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1.
Microb Genom ; 10(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38713194

RESUMO

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.


Assuntos
Genoma Bacteriano , Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Escherichia coli/genética , Staphylococcus aureus/genética , Análise de Sequência de DNA/métodos , Pseudomonas aeruginosa/genética , Sequenciamento por Nanoporos/métodos , DNA Bacteriano/genética , Klebsiella pneumoniae/genética , Sequenciamento Completo do Genoma/métodos , Bactérias/genética , Bactérias/classificação , Humanos
2.
J Phys Chem Lett ; 15(19): 5120-5129, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38709198

RESUMO

In the past few decades, nanometer-scale pores have been employed as powerful tools for sensing biological molecules. Owing to its unique structure and properties, solid-state nanopores provide interesting opportunities for the development of DNA sequencing technology. Controlling DNA translocation in nanopores is an important means of improving the accuracy of sequencing. Here we present a proof of principle study of accelerating DNA captured across targeted graphene nanopores using surface charge density and find the intrinsic mechanism of the combination of electroosmotic flow induced by charges of nanopore and electrostatic attraction/repulsion between the nanopore and ssDNA. The theoretical study performed here provides a new means for controlling DNA transport dynamics and makes better and cheaper application of graphene in molecular sequencing.


Assuntos
DNA , Grafite , Nanoporos , Eletricidade Estática , Grafite/química , DNA/química , DNA de Cadeia Simples/química , Eletro-Osmose , Análise de Sequência de DNA/métodos
3.
J Phys Chem A ; 128(19): 3926-3933, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38712508

RESUMO

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.


Assuntos
DNA , Simulação de Dinâmica Molecular , Nanoporos , Transistores Eletrônicos , DNA/química
4.
Biosensors (Basel) ; 14(5)2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38785729

RESUMO

Glutamic acid decarboxylase antibody (GADAb) has emerged as a significant biomarker for clinical diagnosis and prognosis in type 1 diabetes (T1D). In this study, we investigated the potential utilization of glass capillary solid-state nanopores as a cost-effective and easily preparable platform for the detection of individual antigens, antibodies, and antigen-antibody complexes without necessitating any modifications to the nanopores. Our findings revealed notable characteristic variations in the translocation events of glutamic acid decarboxylase (GAD65) through nanopores under different voltage conditions, discovered that anomalous phenomenon of protein translocation events increasing with voltage may potentially be caused by the crowding of multiple proteins in the nanopores, and demonstrated that there are multiple components in the polyclonal antibodies (GADAb-poly). Furthermore, we achieved successful differentiation between GAD65, GADAb, and GADAb-GAD65 complexes. These results offer promising prospects for the development of a rapid and reliable GADAb detection method, which holds the potential to be applied in patient serum samples, thereby facilitating a label-free, cost-effective, and early diagnosis of type I diabetes.


Assuntos
Diabetes Mellitus Tipo 1 , Glutamato Descarboxilase , Nanoporos , Glutamato Descarboxilase/imunologia , Humanos , Diabetes Mellitus Tipo 1/diagnóstico , Técnicas Biossensoriais , Anticorpos , Vidro
5.
RNA Biol ; 21(1): 1-15, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38758523

RESUMO

2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.


Assuntos
Aprendizado de Máquina , Análise de Sequência de RNA , Transcriptoma , Humanos , Metilação , Análise de Sequência de RNA/métodos , Células HeLa , Sequenciamento por Nanoporos/métodos , Células HEK293 , Biologia Computacional/métodos , Processamento Pós-Transcricional do RNA , Nanoporos , Software , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
ACS Nano ; 18(19): 12412-12426, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38693619

