RESUMO
In this work, a dual-aptamer functionalized magnetic silicon composite was prepared and used to construct a chemiluminescence (CL) sensor for the detection of α-fetoprotein (AFP) and carcinoembryonic antigen (CEA). First, SiO2@Fe3O4 was prepared, and polydiallyl dimethylammonium chloride (PDDA) and AuNPs were sequentially loaded on SiO2@Fe3O4. Subsequently, the complementary strand of CEA aptamer (cDNA2) and the aptamer of AFP (Apt1) were attached to AuNPs/PDDA-SiO2@Fe3O4. Then, the aptamer of CEA (Apt2) and G quadruplex peroxide-mimicking enzyme (G-DNAzyme) were sequentially connected to cDNA2, leading to the final composite. Then, the composite was used to construct a CL sensor. When AFP is present, it will combine with Apt1 on the composite to hinder the catalytic ability of AuNPs to luminol-H2O2, achieving AFP detection. When CEA is present, it will recognize and bind with Apt2, so G-DNAzyme is released to solution and catalyzes the reaction of luminol-H2O2 to achieve CEA determination. After the application of the prepared composite, AFP and CEA were detected in the magnetic medium and supernatant, respectively, after simple magnetic separation. Therefore, the detection of multiple liver cancer markers is realized through the CL technology without additional instruments or technology, which broadens the application range of CL technology. The sensor for detecting AFP and CEA shows wide linear ranges of 1.0 × 10-4 to 1.0 ng·mL-1 and 0.0001-0.5 ng·mL-1 and low detection limits of 6.7 × 10-5 ng·mL-1 and 3.2 × 10-5 ng·mL-1, respectively. Finally, the sensor was successfully used to detect CEA and AFP in serum samples and provides great potential for detection of multiple liver cancer markers in early clinical diagnosis.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Nanopartículas Metálicas , Antígeno Carcinoembrionário , Silício , alfa-Fetoproteínas , Dióxido de Silício , Peróxido de Hidrogênio , Luminescência , DNA Catalítico/metabolismo , DNA Complementar , Ouro , LuminolRESUMO
Sequential control of exogenous chemical events inside cells is a promising way to regulate cell functions and fate. Herein we report a DNA nanocomplex containing cascade DNAzymes and promoter-like Zn-Mn-Ferrite (ZMF), achieving combined gene/chemo-dynamic therapy. The promoter-like ZMF decomposed in response to intratumoral glutathione to release a sufficient quantity of metal ions, thus promoting cascade DNA/RNA cleavage and free radical generation. Two kinds of DNAzymes were designed for sequential cascade enzymatic reaction, in which metal ions functioned as cofactors. The primary DNAzyme self-cleaved the DNA chain with Zn2+ as cofactor, and produced the secondary DNAzyme; the secondary DNAzyme afterwards cleaved the EGR-1 mRNA, and thus downregulated the expression of target EGR-1 protein, achieving DNAzyme-based gene therapy. Meanwhile, the released Zn2+ , Mn2+ and Fe2+ induced Fenton/Fenton-like reactions, during which free radicals were catalytically generated and efficient chemo-dynamic therapy was achieved. In a breast cancer mouse model, the administration of DNA nanocomplex led to a significant therapeutic efficacy of tumor growth suppression.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Fototerapia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , DNA/química , DNA/metabolismo , DNA Catalítico/química , DNA Catalítico/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Compostos Férricos/química , Compostos Férricos/metabolismo , Terapia Genética , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Manganês/química , Manganês/metabolismo , Camundongos , Nanopartículas/química , Nanopartículas/metabolismo , Zinco/química , Zinco/metabolismoRESUMO
Extraction of uranium from seawater is critical for the sustainable development of nuclear energy. However, the currently available uranium adsorbents are hampered by co-existing metal ion interference. DNAzymes exhibit high selectivity to specific metal ions, yet there is no DNA-based adsorbent for extraction of soluble minerals from seawater. Herein, the uranyl-binding DNA strand from the DNAzyme is polymerized into DNA-based uranium extraction hydrogel (DNA-UEH) that exhibits a high uranium adsorption capacity of 6.06 mg g-1 with 18.95 times high selectivity for uranium against vanadium in natural seawater. The uranium is found to be bound by oxygen atoms from the phosphate groups and the carbonyl groups, which formed the specific nano-pocket that empowers DNA-UEH with high selectivity and high binding affinity. This study both provides an adsorbent for uranium extraction from seawater and broadens the application of DNA for being used in recovery of high-value soluble minerals from seawater.
Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , Água do Mar/química , Urânio/isolamento & purificação , Fracionamento Químico/métodos , Hidrogéis , Oxigênio/química , Fosfatos/química , Espectroscopia Fotoeletrônica , Urânio/metabolismoRESUMO
The therapeutic performance of DNAzyme-involved gene silencing is significantly constrained by inefficient conditional activation and insufficient cofactor supply. Herein, a self-sufficient therapeutic nanosystem was realized through the delicate design of DNAzyme prodrugs and MnO2 into a biocompatible nanocapsule with tumor-specific recognition/activation features. The indocyanine green (ICG)-modified DNA prodrugs are designed by splitting the DNAzyme and then reconstituted into the exquisite catalyzed hairpin assembly (CHA) amplification circuit. Based on the photothermal activation of ICG, the nanocapsule was disassembled to expose the MnO2 ingredient which was immediately decomposed into Mn2+ ions to supplement an indispensable DNAzyme cofactor on-demand with a concomitant O2 generation for enhancing the auxiliary phototherapy. The endogenous microRNA catalyzes the amplified assembly of DNA prodrugs via an exquisite CHA principle, leading to the DNAzyme-mediated simultaneous silencing of two key tumor-involved mRNAs. This self-activated theranostic nanocapsule could substantially expand the toolbox for accurate diagnosis and programmable therapeutics.
Assuntos
DNA Catalítico/metabolismo , Terapia Genética , Nanocápsulas/química , Fototerapia , Nanomedicina Teranóstica , Animais , Linhagem Celular Tumoral , DNA Catalítico/química , DNA Catalítico/genética , Inativação Gênica , Humanos , Verde de Indocianina/química , Verde de Indocianina/metabolismo , Compostos de Manganês/química , Compostos de Manganês/metabolismo , Camundongos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Óxidos/química , Óxidos/metabolismo , Tamanho da Partícula , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Propriedades de SuperfícieRESUMO
RNA-cleaving DNAzyme is a very useful biomaterial for metal ions determination. However, parts of DNAzymes can be cleaved by several metal ions, which makes it difficult to distinguish the concentrations of different metal ions. A method was applied to determine the Cu(II) concentration by using electrochemical biosensors combined with a mathematical model. An electrochemical biosensor was fabricated using single carbon nanotubes/field-effect transistor (SWNTs/FET) functionalized with a DNAzyme named PSCu10 and its complementary DNA embedded phosphorothioate RNA (CS-DNA). The CS-DNA with amino groups at the 5' end was immobilized on the SWNTs' surface via the peptide bond and then combined with PSCu10 by identifying bases complementary pairing (Cuzyme/SWNTs/FET). The CS-DNA can be cleaved when Cu(II) bonded with the PSCu10 so that the structural change of Cuzyme improves the electrical conductivity of Cuzyme/SWNTs/FET. But CS-DNA also can be cut-off by the Hg(II) directly, which might interfere with the detection of the Cu(II) concentration using Cuzyme/SWNTs/FET. To solve this problem, Hgzyme/SWNTs/FET was employed to monitor the Hg(II) concentration at the same time, thus serving to determine the Cu(II) content through the Gaussian process regression. The biosensor array can determine the Cu(II) concentration varying from 0.01 to 10,000 nM when the Hg(II) concentration was ranging from 5 to 10,000 nM, and the limits of detection for Cu(II) and Hg(II) were 6.7 pM and 3.43 nM, respectively. Graphical abstract A biosensor array (Cuzyme/SWNTs/FET and Hgzyme/SWNTs/FET) was developed, and the detecting data were processed using Gaussian process regression. It allows determination of Cu(II) and Hg(II) concentrations with high accuracy.
