Zn ions improve the fidelity of metal-mediated primer extension while suppressing intrinsic and Mn-induced mutagenic effects by DNA polymerases.

While Mn ions are well-established for reducing the fidelity of DNA polymerases, leading to the misincorporation of nucleotides, our investigation of the effects of metal ions revealed a contrasting role of Zn. Here, we demonstrate that Zn ions enhance the fidelity of DNA polymerases (the 3' → 5' exonuclease-deficient Klenow fragment and Taq DNA polymerase) by suppressing misincorporation during primer extension reactions. Remarkably, Zn ions inhibit both intrinsic misincorporation and Mn-induced misincorporation of nucleotides.

Hepatocyte Period 1 dictates oxidative substrate selection independent of the core circadian clock.

Organisms integrate circadian and metabolic signals to optimize substrate selection to survive starvation, yet precisely how this occurs is unclear. Here, we show that hepatocyte Period 1 (Per1) is selectively induced during fasting, and mice lacking hepatocyte Per1 fail to initiate autophagic flux, ketogenesis, and lipid accumulation. Transcriptomic analyses show failed induction of the fasting hepatokine Fgf21 in Per1-deficient mice, and single-nucleus multiome sequencing defines a putative responding hepatocyte subpopulation that fails to induce the chromatin accessibility near the Fgf21 locus.

Hepatocyte MMP14 mediates liver and inter-organ inflammatory responses to diet-induced liver injury.

The matrix metalloproteinase MMP14 is a ubiquitously expressed, membrane-bound, secreted endopeptidase that proteolyzes substrates to regulate development, signaling, and metabolism. However, the spatial and contextual events inciting MMP14 activation and its metabolic sequelae are not fully understood. Here, we introduce an inducible, hepatocyte-specific MMP14-deficient model (MMP14 mice) to elucidate cell-intrinsic and systemic MMP14 function.

Rational design of prodrug-type apoB-targeted siRNA for nuclease resistance improvement without compromising gene silencing potency.

Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells.

Prodrug-Type Phosphotriester Oligonucleotides with Linear Disulfide Promoieties Responsive to Reducing Environment.

Various chemical modifications have been developed to create new antisense oligonucleotides (AONs) for clinical applications. Our previously designed prodrug-type phosphotriester-modified oligonucleotide with cyclic disulfides (cyclic SS PTE ON) can be converted into unmodified ON in an intracellular-mimetic reducing environment. However, the conversion rate of the cyclic SS PTE ON was very low, and the AON with cyclic SS PTE modifications showed much weaker antisense activity than corresponding to the fully phosphorothioate-modified AON.

Rediscovery of 4-Trehalosamine as a Biologically Stable, Mass-Producible, and Chemically Modifiable Trehalose Analog.

Nonreducing disaccharide trehalose is used as a stabilizer and humectant in various products and is a potential medicinal drug, showing curative effects on the animal models of various diseases. However, its use is limited as it is hydrolyzed by trehalase, a widely expressed enzyme in multiple organisms. Several trehalose analogs are prepared, including a microbial metabolite 4-trehalosamine, and their high biological stability is confirmed.

Influence of Aib-Containing Amphipathic Helical Chain Length in MAP(Aib)-cRGD as Carrier for siRNA Delivery.

MAP(Aib)-cRGD, which is a conjugate of an α-aminoisobutyric acid (Aib)-containing amphipathic helical peptide [MAP(Aib)] with a α β integrin binding ligand, cRGD, at the C-terminus of the helical peptide, has been developed for siRNA delivery into cells. In this work, we synthesized three peptides containing 19 (PI), 18 (PII), and 17 (PIII) amino acid residues in the helical peptide, which lack Aib, Leu-Aib, and Lys-Leu-Aib residues present in the C-terminus of the helical peptide of the parent MAP(Aib)-cRGD, respectively. MAP(Aib)-cRGD showed the siRNA delivery into cells and the RNAi effect both in the presence and in the absence of serum in reaction media.

Enzymatic formation of consecutive thymine-Hg-thymine base pairs by DNA polymerases.

