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1.
Nucleic Acids Res ; 48(11): 6081-6091, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402089

RESUMO

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.


Assuntos
DNA/química , DNA/metabolismo , Leishmania infantum/metabolismo , Macrófagos/microbiologia , Macrófagos/parasitologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , DNA/análise , Replicação do DNA , Fluorescência , Meia-Vida , Humanos , Leishmaniose/terapia , Macrófagos/citologia , Macrófagos/imunologia , Nanoestruturas/análise , Nanoestruturas/química , Técnicas de Amplificação de Ácido Nucleico , Fagocitose , Fagossomos/química , Fagossomos/microbiologia , Fagossomos/parasitologia , Tuberculose/terapia
2.
Sensors (Basel) ; 21(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34300575

RESUMO

DNA sensors can be used as robust tools for high-throughput drug screening of small molecules with the potential to inhibit specific enzymes. As enzymes work in complex biological pathways, it is important to screen for both desired and undesired inhibitory effects. We here report a screening system utilizing specific sensors for tyrosyl-DNA phosphodiesterase 1 (TDP1) and topoisomerase 1 (TOP1) activity to screen in vitro for drugs inhibiting TDP1 without affecting TOP1. As the main function of TDP1 is repair of TOP1 cleavage-induced DNA damage, inhibition of TOP1 cleavage could thus reduce the biological effect of the TDP1 drugs. We identified three new drug candidates of the 1,5-naphthyridine and 1,2,3,4-tetrahydroquinolinylphosphine sulfide families. All three TDP1 inhibitors had no effect on TOP1 activity and acted synergistically with the TOP1 poison SN-38 to increase the amount of TOP1 cleavage-induced DNA damage. Further, they promoted cell death even with low dose SN-38, thereby establishing two new classes of TDP1 inhibitors with clinical potential. Thus, we here report a dual-sensor screening approach for in vitro selection of TDP1 drugs and three new TDP1 drug candidates that act synergistically with TOP1 poisons.


Assuntos
DNA Topoisomerases Tipo I , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases , DNA , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo
3.
Sensors (Basel) ; 17(6)2017 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-28538659

RESUMO

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.


Assuntos
DNA/química , Ditiotreitol
4.
Exp Mol Pathol ; 99(1): 56-64, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25987486

RESUMO

Topoisomerase I (TOP1) regulates DNA topology during replication and transcription whereas tyrosyl-DNA phosphodiesterase 1 (TDP1) is involved in the repair of several types of DNA damages, including damages from defective TOP1 catalysis. TOP1 is the target of chemotherapeutic drugs of the camptothecin family (CPT). TDP1 has in cell line based assays been shown to counteract the effect of CPT. We have quantified the enzymatic activities of TOP1 and TDP1 in paired (tumor and adjacent non-tumor) samples from non-small cell lung cancer (NSCLC) patients and show that in NSCLC TOP1 and TDP1 activities are significantly upregulated in the tumor tissue. Furthermore, we found a positive correlation between the TDP1 activity and the tumor percentage (TOP1 activity did not correlate with the tumor percentage) as well as between the activities of TOP1 and TDP1 both within the tumor and the non-tumor group. That TDP1 activity was upregulated in all tumor samples and correlated with the tumor percentage suggest that it must play a highly important function in NSCLC. This could be to protect against TOP1 mediated DNA damage as the activity of TOP1 likewise was upregulated in the majority of tumor samples and correlated positively to the TDP1 activity. Regardless, the finding that the TOP1 and TDP1 activities are upregulated and correlate positively suggests that combinatorial treatment targeting both activities could be advantageous in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Técnicas Biossensoriais , Dano ao DNA , Replicação do DNA , Humanos , Nanotecnologia , Sensibilidade e Especificidade
5.
Anal Biochem ; 451: 42-4, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24525043

RESUMO

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage-religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.


Assuntos
DNA Topoisomerases Tipo I/análise , DNA Circular/metabolismo , Magnetismo , Microscopia Confocal , Técnicas de Amplificação de Ácido Nucleico , DNA Topoisomerases Tipo I/metabolismo , DNA Circular/química , Corantes Fluorescentes/química , Humanos , Razão Sinal-Ruído
6.
Sensors (Basel) ; 13(4): 4017-28, 2013 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-23529147

RESUMO

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.


Assuntos
Técnicas Biossensoriais/métodos , Sistemas Computacionais , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Sequência de Bases , Camptotecina/farmacologia , DNA/química , DNA/genética , Corantes Fluorescentes/metabolismo , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Espectrometria de Fluorescência
7.
Nucleic Acids Res ; 35(18): 6170-80, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17827209

RESUMO

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1-206 or 191-206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621-6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation.


Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/química , DNA Super-Helicoidal/metabolismo , Inibidores Enzimáticos/farmacologia , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/química , Humanos , Cinética , Mutação , Plasmídeos/química , Triptofano/química
8.
Nanoscale ; 11(2): 587-597, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30556557

RESUMO

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.


