RESUMEN
Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.
Asunto(s)
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidad por Sustrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/químicaRESUMEN
Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes.
Asunto(s)
Oocitos , Poliadenilación , Oocitos/metabolismo , Animales , Humanos , Femenino , Ratones , Poli A/metabolismo , Técnicas de Maduración In Vitro de los Oocitos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Transcriptoma , ARN Mensajero Almacenado/metabolismo , ARN Mensajero Almacenado/genética , Metafase , Exorribonucleasas , Proteínas Represoras , Proteínas de Ciclo CelularRESUMEN
La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].
Asunto(s)
Poli A , Unión Proteica , Ribonucleoproteínas , Antígeno SS-B , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Poli A/metabolismo , Poli A/química , Humanos , Modelos Moleculares , Sitios de Unión , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Cristalografía por Rayos X , Dominios Proteicos , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , ARN Mensajero/metabolismo , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.
Asunto(s)
Regiones no Traducidas 3' , Estadios del Ciclo de Vida , Proteínas Protozoarias , Proteínas de Unión al ARN , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Estadios del Ciclo de Vida/genética , Regiones no Traducidas 3'/genética , Animales , Transcriptoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Poli A/metabolismo , Poli A/genética , PoliadenilaciónRESUMEN
The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.
Asunto(s)
Poli A , Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Poli A/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , Células HeLa , Caperuzas de ARN/metabolismo , Estabilidad del ARNRESUMEN
Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates but is decoupled from translation. Interestingly, an optimal tail length of â¼100 nt displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability-a feature of endogenous mRNAs-impacts translation efficiency but not mRNA degradation rates. Stochastic modeling combined with single-cell tracking reveals that poly(A) tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.
Asunto(s)
Poli A , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Poli A/metabolismo , Poli A/genética , Biosíntesis de Proteínas/genética , Estabilidad del ARN/genética , Células HEK293 , Regulación de la Expresión Génica/genéticaRESUMEN
Single-cell RNA sequencing (scRNA-seq) enables the measurement of RNA expressed from individual cells within a tissue or population. RNA expression profiles may be used to draw conclusions about cellular states, cell subtypes within the population, responses to perturbations, and cellular behavior in the context of disease. Here we describe a method for scRNA-seq via single-cell encapsulation and capture of the polyadenosine tails at the 3' end of mRNA transcripts combined with cell and molecular barcoding, allowing for the sequencing of 3' untranslated regions in order to identify expressed genes from a cell.
Asunto(s)
Regiones no Traducidas 3' , ARN Mensajero , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/métodos , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Poli A/genética , Transcriptoma/genéticaRESUMEN
Carcinoembryonic antigen (CEA), a key colon biomarker, demands a precise detection method for cancer diagnosis and prognosis. This study introduces a novel electrochemical aptasensor using a triblock polyadenine probe for ultra-sensitive detection of CEA. The method leverages Exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction. The triblock polyadenine probe self-assembles on the bare gold electrode through the strong affinity between adenine and gold electrode, blocking CEA diffusion and providing a large immobilization surface. CEA binding to hairpin probe 1 (HP1), followed by the hybridization between HP1 and hairpin probe 2 (HP2), triggers DNA cleavage by Exo III, amplifying the signal via a hybridization chain reaction and producing numerous dsDNA walkers that generates a dramatic electrochemical impedance signal. Under optimized conditions, the aptasensor achieved two ultra-low detection limits: 0.39 agâmL-1 within the concentration range of 5 agâmL-1 to 5 × 106 agâmL-1, and 1.5 agâmL-1 within the concentration range of 5 × 106 agâmL-1 to 1 × 1010 agâmL-1. Its performance in human serum samples meets the practical standards, offering a promising new tool for ultrasensitive tumor marker detection, potentially revolutionizing early cancer diagnosis.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Antígeno Carcinoembrionario , Técnicas Electroquímicas , Exodesoxirribonucleasas , Límite de Detección , Hibridación de Ácido Nucleico , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Antígeno Carcinoembrionario/sangre , Humanos , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Poli A/química , Oro/química , ElectrodosRESUMEN
Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.
Asunto(s)
Poli A , Poliadenilación , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , Poli A/metabolismo , Animales , Ratones , Intrones/genética , Transcriptoma/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Regulación de la Expresión GénicaRESUMEN
Microscopic understanding of protein-RNA interactions is important for different biological activities, such as RNA transport, translation, splicing, silencing, etc. Polyadenine (Poly(A)) binding proteins (PABPs) make up a class of regulatory proteins that play critical roles in protecting the poly(A) tails of cellular mRNAs from nuclease degradation. In this work, we performed molecular dynamics simulations to investigate the conformational modifications of human PABP protein and poly(A) RNA that occur during complexation. It is demonstrated that the intermediate linker domain of the protein transforms from a disordered coil-like structure to a helical form during the recognition process, leading to the formation of the complex. On the other hand, disordered collapsed coil-like RNA on complexation has been found to transform into a rigid extended conformation. Importantly, the binding free energy calculation showed that the thermodynamic stability of the complex is primarily guided by favorable hydrophobic interactions between the protein and the RNA.
