Motor protein-assisted glycan translocation and sequencing using nanopores DOI Open Access
Zhaobing Gao, Wenjun Ke, Bingqing Xia

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 29, 2024

The glycan sequencing remains a significant bottleneck in glycoscience. While nanopore platforms have achieved substantial progress single-molecule nucleic acid sequencing, their application to has faced considerable challenges, with limited advancements date. In this study, we propose novel strategy for controlling translocation through the MspA as an initial step toward sequencing. By conjugating target helicase-controlled single-stranded DNA, successfully reads of up eleven glycans. For first time, isolated glycan-associated electrical signals, enabling translocation, stretching, and controlled speed neutral glycans nanopore. This method provides platform obtaining read lengths identifying different modifications, demonstrating capability resolve monosaccharide composition glycosidic linkages. To further improve resolution, engineered M2-MspA reduce pore constriction size enhance precision by minimizing random thermal motion translocating glycan. These modifications are expected increase accuracy reliability. work represents proof-of-concept demonstration chain lays promising foundation development fingerprinting technologies. We anticipate that approach will significantly advance commercialization nanopore-based techniques.

Language: Английский

Aerolysin Nanopore Electrochemistry DOI

Jun‐Ge Li,

Yi‐Lun Ying, Yi‐Tao Long

et al.

Accounts of Chemical Research, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 28, 2025

ConspectusIons are the crucial signaling components for living organisms. In cells, their transportation across pore-forming membrane proteins is vital regulating physiological functions, such as generating ionic current signals in response to target molecule recognition. This ion transport affected by confined interactions and local environments within protein pore. Therefore, can efficiently transduce characteristics of each into ion-transport-mediated with high sensitivity. Inspired nature, various pores have been developed high-throughput label-free nanopore sensors single-molecule detection, enabling rapid accurate readouts. particular, aerolysin, a key virulence factor Aeromonas hydrophila, exhibits sensitivity fingerprints detecting subtle differences sequence, conformation, structure DNA, proteins, polypeptides, oligosaccharides, other molecules. Aerolysin features cap that approximately 14 nm wide on cis side central pore about 10 long minimum diameter around 1 nm. Its lumen, 11 charged rings at two entrances neutral amino acids between, facilitates dwelling single analyte characteristic enables rich between well-defined residues analyte. As result, signal offers unique molecular fingerprint, extending beyond traditional volume exclusion model sensing. 2006, aerolysin was first reported discriminate conformational peptides, opening door rapidly growing field electrochemistry. Over years, mutant nanopores emerged, associated advanced instrumentation data analysis algorithms, simultaneous identification over 30 targets number still increasing. electrochemistry particular allows time-resolved qualitative quantitative ranging from DNA sequencing, proteomics, enzyme kinetics, reactions potential clinical diagnostics. Especially, feasibility dynamic would revolutionize omics studies level, paving way promising temporal omics. Despite success this approach so far, it remains challenging understand how correlate distinguishable signatures. Recent attempts added correction terms account variations mobility caused Account, we revisit origin blockade induced molecules inside nanopore. We highlight contributions noncovalent sensing ability through corrected conductance model. Account then describes design interaction networks nanopore, including electrostatic, hydrophobic, hydrogen-bonding, cation−π, ion–charged acid interactions, ultrasensitive biomolecular quantification. Finally, provide an outlook further understanding network improving manipulating fine-tuning toward broad range practical applications.

Language: Английский

Citations

1
Aerolysin Nanopore Structures Revealed at High Resolution in a Lipid Environment DOI Creative Commons

Jana S. Anton,

Ioan Iacovache, Juan F. Bada Juarez

et al.

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 3, 2025

Aerolysin is a β-pore-forming toxin produced by most Aeromonas bacteria, which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging aerolysin-like family, are present throughout all kingdoms life, but very few them have been structurally characterized lipid environment. Here, we first high-resolution atomic cryo-EM structures aerolysin prepore membrane-like These allow identification key interactions, relevant for understanding formation mechanism correctly positioning β-barrel anchoring β-turn motif membrane. Moreover, elucidate at high resolution architecture mutations precisely identify four constriction rings lumen that highly experiments.

