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A series of fluorescent ligands, which were systematically constructed from thiazole orange scaffold, was investigated for their interactions with G-quadruplex structures and antitumor activity. Among the ligands, compound 3 was identified to exhibit excellent specificity toward telomere G4-DNA over other nucleic acids. The affinity of 3-Htg24 was almost 5 times higher than that of double-stranded DNA and promoter G4-DNA. Interaction studies showed that 3 may bind to both G-tetrad and the lateral loop near the 5'-end. The intracellular colocalization with BG4 and competition studies with BRACO19 reveal that 3 may interact with G4-structures. Moreover, 3 reduces the telomere length and downregulates hTERC and hTERT mRNA expression in HeLa cells. The cytotoxicity of 3 against cancer cells (IC50 = 12.7-16.2 μM) was found to be generally higher than noncancer cells (IC50 = 52.3 μM). The findings may support that the ligand is telomere G4-DNA specific and may provide meaningful insights for anticancer drug design.

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson–Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop., Funding from Spanish government [CTQ2014-61758-EXP, CTQ2014-52588-R, CTQ2015-66254-C2-2-P]; Autonomous Catalan government [2017SGR114]. Funding for open access charge: University of Bracelona [CTQ2015-66254-C2-2-P] and Université de Lille, LASIR CNRS.

A (3+1)-hybrid-type G-quadruplex was substituted within its central tetrad by a single 2'-fluoro-modified guanosine. Driven by the anti-favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G-tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra- and inter-residual F⋅⋅⋅H8-C8 pseudo-hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.

A novel homogenous fluorescent sensor for signal-on detection of Cu2+ has been developed based on intra-molecular G-quadruplex formed by DNA-templated click reaction and crystal violet (CV) as label-free signal reporter. The clickable DNA probe consists of two G-rich strands (A and B) bearing azide and alkyne group, respectively, and a template strand (C) locating two proximate reactants by pairing with A and B. The sequences of A and B are derived from asymmetric split of the G-quadruplex sequence (TTAGGG)4. In the presence of Cu2+, the whole G-quadruplex sequence A–B is generated by chemical ligation of A and B via copper ion-catalyzed alkyne-azide cycloaddition, then released from template by toehold strand displacement, and consequently forming a stable intra-molecular G-quadruplex, which binds with CV to generate a strong fluorescent signal. Oppositely, weak fluorescence was obtained without Cu2+ because of unstable intermolecular G-quadruplex formed by A and B and lack of lateral loop connection. Therefore, the Cu2+ can be sensitively and specifically detected by the fluorescence of the CV-stained G-quadruplex with a low detection limit of 65 nM and a linear range of 0.1–3 µM. This method rationally integrated the DNA-templated synthesis and G-quadruplex structure-switch, presenting a simple and promising approach for biosensor development.

Platelet-derived growth factor receptor beta (PDGFR-β) is a cell-surface-receptor tyrosine kinase implicated in platelet-derived growth factor (PDGF) signaling pathways, and overexpression of PDGFR-β is associated with tumor growth, angiogenesis, and migration, making PDGFR-β an attractive target for cancer therapy. The formation of DNA G-quadruplexes in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter has been found to inhibit PDGFR-β transcriptional activity. We previously determined the major G-quadruplex structure formed in the PDGFR-β promoter using a 22-mer sequence of pu22m1, which adopts a folding pattern containing a unique broken G-strand with the 3' tetrad-guanine coming from a distant site connected by a five nucleotide lateral loop. However, extension of the 3' end flanking segment of pu22m1 results in destabilization of the G-quadruplex, suggesting that this structure may be unstable in vivo. Recently, it has been reported that a unique type of intramolecular G-vacancy-bearing G-quadruplex (GVBQ) can be formed with three G3 tracts and one G2 tract and has an incomplete G-tetrad with a G-vacancy. A guanine derivative, such as GMP or GTP, can fill the G-vacancy with Hoogsteen hydrogen bonding and complete the G-tetrad, resulting in G-quadruplex stabilization. The unique broken-strand structure of PDGFR-β pu22m1 suggests a possibility of the formation of a GVBQ structure in this sequence, which can be completed and stabilized by guanine and guanine derivatives. To test this hypothesis, we examined the formation and stability of G-quadruplex formed in pu22m1 variants and the potential of guanine derivatives to fill in the incomplete G-tetrad and stabilize the overall G-quadruplex, using nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy and DMS footprinting experiments. Our results showed that a GVBQ could form in in the PDGFR-β promoter sequence and that dGMP could insert and complete the incomplete G-tetrad to induce the formation of a stable and well-defined dGMP-fill-in-GVBQ in potassium solution. In addition, we discovered two natural plant alkaloids, berberine and chelerythrine, which could stabilize this dGMP-fill-in-GVBQ, with an increase in melting temperature by more than 10 °C. Therefore, it appears that dGMP could interact and stabilize the broken-stranded PDGFR-β promoter G-quadruplex, which may play a role in PDGFR-β transcriptional regulation. Elevated levels of guanine and guanine derivatives/metabolites are found in tumor cells, suggesting that the GVBQs may play important regulatory roles in tumor cell pathological processes, such as PDGFR-β transcriptional regulation. Furthermore, our results suggest that this unique dGMP-fill-in G-quadruplex in the PDGFR-β promoter may serve as a novel anticancer drug target for modulating PDGFR-β gene regulation. Citation Format: Kaibo Wang, Clement Lin, Guanhui Wu, Danzhou Yang. A dGMP fill-in G-quadruplex forms in the PDGFR-β promoter that serves as a unique target for drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 687.

