Translocation Velocity Research Articles

Transport of a comb-like polymer across a nanochannel subject to a pulling force.

We investigate the dynamics of comb-like polymer translocation through a nanochannel using three-dimensional Langevin dynamics simulations based on a coarse-grained chain model. A comprehensive set of simulations are performed to examine the effects of system parameters such as the grafting densityρof the side chains, the polymer chain length, the nanochannel dimensions, and the magnitude of the pulling force on the translocation dynamics. For a given polymer chain length, keeping the backbone length is constant while varyingρ, we have found that the dependence of the mean translocation time⟨τ⟩onρis non-monotonic, with a maximum translocation time for a specificρat which the translocation is the slowest. The simulation results also show that⟨τ⟩is not significantly affected by the channel width above a certain radius, while the comb-like polymer translocation is hindered by a narrower channel due to increased interactions between the chain monomers and the channel. In addition,⟨τ⟩increases linearly with the nanochannel length. A linear scaling relationship between the mean translocation time⟨τ⟩and the chain lengthNof polymer is obtained,⟨τ⟩∼N. Similarly, the dependence of⟨τ⟩on the backbone chain sizeNbbhas a quasi-linear dependence,⟨τ⟩∼Nbb. On the other hand, the translocation velocityvfollows a power-law relationship with the polymer chain lengthNasv∼N-1. The mean translocation time also shows an inverse linear relationship with the magnitude of the pulling forceF,⟨τ⟩∼F-1. The power-law relationships discovered in this study contribute to the fundamental understanding of the comb polymer translocation dynamics and to establishing a framework for further investigations in this field.

Blobs form during the single-file transport of proteins across nanopores

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis.

Ten Thousand Recaptures of a Single DNA Molecule in a Nanopore and Variance Reduction of Translocation Characteristics.

Nanopores have emerged as highly sensitive biosensors operating at the single-molecule level. However, the majority of nanopore experiments still rely on averaging signals from multiple molecules, introducing systematic errors. To overcome this limitation and obtain accurate information from a single molecule, the molecular ping-pong methodology provides a precise approach involving repeated captures of a single molecule. In this study, we have enhanced the molecular ping-pong technique by incorporating a customized electronic system and control algorithm, resulting in a recapture number exceeding 10,000. During the ping-pong process, we observed a significant reduction in the variance of translocation characteristics, providing fresh insights into single-molecule translocation dynamics. An inhomogeneous translocation velocity of folded DNA has been revealed, illustrating a strong interaction between the molecule and the solid-state nanopore. The results not only promise heightened experimental efficiency with reduced sample volume but also increase the precision in statistical analysis of translocation events, marking a significant stride toward authentic single-molecule nanopore sensing.

Response of carbon fixation, allocation, and growth to source-sink manipulation by defoliation in vegetative citrus trees.

Source-sink balance in plants determines carbon distribution, and altering it can impact carbon fixation, transport, and allocation. We aimed to investigate the effect of altered source-sink ratios on carbon fixation, transport, and distribution in 'Valencia' sweet orange (Citrus x sinensis) by various defoliation treatments (0%, 33%, 66%, and 83% leaf removal). Gas exchange parameters were measured on 0 and 10 days after defoliation using A/Ci response curves, and leaf export was measured two days after defoliation using radioisotope tracer techniques. Greater defoliation increased the maximum rate of carboxylation (Vcmax), electron transport rate (J1200), and triose-phosphate utilization rate (TPU). Leaf export was unaffected by defoliation but increased in leaves closer to the shoot apex. Basipetal translocation velocity in the trunk remained unaltered, indicating that more photosynthates remained in the shoot rather than being transported directly to the root sink. Defoliated plants initiated more new flush shoots but accumulated less shoot biomass per plant after 8 weeks. Carbon allocation to fine roots was smaller in defoliated plants, suggesting defoliation led to retention of carbohydrates in aboveground organs such as the trunk and other shoots from previous growing cycles. In conclusion, the low source-sink ratio increased carbon fixation without impacting individual leaf export in citrus. The results suggest that intermediate sinks such as the aboveground perennial organs play a role in mediating the translocation velocity. Further research is necessary to better understand the dynamics of source-sink regulation in citrus trees.

