lower background signal.     

Toward the Design of a Catalytic Metallodrug: Selective Cleavage of G‐Quadruplex Telomeric DNA by an Anticancer Copper–Acridine–ATCUN Complex

Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper–acridine–ATCUN complex (CuGGHK‐Acr) has been designed that targets G‐quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK‐Acr catalyst to selectively bind and cleave the G‐quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) experiments. CuGGHK‐Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7.     

Structural basis for telomeric G-quadruplex targeting by naphthalene diimide ligands

The folding of the single-stranded 3' end of the human telomere into G-quadruplex arrangements inhibits the overhang from hybridizing with the RNA template of telomerase and halts telomere maintenance in cancer cells. The ability to thermally stabilize human telomeric DNA as a four-stranded G-quadruplex structure by developing selective small molecule compounds is a therapeutic path to regulating telomerase activity and thereby selectively inhibit cancer cell growth. The development of compounds with the necessary selectivity and affinity to target parallel-stranded G-quadruplex structures has proved particularly challenging to date, relying heavily upon limited structural data. We report here on a structure-based approach to the design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA. Crystal structures have been determined of complexes between a 22-mer intramolecular human telomeric quadruplex and two potent tetra-substituted naphthalene diimide compounds, functionalized with positively charged N-methyl-piperazine side-chains. These compounds promote parallel-stranded quadruplex topology, binding exclusively to the 3' surface of each quadruplex. There are significant differences between the complexes in terms of ligand mobility and in the interactions with quadruplex grooves. One of the two ligands is markedly less mobile in the crystal complex and is more quadruplex-stabilizing, forming multiple electrostatic/hydrogen bond contacts with quadruplex phosphate groups. The data presented here provides a structural rationale for the biophysical (effects on quadruplex thermal stabilization) and biological data (inhibition of proliferation in cancer cell lines and evidence of in vivo antitumor activity) on compounds in this series and, thus, for the concept of telomere targeting with DNA quadruplex-binding small molecules.     

(3 + 1) Assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex

We present an NMR study on the structure of a DNA fragment of the human telomere containing three guanine-tracts, d(GGGTTAGGGTTAGGGT). This sequence forms in Na(+) solution a unique asymmetric dimeric quadruplex, in which the G-tetrad core involves all three G-tracts of one strand and only the last 3'-end G-tract of the other strand. We show that a three-repeat human telomeric sequence can also associate with a single-repeat human telomeric sequence into a structure with the same topology that we name (3 + 1) quadruplex assembly. In this G-quadruplex assembly, there are one syn.syn.syn.anti and two anti.anti.anti.syn G-tetrads, two edgewise loops, three G-tracts oriented in one direction and the fourth oriented in the opposite direction. We discuss the possible implications of the new folding topology for understanding the structure of telomeric DNA, including t-loop formation, and for targeting G-quadruplexes in the telomeres.     

Side chain-dependent binding of antitumor indoloquinoxaline derivatives to DNA: comparative spectroscopic and viscometric measurements

NCA0424 (1) and its side chain positional isomer, NCA0465 (2), are indoloquinoxaline derivatives with potent antitumor activity. To investigate the effect of side chain position for binding with DNA, the interactions of 2 with various B-form DNAs were studied by spectroscopic (circular dichroism (CD), fluorescence and UV) and viscosity measurements and were compared with those of 1. The binding preference for the base sequence was different in each case. The CD spectra showed that 2 formed an asymmetric binding of indoloquinoxaline ring with adenine in DNA, whereas such a base selectivity was not found with 1. The binding features are discussed based on association constants and thermodynamic parameters, indicating the importance of the side chain position for binding specificity for DNA.     

Sequence-specific alkylation of double-strand human telomere repeat sequence by pyrrole-imidazole polyamides with indole linkers

We designed and synthesized pyrrole (Py)-imidazole (Im) hairpin polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates 1 and 2, which target both strands of the double-stranded region of the human telomere repeat sequences, 5'-d(TTAGGG)(n)-3'/5'-d(CCCTAA)(n)-3'. High-resolution denaturing polyacrylamide gel electrophoresis demonstrated that conjugates 1 and 2 alkylated DNA at the 3' A of 5'-ACCCTA-3' and 5'-AGGGTTA-3', respectively. Cytotoxicities of conjugates 1 and 2 were evaluated using 39 human cancer cell lines; averages of log IC(50) values for conjugates 1 and 2 were -6.96 (110 nM) and -7.24 (57.5 nM), respectively. Conjugates 1 and 2 have potential as antitumor drugs capable of targeting telomere repeat sequence.     