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K(+). The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel-antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5'- and 3'-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K(+). Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K(+). The unique human telomeric G-quadruplex structure formed in K(+) suggests that it can be specifically targeted for anticancer drug design.

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is associated with multiple cancers, making PDGFR-β an attractive target for anticancer drugs. Few strategies other than molecular targeting of the PDGFR-β protein or its cognate ligand have been reported for developing inhibitors for the PDGFR-β signaling pathway. DNA G-quadruplexes (G4s) formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity, and stabilization of these G4s by small-molecule compounds could serve as a mechanism for cancer therapeutics. We have shown that the major G4 formed in the PDGFR-β promoter is from the central four G-runs, adopting an intramolecular parallel-stranded structure with a broken G-strand and contains three 1-nucleotide (nt) chain-reversal loops and one lateral loop. The novel folding of the PDGFR-β G4 highlights the inherent stability of the 1-nt loops in parallel-stranded G4 structures. Elucidating the structure of the major PDGFR-β promoter G4 is important for designing small-molecule drugs to specifically target this structure to inhibit gene transcription. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the potassium solution structure of this major G4 formed in the PDGFR-β promoter. Our structure showed a unique 3’ capping structure involving three guanine nucleotides in the lateral loop. This novel capping structure is determined by the specific loop sequence as well as the broken-stranded PDGFR-β G4 folding pattern. The unique 3’ capping structure could be specifically recognized by proteins and small molecule ligands. Unrestrained molecular dynamic calculations showed that this major PDGFR-β G4 and its 3’ capping structure are stable in aqueous environment on the ns time-scale. We also investigated the binding of small-molecules to the PDGFR-β G4 using a combination of NMR, circular dichroism (CD), and fluorescence based methods to identify compounds that can selectively bind the PDGFR-β G4 over other classic parallel-stranded G-quadruplexes, such as the c-MYC promoter G4. Our study demonstrates the structural diversity in promoter G4s which may enable specific recognition and the modulation of gene expression by small-molecule drugs. Citation Format: Clement Lin, Prashansa Agrawal, Yuwei Chen, Salil Kalarn, Nanjie Deng, Laurence Hurley, Danzhou Yang. Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3090.

