Toshikazu Ono

Direct Fluorescence Monitoring of DNA Base Excision Repair

Uracil is an undesired component of DNA, as it arises from spontaneous deamination of cytosine.[1] This hydrolysis reaction promotes mutations, since the resulting U-G pair can be misread during DNA replication. As a result, multiple cellular enzymes have evolved to detect uracil in DNA and remove it prior to replication.[2] In E. coli uracil DNA glycosylase (UDG) enzyme functions to guard the bacterial genome. In humans, similar enzyme activities exist, including the proteins UNG1/2, SMUG, and TDG.[3] These enzymes flip uracil out of the DNA helix and cleave it from its deoxyribose sugar, leaving an abasic site in its place.[4]

Multicomponent molecular puzzles for photofunction design: Emission color variation in lewis acid-base pair crystals coupled with guest-to-host charge transfer excitation

Simple yet ubiquitous multimolecular assembly systems with color-tunable emissions are realized by cooperative electron donor-acceptor interactions, such as the boron-nitrogen (B-N) dative bond as a Lewis acid-base pair and charge transfer (CT) interactions. These are ternary-component systems consisting of a naphthalenediimide derivative (NDI), tris(pentafluorophenyl)borane (TPFB), and aromatic molecules (guest) with an NDI:TPFB:guest ratio of 1:2:2. The crystal shows guest-dependent color-tunable emissions such as deep blue to orange when a guest molecule of benzene is replaced with other π-conjugated systems. A good correlation between the emission wavelength and ionization potential of the guest and electronic structure calculations indicated that the emission is due to the CT transition from the guest to the NDI. The present study suggests that a rational solution of multcomponent molecular puzzles would be useful for obtaining novel photofunctional solid-state systems.

Monitoring eukaryotic and bacterial UDG repair activity with DNA-multifluorophore sensors

We report the development of simple fluorogenic probes that report on the activity of both bacterial and mammalian uracil–DNA glycosylase (UDG) enzymes. The probes are built from short, modified single-stranded oligonucleotides containing natural and unnatural bases. The combination of multiple fluorescent pyrene and/or quinacridone nucleobases yields fluorescence at 480 and 540 nm (excitation 340 nm), with large Stokes shifts of 140–200 nm, considerably greater than previous probes. They are strongly quenched by uracil bases incorporated into the sequence, and they yield light-up signals of up to 40-fold, or ratiometric signals with ratio changes of 82-fold, on enzymatic removal of these quenching uracils. We find that the probes are efficient reporters of bacterial UDG, human UNG2, and human SMUG1 enzymes in vitro, yielding complete signals in minutes. Further experiments establish that a probe can be used to image UDG activity by laser confocal microscopy in bacterial cells and in a human cell line, and that signals from a probe signalling UDG activity in human cells can be quantified by flow cytometry. Such probes may prove generally useful both in basic studies of these enzymes and in biomedical applications as well.

DNA-Polyfluorophores for Real-Time Multicolor Tracking of Dynamic Biological Systems

Dye-ing to live: Spectral limitations of common organic dyes make it difficult or impossible to visualize and follow multiple biological components in rapidly moving systems. The development of a multispectral set of improved DNA-scaffolded fluorophores is described. Their use in multicolor cellular imaging (see scheme) and in tracking of biological motions on the subsecond timescale is demonstrated.

High swelling ability of polystyrene-based polyelectrolyte gels at low temperature

Molecular design of polymer gels that exhibit high swelling degrees at low temperature was demonstrated with the aid of repulsive interaction among the polymer chains in the ionic polymer gels and the utility of organic solvents with relative low melting points. A small amount of tetraalkylammonium tetraphenylborate as ionic groups was incorporated into cross-linked polystyrene to yield novel ionic polymer gels. The swelling degrees at room temperature (23 °C) were about 100 times as much as their dried weights in various organic solvents. Moreover, they swelled and absorbed some organic solvents such as THF and dichloromethane at low temperature (−80 °C), and the swelling degrees were similar to those at room temperature.

Toward the design of superabsorbent materials for non-polar organic solvents and oils: ionic content dependent swelling behaviour of cross-linked poly(octadecyl acrylate)-based lipophilic polyelectrolytes

Oil spills and other industrial chemical leaks to natural environments can cause long-term impact on ecosystems, and the use of polymer-based absorbent materials for such media is a promising technique for cleaning up contaminated sites. Here, we prepared a set of poly(octadecyl acrylate)-based lipophilic polyelectrolyte gels bearing tetraalkylammonium tetrakis(3,5-bis(trifluoromethyl)phenyl) borate (TFPB) with different contents of the ionic groups from 1.0 mol% (EG18(1%)) to 10 mol% (EG18(10%)), and investigated their swelling behaviours in organic solvents and oils. The swelling degrees of the polymer gels in non- or less-polar media (3 < e < 14) increased both with increasing the contents of TFPB salt on the polymer chain and increasing the dielectric constants of the solvents. The swelling degrees of EG18(10%) in dichloromethane and 1,2-dichloroethane showed over 300 times as much as its dry weight. In nonpolar media (e < 3) such as toluene and hexane, however, EG18(10%) collapsed and showed low swelling degrees owing to their low dielectric constants. But, the addition of ethanol (e = 24.6) to them increased the solvent polarities as mixtures, resulting in the high swelling degrees over 100 times by polyelectrolyte effects. The lipophilic polyelectrolyte gels could find applications as protective barriers for volatile organic compounds (VOCs) spilled in the environment and as absorbents for waste oil.