Language: Английский

Citations

1
Probing Nanopores: Molecular Dynamics Insights into the Mechanisms of DNA and Protein Translocation through Solid-State and Biological Nanopores DOI

Yuan‐Shuo Zhang,

Mingming Ding

Soft Matter, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Nanopore sequencing technology has revolutionized single-molecule analysis through its unique capability to detect and characterize individual biomolecules with unprecedented precision.

Language: Английский

Citations

1
Single-Molecule Identification and Quantification of Steviol Glycosides with a Deep Learning-Powered Nanopore Sensor DOI
Minmin Li, Jing Wang, Chen Zhang

et al.

ACS Nano, Journal Year: 2024, Volume and Issue: 18(36), P. 25155 - 25169

Published: Aug. 27, 2024

Steviol glycosides (SGs) are a class of high-potency noncalorie natural sweeteners made up common diterpenoid core and varying glycans. Thus, the diversity glycans in composition, linkage, isomerism results tremendous structural complexity SG family, which poses challenges for precise identification leads to fact that SGs frequently used mixtures their variances biological activity remain largely unexplored. Here we show wild-type aerolysin nanopore can detect discriminate diverse species through modulable electro-osmotic flow effect at varied applied voltages. At low voltages, neutral molecule was drawn stuck pore entrance due an energy barrier around R220 sites. The ensuing binding events enable majority species. Increasing voltage break cause translocation events, allowing unambiguous several pairs differing by only one hydroxyl group recognition accumulation from multiple sensing regions Based on data 15 SGs, deep learning-based artificial intelligence (AI) model created process individual blockage achieving rapid, automated, single-molecule quantification real samples. This work highlights value analysis complex glycans-containing glycosides, as well potential sensitive rapid quality assurance glycoside products with use AI.

Language: Английский

Citations

6
Glycan Sequencing Based on Glycosidase-Assisted Nanopore Sensing DOI

Guangda Yao,

Bingqing Xia,

Fangyu Wei

et al.

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 2, 2025

Nanopores are promising sensors for glycan analysis with the accurate identification of complex glycans laying foundation nanopore-based sequencing. However, their applicability toward continuous sequencing has not yet been demonstrated. Here, we present a proof-of-concept by combining nanopore technology glycosidase-hydrolyzing reactions. By continuously monitoring changes in characteristic current generated translocation hydrolysis products through nanopore, sequence can be accurately identified based on specificity glycosidases. With machine learning, improved accuracy to over 98%, allowing reliable determination consecutive building blocks and glycosidic linkages chains while reducing need operator expertise. This approach was validated real samples, calibrated using hydrophilic interaction chromatography-high-performance liquid chromatography (HILIC-HPLC) mass spectrometry (MS). We achieved ten units natural chains, which provided first evidence feasibility nanopore-glycosidase-compatible system Compared traditional methods, this strategy enhances efficiency 5-fold. Additionally, introduced concept 'inverse sequencing', focuses electrical signal rather than monosaccharide identification. eliminates reliance fingerprint libraries typically required putative 'forward hydrolysis' strategies. When challenges both inverse strategies' addressed, will pave way establishing at single-molecule level.

Language: Английский

Citations

0
Nanopore toward Genuine Single-Molecule Sensing: Molecular Ping-Pong Technology DOI
Xinjia Zhao, Yahui Zhang, Guangyan Qing

et al.

Nano Letters, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 26, 2025

Nanopore sensing is a so-called label-free, single-molecule technology; however, multiple events of different molecules are recorded to obtain statistically robust data, which can limit both efficiency and sample use. To overcome these challenges, nanopore molecular ping-pong technology enables precise manipulation, reducing systematic stochastic errors by repeatedly measuring the same molecule. This review introduces fundamentals advancements technology, highlighting recent breakthrough achieving over 10,000 recaptures single dsDNA molecule within minutes. innovation not only minimizes requirements, critical for nonamplifiable samples, but also significantly enhances experimental precision. While current applications focus on dsDNA, extending this protein glycan analysis could transform research. Just as revolutionized DNA sequencing, it holds potential drive development nanopore-based sequencers, paving way groundbreaking in biology biomedicine.