Assuntos
Proteínas de Bactérias/análise , Técnicas Biossensoriais/métodos , DNA Topoisomerases Tipo I/análise , Ácidos Nucleicos Imobilizados/metabolismo , Mycobacterium/isolamento & purificação , Patologia Molecular/métodos , Proteínas de Bactérias/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Corantes Fluorescentes/química , Humanos , Ácidos Nucleicos Imobilizados/química , Mycobacterium/enzimologia , Sensibilidade e Especificidade
9.
Sci Rep ; 8(1): 4122, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29515150

RESUMO

Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3',5,5'-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/química , Malária Falciparum , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Saliva/metabolismo , Biomarcadores/metabolismo , Colorimetria/métodos , Humanos , Malária Falciparum/diagnóstico , Malária Falciparum/enzimologia
10.
Nanoscale ; 9(36): 13546-13553, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28872165

RESUMO

With increasing recognition of the importance in addressing cell-to-cell heterogeneity for the understanding of complex biological systems, there is a growing need for assays capable of single cell analyses. In the current study, we describe the measurement of human topoisomerase I activity in single CD44 positive Caco2 cells specifically captured from a mixed population on glass slides, which were dual functionalized with anti-CD44-antibodies and specific DNA primers. On-slide lysis of captured CD44 positive cells, resulted in the release of human topoisomerase I, allowing the enzyme to circularize a specific linear DNA substrate added to the slides. The generated circles hybridized to the anchored DNA primers and acted as templates for a solid support rolling circle amplification reaction leading to the generation of long tandem repeat products that were detected at the single molecule level in a fluorescent microscope upon hybridization of fluorescent labelled probes. The on-slide detection system was demonstrated to be directly quantitative and specific towards CD44 positive cells. Moreover, it allowed reproducible detection of human topoisomerase I activity in single cells.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Análise de Célula Única/métodos , Células CACO-2 , Primers do DNA , Corantes Fluorescentes , Humanos , Receptores de Hialuronatos
11.
J Mol Biol ; 336(1): 93-103, 2004 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-14741206

RESUMO

The human topoisomerase I N-terminal domain is the only part of the enzyme still not crystallized and the function of this domain remains enigmatical. In the present study, we have addressed the specific functions of individual N-terminal regions of topoisomerase I by characterizing mutants lacking amino acid residues 1-202 or 191-206 or having tryptophane-205 substituted by glycine in a broad variety of in vitro activity assays. As a result of these investigations we find that mutants altered in the region 191-206 distinguished themselves from the wild-type enzyme by a faster strand rotation step, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments. The mutant lacking amino acid residues 1-202 was impaired in blunt end DNA ligation and showed wild-type sensitivity towards camptothecin in relaxation. Taken together, the presented data support a model according to which tryptophane-205 and possibly other residues located between position 191-206 coordinates the restriction of free strand rotation during the topoisomerization step of catalysis. Moreover, tryptophane-205 appears important for the function of the bulk part of the N-terminal domain in direct DNA interaction.


Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Estrutura Terciária de Proteína , Antineoplásicos/metabolismo , Sequência de Bases , Camptotecina/metabolismo , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , Inibidores Enzimáticos/metabolismo , Humanos , Mutação
12.
Mol Biol Int ; 2011: 854626, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22091414

RESUMO

Control of diseases inflicted by protozoan parasites such as Leishmania, Trypanosoma, and Plasmodium, which pose a serious threat to human health worldwide, depends on a rather small number of antiparasite drugs, of which many are toxic and/or inefficient. Moreover, the increasing occurrence of drug-resistant parasites emphasizes the need for new and effective antiprotozoan drugs. In the current study, we describe a synthetic peptide, WRWYCRCK, with inhibitory effect on the essential enzyme topoisomerase I from the malaria-causing parasite Plasmodium falciparum. The peptide inhibits specifically the transition from noncovalent to covalent DNA binding of P. falciparum topoisomerase I, while it does not affect the ligation step of catalysis. A mechanistic explanation for this inhibition is provided by molecular docking analyses. Taken together the presented results suggest that synthetic peptides may represent a new class of potential antiprotozoan drugs.

13.
ACS Nano ; 4(3): 1367-76, 2010 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-20146442

RESUMO

The assembly, structure, and stability of DNA nanocages with the shape of truncated octahedra have been studied. The cages are composed of 12 double-stranded B-DNA helices interrupted by single-stranded linkers of thymidines of varying length that constitute the truncated corners of the structure. The structures assemble with a high efficiency in a one-step procedure, compared to previously published structures of similar complexity. The structures of the cages were determined by small-angle X-ray scattering. With increasing linker length, there is a systematic increase of the cage size and decrease of the twist angle of the double helices with respect to the symmetry planes of the cage structure. In the present study, we demonstrate the length of the single-stranded linker regions, which impose a certain degree of flexibility to the structure, to be the important determinant for efficient assembly. The linker length can be decreased to three thymidines without affecting assembly yield or the overall structural characteristics of the DNA cages. A linker length of two thymidines represents a sharp cutoff abolishing cage assembly. This is supported by energy minimization calculations suggesting substantial hydrogen bond deformation in a cage with linkers of two thymidines.


Assuntos
DNA de Cadeia Simples/química , Nanoestruturas/química , Sequência de Bases , DNA de Cadeia Simples/genética , Eletroforese em Gel de Poliacrilamida , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Termodinâmica , Timidina/química , Difração de Raios X
14.
ACS Nano ; 3(12): 4043-54, 2009 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-19950974

RESUMO

We previously demonstrated the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.


Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Rim/enzimologia , Rim/metabolismo , Microscopia de Fluorescência/métodos , Técnicas de Sonda Molecular , Recombinases/química , Recombinases/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos
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