Herding cats: Label-based approaches in protein translocation through nanopore sensors for single-molecule protein sequence analysis

Motone, Keisuke; Cardozo, Nicolas; Nivala, Jeff

doi:10.1016/j.isci.2021.103032

Cited by 13 publications

(16 citation statements)

References 121 publications

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“…In response to the pressing need for a deeper understanding of the intrinsic heterogeneity and internal dynamics of individual molecules, nanopores have emerged as highly sensitive and versatile analytical tools enabling label-free, high-throughput, and low-cost characterization of individual molecules. Nanopore sensors are extremely versatile single-molecule sensors employed for both qualitative and quantitative analysis, and representative applications include polynucleotide detection and gene sequencing [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ], polypeptide secondary structure recognition [ 15 , 16 , 17 , 18 ], protein structure analysis [ 19 , 20 , 21 , 22 , 23 ], small molecule and metal ion detection [ 24 , 25 , 26 , 27 , 28 ], polymer analysis [ 29 , 30 ], and virus and bacteria detection [ 31 , 32 , 33 , 34 , 35 , 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis

et al. 2022

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Real-time monitoring, simple operation, and cheaper methods for detecting immunological proteins hold the potential for a solid influence on proteomics and human biology, as they can promote the onset of timely diagnoses and adequate treatment protocols. In this work we present an exploratory study suggesting the applicability of resistive-pulse sensing technology in conjunction with the α-hemolysin (α-HL) protein nanopore, for the detection of the chronic hepatitis B virus (HBV) e-antigen (HBeAg). In this approach, the recognition between HBeAg and a purified monoclonal hepatitis B e antibody (Ab(HBeAg)) was detected via transient ionic current spikes generated by partial occlusions of the α-HL nanopore by protein aggregates electrophoretically driven toward the nanopore’s vestibule entrance. Despite the steric hindrance precluding antigen, antibody, or antigen–antibody complex capture inside the nanopore, their stochastic bumping with the nanopore generated clear transient blockade events. The subsequent analysis suggested the detection of protein subpopulations in solution, rendering the approach a potentially valuable label-free platform for the sensitive, submicromolar-scale screening of HBeAg targets.

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Section: Introductionmentioning

confidence: 99%

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis

et al. 2022

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“…However, it is limited to the analysis of homogeneous protein samples and read lengths typically <50 AAs, which falls far below the median protein length in eukarya (419 AAs), bacteria (306 AAs), and archaea (288 AAs). 7,8 Edman degradation also relies on the presence of a free α-amino group at the peptide's N-terminus, making accurate detection of PTMs not always possible. 9 In addition, each ∼45 min degradation cycle makes the total process quite time-consuming.…”

Section: Introductionmentioning

confidence: 99%

Engineering Biological Nanopore Approaches toward Protein Sequencing

Wei,

Penkauskas,

Reiner

et al. 2023

ACS Nano

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Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

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“…Starting with a seminal paper appeared in 1996, where nanopore sensing of polynucleotides with a protein nanopore was proposed as a paradigm for the sequencing of nucleic acid strands, nanopore sensing has become over the past decades an attractive method for fingerprinting, analyzing, and even controlling various chemistries, biophysical or biochemical processes, by sampling and manipulating continuously one molecule at a time, from the entire population. − …”

Section: Introductionmentioning

confidence: 99%

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection

Mereuta

Asandei

Schiopu

et al. 2023

ACS Appl. Mater. Interfaces

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Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially proteinbased nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channelforming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

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Herding cats: Label-based approaches in protein translocation through nanopore sensors for single-molecule protein sequence analysis

Cited by 13 publications

References 121 publications

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis

Engineering Biological Nanopore Approaches toward Protein Sequencing

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection

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