A crowded
cellular environment is highly associated with many significant
biological processes. However, the effect of molecular crowding on
the translocation behavior of DNA through a pore has not been explored.
Here, we use nanopore single-molecule analytical technique to quantify
the thermodynamics and kinetics of DNA transport under heterogeneous
cosolute PEGs. The results demonstrate that the frequency of the translocation
event exhibits a nonmonotonic dependence on the crowding agent size,
while both the event frequency and translocation time increase monotonically
with increasing crowder concentration. In the presence of PEGs, the
rate of DNA capture into the nanopore elevates 118.27-fold, and at
the same time the translocation velocity decreases from 20 to 120
μs/base. Interestingly, the impact of PEG 4k on the DNA-nanopore
interaction is the most notable, with up to ΔΔG = 16.27 kJ mol–1 change in free energy
and 764.50-fold increase in the binding constant at concentration
of 40% (w/v). The molecular crowding effect will has broad applications
in nanopore biosensing and nanopore DNA sequencing in which the strategy
to capture analyte and to control the transport is urgently required.
Chiral recognition at single-molecule level for small active molecules is important, as exhibited by many nanostructures and molecular assemblies in biological systems, but it presents a significant challenge. We report a simple and rapid sensing strategy to discriminate all enantiomers of natural aromatic amino acids (AAA) using a metal-organic complex-functionalized protein nanopore, in which a chiral recognition element and a chiral recognition valve were equipped. A trifunctional molecule, heptakis-(6-deoxy-6-amino)-β-cyclodextrin (amβCD), was non-covalently lodged within the nanopore of an α-hemolysin (αHL) mutant, (M113R)-αHL. Copper(ii) ion reversibly bonds to the amino group of amβCD to form an amβCD-Cu complex, which allowed chiral recognition for each enantiomer in the mixture of AAA by distinct current signals. The Cu plugging valve plays a crucial rule that holds chiral molecules in the nanocavity for a sufficient registering time. Importantly, six enantiomers of all nature AAA could be simultaneously recognized at one time. Enantiomeric excess (ee) could also be accurately detected by this approach. It should be possible to generalize this approach for sensing of other chiral molecules.
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