Voltage-driven Translocation Research Articles

Effect of charge distribution on the translocation of an inhomogeneously charged polymer through a nanopore

We investigate the voltage-driven translocation of an inhomogeneously charged polymer through a nanopore by utilizing discrete and continuous stochastic models. As a simplified illustration of the effect of charge distribution on translocation, we consider the translocation of a polymer with a single charged site in the presence and absence of interactions between the charge and the pore. We find that the position of the charge that minimizes the translocation time in the absence of pore-polymer interactions is determined by the entropic cost of translocation, with the optimum charge position being at the midpoint of the chain for a rodlike polymer and close to the leading chain end for an ideal chain. The presence of attractive and repulsive pore-charge interactions yields a shift in the optimum charge position toward the trailing end and the leading end of the chain, respectively. Moreover, our results show that strong attractive or repulsive interactions between the charge and the pore lengthen the translocation time relative to translocation through an inert pore. We generalize our results to accommodate the presence of multiple charged sites on the polymer. Our results provide insight into the effect of charge inhomogeneity on protein translocation through biological membranes.

Progress toward Ultrafast DNA Sequencing Using Solid-State Nanopores

Measurements of the ionic current flowing through nanometer-scale pores (nanopores) have been used to analyze single DNA and RNA molecules, with the ultimate goal of achieving ultrafast DNA sequencing. However, attempts at purely electronic measurements have not achieved the signal contrast required for single nucleotide differentiation. In this report we propose a novel method of optical detection of DNA sequence translocating through a nanopore. Each base of the target DNA sequence is 1st mapped onto a 2-unit code, 2 10-bp nucleotide sequence, by biochemical conversion into Designed DNA Polymers. These 2-unit codes are then hybridized to complementary, fluorescently labeled, and self-quenching molecular beacons. As the molecular beacons are sequentially unzipped during translocation through a <2-nm-wide nanopore, their fluorescent tags are unquenched and are detected by a custom-built dual-color total internal reflection fluorescence (TIRF) microscope. The 2-color optical signal is then correlated to the target DNA sequence. A dual-color TIRFM microscope with single-molecule resolution was constructed, and controlled fabrication of 1-dimensional and 2-dimensional arrays of solid-state nanopores was performed. A nanofluidic cell assembly was constructed for TIRF-based optical detection of voltage-driven DNA translocation through a nanopore. We present a novel nanopore-based DNA sequencing technique that uses an optical readout of DNA translocating unzipping through a nanopore. Our technique offers better single nucleotide differentiation in sequence readout, as well as the possibility of large-scale parallelism using nanopore arrays.

Stochastic Detection of Motor Protein–RNA Complexes by Single‐Channel Current Recording

A label- and immobilization-free approach to detecting the reversible formation of complexes between nucleic acids and proteins at the single-molecule level is described. The voltage-driven translocation of individual oligoribonucleotides through a nanoscale protein pore is observed by single-channel current recordings. The oligoribonucleotide 5'-C25A(25)-3' gives rise to current blockades with an average duration of approximately 0.5 ms. In the presence of the RNA-binding ATPase P4, a viral packaging motor from bacteriophage phi8, longer events of tens to hundreds of milliseconds are observed. Upon addition of ATP the long events disappear, indicating the dissociation of the P4RNA complex. The frequency of events also depends on the concentration of P4 and the length of the oligoribonucleotide, thereby confirming the specificity of the P4RNA events. This study shows that single-channel current recordings can be used to monitor RNA-protein complex formation, thus opening up a new means to examine the motor activity of RNA- or DNA-processing enzymes.

Motor-free mitochondrial presequence translocase drives membrane integration of preproteins

The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions. However, the major preprotein transport machinery of the inner membrane, termed the presequence translocase, translocates polypeptide chains into or across the membrane. Different views exist of the molecular mechanism of the translocase, in particular of the coupling with the import motor of the matrix. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential.