RESUMO

Glycans play vital roles in nearly all life processes of multicellular organisms, and understanding these activities is inseparable from elucidating the biological significance of glycans. However, glycan research has lagged behind that of DNA and protein due to the challenges posed by structural heterogeneity and isomerism (i.e., structures with equal molecular weights) the lack of high-efficiency structural analysis techniques. Nanopore technology has emerged as a sensitive single-molecule biosensor, shining a light on glycan analysis. However, a significant number of glycans are small and uncharged, making it challenging to elicit identifiable nanopore signals. Here we introduce a R-binaphthyl tag into glycans, which enhances the cation-π interaction between the derivatized glycan molecules and the nanopore interface, enabling the detection of neutral glycans with an aerolysin nanopore. This approach allows for the distinction of di-, tri-, and tetrasaccharides with monosaccharide resolution and has the potential for group discrimination, the monitoring of enzymatic transglycosylation reactions. Notably, the aerolysin mutant T240R achieves unambiguous identification of six disaccharide isomers, trisaccharide and tetrasaccharide linkage isomers. Molecular docking simulations reveal that multiple noncovalent interactions occur between residues R282, K238, and R240 and the glycans and R-binaphthyl tag, significantly slowing down their translocation across the nanopore. Importantly, we provide a demonstration of the kinetic translocation process of neutral glycan isomers, establishing a solid theoretical foundation for glycan nanopore analysis. The development of our technology could promote the analysis of glycan structural isomers and has the potential for nanopore-based glycan structural determination and sequencing.


Assuntos
Toxinas Bacterianas , Nanoporos , Polissacarídeos , Proteínas Citotóxicas Formadoras de Poros , Polissacarídeos/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Simulação de Acoplamento Molecular , Mutação
7.
J Am Chem Soc ; 146(19): 12919-12924, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38691627

RESUMO

RNA is a key biochemical marker, yet its chemical instability and complex secondary structure hamper its integration into DNA nanotechnology-based sensing platforms. Relying on the denaturation of the native RNA structure using urea, we show that restructured DNA/RNA hybrids can readily be prepared at room temperature. Using solid-state nanopore sensing, we demonstrate that the structures of our DNA/RNA hybrids conform to the design at the single-molecule level. Employing this chemical annealing procedure, we mitigate RNA self-cleavage, enabling the direct detection of restructured RNA molecules for biosensing applications.


Assuntos
DNA , Nanoporos , RNA , RNA/química , RNA/análise , DNA/química , Técnicas Biossensoriais/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Nanotecnologia/métodos , Ureia/química
8.
Nat Commun ; 15(1): 4049, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38744925

RESUMO

Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.


Assuntos
Oryza , Análise de Sequência de RNA , Humanos , Análise de Sequência de RNA/métodos , Oryza/genética , Processamento Pós-Transcricional do RNA , Nanoporos , RNA/genética , RNA/metabolismo , Sequenciamento por Nanoporos/métodos , Aprendizado Profundo , Inosina/metabolismo , Inosina/genética , Transcriptoma/genética
9.
Nanotechnology ; 35(32)2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38692268

RESUMO

An ion detection device that combines a DNA-origami nanopore and a field-effect transistor (FET) was designed and modeled to determine sensitivity of the nanodevice to the local cellular environment. Such devices could be integrated into a live cell, creating an abiotic-biotic interface integrated with semiconductor electronics. A continuum model is used to describe the behavior of ions in an electrolyte solution. The drift-diffusion equations are employed to model the ion distribution, taking into account the electric fields and concentration gradients. This was matched to the results from electric double layer theory to verify applicability of the model to a bio-sensing environment. The FET device combined with the nanopore is shown to have high sensitivity to ion concentration and nanopore geometry, with the electrical double layer behavior governing the device characteristics. A logarithmic relationship was found between ion concentration and a single FET current, generating up to 200 nA of current difference with a small applied bias.