Electro-osmotic flow in nanoconfinement: Solid-state and protein nanopores.

Electro-osmotic flow (EOF) is a phenomenon where fluid motion occurs in porous materials or micro/nano-channels when an external electric field is applied. In the particular example of single-molecule electrophoresis using single nanopores, the role of EOF on the translocation velocity of the analyte molecule through the nanopore is not fully understood. The complexity arises from a combination of effects from hydrodynamics in restricted environments, electrostatics emanating from charge decorations and geometry of the pores. We address this fundamental issue using the Poisson-Nernst-Planck and Navier-Stokes (PNP-NS) equations for cylindrical solid-state nanopores and three representative protein nanopores (α-hemolysin, MspA, and CsgG). We present the velocity profiles inside the nanopores as a function of charge decoration and geometry of the pore and applied electric field. We report several unexpected results: (a) The apparent charges of the protein nanopores are different from their net charge and the surface charge of the whole protein geometry, and the net charge of inner surface is consistent with the apparent charge. (b) The fluid velocity depends non-monotonically on voltage. The three protein nanopores exhibit unique EOF and velocity-voltage relations, which cannot be simply deduced from their net charge. Furthermore, effective point mutations can significantly change both the direction and the magnitude of EOF. The present computational analysis offers an opportunity to further understand the origins of the speed of transport of charged macromolecules in restricted space and to design desirable nanopores for tuning the speed of macromolecules through nanopores.

Protein Deceleration and Sequencing Using Si3N4-CNT Hybrid Nanopores.

Protein sequencing is crucial for understanding the complex mechanisms driving biological functions and is of utmost importance in molecular diagnostics and medication development. Nanopores have become an effective tool for single molecule sensing, however, the weak charge and non-uniform charge distribution of protein make capturing and sensing very challenging, which poses a significant obstacle to the development of nanopore-based protein sequencing. In this study, to facilitate capturing of the unfolded protein, highly charged peptide was employed in our simulations, we found that the velocity of unfolded peptide translocating through a hybrid nanopore composed of silicon nitride membrane and carbon nanotube is much slower compared to bare silicon nitride nanopore, it is due to the significant interaction between amino acids and the surface of carbon nanotube. Moreover, by introducing variations in the charge states at the boundaries of carbon nanotube nanopores, the competition and combination of the electrophoretic and electroosmotic flows through the nanopores could be controlled, we then successfully regulated the translocation velocity of unfolded proteins through the hybrid nanopores. The proposed hybrid nanopore effectively retards the translocation velocity of protein through it, facilitates the acquisition of ample information for accurate amino acid identification.

Experimental investigation of the forces dictating polymer translocation through solid-state nanopores.

After realizing direct long-read DNA and RNA sequencing, nanopores show also potential for decoding information on other polymers, including proteins and nanostructured DNA. Understanding the process by which these biopolymers are captured and traverse nanopores however remains a challenging task due to the complex nature of the non-equilibrium translocation dynamics which occur on multiple timescales and are dictated mainly by forces over which full experimental control is challenging. This unfortunately also makes verification of theoretical ideas difficult. In this work, we present solid-state nanopore data of DNA fragments for a wide range of experimental conditions in hopes of showcasing the various underlying forces and processes during capture and translocation. Namely, we specifically attempt to establish the absolute and relative magnitude of electrophoretic forces, and frictional contributions originating from hydrodynamic interactions both inside and outside nanopores. This is achieved by studying the dependence of translocation times on nanopore size (3-20 nm diameter) and DNA length (500-45,000 bp), and by measuring the time-dependent translocation velocity of DNA nanostructures, which consists of a linear DNA polymer with three helix-bundle (3HB) sub-structures patterned along its contour. The velocity profile of the patterned nanostructure is measured under different applied voltages (0.1-1 V), in different salt concentrations (0.5-4 M LiCl), and in different pore geometry. The data allow us to corroborate the predictions of tension propagation and consolidate previously published observations regarding measurements of electrophoretic pulling force and pore-polymer interactions in nanopore systems. In addition to bridging nanopore experiment and theory, these results can be used to inform and design several biosensing and digital storage applications.

