RESUMO
Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn2+-specific near-infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photodecages a substrate strand containing a nitrobenzyl group at the 2'-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn2+-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn2+ sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos and detecting in the visible region. In this study, we introduce a platform that can be used to understand the Zn2+ distribution with spatiotemporal control, thereby giving insights into the dynamical Zn2+ ion distribution in intracellular and in vivo models.
Assuntos
DNA Catalítico/química , Corantes Fluorescentes/química , Nanopartículas/química , Zinco/análise , Alcanossulfonatos/química , Alcanossulfonatos/toxicidade , Animais , Compostos Azo/química , Compostos Azo/toxicidade , Sequência de Bases , DNA Catalítico/síntese química , DNA Catalítico/toxicidade , Fluoresceínas/química , Fluoresceínas/toxicidade , Fluorescência , Corantes Fluorescentes/toxicidade , Fluoretos/química , Fluoretos/toxicidade , Células HeLa , Humanos , Raios Infravermelhos , Microscopia Confocal , Microscopia de Fluorescência , Nanopartículas/efeitos da radiação , Nanopartículas/toxicidade , Túlio/química , Túlio/toxicidade , Itérbio/química , Itérbio/toxicidade , Ítrio/química , Ítrio/toxicidade , Peixe-ZebraRESUMO
DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.
Assuntos
Reatores Biológicos , Biotecnologia/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/síntese química , Nanotecnologia/métodos , Nanotubos/química , Conformação de Ácido Nucleico , Sequência de Bases , Biotecnologia/economia , DNA Catalítico/síntese química , DNA Catalítico/química , DNA Catalítico/economia , DNA Catalítico/metabolismo , DNA de Cadeia Simples/economia , DNA de Cadeia Simples/metabolismo , Nanotecnologia/economiaRESUMO
Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.
Assuntos
DNA Catalítico/química , Conformação de Ácido Nucleico , Sequência de Bases , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , DNA Catalítico/síntese química , DNA Catalítico/metabolismo , Desoxirribose/química , Desoxirribose/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Polinucleotídeo Ligases/química , Polinucleotídeo Ligases/metabolismo , RNA/química , RNA/metabolismo , Dobramento de RNA , Especificidade por SubstratoRESUMO
In this work, a new kind of peroxidase-mimicking DNAzyme (G-quadruplex-hemin DNAzyme, G4-hemin) was constructed by using hemin-modified G-rich DNA (hemin-G-DNA). Experimental results demonstrated that the G-rich DNA can form a G-quadruplex structure by the inducement of terminally modified hemin, rendering the assembly of hemin and G-quadruplex structure spontaneously and efficiently. As a result, G-hemin revealed higher peroxidase activity than traditional G-quadruplex/hemin DNAzyme (G4/hemin). Besides, different from G4/hemin, G4-hemin was constructed in one step without the participation of metal ions and adscititious hemin. Accordingly, the construction procedure was significantly simplified and the background signal from dissociative hemin was remarkably reduced. In a proof-of-concept trial, according to the colorimetric signals of G4-hemin, a novel biosensor for the detection of S1 nuclease activity was established, which provides a novel perspective for designing peroxidase-mimicking DNAzyme-based biosensors.
Assuntos
DNA Catalítico/síntese química , Desoxirribonucleases/análise , Quadruplex G , Hemina/síntese química , Metais/química , Animais , Bovinos , Íons , Peroxidase , Potássio , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
A cascade recycling amplification (CRA) that implements cascade logic circuits with feedback amplification function is developed for label-free chemiluminescence detection of microRNA-122 with an ultrahigh sensitivity of 0.82 fM and excellent specificity, which is applied to construct a series of molecular-scale two-input logic gates by using microRNAs as inputs and CRA products as outputs.