Specific recognition of human telomeric G-quadruplex DNA with small molecules and the conformational analysis by ESI mass spectrometry and circular dichroism spectropolarimetry

Electrospray ionization mass spectrometry (ESI-MS) was utilized to investigate the binding affinity and stoichiometry of small molecules with human telomeric G-quadruplex DNA. The binding-affinity order obtained for the (AGGGTT)(4) quadruplex was: Tel01>ImImImbetaDp>PyPyPygammaImImImbetaDp. The specific binding of Tel01 and PyPyPygammaImImImbetaDp in one system consisting of human telomeric G-quadruplex and duplex DNA was observed directly for the first time. This revealed that PyPyPygammaImImImbetaDp has a binding specificity for the duplex DNA, whereas Tel01 selectively recognizes the G-quadruplex DNA. Moreover, both ESI-MS and circular dichroism (CD) spectra indicated that Tel01 favored the formation and stabilization of the antiparallel G-quadruplex, and a structural transition of the (AGGGTT)(4) sequence from a coexistence of parallel and antiparallel G-quadruplexes to a parallel G-quadruplex induced by annealing.     

Combining G-quadruplex targeting motifs on a single peptide nucleic acid scaffold: a hybrid (3+1) PNA-DNA bimolecular quadruplex

We describe the first G-quadruplex targeting approach that combines intercalation and hybridization strategies by investigating the interaction of a G-rich peptide nucleic acid (PNA) acridone conjugate 1 with a three-repeat fragment of the human telomere G 3 to form a hybrid PNA-DNA quadruplex that mimicks the biologically relevant (3+1) pure DNA dimeric telomeric quadruplex. Using a combination of UV and fluorescence spectroscopy, circular dichroism (CD), and mass-spectrometry, we show that PNA 1 can induce the formation of a bimolecular hybrid quadruplex even at low salt concentration upon interaction with a single-stranded three-repeat fragment of telomeric DNA. However, PNA 1 cannot invade a short fragment of B-DNA even if the latter contains a CCC motif complementary to the PNA sequence. These studies could open up new possibilities for the design of a novel generation of quadruplex ligands that target not only the external features of the quadruplex but also its central core constituted by the tetrads themselves.     

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.     

Decreased expression of DNA repair proteins Ku70 and Mre11 is associated with aging and may contribute to the cellular senescence

The gradual loss of telomeric DNA can contribute to replicative senescence and thus, having longer telomeric DNA is generally considered to provide a longer lifespan. Maintenance and stabilization of telomeric DNA is assisted by binding of multiple DNA-binding proteins, including those involved in double strand break (DSB) repair. We reasoned that declining DSB repair capacity and increased telomere shortening in aged individuals may be associated with decreased expression of DSB repair proteins capable of telomere binding. Our data presented here show that among the DSB repair proteins tested, only the expression of Ku70 and Mre11 showed statistically significant age-dependent changes in human lymphocytes. Furthermore, we found that expressions of Ku70 and Mre11 are statistically correlated, which indicate that the function of Ku70 and Mre11 may be related. All the other DSB repair proteins tested, Sir2, TRF1 and Ku80, did not show any significant differences upon aging. In line with these data, people who live in the regional community (longevity group), which was found to have statistically longer average life span than the rest area, shows higher level of Ku70 expression than those living in the neighboring control community. Taken together, our data show, for the first time, that Ku70 and Mre11 may represent new biomarkers for aging and further suggest that maintenance of higher expression of Ku70 and Mre11 may be responsible for keeping longer life span observed in the longevity group.     

An intramolecular G-quadruplex structure is required for binding of telomeric repeat-containing RNA to the telomeric protein TRF2

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization.     

Interactions of Phenanthroline Compounds with i-Motif DNA

The interactions of each of three phenanthroline derivatives 1, 2 and 3 with the human telomeric/-motif DNA were investigated. The results suggest these compounds are potent binders. The compounds could stabilize the structure of i-motif DNA by π-π stacking. Moreover, the binding constants of the compounds with/-motif DNA were （2.71-8.12）×10^4 L·mol^-1, and the binding stoichiometry ratio was 1：1. CD studies reveal that the binding by phenanthroline comoounds perturbs the conformation of i-motif DNA.     

Intramolecular folding in three tandem guanine repeats of human telomeric DNA

Intramolecular folding in three tandem guanine repeats of human telomeric DNA has been investigated using optical-tweezers, MD simulation and circular dichroism. A mechanically and thermodynamically stable species in this sequence shows a structure consistent with a triplex conformation. A similar species has also been observed to coexist with a G-quadruplex in a DNA sequence with four tandem guanine repeats.     

