Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity DOI Creative Commons
Keisuke Motone,

Daphne Kontogiorgos-Heintz,

Jasmine Wee

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Окт. 20, 2023

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited achieving this goal. Here, we establish method for long-range, single-molecule reading intact strands on commercial nanopore sensor array. By using the ClpX unfoldase ratchet proteins through CsgG nanopore, achieve single-amino acid level sensitivity, enabling sequencing combinations amino substitutions across long strands. For greater accuracy, demonstrate reread individual molecules, spanning hundreds acids length, multiple times, and explore potential high accuracy barcode sequencing. Further, develop biophysical model that can simulate raw signals

Язык: Английский

Full-length single-molecule protein fingerprinting DOI
Mike Filius, Raman van Wee, Carlos de Lannoy

и другие.

Nature Nanotechnology, Год журнала: 2024, Номер 19(5), С. 652 - 659

Опубликована: Фев. 13, 2024

Язык: Английский

Процитировано

9
Dynamic Features Driven by Stochastic Collisions in a Nanopore for Precise Single-Molecule Identification DOI
Jia Wang, Shao-Chuang Liu, Zheng‐Li Hu

и другие.

Journal of the American Chemical Society, Год журнала: 2025, Номер unknown

Опубликована: Янв. 2, 2025

Nanopore technology holds great potential for single-molecule identification. However, extracting meaningful features from ionic current signals and understanding the molecular mechanisms underlying specific remain unresolved. In this study, we uncovered a distinctive pattern in K238Q aerolysin nanopore, characterized by transient spikes superimposed on two stable transition states. By employing neural network model, demonstrated that these previously overlooked dynamic spike exhibit superior discriminative power, improving accuracy 44% to 93%. We identified states result simultaneous interactions of ssDNA with sensitive sites nanopore. The proposed stochastic collision model offers mechanistic framework interpreting generation features. This indicates continuous transitions facilitate iterative, comprehensive snapshots nanopores. Our findings introduce new approach optimizing nanopore capture complex substantially improve

Язык: Английский

Процитировано

1
Large and Stable Nanopores Formed by Complement Component 9 for Characterizing Single Folded Proteins DOI Creative Commons
Wachara Chanakul, Anasua Mukhopadhyay, Saurabh Awasthi

и другие.

ACS Nano, Год журнала: 2025, Номер unknown

Опубликована: Янв. 27, 2025

Biological nanopores offer a promising approach for single-molecule analysis of nucleic acids, peptides, and proteins. The work presented here introduces biological nanopore formed by the self-assembly complement component 9 (C9). This exceptionally large cylindrical protein pore is composed 20 ± 4 monomers C9 resulting in diameter 10 nm an effective length 13 nm. These poly(C9) pores remain stable up to 30 min without indications gating, flickering, or clogging across range transmembrane voltages (−150 +150 mV) ionic strengths (50 1000 mM). At physiologic pH, ring-shaped distribution negative positive surface charges lumen enables capture analyte proteins electro-osmotic flow leads residence times whose most probable values can exceed 300 μs. We used determine volume shape unlabeled folded with molecular weights between 230 kDa unprecedented accuracy context resistive pulse recordings. Finally, made it possible distinguish open closed conformations adenylate kinase based on differences current modulations within pulses corresponding approximations their shape. Thus, enable highly sensitive accurate characterization wide natively single molecule level.

Язык: Английский

Процитировано

1
Nanopore‐Functionalized Hybrid Lipid‐Block Copolymer Membranes Allow Efficient Single‐Molecule Sampling and Stable Sensing of Human Serum DOI Creative Commons
Edo Vreeker, Fabian Grünewald, Nieck Jordy van der Heide

и другие.

Advanced Materials, Год журнала: 2025, Номер unknown

Опубликована: Март 4, 2025

Abstract Biological nanopores are powerful tools for single‐molecule detection, with promising potential as next‐generation biosensors. A major bottleneck in nanopore analysis is the fragility of supporting lipid membranes, that easily rupture after exposure to biological samples. Membranes comprising PMOXA‐PDMS‐PMOXA (poly(2‐methyloxazoline‐b‐dimethylsiloxane‐b‐2‐methyloxazoline)) or PBD‐PEO (poly(1,2‐butadiene)‐b‐poly(ethylene oxide)) polymers may form robust alternatives, but their suitability reconstitution a broad range has not yet been investigated. Here, membranes found be highly toward applied voltages and human serum, while providing poor environment reconstitution. However, hybrid containing similar molar ratio PBD 11 PEO 8 diphytanoyl phosphatidylcholine (DPhPC) lipids show best both worlds: suitable wide variety nanopores. Molecular dynamics simulations reveal ≈12 nm domains interspersed by polymer matrix. Nanopores partition into these nanodomains sequester lipids, possibly offering same binding strength native bilayer. reconstituted yield efficient sampling biomolecules enable sensing high concentrations serum. This work thus shows functionalized allow sensing, forming interfaces, resolving an important novel nanopore‐based

Язык: Английский

Процитировано

1
Electro-osmotic Flow Generation via a Sticky Ion Action DOI
Behzad Mehrafrooz, Luning Yu, Laxmi Pandey

и другие.