TMPyP4 (Mesotetra(N-methyl-4-pyridyl)porphine) is known to have a high affinity for G-quadruplex DNA. However, there is still some controversy over the exact site(s) and mode(s) of TMPyP4 binding to G-quadruplex DNA. We examined TMPyP4 interactions with seven G-quadruplex forming oligonucleotides. The parent oligonucleotide is a 27-mer with a wild-type (WT) G-rich sequence of the Bcl-2 P1 promoter mid-region (5'-d(CGG GCG CGG GAG GAA GGG GGC GGG AGC-3')). This sequence folds into at least three unique loop isomer quadruplexes. The two mutant oligonucleotides used in this study are shorter (23-mer) sequences in which nonquadruplex core bases were eliminated and two different (-G-G-) --(-T-T-) substitutions were made to restrict the folding complexity. The four additional mutant oligonucleotides were labeled by substituting a 2-aminopurine (2-AP) base for an A or G in either the first three-base lateral loop or the second five- or seven-base lateral loop (depending on the G--T mutation positions). Spectroscopic and microcalorimetric studies indicate that four molecules of TMPyP4 can be bound to a single G-quadruplex. Binding of the first two moles of TMPyP4 appears to occur by an end or exterior mode (K approximately 1 x 10(7) M(-1)), whereas binding of the third and fourth moles of TMPyP4 appears to occur by a weaker, intercalative binding mode (K approximately 1 x 10(5) M(-1)). As the mid-loop size decreases from seven to five bases, end binding occurs with significantly increased affinity. 2-AP-labeled Bcl-2 promoter region quadruplexes show increased fluorescence of the 2-AP base on addition of TMPyP4. The change in fluorescence for 2-AP bases in the second half of the TMPyP4 titration lends support to our previous speculation regarding the intercalative nature of the weaker binding mode.

Background: Telomeres in eukaryotic organisms are protein–DNA complexes which are essential for the protection and replication of chromosomal termini. The telomeric DNA of Tetrahymena consists of T 2 G 4 repeats, and models have been previously proposed for the intramolecular folded structure of the d(T 2 G 4 ) 4 sequence based on chemical footprinting and cross-linking data. Ahigh-resolution solution structure of this sequence would allow comparison with the structures of related G–tetraplexes. Results The solution structure of the Na + –stabilized d(T 2 G 4 ) 4 sequence has been determined using a combined NMR–molecular dynamics approach. The sequence folds intramolecularly into a right-handed G–tetraplex containing three stacked G-tetrads connected by linker segments consisting of a G-T-T-G lateral loop, a central T-T-G lateral loop and a T-T segment that spans the groove through a double chain reversal. Te latter T–T connectivity aligns adjacent G-G-G segments in parallel and introduces a new G-tetraplex folding topology with unprecedented combinations of strand directionalities and groove widths, as well as guanine syn/anti distributions along individual strands and around individual G-tetrads. Conclusion The four repeat Tetrahymena and human G-tetraplexes, which differ by a single guanine for adenine substitution, exhibit strikingly different folding topologies. The observed structural polymorphism establishes that G-tetraplexes can adopt topologies which project distinctly different groove dimensions, G-tetrad base edges and linker segments for recognition by, and interactions with, other nucleic acids and proteins.

Denatured 3H-labelled DNAs containing histone gene sequences originating from the echinoderms Psammechinus miliaris, Echinus esculentus and Strongylocentrotus purpuratus have been in situ hybridized to RNA transcripts on newt lampbrush chromosomes. Autoradiographs of the hybridized lampbrush preparations show labelling restricted to four or fewer lateral loop pairs all lying within the heteromorphic regions of chromosome I, also one or two loop pairs on chromosome VI, one loop pair on chromosome X and one loop pair on chromosome XI. For oocytes from a single newt, coincident label distribution is found with DNAs of diverse echinoderm origin; however different newts show some specific individual diversity in label distribution, including heterozygosity in the case of loops on bivalents VI and X. The more conspicuously labelled loops, particularly those on chromosome I, show a pattern of labelling which is explicable if the newt histone DNA sequences are confined to short intercepts of lateral loop axis. Transcription is initiated prior to the histone DNA sequences, proceeds through the histone DNA sequences, and beyond, and the histone RNA sequences are cut from the transcripts before the termination of transcription.