Assuntos
Técnicas Biossensoriais/métodos , Computadores Moleculares , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA/síntese química , DNA Polimerase I/química , DNA Catalítico/síntese química , Eletroforese em Gel de Poliacrilamida , Endonucleases/química , Sequências Repetidas Invertidas , Hibridização de Ácido NucleicoRESUMO
BACKGROUND: The recent discovery of microRNAs (miRNAs) and their extracellular presence suggest a potential role of these regulatory molecules in defining the metastatic potential of cancer cells and mediating the cancer-host communication. This study aims to improve the sensitivity of miRNA detection via DNAzyme-based method and enhance the selectivity by using the DNAzyme-based probe to reduce nonspecific amplification. METHODS: The miRNA probes were chemically synthesized with a phosphate at the 5' end and purified by polyacrylamide gel electrophoresis. Exosomal RNA from peripheral blood was isolated. Carboxylated magnetic microsphere beads (MBs) were functionalized with streptavidin (SA) according to a previously reported method with some modification. T capture probe-coated SA-MBs (DNA-MBs) were also prepared. The fluorescent spectra were measured using a spectrofluorophotometer. RESULTS: We designed an incomplete DNAzyme probe with two stems and one bubble structure as a recognition element for the specific detection of miRNA with high sensitivity. The background effects were decreased with increase of the added of DNA-MBs and capturing times. Therefore, 20 minutes was selected as the optimal concentration in the current study. The fluorescence intensity increases as the hybridization time changed and reached a constant level at 40 minutes, and 1 µM is the optimum signal probe concentration for self-assembled DNA concatemers formation. In the presence of miRNA, the fluorescence of the solution increased with increasing miRNA concentration. There is no obvious fluorescence in the presence of 10 mM of other nontarget DNA. CONCLUSION: A simple, rapid method with high performance has been constructed based on identified circulating miRNA signatures using miRNA-induced DNAzyme. This assay is simple, inexpensive, and sensitive, enabling quantitative detection of as low as 10 fM miRNA.
Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , MicroRNAs/sangue , DNA Catalítico/sangue , DNA Catalítico/síntese química , Corantes Fluorescentes , Humanos , Espectrometria de FluorescênciaRESUMO
10-23 DNAzyme is capable of catalytically cleaving RNA substrates with the preferred cleavage sites rAU and rGU, in which the common base pair U-dA0 forms between the substrate and the DNAzyme in the cleavage reaction. Here its conservation was studied with base modifications on dA and extra functional groups introduced. The nitrogen atom at 7- or 8-position of adenine was demonstrated to be equally important for the cleavage reaction, although it is not related to the thermal stability of the base pair. Deletion of 6-amino group led to decreased stability of the base pair and a slight slower reaction rate. Extra functional groups through 6-amino group were not favorably accommodated in the cleavage site. From these modifications at the level of functional groups, it demonstrated that the base pair U-dA0 not only contributes to the recognition and binding stability, but also it is involved in the active catalytic center by its functional groups and base stacking. This kind of chemical modifications with 7-substituted 8-aza-7-deaza-2'-deoxyadenosine at dA0 is favorable for the introduction of signal molecules for mechanistic studies and biological applications, without significant loss of the catalytic function and structural destruction.
Assuntos
Adenina/química , DNA Catalítico/química , DNA Catalítico/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Pareamento de Bases , Domínio Catalítico , Técnicas de Química Sintética , Dicroísmo Circular , DNA Catalítico/síntese química , DNA de Cadeia Simples/síntese química , Desoxiadenosinas/química , Manganês/química , Uracila/químicaRESUMO
The highly conserved untranslated regions of the hepatitisâ C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2'R)-, (2'S)-2'-deoxy-2'-C-methyl- and -2'-O-methylnucleosides at various positions of the catalytic core were assayed against the 5'-internal ribosome entry site element (5'-IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes' cores were jointly modified with 2'-C-methyl- and 2'-O-methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48â h of transfection. Different regions of the HCV IRES were explored with unmodified 10-23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES-firefly luciferase translation-dependent RNA (IRES-FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh-7 human hepatocarcinoma cell line. Inhibition of IRES-dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , DNA Catalítico/síntese química , DNA Catalítico/farmacologia , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/farmacologia , Hepacivirus/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Ribossomos/efeitos dos fármacos , Regiões não TraduzidasRESUMO
OBJECTIVES: To develop a novel screening method for DNAzymes targeting the LMP1 carboxy region. STUDY DESIGN: To design a method to screen special DNAzymes toward the Epstein-Barr virus (EBV)-associated carcinoma before clinic use. SETTING: Key Laboratory of the Ministry of Education-Molecular Biology of Infectious Diseases in Chongqing Medical University. SUBJECTS AND METHODS: Four novel 10-23 DNAzymes (DZ509, DZ1037, DZ893, and DZ827) targeting the EBV-LMP1 gene were designed and evaluated by detecting enhanced green fluorescence protein (EGFP) expression of LMP1 mRNA and the protein in the nasopharyngeal carcinoma (NPC) cell line CNE2 transfected with the pEGFP-C1-LMP1c vector. The screened specific DNAzymes were then transfected into NPC cell lines C666-1 while a mutant oligonucleotide mutDZ509 and an antisense oligonucleotide ASODN509 were designed as positive and negative controls. Cell proliferation, cell apoptosis, LMP1 mRNA, and the protein were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V-fluorescence isothiocyanate (FITC), reverse transcription polymerase chain reaction (RT-PCR), and Western blots. RESULTS: The inhibition rates of fluorescence expression of the DNAzymes DZ509, DZ1037, DZ893, and DZ827 were 91.25%, 65.84%, 49.02%, and 44.56%, respectively. The results were in accordance with the inhibition effects of mRNA and protein expression. The screened DZ509 could effectively knock down endogenous LMP1 expression in C666-1 cells, inhibit cell proliferation, and induce cell apoptosis compared with mutDZ509 and ASODN509. CONCLUSION: LMP1 could present a potential target for DNAzymes toward the EBV-associated carcinoma, and the EGFP expression vector could be a visible method for screening special DNAzymes before clinic use.
