9 research outputs found
Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition
Detection of DNA
methylation in real human serum is of great importance
to push the development of clinical research and early diagnosis of
human diseases. Herein, taking advantage of stable pore structure
of aerolysin in a harsh environment, we distinguish methylated cytosine
from cytosine using aerolysin nanopore in human serum. Since wild-type
(WT) aerolysin enables high sensitivity detection of DNA, the subtle
difference between methylated cytosine and cytosine could be measured
directly without any specific designs. Methylated cytosine induced
a population of <i>I</i>/<i>I</i><sub>0</sub> =
0.53 while cytosine was focused on <i>I</i>/<i>I</i><sub>0</sub> = 0.56. The dwell time of methylated cytosine (5.3 ±
0.1 ms) was much longer than that of cytosine (3.9 ± 0.1 ms),
which improves the accuracy for the discrimination of the two oligomers.
Moreover, the pore-membrane system could remain stable for more than
2 h and achieve the detection of methylated cytosine with zero-background
signal in the presence of serum. Additionally, event frequency of
methylated cytosine is in correspondence with the relative concentration
and facilitate the quantification of methylation
Enhanced Resolution of Low Molecular Weight Poly(Ethylene Glycol) in Nanopore Analysis
A design with conjugation of DNA
hairpin structure to the poly(ethylene
glycol) molecule was presented to enhance the temporal resolution
of low molecular weight poly(ethylene glycol) in nanopore studies.
By the virtue of this design, detection of an individual PEG with
molecular weight as low as 140 Da was achieved at the single-molecule
level in solution, which provides a novel strategy for characterization
of an individual small molecule within a nanopore. Furthermore, we
found that the current duration time of poly(ethylene glycol) was
scaled with the relative molecular weight, which has a potential application
in single-molecule detection
Accurate Data Process for Nanopore Analysis
Data
analysis for nanopore experiments remains a fundamental and
technological challenge because of the large data volume, the presence
of unavoidable noise, and the filtering effect. Here, we present an
accurate and robust data process that recognizes the current blockades
and enables evaluation of the dwell time and current amplitude through
a novel second-order-differential-based calibration method and an
integration method, respectively. We applied the developed data process
to analyze both generated blockages and experimental data. Compared
to the results obtained using the conventional method, those obtained
using the new method provided a significant increase in the accuracy
of nanopore measurements
Analysis of a Single α‑Synuclein Fibrillation by the Interaction with a Protein Nanopore
The
formation of an α-synuclein fibril is critical in the
pathogenesis of Parkinson’s disease. The native unfolded α-synuclein
monomer will translocate through an α-hemolysin nanopore by
applied potential at physiological conditions in vitro. Applying a
potential transformed α-synuclein into a partially folded intermediate,
which was monitored by capture inside the vestibule of an α-hemolysin
nanopore with a capture current of 20 ± 1.0 pA. The procedure
involves the critical early stage of α-synuclein structural
transformation. Further elongation of the intermediate produces a
block current to 5 ± 0.5 pA. It is revealed that the early stage
fibril of α-synuclein inside the nanopore is affected by intrapeptide
electrostatic interaction. In addition, trehalose cleared the fibrillation
by changing the surface hydrophobic interaction of A53T α-synuclein,
which did not show any inhibition effect from WT α-synuclein.
The results proved that the interpeptide hydrophobic interactions
in the elongation of A53T α-synuclein protofilaments can be
greatly weakened by trehalose. This suggests that trehalose inhibits
the interpeptide interaction involved in protein secondary structure.
The hydrophobic and electrostatic interactions are associated with
an increase in α-synuclein fibrillation propensity. This work
provides unique insights into the earliest steps of the α-synuclein
aggregation pathway and provides the potential basis for the development
of drugs that can prevent α-synuclein aggregation at the initial
stage
Analysis of a Single α‑Synuclein Fibrillation by the Interaction with a Protein Nanopore
The
formation of an α-synuclein fibril is critical in the
pathogenesis of Parkinson’s disease. The native unfolded α-synuclein
monomer will translocate through an α-hemolysin nanopore by
applied potential at physiological conditions in vitro. Applying a
potential transformed α-synuclein into a partially folded intermediate,
which was monitored by capture inside the vestibule of an α-hemolysin
nanopore with a capture current of 20 ± 1.0 pA. The procedure
involves the critical early stage of α-synuclein structural
transformation. Further elongation of the intermediate produces a
block current to 5 ± 0.5 pA. It is revealed that the early stage
fibril of α-synuclein inside the nanopore is affected by intrapeptide
electrostatic interaction. In addition, trehalose cleared the fibrillation
by changing the surface hydrophobic interaction of A53T α-synuclein,
which did not show any inhibition effect from WT α-synuclein.
