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The consumption of foods prepared at high temperatures has been associated with numerous health risks. To date, the chief identified source of risk has been small molecules produced in trace levels by cooking and reacting with healthy DNA upon consumption. Here, we considered whether the DNA in food itself also presents a hazard. We hypothesize that high-temperature cooking may cause significant damage to the DNA in food, and this damage might find its way into cellular DNA by metabolic salvage. We tested cooked and raw foods and found high levels of hydrolytic and oxidative damage to all four DNA bases upon cooking. Exposing cultured cells to damaged 2'-deoxynucleosides (particularly pyrimidines) resulted in elevated DNA damage and repair responses in the cells. Feeding a deaminated 2'-deoxynucleoside (2'-deoxyuridine), and DNA containing it, to mice resulted in substantial uptake into intestinal genomic DNA and promoted double-strand chromosomal breaks there. The results suggest the possibility of a previously unrecognized pathway whereby high-temperature cooking may contribute to genetic risks.
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The presence of a hydroxyl group at the 2'-position in its ribose makes RNA susceptible to hydrolysis. Stabilization of RNAs for storage, transport and biological application thus remains a serious challenge, particularly for larger RNAs that are not accessible by chemical synthesis. Here we present reversible 2'-OH acylation as a general strategy to preserve RNA of any length or origin. High-yield polyacylation of 2'-hydroxyls ('cloaking') by readily accessible acylimidazole reagents effectively shields RNAs from both thermal and enzymatic degradation. Subsequent treatment with water-soluble nucleophilic reagents removes acylation adducts quantitatively ('uncloaking') and recovers a remarkably broad range of RNA functions, including reverse transcription, translation and gene editing. Furthermore, we show that certain α-dimethylamino- and α-alkoxy- acyl adducts are spontaneously removed in human cells, restoring messenger RNA translation with extended functional half-lives. These findings support the potential of reversible 2'-acylation as a simple and general molecular solution for enhancing RNA stability and provide mechanistic insights for stabilizing RNA regardless of length or origin.
Assuntos
Proteínas , RNA , Humanos , RNA/química , Proteínas/metabolismo , Água , Acilação , RNA Mensageiro/genética , Indicadores e ReagentesRESUMO
Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pHâ 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.
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Núcleo Celular , Corantes Fluorescentes , Corantes Fluorescentes/química , Fluorescência , Linhagem Celular , Trifosfato de AdenosinaRESUMO
DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory's work on developing chemical tools to study DNA repair processes in vitro, as well as in cells and tissues, and what we have learned by applying them.We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage.We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both in vitro and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses.To investigate cellular nucleotide sanitation pathways, we designed a series of "two-headed" nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme.We proceed to discuss the development of a "universal" probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.
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Dano ao DNA , Reparo do DNA , Animais , Humanos , DNA/química , Sondas de DNA , NucleotídeosRESUMO
Fluorescence labeling of DNAs is broadly useful, but methods for labeling are expensive and labor-intensive. Here we describe a general method for fluorescence labeling of oligonucleotides readily and cost-efficiently via base excision trapping (BETr), employing deaminated DNA bases to mark label positions, which are excised by base excision repair enzymes generating AP sites. Specially designed aminooxy-substituted rotor dyes trap the AP sites, yielding high emission intensities. BETr is orthogonal to DNA synthesis by polymerases, enabling multi-uracil incorporation into an amplicon and in situ BETr labeling without washing. BETr also enables labeling of dsDNA such as genomic DNA at a high labeling density in a single tube by use of nick translation. Use of two different deaminated bases facilitates two-color site-specific labeling. Use of a multi-labeled DNA construct as a bright fluorescence tag is demonstrated through the conjugation to an antibody for imaging proteins. Finally, double-strand selectivity of a repair enzyme is harnessed in sensitive reporting on the presence of a target DNA or RNA in a mixture with isothermal turnover and single nucleotide specificity. Overall, the results document a convenient and versatile method for general fluorescence labeling of DNAs.
