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1.
Nat Commun ; 11(1): 3847, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737299

RESUMO

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.


Assuntos
Aptâmeros de Nucleotídeos/genética , Arabidopsis/genética , Brassica/genética , Engenharia Genética/métodos , RNA Mensageiro/genética , Tabaco/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Arabidopsis/metabolismo , Compostos de Benzil/química , Brassica/metabolismo , Fluorescência , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Genes Reporter , Imidazolinas/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/metabolismo , Tabaco/metabolismo , Transformação Genética
2.
Nucleic Acids Res ; 48(15): e90, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32609809

RESUMO

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição de Choque Térmico/química , Substâncias Macromoleculares/isolamento & purificação , Espectrometria de Massas , Aptâmeros de Nucleotídeos/genética , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/genética , Histonas/química , Humanos , Imunoprecipitação , Substâncias Macromoleculares/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional
3.
Nucleic Acids Res ; 48(15): 8591-8600, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644133

RESUMO

In nature, allostery is the principal approach for regulating cellular processes and pathways. Inspired by nature, structure-switching aptamer-based nanodevices are widely used in artificial biotechnologies. However, the canonical aptamer structures in the nanodevices usually adopt a duplex form, which limits the flexibility and controllability. Here, a new regulating strategy based on a clamp-like triplex aptamer structure (CLTAS) was proposed for switching DNA polymerase activity via conformational changes. It was demonstrated that the polymerase activity could be regulated by either adjusting structure parameters or dynamic reactions including strand displacement or enzymatic digestion. Compared with the duplex aptamer structure, the CLTAS possesses programmability, excellent affinity and high discrimination efficiency. The CLTAS was successfully applied to distinguish single-base mismatches. The strategy expands the application scope of triplex structures and shows potential in biosensing and programmable nanomachines.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Técnicas Biossensoriais , DNA Polimerase Dirigida por DNA/genética , Taq Polimerase/genética , Aptâmeros de Nucleotídeos/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/farmacologia , Humanos , Nanoestruturas/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Taq Polimerase/antagonistas & inibidores , Taq Polimerase/química
4.
Nucleic Acids Res ; 48(12): 6970-6979, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479610

RESUMO

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.


Assuntos
Aptâmeros de Nucleotídeos/genética , Conformação de Ácido Nucleico , Ribonucleotídeos/genética , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Firmicutes/genética , Firmicutes/ultraestrutura , Ligantes , Purinas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Ribonucleotídeos/química
5.
Nucleic Acids Res ; 48(12): 6811-6823, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32496535

RESUMO

A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.


Assuntos
Sistemas CRISPR-Cas/genética , Clivagem do DNA , Estruturas R-Loop/genética , RNA Guia/genética , Aptâmeros de Nucleotídeos/genética , Edição de Genes , Conformação de Ácido Nucleico , RNA Guia/ultraestrutura , Ribonucleoproteínas/genética , Ribonucleoproteínas/ultraestrutura , Imagem Individual de Molécula , Streptococcus pyogenes/genética
6.
Nucleic Acids Res ; 48(12): 6471-6480, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442296

RESUMO

Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.


Assuntos
Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Simulação de Dinâmica Molecular , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Ligação de Hidrogênio , Mutação
7.
Biochim Biophys Acta Rev Cancer ; 1874(1): 188377, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32418899

RESUMO

Cancer is one of the most prevalent potentially lethal diseases. With the increase in the number of investigations into the uses of nanotechnology, many nucleic acid (NA)-based nanostructures such as small interfering RNA, microRNA, aptamers, and immune adjuvant NA have been applied to treat cancer. Here, we discuss studies on the applications of NA in cancer treatment, recent research trends, and the limitations and prospects of specific NA-mediated gene therapy and immunotherapy for cancer treatment. The NA structures used for cancer therapy consist only of NA or hybrids comprising organic or inorganic substances integrated with functional NA. We also discuss delivery vehicles for therapeutic NA and anti-cancer agents, and recent trends in NA-based gene therapy and immunotherapy against cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Aptâmeros de Nucleotídeos/administração & dosagem , MicroRNAs/administração & dosagem , Nanomedicina/métodos , Neoplasias/terapia , RNA Interferente Pequeno/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Animais , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Portadores de Fármacos/química , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Terapia Genética/métodos , Humanos , Imunoterapia/métodos , MicroRNAs/genética , Nanopartículas/química , Neoplasias/genética , Neoplasias/imunologia , RNA Interferente Pequeno/genética , Resultado do Tratamento
8.
RNA ; 26(8): 960-968, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32345632

