A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA

Hu, Zheng‐Li; Liu, Shao-Chuang; Ying, Yi‐Lun; Long, Yi‐Tao

doi:10.1039/c8sc03927e

Cited by 39 publications

(33 citation statements)

References 36 publications

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“…There exists a positive correlation between PNK concentration and f r ( and ). The strategies which could be further used to enhance the capture rate include optimizing the concentration gradient and the types of electrolytes [46, 47], reducing the volume of the chamber, and designing the mutagenized aerolysin [48]. Therefore, it is possible to achieve a much lower detection limit compared to other various sensitive PNK detection methods.…”

Section: Resultsmentioning

confidence: 99%

A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

et al. 2019

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The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T4 oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.

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Section: Resultsmentioning

confidence: 99%

A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

et al. 2019

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“…The intact native xylans were extracted from the rice cultivar Nipponbare (NP), and their compositions were confirmed by GC-MS analysis ( Supplemental Table 1 ). The molecular skeleton of xylans from NP is displayed in Figure 1 D. Because lithium ions have been confirmed to improve the capture rate and to cause little ionic current noise ( Kowalczyk et al., 2012 ; Hu et al., 2019 ; Yan et al., 2019 ), lithium chloride (LiCl) was loaded as an electrolyte for nanopore translocation measurements. To evaluate the translocation behavior of xylans, ion currents were recorded as sample molecules passed through the nanopore embedded in the buffer-filled flow cell.…”

Section: Resultsmentioning

confidence: 99%

“…Single-molecule nanopores have received much attention due to their many advantages such as being label-free, having a single-molecular resolution, and possessing quantifiability. After being successfully applied in DNA and protein characterization ( Kowalczyk et al., 2012 ; He et al., 2018 ; Hu et al., 2019 ; Tian et al., 2019 ; Yan et al., 2019 ), nanopores have been used to identify structures with more flexible saccharides ( Bacri et al., 2011 ; Im et al., 2016 , 2019 ; Fennouri et al., 2018 ; Karawdeniya et al., 2018 ). However, their application in plant polysaccharide characterization has not yet been reported.…”

Section: Discussionmentioning

confidence: 99%

A solid-state nanopore-based single-molecule approach for label-free characterization of plant polysaccharides

Cai

Zhang

Liang

et al. 2021

Plant Communications

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Polysaccharides are important biomacromolecules existing in all plants, most of which are integrated into a fibrillar structure called the cell wall. In the absence of an effective methodology for polysaccharide analysis that arises from compositional heterogeneity and structural flexibility, our knowledge of cell wall architecture and function is greatly constrained. Here, we develop a single-molecule approach for identifying plant polysaccharides with acetylated modification levels. We designed a solid-state nanopore sensor supported by a free-standing SiN x membrane in fluidic cells. This device was able to detect cell wall polysaccharide xylans at concentrations as low as 5 ng/μL and discriminate xylans with hyperacetylated and unacetylated modifications. We further demonstrated the capability of this method in distinguishing arabinoxylan and glucuronoxylan in monocot and dicot plants. Combining the data for categorizing polysaccharide mixtures, our study establishes a single-molecule platform for polysaccharide analysis, opening a new avenue for understanding cell wall structures, and expanding polysaccharide applications.

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“…Peptide Q3YEQ2 can traverse through the nanopore over the whole voltage range (+ 60 to + 160 mV); a higher energy barrier for YEQ2Q3 entering into the aerolysin can be speculated. [75,76] For the successful translocation of YEQ2Q3 above + 100 mV, the relationship between the dwell time of peptide YEQ2Q3 and the applied potential can be scaled as τ = τ D exp(À z inside eV/ k B T), where τ is the dwell time of YEQ2Q3 traversing through aerolysin, τ D is a diffusive relaxation time associated with the analyte, e is the magnitude of the elementary charge, k B is the Boltzmann constant, T is the temperature, and V is the applied potential. [46,65,74,76] We estimated the number of effective charges per YEQ2Q3 inside the aerolysin pore (z inside ) and the critical potential (V c ) to be 3.2 × 10 À 1 and 78 mV, where V c for YEQ2Q3 is the potential required to overcome the energy barrier traversing inside a confined aerolysin pore, which can be calculated using V c = k B T/z inside e. Similarly, for peptide Q3YEQ2, the larger z inside value of 5.9 × 10 À 1 reveals a stronger interaction between Q3YEQ2 and the aerolysin interface compared with YEQ2Q3.…”

Section: Resultsmentioning

confidence: 99%

Single‐Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore

Angelov

et al. 2020

ChemBioChem

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Nanopores are original sensors employed for highly sensitive peptides/proteins detection. Herein, we describe the use of an aerolysin nanopore to identify two similar model peptides, YEQYEQQDDDRQQQ (YEQ2Q3) and QDDDRQQQYEQYEQ (Q3YEQ2), with the same amino acid composition but different sequences. All‐atom molecular dynamics (MD) simulations reveal that YEQ2Q3 possesses fewer hydrogen bonds and a more extended conformation than Q3YEQ2. These two peptides, which fold differently, exhibit obviously distinct mass‐independent current blockades with characteristic dwell times when entering the aerolysin nanopore. Typically, at +60 mV, the statistical dwell time of 0.630±0.018 ms for peptide Q3YEQ2 is four times longer than the value of 0.160±0.001 ms for peptide YEQ2Q3, and yet peptide YEQ2Q3 induces ∼1.9 % larger blockade current amplitude than peptide Q3YEQ2. The obtained results show the remarkable potential of aerolysin nanopore for peptides/proteins identification, characterization, sequencing and also demonstrate that the mass identification of nonuniformly charged peptides/proteins by using the nanopore technique could be complicated by their folded structure and complex analyte‐pore interaction.

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A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA

Abstract: By developing lithium-ion-active aerolysin, for the first time we have achieved aerolysin detection of single-stranded DNA longer than 100 nt.

Cited by 39 publications

References 36 publications

A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

A solid-state nanopore-based single-molecule approach for label-free characterization of plant polysaccharides

Single‐Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore

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