Identifying G-quadruplex-binding ligands using DNA-functionalized gold nanoparticles

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.     

Visualization of long human telomere mimics by single-molecule fluorescence imaging

Study of long single-stranded telomeric DNA is important for a variety of basic science and biotechnological applications, yet few methods exist for synthesis and visualization of single copies of this DNA in solution at biologically relevant length scales necessary for assessment of heterogeneity in its structure and behavior. We have synthesized kilobase-long single-stranded human telomere mimics in situ by rolling circle replication (RCR) on a microscope coverslip surface and visualized individual strands by staining with SYBR Gold. Under buffer flow, differential extensibility and varying morphology of these long telomere-mimicking DNA sequences were observed at the single-molecule level in real time. Using this procedure, we detected striking differences in the extensibility of individual RCR products based on the human G-rich telomeric sequence in the presence and absence of short, complementary single-stranded oligonucleotides. We also apply this new mode of single-stranded DNA characterization to probe the interaction of kilobase-length telomere mimics with the small-molecule G-quadruplex-binding agent TMPyP4.     

Alteration of the selectivity of DNA cleavage by a deglycobleomycin analogue containing a trithiazole moiety

The bleomycin (BLM) group of antitumor antibiotics effects DNA cleavage in a sequence-selective manner. Previous studies have indicated that the metal-binding and bithiazole moieties of BLM are both involved in the binding of BLM to DNA. The metal-binding domain is normally the predominant structural element in determining the sequence selectivity of DNA binding, but it has been shown that replacement of the bithiazole moiety with a strong DNA binder can alter the sequence selectivity of DNA binding and cleavage. To further explore the mechanism by which BLM and DNA interact, a trithiazole-containing deglycoBLM analogue was synthesized and tested for its ability to relax supercoiled DNA and cleave linear duplex DNA in a sequence-selective fashion. Also studied was cleavage of a novel RNA substrate. Solid-phase synthesis of the trithiazole deglycoBLM A(5) analogue was achieved using a TentaGel resin containing a Dde linker and elaborated from five key intermediates. The ability of the resulting BLM analogue to relax supercoiled DNA was largely unaffected by introduction of the additional thiazole moiety. Remarkably, while no new sites of DNA cleavage were observed for this analogue, there was a strong preference for cleavage at two 5'-GT-3' sites when a 5'-(32)P end-labeled DNA duplex was used as a substrate. The alteration of sequence selectivity of cleavage was accompanied by some decrease in the potency of DNA cleavage, albeit without a dramatic diminution. In common with BLM, the trithiazole analogue of deglycoBLM A(5) effected both hydrolytic cleavage of RNA in the absence of added metal ion and oxidative cleavage in the presence of Fe(2+) and O(2). In comparison with BLM A(5), the relative efficiencies of hydrolytic cleavage at individual sites were altered.     

Inducement and stabilization of G-quadruplex DNA by a thiophene-containing dinuclear ruthenium(II) complex

G-quadruplex structures are attractive targets for the development of anticancer drugs, as their formation in human telomere could impair telomerase activity, thus inducing apoptosis in cancer cells. In this work, a thiophene-containing dinuclear ruthenium(II) complex, [Ru₂(bpy)₄(H₂bipt)]⁴⁺ {bpy = 2,2′-bipyridine, H₂bipt = 2,5-bis[1,10]phenanthrolin[4,5-f]-(imidazol-2-yl)thiophene}, was prepared and the interaction between the complex and human telomeric DNA oligomers 5′-G₃(T₂AG₃)₃-3′ (HTG21) has been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, fluorescent intercalator displacement (FID) titrations, Job plot and color reaction studies. The results indicate that the complex can well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and the ΔT_m value of the G-quadruplex DNA treated with the complex was obtained to be 12.8 °C even at levels of 50-fold molar of duplex DNA (calf-thymus DNA), suggesting that the complex exhibits higher G-quadruplex DNA selectivity over duplex DNA. The complex shows high interaction ability with G-quadruplex DNA at (1.17 ± 0.12) × 10⁷ M^?1 binding affinity using a 2:1 [complex]/[quadruplex] binding mode ratio. A novel visual method has been developed here for making a distinction between G-quadruplex DNA and duplex DNA by our ruthenium complex binding hemin to form the hemin-G-quadruplex DNAzyme.     

Single-walled carbon nanotubes binding to human telomeric i-motif DNA: significant acceleration of S1 nuclease cleavage rate

Single-walled carbon nanotubes (SWNTs) binding to human telomeric i-motif DNA can significantly accelerate S1 nuclease cleavage rate by increasing the enzyme turnover number.