RESUMO

Dozens of candidate orphan riboswitch classes have been discovered previously by using comparative sequence analysis algorithms to search bacterial genomic sequence databases. Each orphan is classified by the presence of distinct conserved nucleotide sequences and secondary structure features, and by its association with particular types of genes. One previously reported orphan riboswitch candidate is the "NMT1 motif," which forms a hairpin structure with an internal bulge that includes numerous highly conserved nucleotides. This motif associates with genes annotated to encode various dioxygenase enzymes, transporters, or proteins that have roles associated with thiamin or histidine metabolism. Biochemical evaluation of numerous ligand candidates revealed that NMT1 motif RNA constructs most tightly bind 8-azaxanthine, xanthine, and uric acid, whereas most other closely related compounds are strongly rejected. Genetic assays revealed that NMT1 motif RNAs function to turn off gene expression upon ligand binding, likely by regulating translation initiation. These results suggest that NMT1 motif RNAs function as aptamer domains for a riboswitch class that specifically responds to high concentrations of oxidized purines. Members of this "xanthine riboswitch" class appear to regulate genes predominantly related to purine transport and oxidation, thus avoiding the effects of overproduction of these common purine derivatives.


Assuntos
Purinas/metabolismo , RNA Bacteriano/genética , Riboswitch/genética , Ácido Úrico/metabolismo , Xantina/metabolismo , Aptâmeros de Nucleotídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Oxirredução , Xantinas/metabolismo
9.
J Med Microbiol ; 69(4): 640-652, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32125966

RESUMO

Introduction. The use of silver as an antimicrobial therapeutic is limited by its toxicity to host cells compared with that required to kill bacterial pathogens.Aim. To use aptamer targeting of DNA scaffolded silver nanoclusters as an antimicrobial agent for treating Pseudomonas aeruginosa infections.Methodology. Antimicrobial activity was assessed in planktonic cultures and in vivo using an invertebrate model of infection.Results. The aptamer conjugates that we call aptabiotics have potent antimicrobial activity. Targeted silver nanoclusters were more effective at killing P. aeruginosa than the equivalent quantity of untargeted silver nanoclusters. The aptabiotics have an IC50 of 1.3-2.6 µM against planktonically grown bacteria. Propidium iodide staining showed that they rapidly depolarize bacterial cells to kill approximately 50 % of the population within 10 min following treatment. In vivo testing in the Galleria mellonella model of infection prolonged survival from an otherwise lethal infection.Conclusion. Using P. aeruginosa as a model, we show that targeting of DNA-scaffolded silver nanoclusters with an aptamer has effective fast-acting antimicrobial activity in vitro and in an in vivo animal model.


Assuntos
Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos/química , Nanoestruturas/química , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Prata/farmacologia , Animais , Antibacterianos/química , Aptâmeros de Nucleotídeos/genética , Humanos , Testes de Sensibilidade Microbiana , Mariposas , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Prata/química
10.
Nat Commun ; 11(1): 1347, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165631

RESUMO

Protein-dominant cellular processes cannot be fully decoded without precise manipulation of their activity and localization in living cells. Advances in optogenetics have allowed spatiotemporal control over cellular proteins with molecular specificity; however, these methods require recombinant expression of fusion proteins, possibly leading to conflicting results. Instead of modifying proteins of interest, in this work, we focus on design of a tunable recognition unit and develop an aptamer-based near-infrared (NIR) light-responsive nanoplatform for manipulating the subcellular localization of specific proteins in their native states. Our results demonstrate that this nanoplatform allows photocontrol over the cytoplasmic-nuclear shuttling behavior of the target RelA protein (a member of the NF-κß family), enabling regulation of RelA-related signaling pathways. With a modular design, this aptamer-based nanoplatform can be readily extended for the manipulation of different proteins (e.g., lysozyme and p53), holding great potential to develop a variety of label-free protein photoregulation strategies for studying complex biological events.


