RESUMEN

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

Asunto(s)

Aptámeros de Nucleótidos/metabolismo , ADN Nucleotidilexotransferasa/fisiología , Evaluación Preclínica de Medicamentos/métodos , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/aislamiento & purificación , Sitios de Unión , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Lactoferrina/metabolismo , Técnicas de Amplificación de Ácido Nucleico , Unión Proteica , Especificidad por Sustrato , Trombina/metabolismo , Recombinación V(D)J

RESUMEN

Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play important roles in human cancers, acting as transcriptional and post-transcriptional regulators, respectively. These phenomena raise questions about the ability of an artificial device to simultaneously regulate miRNAs and TFs. In this study, we aimed to construct artificial long non-coding RNAs, "alncRNAs", and to investigate their therapeutic effects on bladder cancer cell lines. Based on engineering principles of synthetic biology, we combined tandem arrayed aptamer cDNA sequences for TFs with tandem arrayed cDNA copies of binding sites for the miRNAs to construct alncRNAs. In order to prove the utility of this platform, we chose ß-catenin and the miR-183-182-96 cluster as the functional targets and used the bladder cancer cell lines 5637 and SW780 as the test models. Dual-luciferase reporter assay, real-time quantitative PCR (qRT-PCR) and related phenotypic experiments were used to test the expression of related genes and the therapeutic effects of our devices. The result of dual-luciferase reporter assay and qRT-PCR showed that alncRNAs could inhibit transcriptional activity of TFs and expression of corresponding microRNAs. Using functional experiments, we observed decreased cell proliferation, increased apoptosis, and motility inhibition in alncRNA-infected bladder cancer cells. What's more, follow-up mechanism experiments further confirmed the anti-tumor effect of our devices. In summary, our synthetic devices indeed function as anti-tumor regulators, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cells. Most importantly, anti-cancer effects were induced by the synthetic alncRNAs in the bladder cancer lines. Our devices, all in all, provided a novel strategy and methodology for cancer studies, and might show a great potential for cancer therapy if the challenges of in vivo DNA delivery are overcome.

Asunto(s)

Aptámeros de Nucleótidos/farmacología , MicroARNs/antagonistas & inhibidores , ARN Largo no Codificante/farmacología , Neoplasias de la Vejiga Urinaria/genética , beta Catenina/antagonistas & inhibidores , Aptámeros de Nucleótidos/biosíntesis , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Fenotipo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Largo no Codificante/síntesis química , Biología Sintética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico

RESUMEN

The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , ADN de Cadena Simple/biosíntesis , Técnicas de Amplificación de Ácido Nucleico , Oligodesoxirribonucleótidos/biosíntesis , Anticoagulantes/síntesis química , Anticoagulantes/metabolismo , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , ADN de Cadena Simple/genética , G-Cuádruplex , Humanos , Oligodesoxirribonucleótidos/genética

RESUMEN

Asymmetric PCR, a simple method to generate single-stranded DNA (ssDNA) aptamers in systematic evaluation of ligand by exponential enrichments rounds, is coupled with limitations. We investigated the essential strategies for optimization of conditions to perform a high-quality asymmetric PCR. Final concentrations of primers and template, the number of PCR cycles, and annealing temperature were selected as optimizing variables. The qualities of visualized PCR products were analyzed by ImageJ software. The highest proportion of interested DNA than unwanted products was considered as optimum conditions. Results revealed that the best values for primers ratio, final template concentration, annealing temperature, and PCR cycles were, respectively, 30:1, 1 ng/µL, 55 °C, and 20 cycles for the first and 50:1, 2 ng/µL, 59 °C, and 20 cycles for other rounds. No significant difference was found between optimized asymmetric PCR results in the rounds of two to eight (P > 0.05). The ssDNA quality in round 10 was significantly better than other rounds (P < 0.05). Generally, the ssDNA product with less dimers, double-stranded DNA (dsDNA), and smear are preferable. The dsDNA contamination is the worst, because it can act as antidote and inhibits aptameric performance. Therefore, to choose the best conditions, the lower amount of dsDNA is more important than other unwanted products.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/genética , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/genética , Reacción en Cadena de la Polimerasa/métodos