Counter anion dependent swelling behaviour of poly(octadecyl acrylate)-based lipophilic polyelectrolyte gels as superabsorbent polymers for organic solvents

Molecular design of superabsorbent materials for organic solvents has attracted much attention due to their superior applications for the removal of hazardous leakages or spillages of volatile organic compounds (VOCs) and waste oils. One approach for preparing such soft matter is loosely crosslinked lipophilic polyelectrolytes. Here, we demonstrate the counter anion effect for superabsorbency for various organic solvents in a series of poly(octadecyl acrylate)-based lipophilic polyelectrolyte gels bearing tetraalkylammonium with relatively hydrophobic counter-anions: dodecyl sulfate (DS), tetrakis(3,5-bis(trifluoromethyl)phenyl) borate (TFPB) and (μ-(1H-imidazolato κ-N1:κ-N3) hexakis(pentafluorophenyl borate) (IM). The benefit of high-swelling abilities due to dissociation of ionpairs was observed in less- and non-polar media (3 < e < 13), when TFPB and IM were selected as counter anions. These results indicate that molecular design of superabsorbent materials requires that the ionic groups have sufficient bulkiness and lipophilicity for ionic dissociation in nonpolar solvents.

An Electropolymerized Crystalline Film Incorporating Axially-Bound Metalloporphycenes: Remarkable Reversibility, Reproducibility, and Coloration Efficiency of Ruthenium(II/III)-Based Electrochromism

Oxidative electropolymeization of an axially bound, bithiophene-pyridine complex of ruthenium(III)-porphycene [Ru(TPrPc) (btp)2]PF6 (1) gives a submicrometer-thick, polymeric film on an ITO electrode with a crystalline morphology. The polymeric film, the first example of axially linked multimetalloporphycene coordination arrays, exhibits highly stable and reproducible electrochromic response with high electrochromic efficiency upon electrochemical control over the metal-centered electron transfer process (Ru(II)/Ru(III)).

Turn-On Fluorogenic and Chromogenic Detection of Small Aromatic Hydrocarbon Vapors by a Porous Supramolecular Host

Benzene, toluene, ethylbenzene, the isomers of xylene, and trimethylbenzene are harmful volatile organic compounds and pose risks to human health and the environment. However, there are currently no effective chemosensors for vapors of these compounds. A porous supramolecular host for turn-on fluorogenic and chromogenic detection of the vapors of small aromatic hydrocarbons is presented. The host was constructed from a naphthalenediimide derivative that was supramolecularly connected to tris(pentafluorophenyl)borane. The amorphous powder form of the host allowed for effective accommodation of vapors of small aromatic hydrocarbons, resulting in a guest-dependent fluorescence emission. Increases in the fluorescence yield of 76-, 46-, and 37-fold were observed with toluene, benzene, and m-xylene, respectively. Negligible responses were obtained with common organic solvents. This simple supramolecular host could be applied as a useful sensor of small aromatic hydrocarbon vapors.

DNA-polyfluorophore Chemosensors for Environmental Remediation: Vapor-phase Identification of Petroleum Products in Contaminated Soil

Contamination of soil and groundwater by petroleum-based products is an extremely widespread and important environmental problem. Here we have tested a simple optical approach for detecting and identifying such industrial contaminants in soil samples, using a set of fluorescent DNA-based chemosensors in pattern-based sensing. We used a set of diverse industrial volatile chemicals to screen and identify a set of five short oligomeric DNA fluorophores on PEG-polystyrene microbeads that could differentiate the entire set after exposure to their vapors in air. We then tested this set of five fluorescent chemosensor compounds for their ability to respond with fluorescence changes when exposed to headgas over soil samples contaminated with one of ten different samples of crude oil, petroleum distillates, fuels, lubricants and additives. Statistical analysis of the quantitative fluorescence change data (as Δ(R,G,B) emission intensities) revealed that these five chemosensors on beads could differentiate all ten product mixtures at 1000 ppm in soil within 30 minutes. Tests of sensitivity with three of the contaminant mixtures showed that they could be detected and differentiated in amounts at least as low as one part per million in soil. The results establish that DNA-polyfluorophores may have practical utility in monitoring the extent and identity of environmental spills and leaks, while they occur and during their remediation.