Assuntos
Aptâmeros de Nucleotídeos/genética , Fator de Transcrição RelA/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Optogenética , Transporte Proteico , Transdução de Sinais , Fator de Transcrição RelA/genética
11.
Nat Commun ; 11(1): 972, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080195

RESUMO

Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as a prodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Paclitaxel/administração & dosagem , RNA/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/química , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Sistemas de Liberação de Medicamentos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imageamento Tridimensional , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/ultraestrutura , Paclitaxel/química , RNA/química , RNA/genética , Estabilidade de RNA , Imagem Individual de Molécula , Solubilidade , Termodinâmica , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cardiovasc Ther ; 2020: 6869856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32042311

RESUMO

Objectives: To observe the effect of avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Background: Percutaneous transluminal coronary angioplasty (PTCA) is currently the preferred method for the treatment of coronary heart disease. However, vascular restenosis still occurs after PTCA treatment, severely affecting the clinical efficacy of PTCA. Integrin avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Methods: In this experiment, we used systematic evolution of ligands by exponential enrichment (SELEX) to screen out avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Results: In the present study, we found that avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. P < 0.05). Avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. P < 0.05). AvP < 0.05). Av. Conclusions: The findings suggest that avß3 ssDNA inhibited the proliferation and migration of VSMCs by suppressing the activation of Ras-PI3K/MAPK signaling.ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Movimento Celular , Proliferação de Células , DNA de Cadeia Simples/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Aptâmeros de Nucleotídeos/genética , Células Cultivadas , DNA de Cadeia Simples/genética , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Integrina alfaVbeta3/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Osteopontina/genética , Osteopontina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas ras/genética
13.
Nucleic Acids Res ; 48(6): 3366-3378, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32052019

RESUMO

RNAs play major roles in the regulation of gene expression. Hence, designer RNA molecules are increasingly explored as regulatory switches in synthetic biology. Among these, the TetR-binding RNA aptamer was selected by its ability to compete with operator DNA for binding to the bacterial repressor TetR. A fortuitous finding was that induction of TetR by tetracycline abolishes both RNA aptamer and operator DNA binding in TetR. This enabled numerous applications exploiting both the specificity of the RNA aptamer and the efficient gene repressor properties of TetR. Here, we present the crystal structure of the TetR-RNA aptamer complex at 2.7 Å resolution together with a comprehensive characterization of the TetR-RNA aptamer versus TetR-operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The fold of the RNA aptamer bears no resemblance to regular B-DNA, and neither does the thermodynamic characterization of the complex formation reaction. Nevertheless, the functional aptamer-binding epitope of TetR is fully contained within its DNA-binding epitope. In the RNA aptamer complex, TetR adopts the well-characterized DNA-binding-competent conformation of TetR, thus revealing how the synthetic TetR-binding aptamer strikes the chords of the bimodal allosteric behaviour of TetR to function as a synthetic regulator.


Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Conformação Proteica , Aptâmeros de Nucleotídeos/genética , Cristalografia por Raios X , DNA de Forma B/química , DNA de Forma B/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Regulação da Expressão Gênica/genética , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Ligação Proteica/genética , RNA/química , RNA/genética
14.
Nucleic Acids Res ; 48(4): 1669-1680, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31950158

RESUMO

The development of structure-specific RNA binding reagents remains a central challenge in RNA biochemistry and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve l-RNA aptamers that bind tightly to structured d-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of l-DNA, which has several practical advantages compared to l-RNA, remains unknown. Here, we report the discovery and characterization of the first l-DNA aptamers against a structured RNA molecule, precursor microRNA-155, thereby establishing the capacity of DNA and RNA molecules of the opposite handedness to form tight and specific 'cross-chiral' interactions with each other. l-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of l-DNA to interact with native d-RNA via Watson-Crick base pairing. Furthermore, l-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of l-DNA aptamers are distinct from previously reported l-RNA aptamers against pre-miR-155, indicating that l-DNA and l-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that l-DNA may be pursued as an alternative to l-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.