Assuntos
Técnicas Biossensoriais , Cobre/análise , DNA Catalítico/metabolismo , Técnicas Eletroquímicas , Mercúrio/análise , Nanotubos de Carbono/química , Cobre/metabolismo , DNA Catalítico/química , Aprendizado de Máquina , Mercúrio/metabolismoRESUMO
Although photothermal therapy (PTT) is preclinically applied in solid tumor treatment, incomplete tumor removal of PTT and heat endurance of tumor cells induces significant tumor relapse after treatment, therefore lowering the therapeutic efficiency of PTT. Herein, a programmable therapeutic strategy that integrates photothermal therapeutic agents (PTAs), DNAzymes, and artificial engineered natural killer (A-NK) cells for immunotherapy of hepatocellular carcinoma (HCC) is designed. The novel PTAs, termed as Mn-CONASHs, with 2D structure are synthesized by the coordination of tetrahydroxyanthraquinone and Mn2+ ions. By further adsorbing polyetherimide/DNAzymes on the surface, the DNAzymes@Mn-CONASHs exhibit excellent light-to-heat conversion ability, tumor microenvironment enhanced T1 -MRI guiding ability, and antiheat endurance ability. Furthermore, the artificial engineered NK cells with HCC specific targeting TLS11a-aptamer decoration are constructed for specifically eliminating any possible residual tumor cells after PTT, to systematically enhance the therapeutic efficacy of PTT and avoid tumor relapse. Taken together, the potential of A-NK cells combined with antiheat endurance as a powerful strategy for immuno-enhancing photothermal therapy efficiency of solid tumors is highlighted, and the current strategy might provide promising prospects for cancer therapy.
Assuntos
Células Artificiais , Carcinoma Hepatocelular/terapia , Engenharia Celular , Hipertermia Induzida , Imunoterapia , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/terapia , Fototerapia , Carcinoma Hepatocelular/diagnóstico por imagem , DNA Catalítico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Hepáticas/diagnóstico por imagem , Imageamento por Ressonância Magnética , Manganês/química , Nanopartículas/ultraestrutura , Espectrometria de Fluorescência , Microambiente TumoralRESUMO
Telomerase has been regarded as a biomarker for cancer diagnosis as well as the clinical treatment and the reliable detection of intracellular telomerase activity is of great significance. By developing a telomere elongation-based DNA-catalytic amplification strategy, a novel surface-enhanced Raman scattering (SERS) method is proposed for the assay of telomerase activity. In the presence of telomerase and nucleotide mixture dNTPs, the telomerase substrate (TS) primer extended and generated a long single-strand DNA (ssDNA) containing the telomere repeat units (TTAGGG)n, which could catalyze the entropy-driven circuit reaction (EDCR). One of the products of EDCR was ingeniously used as the catalyst of catalytic hairpin assembly (CHA) occured on magnetic beads (MBs). As a result, a large amount of ROX-labeled Raman probes could be anchored on the surface of MBs and used for SERS detection. Using this strategy, the assay can detect telomerase activity from cell extracts equivalent down to single HeLa cell.
Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , Análise Espectral Raman/métodos , Telomerase/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Ensaios Enzimáticos/métodos , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Especificidade por Substrato , Telomerase/antagonistas & inibidores , Telômero/metabolismoRESUMO
A uranyl detection strategy with ultra-sensitivity was developed based on entropy-driven amplification and DNAzyme circular cleavage amplification. The cleavage of UO22+-specific DNAzyme produces a DNA fragment to initiate the entropy-driven amplification. Two DNA sequences released from the entropy-driven amplification are partly complementary. They can form an entire enzyme strand (E-DNA) of Mg2+-specific DNAzyme. The formed E-DNA can circularly cleave FAM-labeled probes on gold nanoparticles (AuNPs), causing the leaving of FAM from AuNPs and recovery of fluorescent signal. A linear relationship was obtained in the range from 30 pM to 5â¯nM between fluorescence intensity and concentration of UO22+. The limit of detection was low to 13 pM. This method showed a promising future for practical application in real water samples.
Assuntos
Técnicas Biossensoriais , DNA Catalítico/química , Entropia , Fluorescência , Técnicas de Amplificação de Ácido Nucleico , Urânio/análise , DNA Catalítico/metabolismoRESUMO
A visual and sensitive Hg2+ detection strategy was developed based on split DNAzyme amplification and hemin-graphene oxide composites (H-GNs). Two split DNAzyme sequences can form two entire enzyme-strands DNA (E-DNA) by T-Hg2+-T interaction. The E-DNA can bind with the loop of molecular beacon (MB) to form Mg2+-dependent DNAzyme structure. The formed DNAzyme can circularly cleave the loop of MB, resulting large amount of DNA fragments. The resultant DNA fragments can prevent H-GNs from aggregation by adsorbing on its surface. Consequently, the supernate with large amount of H-GNs shows dark blue color after chromogenic reaction. This strategy shows a linear range from 50â¯pM to 1200â¯pM. The limit detection can be low to 33â¯pM. This strategy provides a visual and enzyme-free amplification mode for quick and sensitive screen of Hg2+.
