bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Май 15, 2024
The
flaviviral
NS2B/NS3
protease
is
a
conserved
enzyme
required
for
flavivirus
replication.
Its
highly
dynamic
conformation
poses
major
challenges
but
also
offers
opportunities
antiviral
inhibition.
Here,
we
established
nanopore
tweezers-based
platform
to
monitor
conformational
dynamics
in
real-time.
Molecular
simulations
coupled
with
electrophysiology
revealed
that
the
could
be
captured
middle
of
ClyA
lumen,
stabilized
mainly
by
electrostatic
interactions.
We
designed
new
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.
Theoretical and Natural Science,
Год журнала:
2025,
Номер
89(1), С. 1 - 6
Опубликована: Янв. 15, 2025
Transcriptome
sequencing
has
become
one
of
the
mainstream
methods
for
detecting
gene
expression
after
development
high-throughput
technology.
We
present
trajectory
technological
evolution
technologies
between
three
generations
and
discuss
their
technical
implementations,
advantages,
disadvantages.
Next,
we
show
examples
this
technology
through
current
NGS
technology-based
RNA-seq
analysis.
also
look
forward
to
progress
third-generation
in
read
length
variable
splicing
detection.
Advanced Materials,
Год журнала:
2024,
Номер
unknown
Опубликована: Июль 16, 2024
Biological
nanopores
crucially
control
the
import
and
export
of
biomolecules
across
lipid
membranes
in
cells.
They
have
found
widespread
use
biophysics
biotechnology,
where
their
typically
narrow,
fixed
diameters
enable
selective
transport
ions
small
molecules,
as
well
DNA
peptides
for
sequencing
applications.
Yet,
due
to
channel
sizes,
they
preclude
passage
large
macromolecules,
e.g.,
therapeutics.
Here,
unique
combined
properties
origami
nanotechnology,
machine-inspired
design,
synthetic
biology
are
harnessed,
present
a
structurally
reconfigurable
MechanoPore
(MP)
that
features
lumen
is
tuneable
size
through
molecular
triggers.
Controllable
switching
MPs
between
3
stable
states
confirmed
by
3D-DNA-PAINT
super-resolution
imaging
dye-influx
assays,
after
reconstitution
membrane
liposomes
via
an
inverted-emulsion
cDICE
technique.
Confocal
transmembrane
shows
size-selective
behavior
with
adjustable
thresholds.
Importantly,
conformational
changes
fully
reversible,
attesting
robust
mechanical
overcomes
pressure
from
surrounding
molecules.
These
advance
nanopore
technology,
offering
functional
nanostructures
can
be
tuned
on-demand
-
thereby
impacting
fields
diverse
drug
delivery,
biomolecule
sorting,
sensing,
bottom-up
biology.
Journal of the American Chemical Society,
Год журнала:
2025,
Номер
unknown
Опубликована: Янв. 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.