From the perspective of the preparation of a DNA-based nanowire containing an array of metal ions, DNA-polymerase-catalyzed primer extension reactions were investigated and the formation of up to ten consecutive T-Hg-T base pairs was achieved by the Hg-mediated primer extension reaction in the presence of Mn ions. This enzymatic approach may be one of the promising techniques for preparing a DNA-based metal array.

α-Aminoisobutyric Acid-Containing Amphipathic Helical Peptide-Cyclic RGD Conjugation as a Potential Drug Delivery System for MicroRNA Replacement Therapy in Vitro.

Replacement therapy with tumor suppressive microRNA (TS-miRNA) might be the next-generation oligonucleotide therapy; however, a novel drug delivery system (DDS) is required. Recently, we developed the cell-penetrating peptide, model amphipathic peptide with α-aminoisobutyric acid (MAP(Aib)), as a carrier for oligonucleotide delivery to cells. In this study, we examined whether a modified MAP(Aib) analogue, MAP(Aib)-cRGD, could be a DDS for TS-miRNA replacement therapy.

Effects of metal ions on thermal stabilities of DNA duplexes containing homo- and heterochiral mismatched base pairs: comparison of internal and terminal substitutions.

The effects of metal ions on the stabilities of duplexes containing a -homochiral and heterochiral mismatched base pairs were studied. In some duplexes containing an internal mismatched base pair, significant stabilization by Hg and Ag ions was observed. While, in duplexes containing a terminal mismatched base pair, only the duplexes containing T-T and T-T mispairs were significantly stabilized by Hg ions, and the stabilities of the duplexes containing T-T and T-T mispairs exceeded those of the corresponding homochiral matched duplex.

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases.

Spectroscopic characterization of Ag -ion-mediated C-Ag -A and C-Ag -T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by Ag ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of Ag ions is in the order C-A≈C-T>C-C, the stabilizing effect of Ag ions follows the order C-C>C-A≈C-T.

Influence of lysine residue in amphipathic helical peptides on targeted delivery of RNA into cancer cells.

The cRGD-conjugated Aib-containing amphipathic helical peptide, MAP(Aib) derivative (PI), has been reported to be a useful carrier for siRNA delivery into cells. We have conducted a series of structure-activity relationship studies of the influence of the balance between hydrophobicity and basicity on the amphipathicity of PI, and synthesized peptides having a larger number of Lys residues than PI. Increasing the number of basic residues in the amphipathic helix suppressed the ability to deliver siRNA into cells.

Correction: Base recognition by l-nucleotides in heterochiral DNA.

[This corrects the article DOI: 10.1039/C2RA01013E.].

Syntheses of prodrug-type 2'-O-methyldithiomethyl oligonucleotides modified at natural four nucleoside residues and their conversions into natural 2'-hydroxy oligonucleotides under reducing condition.

We previously reported that reducing-environment-responsive prodrug-type small interfering RNA (siRNA) bearing 2'-O-methyldithiomethyl (2'-O-MDTM) uridine exhibits efficient knockdown activity and nuclease resistance. In this report, we describe the preparation of 2'-O-MDTM oligonucleotides modified not only at uridine but also at adenosine, guanosine and cytidine residues by post-synthetic modification. Precursor oligonucleotides bearing 2'-O-(2,4,6-trimethoxybenzylthiomethyl) (2'-O-TMBTM) adenosine, guanosine, and cytidine were reacted with dimethyl(methylthio)sulfonium tetrafluoroborate to form 2'-O-MDTM oligonucleotides in the same manner as the oligonucleotide bearing 2'-O-TMBTM uridine.

Effective gene silencing activity of prodrug-type 2'-O-methyldithiomethyl siRNA compared with non-prodrug-type 2'-O-methyl siRNA.

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC).

Synthesis and biological evaluation of histone deacetylase and DNA topoisomerase II-Targeted inhibitors.

