Oligonucleotides DNA containing 8-trifluoromethyl-2'-deoxyguanosine for observing Z-DNA structure.

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles as well as being related to cancer and other genetic diseases. It is therefore important to investigate Z-DNA structure and related biological events in living cells. However, the development of molecular probes for the observation of Z-DNA structures inside living cells has not yet been realized. Here, we have succeeded in developing site-specific trifluoromethyl oligonucleotide DNA by incorporation of 8-trifluoromethyl-2'-deoxyguanosine (FG). 2D NMR strongly suggested that FG adopted a syn conformation. Trifluoromethyl oligonucleotides dramatically stabilized Z-DNA, even under physiological salt concentrations. Furthermore, the trifluoromethyl DNA can be used to directly observe Z-form DNA structure and interaction of DNA with proteins in vitro, as well as in living human cells by19F NMR spectroscopy for the first time. These results provide valuable information to allow understanding of the structure and function of Z-DNA.

Hybrid-type and two-tetrad antiparallel telomere DNA G-quadruplex structures in living human cells.

Although the telomeric sequence has been reported to form various G-quadruplex topologies in vitro and in Xenopus laevis oocytes, in living human cells, the topology of telomeric DNA G-quadruplex remains a challenge. To investigate the human telomeric DNA G-quadruplex in a more realistic human cell environment, in the present study, we demonstrated that the telomeric DNA sequence can form two hybrid-type and two-tetrad antiparallel G-quadruplex structures by in-cell 19F NMR in living human cells (HELA CELLS). This result provides valuable information for understanding the structures of human telomeric DNA in living human cells and for the design of new drugs that target telomeric DNA.

Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy.

Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.

In Situ Reduction from Uranyl Ion into a Tetravalent Uranium Trimer and Hexamer Featuring Ion-Exchange Properties and the Alexandrite Effect.

By utilizing zinc amalgam as an in situ reductant and pH regulator, mild hydrothermal reaction between UO2(CH3COO)2·2H2O, H2SO4, and Cs2CO3 or between UO2(CH3COO)2·2H2O, C2H4(SO3H)2, and K2CO3 yielded a novel cesium UIV sulfate trimer Cs4[U3O(SO4)7]·2.2H2O (1) and a new potassium UIV disulfonic hexamer K[U6O4(OH)5(H2O)5][C2H4(SO3)2]6·6H2O (2), respectively. Compound 1 features a lamellar structure with a honeycomb lattice, and it represents an unprecedented trimeric UIV cluster composed of purely inorganic moieties. Complex 2 is built from hexanuclear U4+ cores and K+ ions interconnected by µ5-[C2H4(SO3)2]2- groups, leading to the construction of an extended framework rather than commonly observed discrete, neutral molecular sulfonate clusters. The various binding modes of the sulfate and disulfonate groups, especially the multidentate ones, enable additional bridging between metal ions, which promotes oligomerization and isolation of polynuclear species. Furthermore, compound 1 exhibits both ion-exchange properties and the Alexandrite effect, and it is the second example of a uranium complex without chromic functional ligands displaying the latter feature.

2'-O-Methyl-8-methylguanosine as a Z-Form RNA Stabilizer for Structural and Functional Study of Z-RNA.

In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m8Gm, and demonstrated that incorporation of m8Gm into RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m8Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.

Structure-Dependent Binding of hnRNPA1 to Telomere RNA.

Telomeric repeat-containing RNA is a new noncoding RNA molecule that performs various biofunctions. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein involved in the telomere maintenance machinery. To date, little is known about how hnRNPA1 binds to telomeric RNA. In this study, we investigated the binding affinity and recognition mechanism of telomere RNA with the RNA recognition motif of hnRNPA1. Using the photochemical cross-linking method, we showed that the telomere RNA G-quadruplex with loops is important in the interaction of telomere RNA with hnRNPA1. Using small-molecule probes, we directly visualized the complex formed by the telomere RNA G-quadruplex and hnRNPA1 in vitro and in live cells. The results suggested that the structure-dependent binding of hnRNPA1 to telomere RNA regulates the telomere function. Therefore, our study provides new insights into the interactions between the RNA G-quadruplex and proteins at the telomere.

A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

In the present study, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

Photochemically engineering the metal-semiconductor interface for room-temperature transfer hydrogenation of nitroarenes with formic acid.

A mild photochemical approach was applied to construct highly coupled metal-semiconductor dyads, which were found to efficiently facilitate the hydrogenation of nitrobenzene. Aniline was produced in excellent yield (>99 %, TOF: 1183) using formic acid as hydrogen source and water as solvent at room temperature. This general and green catalytic process is applicable to a wide range of nitroarenes without the involvement of high-pressure gases or sacrificial additives.