Langevin dynamics simulations of ds-DNA translocation through synthetic nanopores

We have implemented a coarse-grained model to study voltage-driven as-DNA translocation through nanopores located in synthetic membranes. The simulated trajectory of the DNA through the nanopores was calculated using Langevin dynamics. We present the results based on more than 120,000 individual translocations. We are particularly interested in this work in probing the physical basis of various experimentally observed--yet poorly understood--phenomena. Notably, we observe in our simulations the formation of ds-DNA hairpins, widely suspected to be the basis for quantized blockage. We study the translocation time, a measurable quantity crucially important in polyelectrolyte characterization, as a function of hairpin vertex location along the polymer backbone, finding that this behavior can be tuned to some degree by simulation parameters. We also study the voltage dependence of the tendency of hairpins to serve as the initiators of translocation events. Surprisingly, we find that the resulting probability depends vitally upon whether the events counted are ultimately successful or not. Further details lead us to propose that failed attempts in experimental translocation studies may be more common--and deceptive--than is generally recognized. We find the time taken by successful single file translocations to be directly proportional to the ratio of chain length to the applied voltage. Finally, we address a common yet puzzling phenomenon in translocation experiments: translocation events in which the current through the pore is highly, yet incompletely, blocked. We present the findings that offer a new explanation for such events.

Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers—in cells and in silico

Nanosecond, megavolt-per-meter pulses—higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells—produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.

Optical tweezers for force measurements on DNA in nanopores

We demonstrate the means to integrate two powerful and widely used single-molecule techniques, viz., optical tweezers and solid-state nanopores. This setup permits simultaneous spatial sampling and high-resolution force measurements of nucleic acids and proteins. First, we demonstrate the rapid spatial localization of nanopores using our custom-built inverted microscope and ionic current measurements. This is made possible by including a specialized flow cell for silicon-based nanopores with an optical window for a high-numerical aperture microscope. Subsequently, we can insert individual DNA molecules into a single nanopore and arrest the DNA during voltage-driven translocation. To detect the position of the trapped particle in the optical trap with high accuracy in the presence of the nanopore, the optical tweezers uses reflected light from the bead for detection. Consequently, we can use our setup to directly determine the force on a DNA molecule in a solid-state nanopore. Finally, we suggest a number of new experiments that become possible with this unique technique.

Direct force measurements on DNA in a solid-state nanopore

Among the variety of roles for nanopores in biology, an important one is enabling polymer transport, for example in gene transfer between bacteria1 and transport of RNA through the nuclear membrane2. Recently, this has inspired the use of protein3,4,5 and solid-state6,7,8,9,10 nanopores as single-molecule sensors for the detection and structural analysis of DNA and RNA by voltage-driven translocation. The magnitude of the force involved is of fundamental importance in understanding and exploiting this translocation mechanism, yet so far it has remained unknown. Here, we demonstrate the first measurements of the force on a single DNA molecule in a solid-state nanopore by combining optical tweezers11 with ionic-current detection. The opposing force exerted by the optical tweezers can be used to slow down and even arrest the translocation of the DNA molecules. We obtain a value of 0.24±0.02 pN mV−1 for the force on a single DNA molecule, independent of salt concentration from 0.02 to 1 M KCl. This force corresponds to an effective charge of 0.50±0.05 electrons per base pair equivalent to a 75% reduction of the bare DNA charge.

Microscopic Kinetics of DNA Translocation through Synthetic Nanopores

We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 × 2.6 nm 2 cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.

Monte Carlo simulations of voltage-driven translocation of a signal sequence

Transmembrane potentials play important but poorly understood roles in many biological processes, including signal sequence-mediated protein translocation across bacterial membranes. In this study we applied Monte Carlo techniques to simulate the way the potential acts on a signal sequence. The simulations demonstrate that in the absence of a potential the signal sequence prefers insertion in both helical hairpin and transmembrane α-helical conformations. However, in the presence of a potential only the transmembrane α-helical conformation is the state of lowest energy for the signal sequence. From these results it is concluded that the membrane potential stabilizes the transmembrane orientation of a signal sequence, explaining the membrane potential dependence of preprotein translocation.