Unidirectional single-file transport of full-length proteins through a nanopore.

The electrical current blockade of a peptide or protein threading through a nanopore can be used as a fingerprint of the molecule in biosensor applications. However, threading of full-length proteins has only been achieved using enzymatic unfolding and translocation. Here we describe an enzyme-free approach for unidirectional, slow transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore, α-hemolysin (narrowest part is ~1.4 nm in diameter), and a high concentration guanidinium chloride buffer enables unidirectional, single-file protein transport propelled by an electroosmotic effect. We show that the mean protein translocation velocity depends linearly on the applied voltage and translocation times depend linearly on length, resembling the translocation dynamics of ssDNA. Using a supervised machine-learning classifier, we demonstrate that single-translocation events contain sufficient information to distinguish their threading orientation and identity with accuracies larger than 90%. Capture rates of protein are increased substantially when either a genetically encoded charged peptide tail or a DNA tag is added to a protein.

Single-Molecule Optical Tweezers As a Tool for Delineating the Mechanisms of Protein-Processing Mechanoenzymes.

Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA unwinding to protein degradation. Protein-processing mechanoenzymes either remodel the protein structures or translocate them across cellular compartments in an energy-dependent manner. Optical-tweezer-based single-molecule force spectroscopy assays have divulged information on details of chemo-mechanical coupling, directed motion, as well as mechanical forces these enzymes are capable of generating. In this review, we introduce the working principles of optical tweezers as a single-molecule force spectroscopy tool and assays developed to decipher the properties such as unfolding kinetics, translocation velocities, and step sizes by protein remodeling mechanoenzymes. We focus on molecular motors involved in protein degradation and disaggregation, i.e., ClpXP, ClpAP, and ClpB, and insights provided by single-molecule assays on kinetics and stepping dynamics during protein unfolding and translocation. Cellular activities such as protein synthesis, folding, and translocation across membranes are also energy dependent, and the recent single-molecule studies decoding the role of mechanical forces on these processes have been discussed.

Extrusion of chromatin loops by a composite loop extrusion factor.

Chromatin loop extrusion by structural maintenance of chromosome (SMC) complexes is thought to underlie intermediate-scale chromatin organization inside cells. Motivated by a number of experiments suggesting that nucleosomes may block loop extrusion by SMCs, such as cohesin and condensin complexes, we introduce and characterize theoretically a composite loop extrusion factor (composite LEF) model. In addition to an SMC complex that creates a chromatin loop by encircling two threads of DNA, this model includes a remodeling complex that relocates or removes nucleosomes as it progresses along the chromatin, and nucleosomes that block SMC translocation along the DNA. Loop extrusion is enabled by SMC motion along nucleosome-free DNA, created in the wake of the remodeling complex, while nucleosome rebinding behind the SMC acts as a ratchet, holding the SMC close to the remodeling complex. We show that, for a wide range of parameter values, this collection of factors constitutes a composite LEF that extrudes loops with a velocity, comparable to the velocity of remodeling complex translocation on chromatin in the absence of SMC, and much faster than loop extrusion by an isolated SMC that is blocked by nucleosomes.