Assuntos
DNA , Íons , Nanoporos , Transistores Eletrônicos , DNA/análise , DNA/química , Nanotecnologia/instrumentação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos
10.
J Phys Chem Lett ; 15(20): 5556-5563, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38752895

RESUMO

Solid-state nanopores have been extensively explored as single-molecule sensors, bearing the potential for the sequencing of DNA. Although they offer advantages in terms of high mechanical robustness, tunable geometry, and compatibility with existing semiconductor fabrication techniques in comparison with their biological counterparts, efforts to sequence DNA with these nanopores have been hampered by insufficient spatial resolution and high noise in the measured ionic current signal. Here we show that these limitations can be overcome by the use of solid-state nanopores featuring a thin, narrow constriction as the sensing region, inspired by biological protein nanopores that have achieved notable success in DNA sequencing. Our extensive molecular dynamics simulations show that these bio-inspired nanopores can provide high spatial resolution equivalent to 2D material nanopores and, meanwhile, significantly inhibit noise levels. A theoretical model is also provided to assess the performance of the bio-inspired nanopore, which could guide its design and optimization.


Assuntos
Simulação de Dinâmica Molecular , Nanoporos , DNA/química , Análise de Sequência de DNA/métodos
12.
Lab Chip ; 24(10): 2721-2735, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38656267

RESUMO

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 µm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.


Assuntos
Nanoporos , Ribonucleotídeos/química , Ribonucleotídeos/análise , Temperatura , Polimetil Metacrilato/química
13.
J Am Chem Soc ; 146(19): 13356-13366, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38602480

RESUMO

The crucial roles that glycans play in biological systems are determined by their structures. However, the analysis of glycan structures still has numerous bottlenecks due to their inherent complexities. The nanopore technology has emerged as a powerful sensor for DNA sequencing and peptide detection. This has a significant impact on the development of a related research area. Currently, nanopores are beginning to be applied for the detection of simple glycans, but the analysis of complex glycans by this technology is still challenging. Here, we designed an engineered α-hemolysin nanopore M113R/T115A to achieve the sensing of complex glycans at micromolar concentrations and under label-free conditions. By extracting characteristic features to depict a three-dimensional (3D) scatter plot, glycans with different numbers of functional groups, various chain lengths ranging from disaccharide to decasaccharide, and distinct glycosidic linkages could be distinguished. Molecular dynamics (MD) simulations show different behaviors of glycans with ß1,3- or ß1,4-glycosidic bonds in nanopores. More importantly, the designed nanopore system permitted the discrimination of each glycan isomer with different lengths in a mixture with a separation ratio of over 0.9. This work represents a proof-of-concept demonstration that complex glycans can be analyzed using nanopore sequencing technology.


Assuntos
Simulação de Dinâmica Molecular , Nanoporos , Polissacarídeos , Polissacarídeos/química , Proteínas Hemolisinas/química , Engenharia de Proteínas
14.
Anal Chem ; 96(19): 7411-7420, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38652893

RESUMO

Accurate analysis of microRNAs (miRNAs) at the single-cell level is extremely important for deeply understanding their multiple and intricate biological functions. Despite some advancements in analyzing single-cell miRNAs, challenges such as intracellular interferences and insufficient detection limits still remain. In this work, an ultrasensitive nanopore sensor for quantitative single-cell miRNA-155 detection is constructed based on ionic current rectification (ICR) coupled with enzyme-free catalytic hairpin assembly (CHA). Benefiting from the enzyme-free CHA amplification strategy, the detection limit of the nanopore sensor for miRNA-155 reaches 10 fM and the nanopore sensor is more adaptable to complex intracellular environments. With the nanopore sensor, the concentration of miRNA-155 in living single cells is quantified to realize the early diagnosis of triple-negative breast cancer (TNBC). Furthermore, the nanopore sensor can be applied in screening anticancer drugs by tracking the expression level of miRNA-155. This work provides an adaptive and universal method for quantitatively analyzing intracellular miRNAs, which will greatly improve our understanding of cell heterogeneity and provide a more reliable scientific basis for exploring major diseases at the single-cell level.