Assuntos
Aptâmeros de Nucleotídeos/genética , DNA/genética , Conformação de Ácido Nucleico , RNA/genética , Aptâmeros de Nucleotídeos/química , Pareamento de Bases/genética , Sítios de Ligação , DNA/química , Humanos , MicroRNAs/química , MicroRNAs/genética , RNA/química
15.
J Biomed Sci ; 27(1): 6, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900238

RESUMO

Today, the treatment of bacterial infections is a major challenge, due to growing rate of multidrug-resistant bacteria, complication of treatment and increased healthcare costs. Moreover, new treatments for bacterial infections are limited. Oligonucleotide aptamers are single stranded DNAs or RNAs with target-selective high-affinity feature, which considered as nucleic acid-based affinity ligands, replacing monoclonal antibodies. The aptamer-based systems have been found to be talented tools in the treatment of microbial infections, regarding their promising anti-biofilm and antimicrobial activities; they can reduce or inhibit the effects of bacterial toxins, and inhibit pathogen invasion to immune cell, as well as they can be used in drug delivery systems. The focus of this review is on the therapeutic applications of aptamers in infections. In this regard, an introduction of infections and related challenges were presented, first. Then, aptamer definition and selection, with a brief history of aptamers development against various pathogens and toxins were reviewed. Diverse strategies of aptamer application in drug delivery, as well as, the effect of aptamers on the immune system, as the main natural agents of human defense against pathogens, were also discussed. Finally, the future trends in clinical applications of this technology were discussed.


Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , DNA/uso terapêutico , RNA/uso terapêutico , Aptâmeros de Nucleotídeos/genética , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , DNA/genética , Humanos , Ligantes , RNA/genética
16.
Talanta ; 209: 120510, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892034

RESUMO

Exosomes play important roles in intercellular communications, tumor migration and invasion. However, the specific detection of cancer exosomes remains as a big challenge due to its low concentration in biofluids. Therefore, the sensitive and selective detection of cancer cells-derived exosomes has attracted growing attention owing to their potential in diagnostic and prognostic applications. Activatable strategies have received great attention for the detection of low abundant analytes due to their high sensitivity. Herein, based on molecular recognition between DNA aptamer and exosome surface biomarker (protein tyrosine kinase-7), a novel activatable and label-free strategy was designed for highly sensitive and specific sensing of exosomes. In this work, the target exosomes trigger strand replacement reaction to form G-quadruplex, which result in an obvious fluorescence enhancement of N-methylmesoporphyrin IX due to the bonding between G-quadruplex and N-methylmesoporphyrin IX. Under the optimum experimental conditions, the linear range for exosomes was measured to be 5.0 × 105-5.0 × 107 particles/µL and the detection limit (LOD) was calculated to be 3.4 × 105 particles/µL (3σ). This assay possesses high specificity to distinguish exosomes derived from different cell lines, and has successfully been validated in patient and healthy plasma samples. Furthermore, the probe can effectively detect the exosomes in 30% fetal bovine serum, indicating that the biological matrix has a negligible effect on this method. This developed label-free, convenient and highly sensitive biosensor will offer a great opportunity for exosomes quantification in biological study and clinical application.


Assuntos
Aptâmeros de Nucleotídeos/química , Exossomos/química , Aptâmeros de Nucleotídeos/genética , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Quadruplex G , Humanos , Limite de Detecção , Mesoporfirinas/química , Hibridização de Ácido Nucleico , Receptores Proteína Tirosina Quinases/química , Espectrometria de Fluorescência/métodos
17.
RNA ; 26(5): 564-580, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31992591

RESUMO

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K 1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.