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Reparo do DNA , DNA , Replicação do DNA , Fluorescência , UracilaRESUMO
Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. Here, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in the treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-2'-deoxyguanosine (8-oxo-dG)TP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogues of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 µM and 410 ± 60% at 5 µM. The compounds are found to increase the activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks.
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Enzimas Reparadoras do DNA/metabolismo , Neoplasias , Monoéster Fosfórico Hidrolases/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Carcinogênese , DNA , Dano ao DNA , Humanos , Nucleotídeos , Estresse OxidativoRESUMO
Cancer cells undergo unscheduled proliferation resulting from dysregulation of the cell cycle, and hence, evaluation in tumor is of keen interest to examine the invasiveness and recurrence of cancer in the lesion. Molecular probes capable of discriminating actively growing tumor from resting ones remain unexplored despite their vast importance. Here, we describe a novel strategy to visualize invasive areas in tumor with a fluorescence probe that implements synergistic fluorescence response toward the slightly acidic environment of tumor and an ATP-abundant nature of actively growing cells. The probe has been designed for ultrafast detection of ATP with high specificity. We demonstrate its utility in visualizing invasive areas in tumor by distinguishing basal cell carcinomas and squamous cell carcinomas at their early stages by two-photon microscopy.
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Carcinoma de Células Escamosas , Neoplasias Cutâneas , Trifosfato de Adenosina , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Humanos , Prótons , Pele/metabolismo , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologiaRESUMO
Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity inâ situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.
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DNA Mitocondrial/análise , Corantes Fluorescentes/química , Imagem Óptica , Animais , Células Cultivadas , Reparo do DNA , DNA Mitocondrial/metabolismo , Células HeLa , Humanos , Camundongos , Estresse OxidativoRESUMO
Methods for the site-selective labeling of long, native RNAs are needed for studying mRNA biology and future therapies. Current approaches involve engineering RNA sequences, which may alter folding, or are limited to specific sequences or bases. Here, we describe a versatile strategy for mRNA conjugation via a novel DNA-tiling approach. The method, TRAIL, exploits a pool of "protector" oligodeoxynucleotides to hybridize and block the mRNA, combined with an "inducer" DNA that extrudes a reactive RNA loop for acylation at a predetermined site. Using TRAIL, an azido-acylimidazole reagent was employed for labeling and controlling RNA for multiple applications inâ vitro and in cells, including analysis of RNA-binding proteins, imaging mRNA in cells, and analysis and control of translation. The TRAIL approach offers an efficient and accessible way to label and manipulate RNAs of virtually any length or origin without altering native sequence.
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DNA/metabolismo , RNA Mensageiro/metabolismo , Acilação , DNA/química , RNA Mensageiro/químicaRESUMO
Caging RNA by polyacylation (cloaking) has been developed recently as a simple and rapid method to control the function of RNAs. Previous approaches for chemical reversal of acylation (uncloaking) made use of azide reduction followed by amine cyclization, requiring â¼2-4 h for the completion of cyclization. In new studies aimed at improving reversal rates and yields, we have designed novel acylating reagents that utilize quinone methide (QM) elimination for reversal. The QM de-acylation reactions were tested with two bioorthogonally cleavable motifs, azide and vinyl ether, and their acylation and reversal efficiencies were assessed with NMR and mass spectrometry on model small-molecule substrates as well as on RNAs. Successful reversal both with phosphines and strained alkenes was documented. Among the compounds tested, the azido-QM compound A-3 displayed excellent de-acylation efficiency, with t1/2 for de-acylation of less than an hour using a phosphine trigger. To test its function in RNA caging, A-3 was successfully applied to control EGFP mRNA translation in vitro and in HeLa cells. We expect that this molecular caging strategy can serve as a valuable tool for biological investigation and control of RNAs both in vitro and in cells.