Assuntos
DNA Catalítico/farmacologia , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteínas Oncogênicas Virais/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Carcinoma , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Catalítico/síntese química , Humanos , Microscopia de Fluorescência , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/fisiopatologia , Neoplasias Nasofaríngeas/virologia , Proteínas Oncogênicas Virais/biossíntese , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas da Matriz Viral/biossínteseRESUMO
One of the hallmarks of progression of influenza virus replication is the step involving the virus uncoating that occurs in the host cytoplasm. The BM2 ion channel protein of influenza B virus is highly conserved and is essentially required during the uncoating processes of virus, thus an attractive target for designing antiviral drugs. We screened several DNA enzymes (Dzs) containing the 10-23 catalytic motif against the influenza B virus BM2 RNA. Dzs directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. The Dz209 not only showed significant intracellular reduction of BM2 gene expression in transient-expression system but also provided considerable protection against influenza B virus challenge in MDCK cells. Our findings suggest that the Dz molecule can be used as selective and effective inhibitor of viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza B virus infection.
Assuntos
Antivirais/metabolismo , DNA Catalítico/metabolismo , Vírus da Influenza B/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/genética , Animais , Antivirais/síntese química , Domínio Catalítico , DNA Catalítico/síntese química , Cães , Vírus da Influenza B/genética , Vírus da Influenza B/crescimento & desenvolvimento , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Motivos de Nucleotídeos , Clivagem do RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/química , RNA Viral/genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Influenza A viruses take a significant place in human and animal pathology causing epidemics and epizootics. Therefore, the development of new antiflu drugs has become more and more urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently and highly specifically cleave RNA molecules. In this study, a number ofgenomic sequences of the most relevant influenza A virus subtypes, H5N1, H3N2, and H1N1, were analyzed. Conservative regions were revealed in five the least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with "10-23" deoxyribozymes were determined. 46 virus-specific 33-mer deoxyribozymes with the general structure of 5'N8AGGCTAGCTACAACGAN9 were designed and synthesized. Screening of the antiviral activity of these agents in conjugation with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005 (H5N1) revealed 17 deoxyribozymes, which suppressed the titer of virus cytopathicity by more than 2.5 IgTCID50/mL (i.e. the virus neutralization index was more than 300), with five of them suppressing the virus titer by a factor of 1000 and more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded nucleoprotein (NP).
Assuntos
Antivirais/farmacologia , DNA Catalítico/farmacologia , DNA de Cadeia Simples , Influenza Humana , Animais , Antivirais/síntese química , DNA Catalítico/síntese química , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/farmacologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Humana/tratamento farmacológico , Influenza Humana/genética , Células Madin Darby de Rim Canino/efeitos dos fármacos , RNA Viral/química , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
RNA-cleaving DNAzymes were constructed to target the point mutation in the BCR-ABL transcript that causes imatinib resistance in leukemic cells. We examined the effect of 12mer peptide nucleic acids (PNAs) as facilitator oligonucleotides that bind to RNA substrate at the termini of the DNAzyme to improve DNAzyme-mediated cleavage of full-length RNA. When imatinib-resistant cells were transfected with the facilitator PNA and DNAzyme, DNAzyme activity was enhanced and the cells were sensitized to imatinib treatment. Thus, facilitator PNA may be used to enhance activity of antisense oligonucleotide targeting the full-length transcript.
