Crowding-Induced DNA Translocation through a Protein Nanopore

Abstract

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.

Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore

“…In summary, we demonstrate that [i] a crowded, but not viscous, milieu enhances the sensitivity of a solid-state nanopore; [ii] increasing the concentration of the crowding agent markedly increases the current amplitude for DNA, which is in agreement with a previous study utilizing high concentrations of crowding agents; 51 [iii] the crowding agent enhanced markedly the detection of both globular and fibrillar proteins; and [iv] the increased sensitivity aided the characterization of molecules with different structures and sizes. However, the most remarkable feature of this method is its simplicity, in that adding a crowding agent to the electrolyte increases the detection efficiency of the nanopore by up to 1000-fold, without any need for complex surface modification of the nanopore.…”

“…An entropy-driven model was proposed to explain the observed increase in capture rate, peak amplitude, and dwell time by macromolecular crowding for a α-hemolysin (αHL) protein nanopore, 51 but the same model may not be directly applied to our approach. This is because unlike in Yao et al, where the biomolecules were mixed with the crowded solution and driven to the uncrowded solution, 51 our method delivers the biomolecules from the uncrowded solution into the crowded solution. Interestingly, the pronounced improvement in the detection of both DNAs and proteins occurred only when the solution was highly crowded at 50% (w/v) PEG 8000.…”

“… 53 In 2019, Larimi et al investigated the effect of crowding on the interactions of a polypeptide with a biological nanopore, concluding that crowded conditions resulted in a stronger polypeptide–nanopore interaction. 54 The enhancement of the capture rate of DNA in biological nanopores by crowding 51 could be associated with the disruption of electroosmotic flow in the nanopore. Indeed, Yusko et al demonstrated that the formation of the asymmetric concentration gradient by the addition of various percentages of dimethyl sulfoxide (DMSO) into the electrolyte altered the solution properties as well as the electroosmotic flow in a conical nanopore.…”

Sensing serotonin secreted from human serotonergic neurons using aptamer-modified nanopipettes

“…29 This phenomenon may be due to the capacity of nanopipettes to detect a small number of 5-HT molecules interacting with aptamers confined within the 10 nm orifice [47][48][49] or due to the macromolecular crowding effect in complex media that has been previously reported to increase detection sensitivity significantly. 50,51 What is more, the sensitivity of these 5-HT aptamer-modified nanopipettes that can detect concentrations of 5-HT in the low picomolar regime does not come at the cost of chemical selectivity. While other voltammetric techniques such as FSCV have existed for over five decades and have seen progress to advance rapid, sub-second neurotransmitter monitoring, 52 to date, the sensing surface (i.e.…”

Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants

“…114 However, another approach for increased capture rate (118.27 folds) and the decreased translocation speed (120 ms per base) of ssDNA was certainly achieved by introducing PEG molecules (PEG 4k) to the nanopore system. 115 Importantly, it is used to analyze and point out the position of the mutations in DNA via non-functionalized PNA, 116 short fragments of DNA, 117 sequence-specic ssDNA detection with gold nanoparticles, 118 label-free detection of completely matched from mismatched DNA on the basis of enzymatic reaction 4 and to quantify multiple cancer biomarkers in blood samples at picomolar level. 119 Moreover, detection of micro-RNA 120,121 and identication of methylated cytosine by employing lithium salt-gradient have also been achieved.…”