Oligonucleotide Containing 8-Trifluoromethyl-2'-Deoxyguanosine as a Z-DNA Probe.

Z-DNA structure is a noncanonical left-handed alternative form of DNA, which has been suggested to be biologically important and is related to several genetic diseases and cancer. Therefore, investigation of Z-DNA structure associated with biological events is of great importance to understanding the functions of these molecules. Here, we described the development of a trifluoromethyl labeled deoxyguanosine derivative and employed it as a 19F NMR probe to study Z-form DNA structure in vitro and in living cells.

Observation of Z-DNA Structure via the Synthesis of Oligonucleotide DNA Containing 8-Trifluoromethyl-2-Deoxyguanosine.

This article contains detailed synthetic protocols for the preparation of DNA oligonucleotides containing 8-trifluoromethyl-2'-deoxyguanosine (CF3 dG) and their application to observe Z-DNA structure in vitro and in living HeLa cells. First, using a catalytic system consisting of FeSO4 , H2 SO4 , and H2 O2 in DMSO, we achieved a one-step synthesis of CF3 dG through a radical reaction between deoxyguanosine (dG) and CF3 I, with a yield of 45%. We then obtained the 3'-phosphoramidite of CF3 dG through a routine three-step procedure. Next, we employed the CF3 dG phosphoramidite monomer in the synthesis of oligonucleotides on a solid-phase DNA synthesizer. Finally, we used the CF3 dG-modified DNA oligonucleotides to observe Z-DNA structure in vitro and in living HeLa cells through 19 F NMR spectroscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of CF3 dG phosphoramidites Basic Protocol 2: Preparation of CF3 dG-modified DNA oligonucleotides Basic Protocol 3: Evaluation of CF3 dG stabilization of Z-DNA structure by CD spectroscopy Basic Protocol 4: Investigation of Z-DNA structure in vitro and in HeLa cells with CF3 dG-modified DNA oligonucleotides and 19 F NMR spectroscopy.

Improving Thermodynamic Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Incorporation of 8-trifluoromethyl-2'-deoxyguanosine.

In this study, we incorporated 8-trifluoromethyl-2'-deoxyguanosine (FG) into a thrombin binding aptamer (TBA). Circular dichroism, nuclear magnetic resonance (NMR), electrophoresis, and prothrombin time (PT) assay were performed to investigate the structure, thermodynamic stability, biological stability, and anticoagulant activity of the FG-modified TBA sequences. We found that the replacement of FG into TBA sequences led to a remarkable improvement in the melting temperature up to 30 °C compared with the native sequence. The trifluoromethyl group allowed us to investigate the TBA G-quadruplex structure by 19F NMR spectroscopy. Furthermore, PT assays showed that the modified sequences can significantly improve the anticoagulant activity in comparison with the native TBA. Finally, we demonstrated that the trifluoromethyl-modified TBA sequence could function as an anticoagulant reagent in live rats. Our results strongly suggested that FG is a powerful nucleoside derivative to increase the thermodynamic stability and anticoagulant activity of TBA.

Systematic Approach to DNA Aptamer Design Using Amino Acid-Nucleic Acid Hybrids (ANHs) Targeting Thrombin.

Chemical modifications of innate DNA/RNA aptamers facilitate the improvement of their function. Herein, we report our modular strategy to manipulate a thrombin-binding DNA aptamer (TBA) to improve its anticoagulation activity and binding affinity. A set of amino acid conjugates, termed amino acid-nucleic acid hybrids or ANHs, was synthesized and incorporated into a TBA loop sequences. We found that substitutions with hydrophobic amino acids in the loop region possessed significantly enhanced antithrombin activity, up to 3-fold higher than the native TBA. We investigated the correlations between thrombin-binding affinity and the features of our amino-acid conjugates using experimental techniques including circular dichroism spectroscopy, surface plasmon resonance assay, and molecular modeling. The present study demonstrates a systematic approach to aptamer design based on amino-acid characteristics, allowing the development of advanced aptamers.

Telomeric DNA-RNA-hybrid G-quadruplex exists in environmental conditions of HeLa cells.

In the present study, we employed a 19F NMR approach to study the association of telomere RNA and DNA in vitro and in living human cells. We successfully characterized the DNA-RNA hybrid G-quadruplex (HQ) structure formed by telomeric DNA and RNA. We further demonstrated for the first time that an HQ conformation can exist in the environmental conditions of HeLa cells.

Linear consecutive hexaoxazoles as G4 ligands inducing chair-type anti-parallel topology of a telomeric G-quadruplex.