Assuntos
DNA Catalítico/metabolismo , Genes abl , Ácidos Nucleicos Peptídicos/metabolismo , RNA Mensageiro/metabolismo , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Benzamidas , Linhagem Celular Tumoral , DNA Catalítico/síntese química , DNA Catalítico/genética , Engenharia Genética , Humanos , Mesilato de Imatinib , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/genética , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética , Piperazinas/farmacologia , Polimorfismo de Nucleotídeo Único , Pirimidinas/farmacologia , Clivagem do RNA/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/genética , Especificidade por Substrato , TransfecçãoRESUMO
Ezrin, a membrane organizer and linker between plasma membrane and cytoskeleton, is well documented to play an important role in the metastatic capacity of cancer cells especially for osteosarcoma cells. It has provided an ideal target for cancer gene therapy. RNA-cleaving 10-23 DNAzymes, consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, can cleave the target mRNA at purine-pyrimidine dinucleotide effectively. In the present study, we designed and screened the target sites for 10-23 DNAzymes against ezrin mRNA by using multiple computational methods with combination of secondary structural and hybridization thermodynamic parameters. Then, we testified the activities of the DNAzymes directed against these selected target sites in vitro. Our results show that AU1751 is the most effective target site of ezrin mRNA for DNAzymes because of its ideal secondary structure and hybridization thermodynamics. So, there is a significant correlation between the multiple computational methods and the efficacy of the corresponding DNAzymes. These provide a rational, efficient way for DNAzymes selection.
Assuntos
Proteínas do Citoesqueleto/química , DNA Catalítico/síntese química , DNA de Cadeia Simples/síntese química , Hibridização In Situ/métodos , Termodinâmica , Algoritmos , Proteínas do Citoesqueleto/genética , Humanos , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Especificidade por SubstratoRESUMO
Specific inhibition of P-glycoprotein (Pgp) expression, which is encoded by multidrug resistance gene-1 (MDR1), is considered a well-respected strategy to overcome multidrug resistance (MDR). Deoxyribozymes (DRz) are catalytic nucleic acids that could cleave a target RNA in sequence-specific manner. However, it is difficult to select an effective target site for DRz in living cells. In this study, target sites of DRz were screened according to MDR1 mRNA secondary structure by RNA structure analysis software. Twelve target sites on the surface of MDR1 mRNA were selected. Accordingly, 12 DRzs were synthesized and their suppression effect on the MDR phenotype in breast cancer cells was confirmed. The results showed that 4 (DRz 2, 3, 4, 9) of the 12 DRzs could, in a dose-dependent response, significantly suppress MDR1 mRNA expression and restore chemosensitivity in breast cancer cells with MDR phenotype. This was especially true of DRz 3, which targets the 141 site purine-pyrimidine dinucleotide. Compared with antisense oligonucleotide or anti-miR-27a inhibitor, DRz 3 was more efficient in suppressing MDR1 mRNA and Pgp protein expression or inhibiting Pgp function. The chemosensitivity assay also proved DRz 3 to be the best one to reverse the MDR phenotype. The present study suggests that screening targets of DRzs according to MDR1 mRNA secondary structure could be a useful method to obtain workable ones. We provide evidence that DRzs (DRz 2, 3, 4, 9) are highly efficient at reversing the MDR phenotype in breast carcinoma cells and restoring chemosensitivity.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , DNA Catalítico/síntese química , DNA Catalítico/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Neoplasias da Mama/genética , Carcinoma/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , MicroRNAs/genética , Oligonucleotídeos Fosforotioatos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
Nucleic acid aptamers are generally obtained by in vitro selection. Some have G-rich consensus sequences with ability to fold into the four-stranded structures known as G-quadruplexes. A few G-quadruplex aptamers have proven to bind hemin to form a new class of DNAzyme with the peroxidase-like activity, which can be significantly promoted by appending an appropriate base-pairing duplex onto the G-quadruplex structures of aptamers. Knowing the structural role of base pairing, here we introduce a novel grafting strategy for the design of improved G-quadruplex aptamers and high-activity DNAzymes. To demonstrate this strategy, three existing G-quadruplex aptamers are chosen as the first generation. A base-pairing DNA duplex is grafted onto the G-quadruplex motif of the first generation aptamers. Consequently, three new aptamers with the quadruplex/duplex DNA structures are produced as the second generation. The hemin-binding affinities and DNAzyme functions of the second generation aptamers are characterized and compared with the first generation. The results indicate three G-quadruplex aptamers obtained by the grafting strategy have more excellent properties than the corresponding original aptamers. Our findings suggest that, if the structures and functions of existing aptamers are thoroughly known, the grafting strategy can be facilely utilized to improve the aptamer properties and thereby producing better next-generation aptamers. This provides a simple but effective approach to the design of nucleic acid aptamers and DNAzymes.
