Masayuki Tera

Solution structure of an intramolecular (3 + 1) human telomeric g-quadruplex bound to a telomestatin derivative.

Guanine-rich human telomeric DNA can adopt secondary structures known as G-quadruplexes, which can be targeted by small molecules to achieve anticancer effects. So far, the structural information on complexes between human telomeric DNA and ligands is limited to the parallel G-quadruplex conformation, despite the high structural polymorphism of human telomeric G-quadruplexes. No structure has been yet resolved for the complex with telomestatin, one of the most promising G-quadruplex-targeting anticancer drug candidates. Here we present the first high-resolution structure of the complex between an intramolecular (3 + 1) human telomeric G-quadruplex and a telomestatin derivative, the macrocyclic hexaoxazole L2H2-6M(2)OTD. This compound is observed to interact with the G-quadruplex through π-stacking and electrostatic interactions. This structural information provides a platform for the design of topology-specific G-quadruplex-targeting compounds and is valuable for the development of new potent anticancer drugs.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

This work describes a glucosylation reaction at the 5/7 positions of anthocyanins in the petals of carnations and delphiniums. Unusually, this reaction is catalyzed by acyl-glucose–dependent glucosyltransferases that belong to glycoside hydrolase family 1. This modification mechanism may play an important role in generating variation in anthocyanins. Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose–dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose–dependent glucosyltransferases.

Visualization of G-quadruplexes by using a BODIPY-labeled macrocyclic heptaoxazole

A BODIPY-labeled macrocyclic heptaoxazole, L1BOD-7OTD, was developed as a fluorescent ligand for G-quadruplexes. The results of the study show that L1BOD-7OTD both selectively induces the formation of intramolecular G-quadruplexes from some G-quadruplex forming oligonucleotides (GFOs). In addition, the labelled macrocyclic heptaozaxole strongly binds to and stabilizes intramolecular G-quadruplexes. Moreover, this substance can be used to directly visualize the G-quadruplexes in the form of green fluorescence. Finally, the possibility that G-quadruplexes form in the cells was demonstrated by using of L1BOD-7OTD.

Synthesis of a potent G-quadruplex-binding macrocyclic heptaoxazole.

A novel G‐quadruplex binder, L1H1‐7OTD (shown in color by atom type), was developed. This macrocyclic heptaoxazole potently and selectively stabilizes telomeric DNA in an intramolecular antiparallel G‐quadruplex conformation. L1H1‐7OTD shows selective cytotoxicity toward HeLa cells, a telomerase‐positive cell line.

Fluorescent‐Ligand‐Mediated Screening of G‐Quadruplex Structures Using a DNA Microarray

Cell-cycle-synchronized DNA replication and cellular-environment-dependent gene expression are mainly controlled by proteins and DNAs with higher-order structures, such as G-quadruplexes (G4s). G4s are non-canonical nucleic acid structures that are formed by the stacking of planar Gquartets, and they are in equilibrium with random coils. These structures were originally found in telomeric DNA at the ends of chromosomes. The formation of telomeric G4s induces dysfunctions of telomeres in cancer cells. Many Gquadruplex-forming oligonucleotides (GFOs) have been identified in promoter regions as well as in regions containing clusters of ribosomal RNA genes, 4] and they may be associated with transcriptional repression of oncogenes or inhibition of ribosome biogenesis in cancer cells, respectively. 5] Furthermore, G4s have been reported to be involved in replication, DNA recombination, and splicing processes. Although relatively few GFOs have been identified thus far, bioinformatic studies suggest that a large number of GFOs are present in the genome. These putative GFOs are concentrated in gene-regulatory elements, including gene promoters, nuclease hypersensitive sites, and cytosine—phosphate—guanosine (CpG) islands (CGIs), which are involved in epigenetic transcriptional modulation. Therefore, G4s may be involved in regulatory mechanisms throughout the genome. Various direct or indirect methods for verifying G4 formation of DNA sequences have been reported, but there has been no fast, high-throughput screening method for identifying large numbers of GFO candidates. Herein, we describe a method for a direct screening of GFOs that employs a fluorescent G4 ligand to probe a large microarray of 88737 probes in 16 030 CGIs. We have recently developed a series of macrocyclic G4 ligands that contain polyoxazole structures, inspired by the natural G4 ligand telomestatin (1). During our structural-development studies, one of the core structures, 7OTD, which has seven oxazole units in the macrocycle, was found to interact strongly and selectively with the known GFOs c-myc, c-kit, bcl-2, and K-ras, without requiring a cationic moiety on the side chain. Based on these results, we developed the fluorescent G4 ligand L1BOD-7OTD, which consists of BODIPY linked to 7OTD. This ligand was effective for visualizing GFOs by a conventional electrophoretic mobility shift assay (EMSA) or fluorescent polarization (FP) titration. We realized that a G4-selective fluorescent ligand might also be useful for screening GFOs on a DNA microarray. To evaluate this idea, we designed L1Cy5-7OTD (3), which consists of a Cy5 fluorophore linked to 7OTD (Figure 1), as a suitable ligand for high-throughput screening of GFOs in CGIs, which are major regulatory regions of gene expression and epigenetic transcription. We considered that they would be suitable targets, because they contain many guanine-rich sequences, which are favorable for GFO formation. L1Cy5-7OTD was synthesized by reacting L1H1-7OTD (2) with the N-hydroxysuccinimide (NHS) ester of Cy5 in the presence of sodium bicarbonate; then, its binding ability and selectivity for GFOs were examined. First, an assay on the displacement of thiazole orange by 3 was carried out using five representative GFOs (telo24, c-myc, c-kit, bcl-2, and K-ras) and non-G4-forming double-stranded DNA (dsDNA). In this assay, 3 effectively displaced thiazole orange from all of the G4-forming DNA sequences, whereas no significant interaction was observed with dsDNA (Supporting Information, Table S1 and Figure S2). Subsequently, the binding ability and selectivity of 3 for GFOs were evaluated by EMSA. For EMSA, the same GFOs (telo24, c-myc, c-kit, bcl-2, and K-ras) were used, and dsDNA and poly(thymine) (poly-T) were evaluated as non[*] K. Iida, T. Nakamura, Dr. W. Yoshida, Prof. Dr. K. Ikebukuro, Prof. Dr. K. Nagasawa Department of Biotechnology and Life Science Tokyo University of Agriculture and Technology 2-24-16, Naka-cho, Koganei city, 184-8588 Tokyo (Japan) E-mail: ikebu@cc.tuat.ac.jp knaga@cc.tuat.ac.jp