Assuntos
Colorimetria/métodos , DNA Catalítico/metabolismo , Grafite/química , Hemina/química , Mercúrio/análise , Atractylodes/química , DNA Catalítico/química , Limite de Detecção , Peroxidases/metabolismo , Plantas Medicinais/químicaRESUMO
Catalytic deoxyribozyme has great potential for gene regulation, but the poor efficiency of the cleavage of mRNA and the lack of versatile DNAzyme vehicles remain big challenges for potent gene therapy. By the rational designing of a diverse vehicle of polydopamine-Mn2+ nanoparticles (MnPDA), we demonstrate that MnPDA has integrated functions as an effective DNAzyme delivery vector, a self-generation source of DNAzyme cofactor for catalytic mRNA cleavage, and an inherent therapeutic photothermal agent as well as contrast agent for photoacoustic and magnetic resonance imaging. Specifically, the DNAzyme-MnPDA nanosystem protects catalytic deoxyribozyme from degradation and enhances cellular uptake efficiency. In the presence of intracellular glutathione, the nanoparticles are able to in situ generate free Mn2+ as a cofactor of DNAzyme to effectively trigger the catalytic cleavage of mRNA for gene silencing. In addition, the nanosystem shows high photothermal conversion efficiency and excellent stability against photothermal processing and degradation in complex environments. Unlike previous DNAzyme delivery vehicles, this vehicle exhibits diverse functionalities for potent gene regulation, allowing multimodal imaging-guided synergetic gene regulation and photothermal therapy both in vitro and in vivo.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Meios de Contraste/farmacologia , DNA Catalítico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fototerapia , Animais , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Meios de Contraste/química , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Indóis/química , Indóis/metabolismo , Células MCF-7 , Imageamento por Ressonância Magnética , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Manganês/química , Manganês/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/metabolismo , Técnicas Fotoacústicas , Polímeros/química , Polímeros/metabolismoRESUMO
A DNAzyme-embedded hyperbranched DNA dendrimer is used as a colorimetric signal amplifier in an ultrasensitive detection scheme for nucleic acids. The hyperbranched DNA dendrimers were constructed by single-step autonomous self-assembly of three structure-free DNA monomers. A cascade of self-assembly reactions between the first and second strands leads to the formation of linear DNA concatemers containing overhang flank fragments. The third strand, which bears a peroxidase-mimicking DNAzyme domain, serves as a bridge to trigger self-assembly between the first and second strands across the side chain direction. This results in a chain branching growth of the DNAzyme-embedded DNA dendrimer. This signal amplifier was incorporated into the streptavidin-biotin detection system which comprises an adaptor oligonucleotide and a biotinylated capture probe. The resulting platform is capable of detecting a nucleic acid target with an LOD as low as 0.8 fM. Such sensitivity is comparable if not superior to most of the reported enzyme-free (and even enzyme-assisted) signal amplification strategies. The DNA dendrimer based method is expected to provide a universal platform for extraordinary signal enhancement in detecting other nucleic acid biomarkers by altering the respective sequences of adaptor and capture probe. Graphical abstract Schematic of an assembly of a DNAzyme-embedded hyperbranched DNA dendrimer which operates as a signal amplifier for nucleic acids detection. The nanostructure is constructed by autonomous self-assembly of three DNA monomers. Colored letters represent each domain, and complementary domains are marked by asterisk. Domain d represents the DNAzyme sequence.
Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , DNA/análise , DNA/química , Dendrímeros/química , Colorimetria , DNA Catalítico/química , Limite de DetecçãoRESUMO
In this study, a novel colorimetric aptasensor was prepared by coupling trivalent peroxidase-mimic DNAzyme and magnetic nanoparticles for highly sensitive and selective detection of target proteins. A three G-quadruplex (G4) DNA-hemin complex was employed as the trivalent peroxidase-mimic DNAzyme, in which hemin assisted the G4-DNA to fold into a catalytic conformation and act as an enzyme. The design of the aptasensor includes magnetic nanoparticles (MNPs), complementary DNA (cDNA) modified with biotin, and a label-free single strand DNA (ssDNA) including the aptamer and trivalent peroxidase-mimic DNAzyme. The trivalent DNAzyme, which has the highest catalytic activity among multivalent DNAzymes, catalyzed the H2O2-mediated oxidation of ABTS. The colorless ABTS was oxidized to produce a blue-green product that can be clearly distinguished by the naked eye. The aptamer and trivalent peroxidase-mimic DNAzyme promote the specificity and sensitivity of this detection method, which can be generalized for other targets by simply replacing the corresponding aptamers. To demonstrate the feasible use of the aptasensor for target detection, a well-known tumor biomarker MUC1 was evaluated as the model target. The limits of detection were determined to be 5.08 and 5.60â¯nM in a linear range of 50-1000â¯nM in a buffer solution and 10% serum system, respectively. This colorimetric and label-free aptasensor with excellent sensitivity and strong anti-interference ability has potential application in disease diagnoses, prognosis tracking, and therapeutic evaluation.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/análise , Colorimetria , DNA Catalítico/metabolismo , Nanopartículas de Magnetita/química , Mucina-1/análise , Peroxidase/metabolismo , Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/metabolismo , Biotina/química , Biotina/metabolismo , Colorimetria/instrumentação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Quadruplex G , Humanos , Mucina-1/metabolismoRESUMO
Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10-23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg[Formula: see text] bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg[Formula: see text] ions more strongly and already stoichiometric amounts of Mg[Formula: see text] and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg[Formula: see text] concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg[Formula: see text] concentrations than that available inside the cell.
Assuntos
DNA Catalítico/metabolismo , Magnésio/metabolismo , RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Humanos , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
DNAzymes are catalytically active DNA molecules that are obtained via in vitro selection. RNA-cleaving DNAzymes have attracted significant attention for both therapeutic and diagnostic applications due to their excellent programmability, stability, and activity. They can be designed to cleave a specific mRNA to down-regulate gene expression. At the same time, DNAzymes can sense a broad range of analytes. By combining these two functions, theranostic DNAzymes are obtained. This review summarizes the progress of DNAzyme for theranostic applications. First, in vitro selection of DNAzymes is briefly introduced, and some representative DNAzymes related to biological applications are summarized. Then, the applications of DNAzyme for RNA cleaving are reviewed. DNAzymes have been used to cleave RNA for treating various diseases, such as viral infection, cancer, inflammation and atherosclerosis. Several formulations have entered clinical trials. Next, the use of DNAzymes for detecting metal ions, small molecules and nucleic acids related to disease diagnosis is summarized. Finally, the theranostic applications of DNAzyme are reviewed. The challenges to be addressed include poor DNAzyme activity under biological conditions, mRNA accessibility, delivery, and quantification of gene expression. Possible solutions to overcome these challenges are discussed, and future directions of the field are speculated.
Assuntos
DNA Catalítico/metabolismo , RNA/metabolismo , Nanomedicina Teranóstica/métodos , Animais , Terapia Biológica/métodos , Pesquisa Biomédica/tendências , Testes Diagnósticos de Rotina/métodos , HumanosRESUMO
In the present work, we constructed a new label-free "inter-sandwich" electrochemical aptasensor for thrombin (TB) detection by employing a cleavage-based hybridization chain reaction (HCR). The designed single-stranded DNA (defined as binding DNA), which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence, was first immobilized on the electrode. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu(2+) could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer-protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu(2+)-dependent DNAzyme. As a result, the signal reporter sequence could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6](3+)) was embedded into the double-stranded DNA (dsDNA) to produce a strong electrochemical signal for the quantitative measurement of TB. For further amplification of the electrochemical signal, graphene reduced by dopamine (PDA-rGO) was introduced as a platform in this work. With this strategy, the aptasensor displayed a wide linearity in the range of 0.0001 nM to 50 nM with a low detection limit of 0.05 pM. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA Catalítico/metabolismo , Técnicas Eletroquímicas/métodos , Trombina/análise , Aptâmeros de Nucleotídeos/química , Cobre/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Nanocompostos/química , Nanocompostos/ultraestrutura , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Trombina/metabolismoRESUMO
Rational design of smart MRI contrast agents with high specificity for metal ions remains a challenge. Here, we report a general strategy for the design of smart MRI contrast agents for detecting metal ions based on conjugation of a DNAzyme with a gadolinium complex. The 39E DNAzyme, which has high selectivity for UO2(2+), was conjugated to Gd(III)-DOTA and streptavidin. The binding of UO2(2+) to its 39E DNAzyme resulted in the dissociation of Gd(III)-DOTA from the large streptavidin, leading to a decrease of the T1 correlation time, and a change in the MRI signal.
