RESUMO
Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.
Assuntos
Avidina , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/química , Reparo do DNA , Diagnóstico por Imagem , Endonucleases/metabolismo , Dano ao DNARESUMO
This study develops a simple and label-free biosensor for sensitive and selective detection of microRNA (miRNA) based on the formation of the adenosine2-coralyne-adenosine2 complex mediated by miRNA-specific polyadenosine extension.
Assuntos
Adenosina/química , Alcaloides de Berberina/química , Técnicas Biossensoriais/métodos , MicroRNAs/análise , Polímeros/química , MicroRNAs/química , Modelos Moleculares , Conformação de Ácido Nucleico , Espectrometria de FluorescênciaRESUMO
A novel fluorescent nanosensor has been developed by combining super fluorescence quenching ability of graphene oxide and hybridization chain reaction amplification, which enables highly sensitive detection of base excision repair enzyme activity with a wide dynamic range from 0.0001 to 100 U mL(-1) and a detection limit of 0.00006 U mL(-1).
Assuntos
Reparo do DNA , Ensaios Enzimáticos/métodos , Grafite/química , Limite de Detecção , Óxidos/química , Uracila-DNA Glicosidase/metabolismo , Modelos Moleculares , Conformação Molecular , Nanotecnologia , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência , Uracila-DNA Glicosidase/químicaRESUMO
Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.
Assuntos
MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/química , Fluorescência , Humanos , Limite de Detecção , Sensibilidade e Especificidade , Uracila-DNA Glicosidase/químicaRESUMO
Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in point-of-care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as â¼10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications.
Assuntos
DNA de Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico , Nucleotídeos/genética , DNA de Neoplasias/isolamento & purificação , Células HT29 , Humanos , Mutação , Sistemas Automatizados de Assistência Junto ao Leito , Células Tumorais CultivadasRESUMO
A novel DNAzymatic amplifier nanomachine that is reconstructed in response to an mRNA input has been developed to enable the functions of concurrent sensitive mRNA imaging and activatable gene therapy in living cells.