Assuntos
Aptâmeros de Nucleotídeos/genética , Glicina/genética , RNA Bacteriano/genética , Riboswitch/genética , Bacillus subtilis/genética , Sítios de Ligação/genética , Biologia Computacional , Ligantes , Mutação/genética , Conformação de Ácido Nucleico , Vibrio cholerae/genética
18.
Food Chem ; 314: 126133, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978716

RESUMO

The development of a sensitive and rapid detection approach for allergens in various food matrices is essential to assist patients in managing their allergies. The most common methods used for allergen detection are based on immunoassays, PCR and mass spectrometry. However, all of them are very complex and time-consuming. Herein, an aptamer biosensor for the detection of the major shrimp allergen tropomyosin (TM) was developed. Graphene oxide (GO) was used as a platform for screening of the minimal-length aptamer sequence required for high-affinity target binding. A fluorescein dye labeled GO quenches the truncated aptamer by π-stacking interactions. After the addition of TM, the fluorescence was restored due to the competitive binding of the aptamer to GO. One of the truncated aptamers was found to bind to TM with four-fold higher affinity (30 nM) compared to the full-length aptamer (124 nM), with a limit of detection (LOD) of 2 nM. The aptamer-based sensor demonstrates the sensitive, selective, and specific detection of TM in 30 min. The performance of the sensor was confirmed using TM spiked chicken soup, resulting in a high percentage recovery (~97 ± 10%). The association of GO and labelled aptamer sensor platform has shown the rapid detection of TM in food, which is compared to other methods very sensitive, specific and performs in high throughput application.


Assuntos
Técnicas Biossensoriais/métodos , Crustáceos/química , Tropomiosina/análise , Alérgenos/análise , Alérgenos/genética , Animais , Aptâmeros de Nucleotídeos/genética , Grafite/química , Limite de Detecção , Tropomiosina/genética
19.
J Agric Food Chem ; 68(1): 402-408, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31809034

RESUMO

The functional ingredients of microalgal biomass are receiving substantial recognition as the global demands for health supplements produced from natural sources are on the rise. Paramylon, a conglomerate of ß-1,3-glucans, is one of the major valuable sources derived from Euglena gracilis having multiple applications, thus necessitating the development of an efficient quantification method. Here, we employed a DNA aptamer to quantify the amount of paramylon produced by E. gracilis. Paramylon-specific aptamers were isolated by the systematic evolution of ligands by exponential enrichment (SELEX) process. To evaluate the potential aptamers, the binding affinity between aptamer candidates and paramylon granules was confirmed by a confocal laser scanning microscope and the dissociation constants of the selected aptamers were determined by nonlinear regression analysis. The selected DNA aptamer was successfully used for the quantification of paramylon, and the results were compared to those obtained by the standard methods. The new approach was also used for quantification of paramylon from E. gracilis cells cultured to different cell stages and physiologies. It can be concluded that the aptamer-based protocol for the measurement of paramylon proposed in this study is highly accurate and comparatively less time-consuming.


Assuntos
Aptâmeros de Nucleotídeos/genética , DNA de Cadeia Simples/genética , Euglena gracilis/química , Glucanos/análise , Extratos Vegetais/análise , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Euglena gracilis/genética , Euglena gracilis/metabolismo , Glucanos/metabolismo , Microalgas/química , Microalgas/genética , Microalgas/metabolismo , Extratos Vegetais/metabolismo
20.
Nat Chem Biol ; 16(1): 69-76, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31636432

RESUMO

Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.


Assuntos
Corantes Fluorescentes/química , RNA/química , Espectrometria de Fluorescência/métodos , Aptâmeros de Nucleotídeos/genética , Dimerização , Fluorescência , Biblioteca Gênica , Células HEK293 , Células HeLa , Humanos , Microfluídica/métodos , RNA/análise , Rodaminas/química , Espectrofotometria
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