A detection system using sensing motif-tethered oligodeoxynucleotides for multiplex biomolecular analysis.

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H2O2. After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.

Synthesis of Red-Light-Responsive Pheophorbide-a Tryptamine Conjugated Photosensitizers for Photodynamic Therapy.

Six methyl pheophorbide-a derivatives were prepared by linking a tryptamine side chain at the C-131 , C-152 and C-173 positions of pheophorbide-a. Prepared conjugates were characterized and evaluated for their photocytotoxicity against A549 cells. The conjugate 6 a with strong absorption at 413 nm (Soret band), 663-671 nm (Q bands) and comparable fluorescence quantum yield (0.26) was found to exhibit significant cytotoxicity (659 nM). Molecular integration of pheophorbide-a and tryptamines showed synergistic effects as the most potent conjugate 6 a was identified with enhanced photocytotoxicity when compared to methyl pheophorbide-a. The conjugate 6 a was smoothly taken up by A549 cells and exhibited intracellular localization predominantly to lysosome in the cytoplasm. Upon photoirradiation 6 a generated singlet oxygen to show potent cytotoxicity toward A549 cells.

Synthesis of azafluoranthenes by iridium-catalyzed [2 + 2 + 2] cycloaddition and evaluation of their fluorescence properties.

We report a method for the synthesis of azafluoranthenes under neutral reaction conditions in a highly atom-economical manner by the iridium-catalyzed [2 + 2 + 2] cycloaddition of 1,8-dialkynylnaphthalenes with nitriles. A variety of nitriles react with methyl- or phenyl-substituted 1,8-dialkynylnaphthalenes to give a wide range of azafluoranthenes. Azafluoranthenes bearing an amino group show intense fluorescence at around 500 nm. Comparison of the fluorescence properties of amine-substituted azafluoranthenes with related compounds revealed the importance of the amine moiety for obtaining a high fluorescence quantum yield. The choice of the solvent affected the emission maxima and the fluorescence quantum yield. Azafluoranthenes bearing pyrrolidine exhibited blue-shifted emission bands in a non-polar solvent and gave a fluorescence quantum yield of 0.76 in toluene. A Lippert-Mataga plot and computational studies provide insight into the origin of the fluorescence of azafluoranthenes. Furthermore, cellular experiments using human breast adenocarcinoma cells SK-BR-3 demonstrated the feasibility of using azafluoranthenes as fluorescent probes.

Raman Signal Enhancement by DABCYL-Substitution on DNA Aptamer for Identification of Cellular ATP.

Raman probes have attracted widespread attention for the visualization and identification of biomolecules, because they can be applied to identify detailed chemical structures, detect multiple molecules simultaneously, and visualize cellular functional molecules. However, the biological application of Raman probes is still limited because of their weak signal intensity. Herein, we present a molecular system that shows an enhanced Raman signal using a nonfluorescent dye. We introduced a DABCYL molecule bearing an acetylene unit into thymidine at the 5-position. The resulting modified nucleobase, dDAU, showed a robust signal around 2200 cm-1, which was attributed to the acetylene unit, due to resonance Raman induced by the DABCYL group. We further prepared a DNA aptamer modified with dDAU, and characterized the change of the Raman spectra. Combination with gold nanoparticles, which enhanced the Raman signal by surface-enhanced Raman scattering (SERS), allowed sensitive detection of cellular adenosine derivatives including ATP. Thus, the present system is a promising tool for the detection of biological materials by Raman spectroscopy.

Preparation of a multifunctional photoactivated prodrug on a streptavidin scaffold bearing a DNA aptamer.

Prodrugs that present strong cytotoxicity toward specific cells have been utilized for cell-type selection and purification. In this study, we designed and prepared a multifunctional, photoactivated prodrug based on a streptavidin scaffold. Biotin-labeled DNA aptamer that recognizes the membrane antigen EpCAM, and biotin-labeled photoactivated prodrug bearing the antitumor camptothecin, were prepared. Both molecules were linked to the streptavidin scaffold by simple mixing. The resulting prodrug bound to the EpCAM-overexpressing SK-BR-3 target cells and showed cytotoxic effects upon photoirradiation, corresponding to cytotoxic drug release.

pH-responsive aggregates consisted of oligodeoxynucleotides bearing nitrophenol group that deliver drugs into acidic cells.

A decrease in pH is observed in most solid tumors, thus, the development of drug delivery systems that respond to slightly acidic extracellular pH environment is important in providing tumor-targeted therapies. DNA aggregates can act as useful drug delivery agents, and therefore, we designed an artificial oligodeoxynucleotides (ODNs) that formed an aggregate only under acidic conditions in this study. In other words, we expected that if we could make DNA aggregates that form only in an acidic environment and that encapsulate drugs, it would be possible to transport drugs to tumor tissues selectively. Nitrophenol derivatives, which underwent protonation and deprotonation in response to pH changes, was introduced into ODNs. The ODNs formed aggregates under weakly acidic conditions because of expression of amphiphilicity, which was induced by protonation of nitrophenol unit, and were smoothly taken up into cells. We also found that the aggregates transported anticancer drug, 5FU, into acidified cells to show cytotoxic effects.