RESUMEN

Circulating tumor cells (CTCs) are cells that shed from a primary tumor and freely circulate in the blood, retaining the ability to initiate metastasis and form a secondary tumor in distant organs in the body. CTCs reflect the molecular profile of the primary tumor, therefore studying CTCs can allow for an understanding of the mechanism of metastasis, and an opportunity to monitor the prognosis of cancer. Unfortunately, the detection of CTCs is a considerable challenge due to their low abundance in the bloodstream and the lack of consistent markers present to recognize these cells. The aim of this review is to summarize some of the aptamer-based affinity methods for the detection of CTCs. The basic biological concept of how metastasis occurs and the role of CTCs in this process are presented. Some methods of CTC detection employing antibodies or peptides are mentioned here for comparison. The review of present literature suggests that aptamers are emerging as competitive technology in the detection of CTCs, especially due to their unique properties, but there still remain several challenges to be met, including the need to improve the throughput and sensitivity of such methods.

Asunto(s)

Aptámeros de Nucleótidos/sangre , Biomarcadores de Tumor/análisis , Técnicas Biosensibles/métodos , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Técnica SELEX de Producción de Aptámeros , Anticuerpos/metabolismo , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/síntesis química , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Separación Celular , Molécula de Adhesión Celular Epitelial/análisis , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Receptores ErbB/análisis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Técnicas Analíticas Microfluídicas , Metástasis de la Neoplasia , Neoplasias/sangre , Neoplasias/genética , Células Neoplásicas Circulantes/química , Péptidos/síntesis química , Péptidos/metabolismo

RESUMEN

In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/biosíntesis , Evolución Molecular Dirigida , Reacción en Cadena de la Polimerasa/métodos , ARN Catalítico/biosíntesis , ARN/genética , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/genética , Emparejamiento Base , ARN Polimerasas Dirigidas por ADN/genética , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , ARN Catalítico/genética , Moldes Genéticos

RESUMEN

Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the detailed advantages and disadvantages of antibodies and aptamers in therapeutic applications and summarize recent progress in aptamer selection and modification approaches. We will present therapeutic oligonucleotide aptamers in preclinical studies for skeletal diseases and further discuss oligonucleotide aptamers in different stages of clinical evaluation for various disease therapies including macular degeneration, cancer, inflammation and coagulation to highlight the bright commercial future and potential challenges of therapeutic oligonucleotide aptamers.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/uso terapéutico , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/economía , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Aptámeros de Nucleótidos/economía , Aptámeros de Nucleótidos/inmunología , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Humanos

RESUMEN

The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze-thaw cycle and a maximum of 60 % was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24 % in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , ARN Catalítico/metabolismo , Animales , Secuencia de Bases , Catálisis , Criopreservación , ARN Polimerasas Dirigidas por ADN/genética , Congelación , Concentración de Iones de Hidrógeno , Cinética , Ligadura , Conformación de Ácido Nucleico , ARN , Schistosoma mansoni/metabolismo

RESUMEN

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

Asunto(s)

Anticuerpos/metabolismo , Aptámeros de Nucleótidos/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos/inmunología , Aptámeros de Nucleótidos/genética , Proteínas Recombinantes/inmunología , Técnica SELEX de Producción de Aptámeros

RESUMEN

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.

Asunto(s)

Acetolactato Sintasa/antagonistas & inhibidores , Antituberculosos/química , Aptámeros de Nucleótidos/química , Proteínas Bacterianas/antagonistas & inhibidores , ADN de Cadena Simple/química , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/efectos de los fármacos , Acetolactato Sintasa/química , Acetolactato Sintasa/genética , Animales , Antituberculosos/metabolismo , Antituberculosos/farmacología , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Biblioteca de Genes , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Técnica SELEX de Producción de Aptámeros

RESUMEN

We proposed to use a novel stepwise sequence-constructive SELEX method to develop DNA aptamers that can recognize Globo H which is a tumor-associated carbohydrate antigen. A combinatorial synthetic library that consisted of DNA molecules with randomized regions of 15-bases was used as the starting library for the first SELEX procedure. The input DNA library for the second round of SELEX consisted of the extension of the 5' and 3'-ends with 7-bases that were randomized from four selected aptamers. The third round of SELEX was performed following the same procedures as described for the second round of SELEX. The experimental results indicate that the binding affinity of DNA aptamers to Globo H was enhanced when using the sequence-constructive SELEX approach. The selectivity of the DNA aptamers for related disaccharides, mannose derivatives, and Globo H analogs demonstrated the ability of the DNA aptamers to discriminate the presence of various glycans with different structures.