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IndolquinonasRESUMO
Hypoxia, a condition of oxygen deficiency in tissues, features various diseases including solid tumor. Under hypoxia, several reductases such as nitroreductases are elevated. Based on this fact, we have investigated an indirect way to assess the hypoxia susceptibility of different organ tissues (mouse lung, heart, spleen, kidney, and liver) by detecting nitroreductase present within. Among the organs, the kidney showed a notable susceptibility to hypoxia, which was due to the renal medulla, not due to the renal cortex, as observed by ratiometric fluorescence imaging with a probe. The probe features ratiometric signaling, NIR-emitting, two-photon absorbing, and pH-insensitive emission properties, offering a practical tool for studying the nitroreductase activity and, furthermore, hypoxia-associated biological processes.
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Corantes Fluorescentes , Nitrorredutases , Animais , Hipóxia , Camundongos , Imagem Óptica , FótonsRESUMO
Glycation, the term for non-enzymatic covalent reactions between aldehyde metabolites and nucleophiles on biopolymers, results in deleterious cellular damage and diseases. Since Parkinsonism-associated protein DJ-1 was proposed as a novel deglycase that directly repairs glycated adducts, it has been considered a major contributor to glycation damage repair. Recently, an interesting debate over the mechanism of glycation repair by DJ-1 has emerged, focusing on whether the substrate of DJ-1 is glycated adducts or the free small aldehydes. The physiological significance of DJ-1 on glycation defense also remains in question. This debate is complicated by the fact that glycated biomolecular adducts are in rapid equilibrium with free aldehydes. Here, we summarize experimental evidence for the two possibilities, highlighting both consistencies and conflicts. We discuss the experimental complexities from a mechanistic perspective, and suggest classes of experiments that should help clarify this debate.
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Produtos Finais de Glicação Avançada/metabolismo , Proteína Desglicase DJ-1/metabolismo , Aldeído Pirúvico/metabolismo , Cisteína/metabolismo , Produtos Finais de Glicação Avançada/química , Humanos , Cinética , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Proteína Desglicase DJ-1/química , Proteína Desglicase DJ-1/genética , Proteínas/química , Proteínas/metabolismo , Aldeído Pirúvico/química , Especificidade por SubstratoRESUMO
The benzocoumarin dyes fluoresce negligibly in aqueous media but very strongly in cells, whereas representative conventional dyes display contrasting behaviour; the distinct emission behaviour of the fluorophores in organic solutions, in aqueous media, and in cell convinces the uniqueness of the cellular environment. The in cellulo superbright benzocoumarins also reveal an environment-insensitive emission behaviour, which is required for the reliable analysis via ratiometric imaging.
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Cumarínicos/química , Fluorescência , Corantes Fluorescentes/química , Linhagem Celular Tumoral , Humanos , SoluçõesRESUMO
Near-infrared-emitting hemicyanine dyes are widely used in activatable fluorescent probes for various biological analytes; however, they are chemically unstable and show photoinstability, as shown here with naphthalene-based hemicyanines containing a typical hemicyanine moiety, 2-indolium. These issues can be resolved with a 4-pyridinium derivative, which also has good two-photon imaging capability.
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Photostable and near-infrared (NIR)-emitting organic fluorophores with large Stokes shifts are in great demand for long-term bioimaging at deeper depths with minimal autofluorescence and self-quenching. Herein, a new class of benzorhodamines and their analogues that are photostable and emit in the NIR region (up to 785â nm) with large Stokes shifts (>120â nm) is reported. The synthesis involves condensation of 7-alkylamino-2-naphthols with 2-[4-(dimethylamino)-2-hydroxybenzoyl]benzoic acid, which leads to bent-shaped benzorhodamines that emit orange fluorescence (≈600â nm); however, introduction of steric hindrance near the condensation site switched the regioselectivity, to provide a linear benzorhodamine system for the first time. The linear benzorhodamine derivatives provide bright fluorescence images in cells and in tissue. A carboxy-benzorhodamine was applied for photothermal therapy of cancer cells and xenograft cancer mice.
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Neoplasias , Imagem Óptica , Terapia Fototérmica , Rodaminas , Animais , Compostos de Benzil , Corantes Fluorescentes , Camundongos , Neoplasias/diagnóstico por imagem , Neoplasias/terapiaRESUMO
Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase-induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real-time detection of NTH1 activity inâ vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small-molecule inhibitor with sub-micromolar potency.