The chromomeres of lampbrush chromosomes of Triturus cristatus are Feulgen-positive; they therefore contain DNA. After removal of their DNA in boiling trichloracetic acid, the chromomeres stain with fast green at alkaline pH; they therefore contain basic protein. The lateral loops are Feulgen-negative; they stain with toluidine blue at acid pH, but much less intensely following RNase digestion; they therefore contain RNA. The spheres of chromosomes V and VIII do not contain RNA. Unfixed lampbrush chromosomes retain a life-like appearance in 0.07 M K/NaCl at pH 6.2; in this medium the nuclear sap disperses. As pH is raised to 8.5 the matrices of lateral loops dissolve but chromosome axes remain unbroken. Above pH 8.5 lampbrush chromosomes dissolve. As pH is lowered from 6.2, at between 5.8 and 5.4 coagulation occurs. If pH is rapidly reduced still further, a persistent relaxed condition sets in between 2.5 and 2. In concentrations of K/NaCl above 0.5 M lampbrush chromosomes dissolve. Lateral loop matrices dissolve in 0.25 M K/NaCl but chromosome axes remain unbroken. In concentrations of K/NaCl below 0.05 M lateral loop matrices dissolve, but even in distilled water chromosome axes remain unbroken. Trypsin at pH 6.2 and at pH 7.8 strips the matrices from lateral loops and occasionally breaks matrix fusions. It causes chromomeres to swell and coalesce, but fails to break chromosome axes. The action of ‘pan-protease’ resembles that of trypsin in all respects. Pepsin at pH 6.2 strips the matrices from lateral loops, but does not destroy chromomeres. At low pH peptic digestion is slow: the enzyme is attacking coagulated chromosomes; but if peptic digestion precedes a lowering of pH the overall outcome is a rapid solution of loop matrix, and under these conditions matrix and sphere fusions are broken. If trypsin or ‘pan-protease’ digestion precedes a lowering of pH there is a similarly rapid solution of loop matrix; thus the action is not specifically referable to pepsin. Under no conditions does pepsin break the axes of lampbrush chromosomes. RNase at pH 6.2 strips the matrices from lateral loops; this action is detectable at extreme dilution. RNase does not destroy chromomeres, nor does it break chromosome axes. If tryptic digestion follows RNase digestion this too fails to break chromosome axes. Unlike the proteolytic enzymes and RNase, DNase at pH 6.2 breaks the fibril between adjacent chromomeres, and it also breaks the axes of lateral loops. Contrary to Mazia's experience with salivary gland chromosomes, versene does not break the axes of lampbrush chromosomes even when applied in media of low electrolyte concentration. These results indicate that uninterrupted fibres of DNA run throughout the lengths of lampbrush chromosomes.

A computational analysis of d(GGGGTTTTGGGG)2 guanine quadruplexes containing either lateral or diagonal four-thymidine loops was carried out using molecular dynamics (MD) simulations in explicit solvent, locally enhanced sampling (LES) simulations, systematic conformational search, and free energy molecular-mechanics, Poisson Boltzmann, surface area (MM-PBSA) calculations with explicit inclusion of structural monovalent cations. The study provides, within the approximations of the applied all-atom additive force field, a qualitatively complete analysis of the available loop conformational space. The results are independent of the starting structures. Major conformational transitions not seen in conventional MD simulations are observed when LES is applied. The favored LES structures consistently provide lower free energies (as estimated by molecular-mechanics, Poisson Boltzmann, surface area) than other structures. Unfortunately, the predicted optimal structure for the diagonal loop arrangement differs substantially from the atomic resolution experiments. This result is attributed to force field deficiencies, such as the potential misbalance between solute-cation and solvent-cation terms. The MD simulations are unable to maintain the stable coordination of the monovalent cations inside the diagonal loops as reported in a recent x-ray study. The optimal diagonal and lateral loop arrangements appear to be close in energy although a proper inclusion of the loop monovalent cations could stabilize the diagonal architecture.

Use of the novel but simple preparative techniques developed by Miller and coworkers (1–5) for electron microscopy of nuclear and cytoplasmic material provides preparations suitable for ultrastructural analysis of active genes in a variety of systems. These direct observations, complemented by biochemical and genetic analysis, provide a new approach to the problems of molecular genetics. This chapter compares electron microscopic studies of active genomes in representatives of prokaryotic and eukaryotic cell types.

The largest number of genes in the genetic system of the most complex viruses is not more than 100, and such a system can be represented by a single polycistron DNA macromolecule. In bacteria, more than 1000 genes may be present and their genetic apparatus consists of a new structure, a special type of chromosome consisting of a small or large number of DNA molecules connected into a circular (occasionally linear) system. With the transition to multicellular organisms, the volume of genetic information and the way in which it is translated into action during reproduction differ considerably and a number of fundamentally new genetic problems concerned with differentiation and morphogenesis arise. The number of genes of different types controlling development and providing for differentiation and protein synthesis increases from several thousands in the simplest members of this group of living organisms to several hundreds of thousands or, according to some highly arbitrary calculations, to values exceeding a million (Vogel, 1964) in man. Information equivalent to a million genes can be coded in polynucleotides having a total length equivalent to one billion nucleotides (with a molecular weight of about 600 billions if calculated as double-helical DNA).