Assuntos
Meios de Contraste/química , DNA Catalítico/química , Gadolínio/química , Imageamento por Ressonância Magnética/métodos , Urânio/química , Meios de Contraste/metabolismo , DNA Catalítico/metabolismo , Gadolínio/metabolismo , Urânio/metabolismoRESUMO
DNAzymes are known to bind metal ions specifically to carry out catalytic functions. Despite many studies since DNAzymes were discovered nearly two decades ago, the metal-binding sites in DNAzymes are not fully understood. Herein, we adopt uranyl photocleavage to probe specific uranyl-binding sites in the 39E DNAzyme with catalytically relevant concentrations of uranyl. The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand. Control experiments using two 39E DNAzyme mutants revealed a different cleavage pattern of the mutated region. Another DNAzyme, the 8-17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well. In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated. Together, these experiments suggest that uranyl photocleavage has been successfully used to probe catalytically relevant uranyl-binding sites in the 39E DNAzyme. As uranyl is the cofactor of the 39E DNAzyme as well as the probe, specific uranyl binding has now been identified without disruption of the structure.
Assuntos
Clivagem do DNA , DNA Catalítico/química , Urânio/química , Sítios de Ligação , Cátions Bivalentes , Coenzimas/química , DNA Catalítico/metabolismo , Mutação , Conformação de Ácido Nucleico , FotóliseRESUMO
T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of λ exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5'-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by λ exonuclease. As a result, the blocked 'HRPzyme' sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL(-1) and a wide detection range from 0.06 to 100 U mL(-1). Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs.
Assuntos
Colorimetria , DNA Catalítico/metabolismo , DNA/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Trifosfato de Adenosina/química , Sulfato de Amônio/química , Clivagem do DNA , Exonucleases/metabolismo , Hemina/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Sequências Repetidas Invertidas , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/antagonistas & inibidoresRESUMO
Since the initial discovery of the catalytic capability of short DNA fragments, this peculiar enzyme-like property (termed DNAzyme) has continued to garner much interest in the scientific community because of the virtually unlimited applications in developing new molecular devices. Alongside the exponential rise in the number of DNAzyme applications in the last past years, the search for convenient ways to improve its overall efficiency has only started to emerge. Credence has been lent to this strategy by the recent demonstration that the quadruplex-based DNAzyme proficiency can be enhanced by ATP supplements. Herein, we have made a further leap along this path, trying first of all to decipher the actual DNAzyme catalytic cycle (to gain insights into the steps ATP may influence), and subsequently investigating in detail the influence of all the parameters that govern the catalytic efficiency. We have extended this study to other nucleotides and quadruplexes, thus demonstrating the versatility and broad applicability of such an approach. The defined exquisitely efficient DNAzyme protocols were exploited to highlight the enticing advantages of this method via a 96-well plate experiment that enables the detection of nanomolar DNA concentrations in real-time with the naked-eye (see movie as Supplementary Data).
Assuntos
DNA Catalítico/metabolismo , Quadruplex G , Hemina/química , Nucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Catálise , DNA/análise , DNA Catalítico/química , Peroxidases/metabolismoRESUMO
Portable, low-cost and quantitative detection of a broad range of targets at home and in the field has the potential to revolutionize medical diagnostics and environmental monitoring. Despite many years of research, very few such devices are commercially available. Taking advantage of the wide availability and low cost of the pocket-sized personal glucose meter-used worldwide by diabetes sufferers-we demonstrate a method to use such meters to quantify non-glucose targets, ranging from a recreational drug (cocaine, 3.4 µM detection limit) to an important biological cofactor (adenosine, 18 µM detection limit), to a disease marker (interferon-gamma of tuberculosis, 2.6 nM detection limit) and a toxic metal ion (uranium, 9.1 nM detection limit). The method is based on the target-induced release of invertase from a functional-DNA-invertase conjugate. The released invertase converts sucrose into glucose, which is detectable using the meter. The approach should be easily applicable to the detection of many other targets through the use of suitable functional-DNA partners (aptamers, DNAzymes or aptazymes).