Assuntos
MicroRNAs , Nanoporos , Análise de Célula Única , Neoplasias de Mama Triplo Negativas , MicroRNAs/análise , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Feminino , Linhagem Celular Tumoral , Limite de Detecção
15.
J Clin Microbiol ; 62(5): e0024624, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38563782

RESUMO

Next-generation sequencing has evolved as a powerful tool, with applications that extend from diagnosis to public health surveillance and outbreak investigations. Short-read sequencing, using primarily Illumina chemistry, has been the prevailing approach. Single-molecule sensing and long-read sequencing using Oxford Nanopore Technologies (ONT) has witnessed a breakthrough in the evolution of the technology, performance, and applications in the past few years. In this issue of the Journal of Clinical Microbiology, Bogaerts et al. (https://doi.org/10.1128/jcm.01576-23) describe the utility of the latest ONT sequencing technology, the R10.4.1, in bacterial outbreak investigations. The authors demonstrate that ONT R10.4.1 technology can be comparable to Illumina sequencing for single-nucleotide polymorphism-based phylogeny. The authors emphasize that the reproducibility between ONT and Illumina technologies could facilitate collaborations among laboratories utilizing different sequencing platforms for outbreak investigations.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Surtos de Doenças , Nanoporos , Sequenciamento por Nanoporos/métodos , Saúde Pública , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Polimorfismo de Nucleotídeo Único
16.
Nanoscale ; 16(17): 8352-8360, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38563277

RESUMO

Detection and characterization of protein-protein interactions are essential for many cellular processes, such as cell growth, tissue repair, drug delivery, and other physiological functions. In our research, we have utilized emerging solid-state nanopore sensing technology, which is highly sensitive to better understand heparin and fibroblast growth factor 1 (FGF-1) protein interactions at a single-molecule level without any modifications. Understanding the structure and behavior of heparin-FGF-1 complexes at the single-molecule level is very important. An abnormality in their formation can lead to life-threatening conditions like tumor growth, fibrosis, and neurological disorders. Using a controlled dielectric breakdown pore fabrication approach, we have characterized individual heparin and FGF-1 (one of the 22 known FGFs in humans) proteins through the fabrication of 17 ± 1 nm nanopores. Compared to heparin, the positively charged heparin-binding domains of some FGF-1 proteins translocationally react with the pore walls, giving rise to a distinguishable second peak with higher current blockade. Additionally, we have confirmed that the dynamic FGF-1 is stabilized upon binding with heparin-FGF-1 at the single-molecule level. The larger current blockades from the complexes relative to individual heparin and the FGF-1 recorded during the translocation ensure the binding of heparin-FGF-1 proteins, forming binding complexes with higher excluded volumes. Taken together, we demonstrate that solid-state nanopores can be employed to investigate the properties of individual proteins and their complex interactions, potentially paving the way for innovative medical therapies and advancements.


Assuntos
Fator 1 de Crescimento de Fibroblastos , Heparina , Nanoporos , Ligação Proteica , Fator 1 de Crescimento de Fibroblastos/química , Fator 1 de Crescimento de Fibroblastos/metabolismo , Heparina/química , Heparina/metabolismo , Humanos
17.
Langmuir ; 40(18): 9622-9629, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38652583

RESUMO

The detection and identification of nanoscale molecules are crucial, but traditional technology comes with a high cost and requires skilled operators. Solid-state nanopores are new powerful tools for discerning the three-dimensional shape and size of molecules, enabling the translation of molecular structural information into electric signals. Here, DNA molecules with different shapes were designed to explore the effects of electroosmotic forces (EOF), electrophoretic forces (EPF), and volume exclusion on electric signals within solid-state nanopores. Our results revealed that the electroosmotic force was the main driving force for single-stranded DNA (ssDNA), whereas double-stranded DNA (dsDNA) was primarily dominated by electrophoretic forces in nanopores. Moreover, dsDNA caused greater amplitude signals and moved faster through the nanopore due to its larger diameter and carrying more charges. Furthermore, at the same charge level and amount of bases, circular dsDNA exhibited a tighter structure compared to brush DNA, resulting in a shorter length. Consequently, circular dsDNA caused higher current-blocking amplitudes and faster passage speeds. The characterization approach based on nanopores allows researchers to get molecular information about size and shape in real time. These findings suggest that nanopore detection has the potential to streamline nanoscale characterization and analysis, potentially reducing both the cost and complexity.