Asunto(s)
Simulación de Dinámica Molecular , Poli A , Proteínas de Unión a Poli(A) , Termodinámica , Humanos , Poli A/química , Poli A/metabolismo , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/metabolismo , Conformación Proteica , Unión Proteica , Interacciones Hidrofóbicas e Hidrofílicas , ARN/química , ARN/metabolismoRESUMEN
3'-end cleavage and polyadenylation is an essential process for eukaryotic mRNA maturation. In yeast species, the polyadenylation signals that recruit the processing machinery are degenerate and remain poorly characterized compared with the well-defined regulatory elements in mammals. Here we address this issue by developing deep learning models to deconvolute degenerate cis-regulatory elements and quantify their positional importance in mediating yeast poly(A) site formation, cleavage heterogeneity, and strength. In S. cerevisiae, cleavage heterogeneity is promoted by the depletion of U-rich elements around poly(A) sites as well as multiple occurrences of upstream UA-rich elements. Sites with high cleavage heterogeneity show overall lower strength. The site strength and tandem site distances modulate alternative polyadenylation (APA) under the diauxic stress. Finally, we develop a deep learning model to reveal the distinct motif configuration of S. pombe poly(A) sites, which show more precise cleavage than S. cerevisiae Altogether, our deep learning models provide unprecedented insights into poly(A) site formation of yeast species, and our results highlight divergent poly(A) signals across distantly related species.
Asunto(s)
Aprendizaje Profundo , Poliadenilación , Saccharomyces cerevisiae , Schizosaccharomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Poli A/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.
Asunto(s)
Cromatina , Histonas , Poliadenilación , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Cromatina/metabolismo , Cromatina/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Elongación de la Transcripción Genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Poli A/metabolismoRESUMEN
The effectivity and safety of mRNA vaccines critically depends on the presence of correct 5' caps and poly-A tails. Due to the high molecular mass of full-size mRNAs, however, the direct analysis by mass spectrometry is hardly possible. Here we describe the use of synthetic ribonucleases to cleave off 5' and 3' terminal fragments which can be further analyzed by HPLC or by LC-MS. Compared to existing methods (e. g. RNase H), the new approach uses robust catalysts, is free of sequence limitations, avoids metal ions and combines fast sample preparation with high precision of the cut.
Asunto(s)
Poli A , Ribonucleasas , Vacunas de ARNm , Ribonucleasas/metabolismo , Ribonucleasas/química , Poli A/química , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Control de Calidad , Espectrometría de Masas , Cromatografía Líquida de Alta PresiónRESUMEN
Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.
Asunto(s)
Genoma Viral , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Mutación , ARN Polimerasa II , Transcripción Viral , Animales , Humanos , Línea Celular , Chlorocebus aethiops , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Virales/genética , Genoma Viral/genética , Herpes Simple/virología , Herpes Simple/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Poli A/genética , Poli A/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Células Vero , Transcripción Viral/efectos de los fármacos , Transcripción Viral/genética , Replicación Viral/genéticaRESUMEN
A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids (polyA-SNA) to accurately regulate the densities of DNA tracks, the nanowalker showcases its capability to identify miRNA-21, miRNA-486, and miRNA-155 with quick kinetics and attomolar sensitivity, positioning it as a promising option for cancer clinical surveillance.
Asunto(s)
MicroARNs , MicroARNs/análisis , Humanos , Nanoestructuras/química , Poli A/química , ADN/química , Procesos Estocásticos , Técnicas BiosensiblesRESUMEN
Studies have shown that poly(adenine) DNA and RNA strands protonate at a low pH to form self-associating duplexes; however, the nanoscopic morphology of these structures is unclear. Here, we use Transition Electron Microscopy (TEM), Atomic Force Microscopy (AFM), dynamic light scattering (DLS), and fluorescence spectroscopy to show that both ribose identity (DNA or RNA) and assembly conditions (thermal or room-temperature annealing) dictate unique hierarchical structures for poly(adenine) sequences at a low pH. We show that while the thermodynamic product of protonating poly(adenine) DNA is a discrete dimer of two DNA strands, the kinetic product is a supramolecular polymer that branches and aggregates to form micron-diameter superstructures. In contrast, we find that protonated poly(A) RNA polymerizes into micrometer-length, twisted fibers under the same conditions. These divergent hierarchical morphologies highlight the amplification of subtle chemical differences between RNA and DNA into unique nanoscale behaviors. With the use of poly(adenine) strands spanning vaccine technologies, sensing, and dynamic biotechnology, understanding and controlling the underlying assembly pathways of these structures are critical to developing robust, programmable nanotechnologies.