G-quadruplex structures (G4s) in guanine-rich regions of DNA play critical roles in various biological phenomena, including replication, translation, and gene expression. There are three types of G4 topology, i.e., parallel, anti-parallel, and hybrid, and ligands that selectively interact with or stabilize a specific topology have been extensively explored to enable studies of topology-related functions. Here, we describe the synthesis of a new series of G4 ligands based on 6LCOs (6-linear consecutive oxazoles), i.e., L2H2-2M2EA-6LCO (2), L2A2-2M2EAc-6LCO (3), and L2G2-2M2EG-6LCO (4), which bear four aminoalkyl, acetamidealkyl, and guanidinylalkyl side chains, respectively. Among them, ligand 2 stabilized telomeric G4 and induced anti-parallel topology independently of the presence of cations. The anti-parallel topology induced by 2 was identified as chair-type by means of 19F NMR spectroscopy and fluorescence experiments with 2-aminopurine-labeled DNA.

19F NMR Spectroscopy for the Analysis of DNA G-Quadruplex Structures Using 19F-Labeled Nucleobase.

G-quadruplex structures have been suggested to be biologically important in processes such as transcription and translation, gene expression and regulation in human cancer cells, and regulation of telomere length. Investigation of G-quadruplex structures associated with biological events is therefore essential to understanding the functions of these molecules. We developed the 19F-labeled nucleobases and introduced them into DNA sequences for the 19F NMR spectroscopy analysis. We present the 19F NMR methodology used in our research group for the study of G-quadruplex structures in vitro and in living cells.

Investigation of higher-order RNA G-quadruplex structures in vitro and in living cells by 19F NMR spectroscopy.

Growing evidence indicates that RNA G-quadruplexes have important roles in various processes such as transcription, translation, regulation of telomere length, and formation of telomeric heterochromatin. Investigation of RNA G-quadruplex structures associated with biological events is therefore essential to understanding the functions of these RNA molecules. We recently demonstrated that the sensitivity and simplicity of 19F NMR can be used to directly observe higher-order telomeric G-quadruplexes of labeled RNA molecules in vitro and in living cells, as well as their interactions with ligands and proteins. This protocol describes detailed procedures for preparing 19F-labeled RNA, the evaluation of 19F-labeled RNA G-quadruplexes in vitro and in living Xenopus laevis oocytes by 19F NMR spectroscopy, the quantitative characterization of thermodynamic properties of the G-quadruplexes, and monitoring of RNA G-quadruplex interactions with ligand molecules and proteins. This approach has several advantages over existing techniques. First, it is relatively easy to prepare 19F-labeled RNA molecules by introducing a 3,5-bis(trifluoromethyl) benzene moiety into its 5' terminus. Second, the absence of any natural fluorine background signal in RNA and cells results in a simple and clear 19F NMR spectrum and does not suffer from high background signals as does 1H NMR. Finally, the simplicity and sensitivity of 19F NMR can be used to easily distinguish different RNA G-quadruplex conformations under various conditions, even in living cells, and to obtain the precise thermodynamic parameters of higher-order G-quadruplexes. This protocol can be completed in 2 weeks.

Direct Methylation of Amines with Carbon Dioxide and Molecular Hydrogen using Supported Gold Catalysts.

The N-methylation of amines with CO2 and H2 is an important step in the synthesis of bioactive compounds and chemical intermediates. The first heterogeneous Au catalyst is reported for this methylation reaction with good to excellent yields. The average turnover frequency (TOF) based on surface Au atoms is 45 h(-1) , which is the highest TOF value ever reported for the methylation of aniline with CO2 and H2 . Furthermore, the catalyst is tolerant toward a variety of amines, which includes aromatic, aliphatic, secondary, and primary amines. Preliminary mechanistic studies suggest that the N-alkyl formamide might be an intermediate in the N-methylation of amine process. Moreover, through a one-pot process, it is possible to convert primary amines, aldehydes, and CO2 into unsymmetrical tertiary amines with H2 as a reductant in the presence of the Au catalyst.

Synergistic effect of Brønsted acid and platinum on purification of automobile exhaust gases.

The catalytic purification of automobile exhaust gases (CO, NOx and hydrocarbons) is one of the most practiced conversion processes used to lower the emissions and to reduce the air pollution. Nevertheless, the good performance of exhaust gas purification catalysts often requires the high consumption of noble metals such as platinum. Here we report that the Brønsted acid sites on the external surface of a microporous silicoaluminophosphate (SAPO) act as a promoter for exhaust gas purification, effectively cutting the loading amount of platinum in the catalyst without sacrifice of performance. It is revealed that in the Pt-loaded SAPO-CHA catalyst, there exists a remarkable synergistic effect between the Brønsted acid sites and the Pt nanoparticles, the former helping to adsorb and activate the hydrocarbon molecules for NO reduction during the catalytic process. The thermal stability of SAPO-CHA also makes the composite catalyst stable and reusable without activity decay.