Magnetic control of particle trapping in a hybrid plasmonic nanopore

Plasmonic nanopores are extensively investigated as single molecules detectors. The main limitations in plasmonic nanopore technology are the too fast translocation velocity of the molecule through the pore and the consequent very short analysis times, as well as the possible instabilities due to local heating. An interesting approach to enable longer acquisition times is represented by the ability to stably trap the nanoparticles used to tag molecules close to the nanopore. Here, we theoretically investigate the performance of a magneto-plasmonic nanopore prepared with a thin layer of cobalt sandwiched between two gold layers. A nanopore is then coupled with a bifunctional (magnetic and plasmonic) core–shell nanoparticle made of magnetite (core) covered with a thin layer of gold (shell). By setting the magnetic configuration of the cobalt layer around the pore by an external magnetic field, it is possible to generate a nanoscale magnetic tweezer to trap the nanoparticle at a specific point. Considering a ∼10 nm diameter magnetite nanoparticle, we calculate a trapping force up to 28 pN, an order of magnitude above the force that can be obtained with standard optical or plasmonic trapping approaches. Moreover, the magnetic force pulls the nanoparticle in close contact with the plasmonic nanopore's wall, thus enabling the formation of a nanocavity enclosing a sub-10 nm3 confined electromagnetic field with an average field intensity enhancement up to 230 at near-infrared wavelengths. The presented hybrid magneto-plasmonic system points toward a strategy to improve nanopore-based biosensors for single-molecule detection and potentially for the analysis of various biomolecules.

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that includecell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Velocity control of protein translocation through a nanopore by tuning the fraction of benzenoid residues.

Protein sequencing is essential to unveil the mechanism of cellular processes that govern the function of living organisms, and which play a crucial role in the field of drug design and molecular diagnostics. Nanopores have been proved to be effective tools in single molecule sensing, but the fast translocation speed of a peptide through a nanopore is one of the major obstacles that hinders the development of nanopore-based protein sequencing. In this work, by using molecular dynamics simulations (MDS) it is found that the peptide containing more hydrophobic residues permeates slower through a molybdenum disulfide nanopore, which originates from the strong interaction between the membrane surface and the hydrophobic residues. The binding affinity is remarkable especially for benzenoid residues as they contain a hydrophobic aromatic ring that is composed of relatively non-polar C-C and C-H bonds. By tuning the fraction of benzenoid residues of the peptide, the velocity of the protein translocation through the nanopore is well controlled. The peptide with all the hydrophobic residues being benzenoid residues is found to translocate through the nanopore almost ten times slower than the one without any benzenoid residues, which is beneficial for gathering adequate information for precise amino acid identification.

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

Pore shapes effects on polymer translocation.

We translocated polymers through pores of different shapes and interaction patterns in three dimensions by Langevin molecular dynamics. There were four simple cylindrical pores of the same length but with different diameters. The results showed that even though decreasing the pore diameter would always decrease the translocation velocity, it was strongly dependent on the shape of the increased pore diameter. Although increasing the pore diameter made the translocation faster in simple cylindrical pores, it was complicated in different pore shapes, e.g. increasing the diameter in the middle decreased the translocation velocity. Investigating polymer shapes through the translocation process and comparing the shapes by the cumulative waiting time for different pore structures reveals the non-equilibrium properties of translocation. Moreover, polymer shape parameters such as gyration radius, polymer center of mass, and average aspect ratio help us to distinguish different pore shapes and/or different polymers.

Nanoparticle-assisted polymer translocation through a nanopore

The effect of interaction between immobile nanoparticle and polymer on the translocation process was investigated using a three dimensional Langevin molecular dynamics. Several parameters for describing the translocation time, the polymer shape, and the dispersion of polymer were calculated. Placing a purely repulsive nanoparticle in front of the nanopore not only increases the translocation time but also cause the translocation process to be longer than the translocation process without the presence of nanoparticle. On the other hand, the attractive–repulsive nanoparticle decreases the translocation time for both having a bigger nanoparticle and stronger interaction between polymer and nanoparticle. Simulation results show that the nanoparticle effect on the translocation velocity depends on the binding energy (BE) and by increasing the BE it changes from a decelerator to an accelerator.Moreover, the waiting time plots show a significant difference between the translocation with or without the nanoparticle and change from step-like to the bell-shaped.Analyzing the center of the mass and shape factor, show that increasing the nanoparticle–nanopore distance will elongate the polymer and manifest itself on the shape of the polymer.