Asunto(s)

Antígenos de Carbohidratos Asociados a Tumores/química , Aptámeros de Nucleótidos/biosíntesis , Técnica SELEX de Producción de Aptámeros/métodos , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Aptámeros de Nucleótidos/química , Secuencia de Bases , Sitios de Unión , Disacáridos/química , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica

RESUMEN

Certain DNA polymerases, such as ϕ29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of Ï•29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein-aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/química , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/química , Oligonucleótidos/biosíntesis , Oligonucleótidos/química , Técnicas de Amplificación de Ácido Nucleico/métodos

RESUMEN

NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-ß) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies.

Asunto(s)

Antivirales/farmacología , Aptámeros de Nucleótidos/farmacología , Regulación Viral de la Expresión Génica , Hepacivirus/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Proteínas no Estructurales Virales/genética , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Hepacivirus/fisiología , Hepatocitos/inmunología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Interferón beta , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación Viral

RESUMEN

Aptamer microarrays are a promising high-throughput method for ultrasensitive detection of multiple analytes, but although much is known about the optimal synthesis of oligonucleotide microarrays used in hybridization-based genomics applications, the bioaffinity interactions between aptamers and their targets is qualitatively different and requires significant changes to synthesis parameters. Focusing on streptavidin-binding DNA aptamers, we employed light-directed in situ synthesis of microarrays to analyze the effects of sequence fidelity, linker length, surface probe density, and substrate functionalization on detection sensitivity. Direct comparison with oligonucleotide hybridization experiments indicates that aptamer microarrays are significantly more sensitive to sequence fidelity and substrate functionalization and have different optimal linker length and surface probe density requirements. Whereas microarray hybridization probes generate maximum signal with multiple deletions, aptamer sequences with the same deletion rate result in a 3-fold binding signal reduction compared with the same sequences synthesized for maximized sequence fidelity. The highest hybridization signal was obtained with dT 5mer linkers, and the highest aptamer signal was obtained with dT 11mers, with shorter aptamer linkers significantly reducing the binding signal. The probe hybridization signal was found to be more sensitive to molecular crowding, whereas the aptamer probe signal does not appear to be constrained within the density of functional surface groups commonly used to synthesize microarrays.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Estimulación Luminosa/métodos , Aptámeros de Nucleótidos/genética , Unión Proteica/fisiología

RESUMEN

RNA aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. They are an effective tool for pharmaceutical research and development of antiviral agents. Here, we describe four Hirame rhabdovirus (HIRRV)-RNA aptamers (H1, H2, H3 and H4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (SELEX). The HIRRV-RNA aptamers specifically bind to HIRRV. Hirame natural embryo (HINAE) cells treated with virus and the RNA aptamer showed a decrease in appearance of cytopathic effect when compared with control (treated only with virus). Rhodovulum sulfidophilum was transformed with genes for the RNA aptamers, and the aptamers were detected in the culture medium, indicating that they were secreted from the cells. Thus, the recombinant R. sulfidophilum might be a powerful tool for the prevention of HIRRV in aquaculture.

Asunto(s)

Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/farmacología , Novirhabdovirus/crecimiento & desarrollo , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Aptámeros de Nucleótidos/biosíntesis , Línea Celular , ADN Recombinante/genética , Lenguado , Plásmidos/genética , Rhodovulum/genética

RESUMEN

Aptamers are a group of artificial oligonucleotides identified by exponential enrichment system evolution technology (Selective expansion of ligands by exponential enrichment, SELEX). Aptamers have been widely used in basic research, clinical diagnostics, and nano-technology. In this article we will introduce the technology of aptamer and summarize its applications in medical microbiology.