Excited States of Thio-2'-deoxyuridine Bearing an Extended π-Conjugated System: 3',5'-Di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine.

A new thio-2'-deoxyuridine with an extended π-conjugated group was successfully synthesized: 3',5'-di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine. The thio-2'-deoxyuridine derivative has a large red-shifted absorption band in the UVA region and also shows fluorescence, a rare photo-property among thionucleobases/thionucleosides. The triplet-triplet absorption spectrum and the rate constants (the intrinsic decay rate constant of the triplet state, the self-quenching rate constant, and the quenching rate constant of the triplet state by an oxygen molecule) of the thio-2'-deoxyuridine were obtained by transient absorption spectroscopy. The quantum yield of intersystem crossing and the quantum yield of singlet molecular oxygen formation (ϕΔ) under an oxygen atmosphere were also determined. The Ï•Δ value of the new thio-2'-deoxyuridine was found to be substantially higher than all reported values of other thio-2'-deoxyribonucleosides in low oxygen concentrations similar to cancer cell environments. The fluorescence quantum yield depended on the excitation wavelength, revealing certain photochemical reactions in the higher excited singlet states. However, when excited into the higher excited state with non-resonant two-photon absorption, the Ï•Δ of the thio-2'-deoxyuridine derivative was found to remain sufficiently large. These findings should be very useful for the development of thio-2'-deoxyribonucleoside-based pharmaceuticals as DNA-specific photosensitizers for photochemotherapy.

Modulation of cell membrane functionalization with aggregates of oligodeoxynucleotides containing alkyl chain-modified uridines.

In this study, we prepared oligodeoxynucleotides (ODNs) containing the uridine base modified by an alkyl chain at the 5-position (AU) and characterized their aggregate formation, localization, and functions in cells. These experiments revealed that aggregates of these ODNs were readily transported into cells, but their localization was dependent upon the number of hydrophobic units. ODNs with one modified AU were transported in the cytosol, while ODNs with multiple AU modifications resulted in their accumulation at the cell membrane. We also examined the ability of the AU-modified ODNs to capture small molecules at the cell membrane and their cellular uptake. We positioned a thioflavin-T (ThT)-binding aptamer on the cell membrane by means of hybridization with ODNs with three AUs at the strand end. Treatment with ThT resulted in its efficient uptake into cells, due to the capture of the ThT by the aptamers on the cell membrane. Thus, we demonstrated the functionalization of cell membranes with modified ODNs and the efficient delivery of small molecules into the cells.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells.

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

Biological reduction of nitroimidazole-functionalized gold nanorods for photoacoustic imaging of tumor hypoxia.

Tumor-selective accumulation of gold nanorods (GNR) has been demonstrated for visualization of tumor hypoxia by photoacoustic imaging. We prepared GNRs with hypoxia-targeting nitroimidazole units (G-NI) on their surface. Biological experiments revealed that G-NI produced a strong photoacoustic signal in hypoxic tumor cells and tissues.

Aggregate Formation of BODIPY-Tethered Oligonucleotides That Led to Efficient Intracellular Penetration and Gene Regulation.

Exogenous nucleic acids showed low efficiency regarding cellular uptake and low stability in biological conditions; therefore, a number of techniques have been developed to improve their basic properties. One of the best solutions is the application of nanosized particles consisting of oligonucleotides that penetrate the cell membrane without any additives and exhibit high stability in cells. In this report, we employed a simple approach to address the basic properties of nanoparticles of oligonucleotides in biological systems. We prepared BODIPY-labeled oligonucleotides that carried an exclusive modification at the strand end. BODIPY shows high hydrophobicity and fluorescent emission; therefore, the oligonucleotides formed nanosized aggregates in aqueous solution and their behaviors in cells or tissues were easily tracked. Detailed experiments revealed that aggregate formation was indispensable for the high cellular uptake of the oligonucleotides via scavenger-receptor-mediated endocytosis. In addition, the aggregates provided an efficient gene regulation in living cells and tumor tissues transplanted into mice.

Aggregate Formation of Oligonucleotides that Assist Molecular Imaging for Tracking of the Oxygen Status in Tumor Tissue.

The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.

Preparation of alkyne-labeled 2-nitroimidazoles for identification of tumor hypoxia by Raman spectroscopy.

Hypoxia is a characteristic feature of solid tumors. Herein, we have developed novel hypoxia-sensitive probes (IM-ACs) for Raman spectroscopic analysis, consisting of nitroimidazole as a hypoxia-targeting unit and acetylene group as the signal-emitting unit. Among IM-ACs synthesized in this study, IM-AC possessing a diacetylene group (IM-AC 3), showed suitable properties as a hypoxia indicator. When administered to A549 cells, we observed a strong signal of IM-AC 3 around 2200cm-1 in the Raman spectra from hypoxic cells. Ex vivo experiments suggest that IM-AC 3 remained in hypoxic tumor tissue and emitted a strong signal.

Confined Singlet Oxygen in Mesoporous Silica Nanoparticles: Selective Photochemical Oxidation of Small Molecules in Living Cells.