Translocation of a Polyelectrolyte through a Nanopore in the Presence of Trivalent Counterions: A Comparison with the Cases in Monovalent and Divalent Salt Solutions.

A polyelectrolyte threading through a nanopore in a trivalent salt solution is investigated by means of molecular dynamics simulations under a reflective wall boundary. By varying the chain length N and the strength E of the driving electric field applied inside the pore, the translocation time is carefully calculated to get rid of the bouncing effect because of the boundary. The results are analyzed under the scaling form ⟨τ⟩ ∼ NαE–δ and four driving force regimes; namely, the unbiased, the weakly driven, the strongly driven trumpet, and the strongly driven isoflux regime, are distinguished. The exponents are calculated in each regime and compared with the cases in the monovalent and divalent salt solutions. Owing to strong condensation of counter ions, the changes of the exponents in the force regimes are found to be nontrivial. A large increase in translocation time can be, however, achieved as the driving field is weak. The variations of the chain size, the ion condensation, and the effective chain charge show that the process is proceeded in a quasi-equilibrium way in the unbiased regime and deviated to exhibit strong nonequilibrium characteristics as E increases. Several astonishing scaling behaviors of the waiting time function, the translocation velocity, and the diffusion properties are discovered in the study. The results provide deep insights into the phenomena of polyelectrolyte translocation in various salt solutions at different driving forces.

Polyelectrolyte Translocation through a Corrugated Nanopore

AbstractThe problem of slowing down the rate of DNA translocation through a nanopore so as to facilitate detection of the genetic information remains a major challenge. In this study, the effectiveness of employing corrugated nanopores to reduce the polymer translocation velocity is investigated using dissipative particle dynamics (DPD) simulations. The average translocation time, <τ>, increases with the pore length and extent of variation of the pore cross‐sectional area. A systematic comparison of <τ> between flat and corrugated nanopores with walls that are either neutral or attractive for the polymer is performed. The value of <τ> for a corrugated nanopore with an attractive wall is 158% greater than that for a flat pore with a neutral wall. The extent of increase in <τ> obtained by manipulating either the geometry of the pore or the nature of its interaction with the polymer alone is significantly smaller. This is because the combined effect of monomer‐pore interactions and the spatial variation of the electric field strength significantly slows down the entry and escape of the chain from the pore.

Crowding-Induced DNA Translocation through a Protein Nanopore.

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.

Mean velocity and effective diffusion constant for translocation of biopolymer chains across membrane

Chaperone-assisted translocation through a nanopore embedded in membrane holds a prominent role in the transport of biopolymers. Inspired by classical Brownian ratchet, we develop a theoretical framework characterizing such a translocation process through a master equation approach. In this framework, the polymer chain, provided with reversible binding of chaperones, undergoes forward/backward diffusion, which is rectified by chaperones. We drop the assumption of timescale separation and keep the length of a polymer chain finite, both of which happen to be key points in most of the previous studies. Our framework makes it accessible to derive analytical expressions for mean translocation velocity and an effective diffusion constant in a stationary state, which is the basis of a comprehensive understanding of the dynamics of such a process. Generally, the translocation of polymer chains across a membrane consist of three subprocesses: initiation, termination, and translocation of the main body part of a polymer chain, where the translocation of the main body part depends on both the binding/unbinding kinetics of chaperones and the diffusion of the biopolymer chain. This is the main concern of this study. Our results show that the increase of the forward/backward diffusion rate of a polymer chain and the binding/unbinding ratio of chaperones both raise the mean translocation velocity of a polymer chain, and the mean velocity finally reaches a saturation amount with an extremely rapid diffusion or extremely high binding/unbinding ratio. Roughly speaking, the dependence of effective diffusion constant on these two major processes achieves similar behavior. Besides, longer polymer chains employ higher velocity when the diffusion rate and binding/unbinding ratio are both large and similar results hold for polymer chains that are not too long in terms of the effects on the effective diffusion constant.