Assuntos
DNA , Nanoporos , DNA/química , Conformação de Ácido Nucleico , DNA de Cadeia Simples/química , Eletro-Osmose/métodos
18.
Anal Chem ; 96(18): 7163-7171, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38664895

RESUMO

Biological nanopores feature functional elements on the outer surfaces (FEOS) and inner walls (FEIW), enabling precise control over ions and molecules with exceptional sensitivity and specificity. This provides valuable inspiration to scientists for the development of intelligent artificial nanochannel-based platforms, with a wide range of potential applications, including biosensors. Much effort has been dedicated to investigating the distinct contribution of FEOS and FEIW of multichannel membrane biosensors. However, the intricate interactions among neighboring pores in multichannel biosensors have presented challenges. This underscores the untapped potential of single nanochannels as ideal candidates in this field. Here, we employed single nanochannel membranes with different pore sizes to investigate the distinct contributions of FEIW and FEOS to single-nanochannel biosensors, combined with numerical simulations. Our findings revealed that alterations in the negative charges of FEIW and FEOS, induced by target binding, have differential effects on ion transport, contingent upon the degree of nanoconfinement. In the case of smaller pores, such as 20 nm, the ion concentration polarization driven by FEIW can independently control ion transport through the surface's electric double layer. However, as the pore size increases to 40-60 nm, both FEIW and FEOS become essential for effective ion concentration polarization. When the pore size reaches 100 nm, both FEIW and FEOS are ineffective and thus unsuitable for biosensors. Simulations demonstrate that the observed phenomena can be attributed to the interactions between the charges of FEIW and FEOS within the overlapping electric double layer under confinement. These results underscore the critical role of pore size as a key parameter in governing the functionality of probes within or on nanopore-based biosensors as well as in the design of nanopore-based devices.


Assuntos
Técnicas Biossensoriais , Nanoporos , Propriedades de Superfície , Tamanho da Partícula , Porosidade
19.
Int J Biol Macromol ; 267(Pt 1): 131273, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38569994

RESUMO

The nanopore-based translocation of a single-stranded RNA (ssRNA) in mixed salt solution has garnered increasing interest for its biological and technological significance. However, it is challenging to comprehensively understand the effects of the mixed ion species on the translocation dynamics due to their cooperation and competition, which can be directly reflected by the ion screening and neutralizing effects, respectively. In this study, Langevin dynamics simulation is employed to investigate the properties of ssRNA conformation and translocation in mixed Na+-Mg2+ ion environments. Simulation results reveal that the ion screening effect dominates the change in the ssRNA conformational size, the ion neutralizing effect controls the capture rate of the ssRNA by the nanopore, and both of them take charge of the different changes in translocation time of the ssRNA under various mixed ion environments. Under high Na+ ion concentration, as Mg2+ concentration increases, the ion neutralizing effect strengthens, weakening the driving force inside the nanopore, leading to longer translocation time. Conversely, at low Na+ concentration, an increase in Mg2+ concentration enhances the ion screening effect, aiding in faster translocation. Furthermore, these simulation results will be explained by quantitative analysis, advancing a deeper understanding of the complicated effects of the mixed Na+-Mg2+ ions.


Assuntos
Magnésio , Conformação de Ácido Nucleico , Sódio , Sódio/química , Sódio/metabolismo , Magnésio/química , Nanoporos , Simulação de Dinâmica Molecular , Íons/química , RNA/química
20.
Genome Res ; 34(3): 454-468, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38627094

RESUMO

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.


Assuntos
Nanoporos , Humanos , Análise de Sequência de DNA/métodos , Sequenciamento por Nanoporos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Genômica/métodos
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