Chemical conversion of specific bioactive molecules by external stimuli in living cells is a powerful noninvasive tool for clarification of biomolecular interactions and to control cellular functions. However, in chaotic biological environments, it has been difficult to induce arbitrary photochemical reactions on specific molecules because of their poor molecular selectivity. Here we report a selective and nontoxic photochemical reaction system utilizing photoactivated mesoporous silica nanoparticles to control biological functions. Methylene blue modification within nanoparticle pores for photosensitization produced singlet oxygen confined to the pore that could mediate selective oxidation of small molecules without any damage to living cells. This intracellular photochemical system produced bioactive molecules in situ and remotely controlled the cell cycle phase. We also confirmed that this photoreaction could be applied to control cell cycle phase in tumor tissue transplanted in mice. The cell cycle phase in the cells in mice, to which our system was administered, was arrested at the G2/M phase upon photoirradiation. We demonstrate a simple and promising method for the exogenous conversion of an intracellular biomolecule to another functional compound.

Water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit for ratiometric sensing of oxygen levels in living cells.

Dual emission was applied to a molecular probe for the ratiometric sensing of oxygen concentration in a living system. We prepared ruthenium complexes possessing a coumarin unit (Ru-Cou), in which the (3)MLCT phosphorescence of the ruthenium complex was efficiently quenched by molecular oxygen, whereas the coumarin unit emitted constant fluorescence independent of the oxygen concentration. The oxygen status could be determined precisely from the ratio of phosphorescence to fluorescence. We achieved the molecular imaging of cellular oxygen levels using Ru-Cou possessing an alkyl chain, which provided appropriate lipophilicity to increase cellular uptake.

Phosphorescent ruthenium complexes with a nitroimidazole unit that image oxygen fluctuation in tumor tissue.

Understanding oxygen fluctuation in a cancerous tumor is important for effective treatment, especially during radiotherapy. In this paper, ruthenium complexes bearing a nitroimidazole group are shown to report the oxygen status in tumor tissue directly. The nitroimidazole group was known to be accumulated in hypoxic tumor tissues. On the other hand, the ruthenium complex showed strong phosphorescence around 600 nm. The emission of ruthenium is quenched instantaneously by molecular oxygen due to energy transfer between triplet states of oxygen and ruthenium complex, but the emission is then recovered by the removal of oxygen. Thus, we could observe oxygen fluctuation in tumor tissue in a real-time manner by monitoring the phosphorescence of the ruthenium complex. The versatility of the probe is demonstrated by monitoring oxygen fluctuation in living cells and tumor tissue planted in mice. The ruthenium complex promptly penetrated plasma membrane and accumulated in cells to emit its oxygen-dependent phosphorescence. In vivo experiments revealed that the oxygen level in tumor tissue seems to fluctuate at the sub-minute timescale.

Enzyme-catalyzed conversion of chemical structures on the surface of gold nanorods.

For developing metal nanoparticles of which surface chemical structures could be altered by enzymes in the cells, functional linkers caged by coumaric acids have been synthesized. Synthesized gold nanorod (GNR) conjugates possessing coumaric acid precursors underwent (porcine liver) esterase-catalyzed hydrolysis on their surface to afford GNRs coated with amino-functionalized polyethylene glycol and fluorescent coumarins as reporter molecules for monitoring the conversion. The chemical structural conversion on the GNR surfaces was successfully observed inside cells by fluorescence microscopy when GNR conjugates were incubated with tumor cells.

Synthesis and one-electron reduction characteristics of radiation-activated prodrugs possessing two 5-fluorodeoxyuridine units.

Two molecules of an antitumor agent, 5-fluorodeoxyuridine (5-FdUrd), were connected by a 2-oxoalkyl linker (Oxo-linker) at the N(3) position to obtain radiation-activated prodrugs, FdUrd(2) A and FdUrd(2) B. The prodrugs in this study released 5-FdUrd via one-electron reduction initiated by hypoxic X-irradiation. The release of 5-FdUrd from FdUrd(2) A and FdUrd(2) B proceeded more efficiently than that of previous prodrug, Oxo-FdUrd, which possessed one molecule of 5-FdUrd. FdUrd(2) A exhibited increased cytotoxicity against A549 cells when the FdUrd(2) A solution had been irradiated with a large dose of X-rays before administration to the cells. However, we observed no effect on cytotoxicity when the cells were X-irradiated under hypoxic conditions in the presence of FdUrd(2) A because the amount of 5-FdUrd released in the cells seemed to be too low to induce cytotoxic activity.

Reductive activation of 5-fluorodeoxyuridine prodrug possessing azide methyl group by hypoxic X-irradiation.

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation.

Radiolytic reduction characteristics of artificial oligodeoxynucleotides possessing 2-oxoalkyl group or disulfide bonds.

A number of advances have been made in the development of modified oligodeoxynucleotides (ODNs), and chemical or physical properties of which are controlled by external stimuli. These intelligent ODNs are promising for the next generation of gene diagnostics and therapy. This paper focuses on the molecular design of artificial ODNs that are activated by X-irradiation and their applications to regulation of hybridization properties, conformation change, radiation-activated DNAzyme, and decoy molecules.