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Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/química , Antimutagênicos/farmacologia , Dano ao DNA , Desoxirribonuclease (Dímero de Pirimidina)/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Oxirredução , Estresse Oxidativo , Pirimidinas/químicaRESUMO
Abnormal vitamin B6 status, marked by deficient intracellular concentrations of pyridoxal phosphate (PLP), is classified as a direct biomarker based on its biomedical significance. However, there exist no direct methods for measuring vitamin B6 status in intact cells. Here we describe the development of a fluorogenic probe, RAB6, which shows remarkable selectivity for PLP among the B6 vitamers and other cellular aldehydes.
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Corantes Fluorescentes/química , Hidrazonas/química , Fosfato de Piridoxal/análise , Rodaminas/química , Biomarcadores/análise , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Hidrazonas/síntese química , Microscopia de Fluorescência/métodos , Rodaminas/síntese química , Espectrometria de Fluorescência/métodosRESUMO
Organic fluorophores emitting in the far-red/near-infrared (NIR) wavelength region are in great demand for minimal autofluorescence and reduced light scattering in deep tissue or whole body imaging. Currently, only a few classes of far-red/NIR fluorophores are available including widely used cyanine dyes, which are susceptible to photobleaching and form nonfluorescent aggregates. Even rare are those far-red/NIR emitting dyes that have two-photon imaging capability. Here we report a new class of far-red/NIR-emitting dyes that are photo-stable, very bright, biocompatible, and also two-photon absorbing. The introduction of an electron-withdrawing group such as N-acyl or N-alkoxycarbonyl groups on the C-10-amino substituent of the new julolidine-derived amino-Si-pyronin dyes (ASiPj), which emit in the far-red region, causes large bathochromic shifts, leading to NIR-emitting amino-Si-pyronin dyes (NIR-ASiPj) having high cellular stability. Furthermore, the ASiPj-NIR-ASiPj couple offers a novel ratiometric bioimaging platform with a large spectral gap, as demonstrated here with a boronate-containing NIR-ASiPj derivative that is converted to the corresponding ASiPj dye upon reaction with hydrogen peroxide.
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γ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations; however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity levels between different mouse organ tissues are quantitatively compared with the highest level in the kidney. The probe with practicality holds great promise for studying GGT-associated biological processes directly through ratiometric imaging by two-photon microscopy.
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Cumarínicos/química , Corantes Fluorescentes/química , Imagem Óptica , Fótons , gama-Glutamiltransferase/análise , Cumarínicos/síntese química , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Células Tumorais Cultivadas , Viscosidade , gama-Glutamiltransferase/metabolismoRESUMO
The promising features of fluorescence spectroscopy have inspired a quest for fluorescent probes for analysis and monitoring of molecular interactions in biochemical, medical, and environmental sciences. To overcome the competitive supramolecular interactions in aqueous media encountered with conventional molecular-recognition-based probes, the use of reaction-based probes that involve making or breaking of covalent bonds has emerged as a complementary sensing strategy to realize higher selectivity and sensitivity with larger spectroscopic changes. In spite of the enormous efforts, the development of reaction-based fluorescent probes meets with certain challenges in terms of their practical applications, demanding "intelligent design" of probes with an appropriate fluorophore attached to an efficient reactive moiety at the right place. This Account summarizes the results of our efforts made in the development and fine-tuning of reaction-based fluorescent probes toward those goals, classified by the type of analyte (anions, metal cations, and biomolecules) with notes on the challenges and achievements. The reaction-based approach was demonstrated to be powerful for the selective sensing of anions (cyanide and (amino)carboxylates) for the first time, and later it was extended to develop two-photon probes for bisulfite and fluoride ions. The reaction-based approach also enabled selective sensing of noble metal ions such as silver, gold, and palladium along with toxic (methyl)mercury species and paramagnetic copper ions. Furthermore, microscopic imaging and monitoring of biologically relevant species with reaction-based two-photon probes were explored for hydrogen sulfide, hypochlorous acid, formaldehyde, monoamine oxidase enzyme, and ATP.