The results proved that the interpeptide hydrophobic interactions
in the elongation of A53T α-synuclein protofilaments can be
greatly weakened by trehalose. This suggests that trehalose inhibits
the interpeptide interaction involved in protein secondary structure.
The hydrophobic and electrostatic interactions are associated with
an increase in α-synuclein fibrillation propensity. This work
provides unique insights into the earliest steps of the α-synuclein
aggregation pathway and provides the potential basis for the development
of drugs that can prevent α-synuclein aggregation at the initial
stage
Driven Translocation of Polynucleotides Through an Aerolysin Nanopore
Aerolysin
has been used as a biological nanopore for studying peptides,
proteins, and oligosaccharides in the past two decades. Here, we report
that wild-type aerolysin could be utilized for polynucleotide analysis.
Driven a short polynucleotide of four nucleotides length through aerolysin
occludes nearly 50% amplitude of the open pore current. Furthermore,
the result of total internal reflection fluorescence measurement provides
direct evidence for the driven translocation of single polynucleotide
through aerolysin
Driven Translocation of Polynucleotides Through an Aerolysin Nanopore
Aerolysin
has been used as a biological nanopore for studying peptides,
proteins, and oligosaccharides in the past two decades. Here, we report
that wild-type aerolysin could be utilized for polynucleotide analysis.
Driven a short polynucleotide of four nucleotides length through aerolysin
occludes nearly 50% amplitude of the open pore current. Furthermore,
the result of total internal reflection fluorescence measurement provides
direct evidence for the driven translocation of single polynucleotide
through aerolysin
Real-Time and Accurate Identification of Single Oligonucleotide Photoisomers via an Aerolysin Nanopore
Identification of
the configuration for the photoresponsive oligonucleotide
plays an important role in the ingenious design of DNA nanomolecules
and nanodevices. Due to the limited resolution and sensitivity of
present methods, it remains a challenge to determine the accurate
configuration of photoresponsive oligonucleotides, much less a precise
description of their photoconversion process. Here, we used an aerolysin
(AeL) nanopore-based confined space for real-time determination and
quantification of the absolute <i>cis</i>/<i>trans</i> configuration of each azobenzene-modified oligonucleotide (Azo-ODN)
with a single molecule resolution. The two completely separated current
distributions with narrow peak widths at half height (<0.62 pA)
are assigned to <i>cis</i>/<i>trans</i>-Azo-ODN
isomers, respectively. Due to the high current sensitivity, each isomer
of Azo-ODN could be undoubtedly identified, which gives the accurate
photostationary conversion values of 82.7% for <i>trans</i>-to-<i>cis</i> under UV irradiation and 82.5% for <i>cis</i>-to-<i>trans</i> under vis irradiation. Further
real-time kinetic evaluation reveals that the photoresponsive rate
constants of Azo-ODN from <i>trans-</i>to-<i>cis</i> and <i>cis</i>-to<i>-trans</i> are 0.43 and
0.20 min<sup>–1</sup>, respectively. This study will promote
the sophisticated design of photoresponsive ODN to achieve an efficient
and applicable photocontrollable process
Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis
Selectivity and sensitivity
are two key parameters utilized to
describe the performance of a sensor. In order to investigate selectivity
and sensitivity of the aerolysin nanosensor, we manipulated its surface
charge at different locations via single site-directed mutagenesis.
To study the selectivity, we replaced the positively charged R220
at the entrance of the pore with negatively charged glutamic acid,
resulting in barely no current blockages for sensing negatively charged
oligonucleotides. For the sensitivity, we substituted the positively
charged lumen-exposed amino acid K238 located at <i>trans</i>-ward third of the β-barrel stem with glutamic acid. This leads
to a surprisingly longer duration time at +140 mV, which is about
20 times slower in translocation speed for Poly(dA)<sub>4</sub> compared
to that of wild-type aerolysin, indicating the stronger pore–analyte
interactions and enhanced sensitivity. Therefore, it is both feasible
and understandable to rationally design confined biological nanosensors
for single molecule detection with high selectivity and sensitivity