Assuntos
Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , DNA Catalítico/síntese química , DNA Catalítico/metabolismo , Quadruplex G , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , DNA Catalítico/química , DNA Catalítico/genética , Hemina/metabolismo , Análise Espectral/métodosRESUMO
In cells, DNA typically consists of two antiparallel strands arranged in a double-helical structure, which is central to its fundamental role in storing and transmitting genetic information. In laboratories, however, DNA can be readily synthesized as a single-stranded polymer that can adopt many other types of structures, including some that have been shown to catalyze chemical transformations. These catalytic DNA molecules are commonly referred to as DNAzymes, or deoxyribozymes. Thus far, DNAzymes have not been found in cells, but hundreds of structural and functional variations have been created in the laboratory. This alternative catalytic platform has piqued the curiosity of many researchers, including those who seek to exploit them in potential applications ranging from analytical tools to therapeutic agents. In this review, we explore the unconventional role of DNA as a biologically inspired synthetic enzyme.
Assuntos
Biocatálise , Biomimética , DNA Catalítico/síntese química , DNA de Cadeia Simples/síntese química , Técnicas Biossensoriais , DNA Catalítico/genética , DNA Catalítico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Engenharia Genética , Nanotecnologia , Técnica de Seleção de AptâmerosRESUMO
The protein enzyme ribonuclease A (RNaseA) cleaves RNA with catalytic perfection, although with little sequence specificity, by a divalent metal ion (M(2+))-independent mechanism in which a pair of imidazoles provides general acid and base catalysis, while a cationic amine provides electrostatic stabilization of the transition state. Synthetic imitation of this remarkable organo-catalyst ("RNaseA mimicry") has been a longstanding goal in biomimetic chemistry. The 9(25)-11 DNAzyme contains synthetically modified nucleotides presenting both imidazole and cationic amine side chains, and catalyzes RNA cleavage with turnover in the absence of M(2+) similarly to RNaseA. Nevertheless, the catalytic roles, if any, of the "protein-like" functional groups have not been defined, and hence the question remains whether 9(25)-11 engages any of these functionalities to mimic aspects of the mechanism of RNaseA. To address this question, we report a mechanistic investigation of 9(25)-11 catalysis wherein we have employed a variety of experiments, such as DNAzyme functional group deletion, mechanism-based affinity labeling, and bridging and nonbridging phosphorothioate substitution of the scissile phosphate. Several striking parallels exist between the results presented here for 9(25)-11 and the results of analogous experiments applied previously to RNaseA. Specifically, our results implicate two particular imidazoles in general acid and base catalysis and suggest that a specific cationic amine stabilizes the transition state via diastereoselective interaction with the scissile phosphate. Overall, 9(25)-11 appears to meet the minimal criteria of an RNaseA mimic; this demonstrates how added synthetic functionality can expand the mechanistic repertoire available to a synthetic DNA-based catalyst.
Assuntos
DNA Catalítico/síntese química , Mimetismo Molecular , Aminas , Catálise , DNA Catalítico/química , DNA Catalítico/metabolismo , Imidazóis , Fosfatos , Ribonuclease PancreáticoRESUMO
Photochemical activation of a deoxyribozyme with peroxidase activity was achieved by the synthesis and incorporation of a caged deoxyguanosine.
Assuntos
Desoxiguanosina/metabolismo , Peroxidase/metabolismo , Raios Ultravioleta , Biocatálise/efeitos da radiação , DNA Catalítico/síntese química , DNA Catalítico/química , DNA Catalítico/efeitos da radiação , Desoxiguanosina/síntese química , Desoxiguanosina/química , Ativação Enzimática/efeitos da radiação , Estrutura Molecular , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Fotoquímica , Fatores de TempoRESUMO
Novel transition metal catalysts based on oligonucleotides can be easily obtained by functionalization of 5-iodouridine with phosphine ligands, resulting in good asymmetric induction in palladium catalyzed allylic nucleophilic substitution.