HDAC inhibitors enable histones to maintain a high degree of acetylation. The resulting looser state of chromatin DNA may increase the accessibility of DNA drug targets and consequently improve the efficiency of anticancer drugs targeting DNA, such as Topo II inhibitors. A novel class of nucleoside-SAHA derivatives has been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors.

Corrigendum: Structures and biological activities of novel 4'-acetylated analogs of chrysomycins A and B.

This corrects the article DOI: 10.1038/ja.2017.

Structure-activity relationship study of Aib-containing amphipathic helical peptide-cyclic RGD conjugates as carriers for siRNA delivery.

The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp-d-Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide (PI) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure-activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus (PII) and both the N- and C-termini (PIII) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 (PIV), 12 (PV), and 8 (PVI) amino acid residues at the N-terminus of the helical chain were synthesized.

Valgamicin C, a novel cyclic depsipeptide containing the unusual amino acid cleonine, and related valgamicins A, T and V produced by Amycolatopsis sp. ML1-hF4.

In the course of optimizing pargamicin A production in Amycolatopsis sp. ML1-hF4, we discovered novel cyclic depsipeptide compounds in the broth and designated them valgamicins A, C, T and V. The structures of these molecules were determined by spectroscopic studies, advanced Marfey's method and X-ray crystal structural analysis.

Structures and biological activities of novel 4'-acetylated analogs of chrysomycins A and B.

Two new 4'-acetylated analogs of chrysomycin were discovered during the screening for antitumor agents from the metabolites of actinomycetes. Their structures and physicochemical properties were determined by standard spectrometric analyses. Their cytotoxicities and antimicrobial activities were evaluated against a panel of cancer cell lines and microbes.

Novel approaches for identification of anti-tumor drugs and new bioactive compounds.

Thanks to the pioneering work done by Professor Hamao Umezawa, bioactive compounds have been used in treatment of several diseases including cancer. In this review, we discuss our work, which focuses on developing new candidates for anti-tumor drugs by screening for bioactive natural compounds in microbial cultures using unique experimental systems. We summarize our recent progress including the following: (1) small-molecule modulators of tumor-stromal cell interactions, (2) inhibitors of three-dimensional spheroid formation of cancer cells, (3) multi-cancer cell panel screening and (4) new experimental animal models for cancer metastasis.

ATP Depletion Assay Led to the Isolation of New 36-Membered Polyol Macrolides Deplelides A and B from Streptomyces sp. MM581-NF15.

New 36-membered polyol macrolides deplelides A and B were isolated from the culture of Streptomyces MM581-NF15 by bioassay-guided fractionation using an ATP depletion assay. The planar structures of these novel compounds were identified by interpretation of the spectroscopic data (1D/2D NMR, MS, and IR). The relative stereochemistry was partially established using the universal NMR database method and J-based configuration analysis using H-H and long-range H-C coupling constants determined by H NMR or E.

Design, synthesis, and evaluation of DNA topoisomerase II-targeted nucleosides.

We developed novel nucleoside-based topoisomerase II selective inhibitors and showed that small structural units, such as catechols, are essential for DNA topoisomerase II inhibitory activity. Moreover, nucleoside analogues containing TBS and 1,3-dithian moieties had potent and selective DNA topoisomerase II inhibitory activities. In further experiments, compound 25b having a beta configuration of the thymine moiety showed relatively strong growth inhibitory activity against cancer cell lines, and was more potent against all cancer cell lines than compound 26b, which carries a thymine moiety in the alpha configuration.

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability.

Stability and Bioavailability of Lentztrehaloses A, B, and C as Replacements for Trehalose.

Trehalose is widely used as a sweetener, humectant, and stabilizer, but is ubiquitously degraded by the enzyme trehalase expressed in a broad variety of organisms. The stability of the new trehalose analogues lentztrehaloses A, B, and C in microbial and mammalian cell cultures and their pharmacokinetics in mice were analyzed to evaluate their potential as successors of trehalose. Among the 12 species of microbes and 2 cancer cell lines tested, 7 digested trehalose, whereas no definitive digestion of the lentztrehaloses was observed in any of them.