Detection of 1-O-malylglucose: Pelargonidin 3-O-glucose-6′′-O-malyltransferase activity in carnation (Dianthus caryophyllus)

Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-beta-D-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.

A Caged Ligand for a Telomeric G‐Quadruplex

Human telomeric DNA, located at the ends of chromosomes, contains repeating single-strand (TTAGGG)n sequences. [1] These G-rich strands form a characteristic stacked three-dimensional structure, called the G-quadruplex (G4). Stabilization of telomeric G4 has been shown to inhibit the activity of telomerase, an enzyme that is selectively expressed in most tumor cells and plays key roles in telomere maintenance and cellular immortalization. Furthermore, telomeric G4 stabilization also stimulates dissociation of POT1 and TRF2 (telomere-related proteins), which protect chromosome ends from degradation and inappropriate DNA repair and induce apoptosis of tumor cells. Stabilization of the telomeric G4 structure is thus considered to be a promising anticancer strategy. Various natural and synthetic small molecules that stabilize telomeric G4 have been investigated. However, additional features—that is, selectivity between telomeric G4 and duplex DNA, as well as selectivity between normal cells and cancer cells—are required for practically useful molecules. Although there has been recent progress in selective structural recognition of G4 by small molecules, stabilization of the G4 structure in specific target cells—cancer cells, for example—is still difficult. We considered that a caging strategy might be useful for overcoming these specificity issues and to aid the development of candidate drugs. Caged compounds are light-sensitive ligands in which one or more significant functional group(s) required for biological activity is/are masked with one or more photolabile group(s). Caged compounds are therefore biologically inactive. When they are “uncaged” by irradiation, however, the biological activity is unmasked, and so the latent biological activities of caged compounds can in principle be activated with high spatial and temporal specificity, or at specific molecular targets, by means of light irradiation. Here we describe the design, synthesis, and characteristics of the first example of a caged compound targeting telomeric G4. Uncaging by irradiation was confirmed to result in inhibition of telomerase activity in vitro and in growth inhibition of several cancer cell lines. We have recently reported a series of macrocyclic polyoxazoles—6OTDs (Scheme 1), b] 6M(4)OTDs, and 7OTD—as G4 ligands inspired by the highly potent natural G4 ligand

Development of a novel biosensing system based on the structural change of a polymerized guanine-quadruplex DNA nanostructure

By inserting an adenosine aptamer into an aptamer that forms a G-quadruplex, we developed an adaptor molecule, named the Gq-switch, which links an electrode with flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) that is capable of transferring electron to a electrode directly. First, we selected an FADGDH-binding aptamer and identified that its sequence is composed of two blocks of consecutive six guanine bases and it forms a polymerized G-quadruplex structure. Then, we inserted a sequence of an adenosine aptamer between the two blocks of consecutive guanine bases, and we found it also bound to adenosine. Then we named it as Gq-switch. In the absence of adenosine, the Gq-switch-FADGDH complex forms a 30-nm high bulb-shaped structure that changes in the presence of adenosine to give an 8-nm high wire-shaped structure. This structural change brings the FADGDH sufficiently close to the electrode for electron transfer to occur, and the adenosine can be detected from the current produced by the FADGDH. Adenosine was successfully detected with a concentration dependency using the Gq-switch-FADGDH complex immobilized Au electrode by measuring response current to the addition of glucose.

Analysis of the unbinding force between telomestatin derivatives and human telomeric G-quadruplex by atomic force microscopy

The force analysis between a macrocyclic hexazole (6OTD) monomer/dimer and telomeric DNA using atomic force microscopy revealed the difference in their binding modes. The 6OTD dimer bound to the G-quadruplex more strongly than the monomer by sandwiching the G-quadruplex.

Synthesis of Macrocyclic Hexaoxazole (6OTD) Dimers, Containing Guanidine and Amine Functionalized Side Chains, and an Evaluation of Their Telomeric G4 Stabilizing Properties

Structure-activity relationship studies were carried out on macrocyclic hexaoxazole (6OTD) dimers, whose core structure stabilizes telomeric G-quadruplexes (G4). Two new 6OTD dimers having side chain amine and guanidine functional groups were synthesized and evaluated for their stabilizing ability against a telomeric G4 DNA sequence. The results show that the 6OTD dimers interact with the DNA to form 1:1 complexes and stabilize the antiparallel G4 structure of DNA in the presence of potassium cation. The guanidine functionalized dimer displays a potent stabilizing ability of the G4 structure, as determined by using a FRET melting assay (ΔTm = 14°C).