Asunto(s)

Aptámeros de Nucleótidos , Técnicas Microbiológicas/métodos , Técnica SELEX de Producción de Aptámeros/métodos , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/genética , Microbiología , Técnica SELEX de Producción de Aptámeros/tendencias

RESUMEN

Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in a large percentage of breast cancers. Monoclonal antibodies targeting HER2 are vastly used for both diagnostic and therapeutic aims. However, identifying a new molecular probe against HER2 with improved diagnostic and therapeutic features is of great importance. In this report, we have applied the cell systematic evolution of ligands by exponential enrichment (SELEX) strategy for 16 selection rounds to generate an enriched pool of aptamers that specifically recognize the HER2 positive cell line. During the Cell SELEX procedure, a human HER2-overexpressing breast cancer cell line and a human HER2 negative breast cancer cell line were used. Our results reveal that polymerase chain reaction (PCR) amplification of random DNA libraries and the selected single-stranded DNA pool in different Cell SELEX rounds are different from what we expect from PCR amplification of homologous DNA. Our results also confirmed previous studies describing positive HER2 status of SK-BR3 and the absence of the HER2 expression in the MDA-MB468. We also developed a new method, Cell enzyme-linked assay, to monitor the enrichment of aptamers in a given round of Cell SELEX. This method would also be useful in other experiments using live cell enzyme-linked immunosorbent assay on adherent cells.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Neoplasias de la Mama/diagnóstico , Sondas de ADN/biosíntesis , Receptor ErbB-2/análisis , Técnica SELEX de Producción de Aptámeros , Aptámeros de Nucleótidos/química , Línea Celular Tumoral , Sondas de ADN/química , Femenino , Humanos , Ligandos , Receptor ErbB-2/química , Receptor ErbB-2/genética

RESUMEN

Nucleic acid aptamers, generated by an in vitro selection procedure named SELEX (Systematic Evolution of Ligands by Exponential enrichment), have been popularly used in various biomedical implementations so far. An adaptation of the conventional SELEX practice to whole living cells was referred to as cell-SELEX, which is very promising in ample sophisticated analytical, therapeutic and diagnostic applications. The current review article would like to provide a survey of contemporary developments in cell-specific aptamers, including methodology, applications and optimization of cell-SELEX.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Técnica SELEX de Producción de Aptámeros/métodos , Aptámeros de Nucleótidos/química , Humanos , Modelos Genéticos , Estructura Molecular

RESUMEN

Aptamers are ssDNA, RNA, or modified nucleic acids, usually consisting of short strands of oligonucleotides. Aptamers have the ability to bind specifically to a range of targets, from small organic molecules to proteins. However, by using cell-based aptamer selection, we have developed a strategy to identify the molecular signatures on the surface of targeted cells by exploiting the differences at the molecular level between any two given cell types. By applying this method, we have generated a panel of aptamers for the specific recognition of leukemia cells, and we report the results in this study. The selected aptamers were found to bind to target cells with an equilibrium dissociation constant (K (d)) in the nanomolar-to-picomolar range. Overall, the cell-based selection process is simple, fast, straightforward, and reproducible. Most importantly, since this strategy can be implemented without prior knowledge of a target's specific molecular signature, cell-based aptamer selection holds great promise for the development of specific molecular probes for cancer diagnosis and cancer biomarker discovery.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Neoplasias/metabolismo , Técnica SELEX de Producción de Aptámeros/métodos , Línea Celular Tumoral , Supervivencia Celular , Cartilla de ADN/metabolismo , Citometría de Flujo , Biblioteca de Genes , Humanos , Microscopía Confocal

RESUMEN

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs.

Asunto(s)

Aptámeros de Nucleótidos/biosíntesis , Plásmidos/genética , ARN/biosíntesis , Rhodovulum/genética , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Clonación Molecular , Medios de Cultivo/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Ingeniería Genética , Microbiología Industrial , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/genética , ARN/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Rhodovulum/enzimología , Rhodovulum/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismo , Análisis de Secuencia de ARN , Estreptavidina/metabolismo , Microbiología del Agua