Language: Английский

Citations

0
Thermus thermophilus Argonaute-Assisted Nanopore-Encoding Strategy for Multiplexed Mycotoxin Detection DOI
Jianing Chen, Zhuoqun Su, Jianghua Liu

et al.

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 16, 2025

Multiplexed and portable detection of small molecular food contaminants in complex matrices remains a challenge analytical chemistry. Herein, inspired by the multiplexed detection, nucleic-acid-targeting capability, programmability Thermus thermophilus argonaute (TtAgo), fingerprint recognition nanopore technology, we proposed TtAgo-assisted encoding strategy to detect three mycotoxins simultaneously (AFB1, OTA, ZEN). In this method, effective signal conversion turns into DNA reporters that were observed via technology . general, guide DNAs (gDNAs) released presence mycotoxins, followed specific cleavage mediated TtAgo release reporters. The translocation produces highly distinguishable information due favorable resolution nanopore. Owing high nanopore, method displayed low LODs (AFB1 67.18 fM, OTA 13.32 ZEN 11.69 fM) with wide linear ranges. Furthermore, specificity practical application also verified showed satisfactory results. conclusion, offers novel avenue for molecules.

Language: Английский

Citations

0
Aerolysin nanopore structure revealed at high resolution in lipid environment DOI Creative Commons

Jana S. Anton,

Ioan Iacovache, Juan F. Bada Juarez

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 12, 2024

Abstract Aerolysin is a β-pore-forming toxin produced by most Aeromonas bacteria which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging aerolysin-like family, are present throughout all kingdoms life, but very few them have been structurally characterized lipid environment. Here we first high-resolution atomic cryo-EM structures aerolysin pre-pore membrane-like These allow identification key interactions, relevant for formation positioning barrel into membrane with anchoring β-turn motif now finally observed. Moreover, elucidate at high resolution architecture mutations precisely identify four constriction rings lumen that highly experiments.

Language: Английский

Citations

1
Unraveling Clinical Glycoproteome by Integrating Affinity Enrichment with Nanopore Sequencing DOI
İrshad Ali, Muhammad Mujahid Ali, Quanjun Liu

et al.

ChemBioChem, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 5, 2024

Abstract This prospect explores the integration of enrichment strategies with nanopore detection to advance clinical glycoproteomics. Glycoproteins, crucial for understanding biological processes, pose challenges due their low abundance and structural diversity. Enrichment techniques using lectin affinity, boronate hydrazide chemistry especially molecular imprinted polymers may selectively specifically isolate glycoproteins from complex samples, while technology enables label‐free, real‐time, single‐molecule analysis. approach holds promise disease‐related glycosylation studies, biomarker discovery, personalized medicine, streamlined Standardization, optimization, data analysis remain challenges, requiring interdisciplinary collaborations technological advancements. Overall, this offer transformative potential glycoproteomics innovative diagnostic therapeutic strategies.

Language: Английский

Citations

0
Motor protein-assisted glycan translocation and sequencing using nanopores DOI Open Access
Zhaobing Gao, Wenjun Ke, Bingqing Xia

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 29, 2024

The glycan sequencing remains a significant bottleneck in glycoscience. While nanopore platforms have achieved substantial progress single-molecule nucleic acid sequencing, their application to has faced considerable challenges, with limited advancements date. In this study, we propose novel strategy for controlling translocation through the MspA as an initial step toward sequencing. By conjugating target helicase-controlled single-stranded DNA, successfully reads of up eleven glycans. For first time, isolated glycan-associated electrical signals, enabling translocation, stretching, and controlled speed neutral glycans nanopore. This method provides platform obtaining read lengths identifying different modifications, demonstrating capability resolve monosaccharide composition glycosidic linkages. To further improve resolution, engineered M2-MspA reduce pore constriction size enhance precision by minimizing random thermal motion translocating glycan. These modifications are expected increase accuracy reliability. work represents proof-of-concept demonstration chain lays promising foundation development fingerprinting technologies. We anticipate that approach will significantly advance commercialization nanopore-based techniques.

Language: Английский

Citations

0