Asunto(s)
ADN , Poli A , ARN , ARN/química , ADN/química , Poli A/química , Protones , Polímeros/química , Microscopía de Fuerza Atómica , Concentración de Iones de HidrógenoRESUMEN
Gold nanorods (AuNRs) with unique optical properties play a pivotal role in applications in plasmonic imaging, small molecule detection, and photothermal therapy. However, challenges in DNA functionalization of AuNRs hinder their full potential due to the presence of a dense cetyltrimethylammonium bromide (CTAB) bilayer, impeding close DNA contact. In this study, we introduced a convenient approach for the rapid assembly of polyadenine (polyA) tailed DNA on AuNRs with control of DNA density, rigidity, and valence. We explored the impact of DNA with designed properties on the construction of core-satellite structures by employing AuNRs as cores and spherical gold nanoparticles (AuNSs) as satellites. Density, rigidity, and valence are identified as crucial factors for efficient construction. Specifically, polyA-tailed DNA modulated DNA density and reduced spatial hindrance and electrostatic repulsion, thereby facilitating the construction. Enhancing the rigidity of DNA and incorporating multiple binding sites can further improve the efficiency.
Asunto(s)
ADN , Oro , Nanotubos , Poli A , Oro/química , Nanotubos/química , ADN/química , Poli A/química , Nanopartículas del Metal/química , Cetrimonio/químicaRESUMEN
In this work, a sensitive colorimetric bioassay method based on a poly(adenine) aptamer (polyA apt) and gold nanoparticles (AuNPs) was developed for the determination of aflatoxin B1 (AFB1). The polyA apt, adsorbed on the AuNPs, especially can bind to the analyte while deterring non-specific interactions. This nano aptasensor uses cationic polymer poly(diallyl dimethyl ammonium chloride) (PDDA), as an aggregating agent, to aggregate gold nanoparticles. PolyA apt-decorated gold nanoparticles (AuNPs/polyA apt) show resistance to PDDA-induced aggregation and maintains their dispersed state (red color) with the optical absorbance signal at λ = 520 nm. However, in the presence of AFB1 in the assay solution, the specific aptamer reacts with high affinity and folds into its three-dimensional form. Aggregation of AuNPs induced by PDDA caused their optical signal shift to λ = 620 nm (blue color). AFB1 concentration in the bioassay solution determines the amount of optical signal shift. Therefore, optical density ratio in two wavelengths (A620/520) can be used as a sturdy colorimetric signal to detect the concentration of aflatoxin B1. AFB1 was linearly detected between 0.5 and 20 ng mL-1, with a detection limit of 0.09 ng mL-1 (S/N = 3). The fabricated aptasensor was applied to the detection of AFB1 in real corn samples.
Asunto(s)
Aflatoxina B1 , Aptámeros de Nucleótidos , Colorimetría , Oro , Nanopartículas del Metal , Zea mays , Aflatoxina B1/análisis , Aflatoxina B1/química , Oro/química , Colorimetría/métodos , Zea mays/química , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Poli A/química , Límite de Detección , Contaminación de Alimentos/análisis , Compuestos de Amonio Cuaternario/química , PolietilenosRESUMEN
A fluorescent immunosorbent assay incorporating signal amplification away from the surface of spherical nucleic acid (SNA) was developed for the detection of chloramphenicol (CAP). Through the conjugation of antibodies and poly-adenine (polyA) DNA onto the surface of gold nanoparticles (AuNPs), the fabrication of the nano-immunoprobe was achieved in a more straightforward and cost-effective manner. Moreover, a strategy utilizing the hybridization chain reaction (HCR) in the amplification step was devised, with particular attention given to the enzyme inhibition associated with SNA. The results demonstrated good performance on CAP detection with a linear range of 0.01-5 ng/L with a detection limit of 0.005 ng/L. The significance of this work mainly lies in the polyA-SNA-based immunoprobe and the thoughtful design to prevent enzyme inhibition.
Asunto(s)
Cloranfenicol , Oro , Nanopartículas del Metal , Cloranfenicol/análisis , Oro/química , Nanopartículas del Metal/química , Poli A/química , Inmunoensayo/métodos , Límite de DetecciónRESUMEN
The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.