ACS Nano, Год журнала: 2024, Номер 18(27), С. 17521 - 17533

Опубликована: Июнь 4, 2024

Selective transport of ions through nanometer-sized pores is fundamental to cell biology and central many technological processes such as water desalination electrical energy storage. Conventional methods for generating ion selectivity include placement fixed charges at the inner surface a nanopore either point mutations in protein pore or chemical treatment solid-state surface, with each type requiring custom approach. Here, we describe general method transforming nanoscale into highly selective, anion-conducting channel capable giant electro-osmotic effect. Our molecular dynamics simulations reverse potential measurements show that exposure biological high concentrations guanidinium chloride renders positively charged due transient binding cations surface. A comparison four nanopores reveals relationship between selectivity, shape, composition flow. Guanidinium are also found produce anion flow via same mechanism. sticky-ion approach generate can have numerous applications controlling detection, identification, sequencing individual proteins.

Язык: Английский

Процитировано

6
A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples DOI Creative Commons
Xiaojun Wei, Xiaoqin Wang, Zehui Zhang

и другие.

Nature Communications, Год журнала: 2022, Номер 13(1)

Опубликована: Ноя. 11, 2022

Abstract Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an virological biomarker HIV-1 infection. Here, To improve p24 patients missed by immunological tests that dominate diagnostics market, we show a click chemistry amplified nanopore (CAN) assay quantitative detection. This strategy achieves 20.8 fM (0.5 pg/ml) limit antigen human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation CAN pilot cohort shows quantification at ultra-low concentration range correlation with CD4 count load. We believe this can utility detecting monitoring progression treatment efficacy, also be readily modified to detect other infectious diseases.

Язык: Английский

Процитировано

22
Functional Nanopores Enabled with DNA DOI Creative Commons
Yongzheng Xing, Alexia Rottensteiner, Jonah Ciccone

и другие.

Angewandte Chemie International Edition, Год журнала: 2023, Номер 62(33)

Опубликована: Апрель 26, 2023

Abstract Membrane‐spanning nanopores are used in label‐free single‐molecule sensing and next‐generation portable nucleic acid sequencing, as powerful research tools biology, biophysics, synthetic biology. Naturally occurring protein peptide pores, well inorganic nanopores, these applications, with their limitations. The structural functional repertoire of can be considerably expanded by functionalising existing pores DNA strands creating an entirely new class nanotechnology. This review outlines progress this area developments to open up applications.

Язык: Английский

Процитировано

14
Narrowing Signal Distribution by Adamantane Derivatization for Amino Acid Identification Using an α-Hemolysin Nanopore DOI
Xiaojun Wei, Dumei Ma, Junlin Ou

и другие.

Nano Letters, Год журнала: 2024, Номер 24(5), С. 1494 - 1501

Опубликована: Янв. 24, 2024

The rapid progress in nanopore sensing has sparked interest protein sequencing. Despite recent notable advancements amino acid recognition using nanopores, chemical modifications usually employed this process still need further refinements. One of the challenges is to enhance specificity avoid downstream misidentification acids. By employing adamantane label proteinogenic acids, we developed an approach fingerprint individual acids wild-type α-hemolysin nanopore. unique structure adamantane-labeled (ALAAs) improved spatial resolution, resulting distinctive current signals. Various parameters were explored a machine-learning algorithm and achieved validation accuracy 81.3% for distinguishing nine selected Our results not only advance effort single-molecule characterization nanopores but also offer potential platform studying intrinsic variant structures molecules.

Язык: Английский

Процитировано

5
Direct and Continuous Monitoring of Multicomponent Antibiotic Gentamicin in Blood at Single-Molecule Resolution DOI
Changjian Zhao, Yu Wang, Chen Chen

и другие.

ACS Nano, Год журнала: 2024, Номер 18(12), С. 9137 - 9149

Опубликована: Март 12, 2024

Point-of-care monitoring of small molecules in biofluids is crucial for clinical diagnosis and treatment. However, the inherent low degree recognition complex composition present significant obstacles current detection technologies. Although nanopore sensing excels analysis molecules, direct remains a challenge. In this study, we method molecule drug gentamicin whole blood based on mechanosensitive channel conductance Pseudomonas aeruginosa (PaMscS) nanopore. PaMscS can directly detect distinguish its main components with only monomethyl difference. The 'molecular sieve' structure enables measurement human within 10 min. Furthermore, continuous device constructed achieved live rats approximately 2.5 h without consumption, while be analyzed situ. This approach rapid convenient single-molecule level resolution, which significantly lower threshold concentration promote more efficient use. Moreover, work also lays foundation future development technology resolution living body.

Язык: Английский

Процитировано

5
Location of Phosphorylation Sites within Long Polypeptide Chains by Binder-Assisted Nanopore Detection DOI Creative Commons
Wei-Hsuan Lan,

Hanxiao He,

Hagan Bayley

и другие.

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(35), С. 24265 - 24270

Опубликована: Июль 10, 2024

The detection and mapping of protein phosphorylation sites are essential for understanding the mechanisms various cellular processes identifying targets drug development. study biopolymers at single-molecule level has been revolutionized by nanopore technology. In this study, we detect within long polypeptides (>700 amino acids), after attachment binders that interact with phosphate monoesters; electro-osmosis is used to drive tagged chains through engineered nanopores. By monitoring ionic current carried a nanopore, located individual polypeptide chains, providing valuable step toward proteomics.

Язык: Английский

Процитировано

5