@article {396, title = {The structure and physical properties of a packaged bacteriophage particle}, journal = {Nature}, volume = {627}, year = {2024}, month = {03/2024}, pages = {905-914}, chapter = {905}, abstract = {

A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species, the three-dimensional organization of viral genomes has mostly eluded experimental probes. Here we report all-atom structural models of an HK97 virion, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome{\textendash}capsid interface.

}, keywords = {DNA packaging, Multiresolution simulation, virology}, doi = {10.1038/s41586-024-07150-4}, url = {https://www.nature.com/articles/s41586-024-07150-4$\#$Abs1}, author = {Kush Coshic and Christopher Maffeo and David Winogradoff and Aleksei Aksimentiev} } @article {389, title = {A DNA turbine powered by a transmembrane potential across a nanopore}, journal = {Nature Nanotechnology}, volume = {19}, year = {2024}, month = {03/2024}, pages = {338--344}, chapter = {338}, abstract = {

Rotary motors play key roles in energy transduction, from macroscale windmills to nanoscale turbines such as ATP synthase in cells. Despite our abilities to construct engines at many scales, developing functional synthetic turbines at the nanoscale has remained challenging. Here, we experimentally demonstrate rationally designed nanoscale DNA origami turbines with three chiral blades. These DNA nanoturbines are 24{\textendash}27 nm in height and diameter and can utilize transmembrane electrochemical potentials across nanopores to drive DNA bundles into sustained unidirectional rotations of up to 10 revolutions s-1. The rotation direction is set by the designed chirality of the turbine. All-atom molecular dynamics simulations show how hydrodynamic flows drive this turbine. At high salt concentrations, the rotation direction of turbines with the same chirality is reversed, which is explained by a change in the anisotropy of the electrophoretic mobility. Our artificial turbines operate autonomously in physiological conditions, converting energy from naturally abundant electrochemical potentials into mechanical work. The results open new possibilities for engineering active robotics at the nanoscale.

}, issn = {1748-3395}, doi = {10.1038/s41565-023-01527-8}, url = {https://doi.org/10.1038/s41565-023-01527-8}, author = {Shi, Xin and Pumm, Anna-Katharina and Maffeo, Christopher and Kohler, Fabian and Feigl, Elija and Zhao, Wenxuan and Verschueren, Daniel and Golestanian, Ramin and Aksimentiev, Aleksei and Dietz, Hendrik and Dekker, Cees} } @article {399, title = {Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein}, journal = {Biochemistry}, year = {2024}, abstract = {

Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound\ holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.

}, doi = {10.1021/acs.biochem.4c00209}, url = {https://doi.org/10.1021/acs.biochem.4c00209}, author = {Fatima, Saman and Mehrafrooz, Behzad and Boggs, David G. and Ali, Noor and Singh, Swapnil and Thielges, Megan C. and Bridwell-Rabb, Jennifer and Aksimentiev, Aleksei and Olshansky, Lisa} } @article {398, title = {Electro-Osmotic Flow Generation via a Sticky Ion Action}, journal = {ACS nano}, volume = {18}, year = {2024}, pages = {17521-17533}, chapter = {17521-17533}, abstract = {

Selective transport of ions through nanometer-sized pores is fundamental to cell biology and central to many technological processes such as water desalination and electrical energy storage. Conventional methods for generating ion selectivity include placement of fixed electrical charges at the inner surface of a nanopore through either point mutations in a protein pore or chemical treatment of a solid-state nanopore surface, with each nanopore type requiring a custom approach. Here, we describe a general method for transforming a nanoscale pore into a highly selective, anion-conducting channel capable of generating a giant electro-osmotic effect. Our molecular dynamics simulations and reverse potential measurements show that exposure of a biological nanopore to high concentrations of guanidinium chloride renders the nanopore surface positively charged due to transient binding of guanidinium cations to the protein surface. A comparison of four biological nanopores reveals the relationship between ion selectivity, nanopore shape, composition of the nanopore surface, and electro-osmotic flow. Guanidinium ions are also found to produce anion selectivity and a giant electro-osmotic flow in solid-state nanopores via the same mechanism. Our sticky-ion approach to generate electro-osmotic flow can have numerous applications in controlling molecular transport at the nanoscale and for detection, identification, and sequencing of individual proteins.

}, doi = {10.1021/acsnano.4c00829}, author = {Mehrafrooz, Behzad and Yu, Luning and Pandey, Laxmi and Siwy, Zuzanna S and Wanunu, Meni and Aksimentiev, Aleksei} } @article {397, title = {Dehydrated Biomimetic Membranes with Skinlike Structure and Function}, journal = {ACS Applied Materials \& Interfaces}, volume = {16}, year = {2024}, pages = {20865-20877}, abstract = {

Novel vapor-permeable materials are sought after for applications in protective wear, energy generation, and water treatment. Current impermeable protective materials effectively block harmful agents but trap heat due to poor water vapor transfer. Here we present a new class of materials, vapor permeable dehydrated nanoporous biomimetic membranes (DBMs), based on channel proteins. This application for biomimetic membranes is unexpected as channel proteins and biomimetic membranes were assumed to be unstable under dry conditions. DBMs mimic human skin{\textquoteright}s structure to offer both high vapor transport and small molecule exclusion under dry conditions. DBMs feature highly organized pores resembling sweat pores in human skin, but at super high densities (\>1012\ pores/cm2). These DBMs achieved exceptional water vapor transport rates, surpassing commercial breathable fabrics by up to 6.2 times, despite containing \>2 orders of magnitude smaller pores (1 nm vs \>700 nm). These DBMs effectively excluded model biological agents and harmful chemicals both in liquid and vapor phases, again in contrast with the commercial breathable fabrics. Remarkably, while hydrated biomimetic membranes were highly permeable to liquid water, they exhibited higher water resistances after dehydration at values \>38 times that of commercial breathable fabrics. Molecular dynamics simulations support our hypothesis that dehydration induced protein hydrophobicity increases which enhanced DBM performance. DBMs hold promise for various applications, including membrane distillation, dehumidification, and protective barriers for atmospheric water harvesting materials.

}, doi = {10.1021/acsami.3c19572}, url = {https://doi.org/10.1021/acsami.3c19572}, author = {Oh, Hyeonji and Tu, Yu-Ming and Samineni, Laximicharan and De Respino, Sophie and Mehrafrooz, Behzad and Joshi, Himanshu and Massenburg, Lynnicia and Lopez-Marques, Horacio and Elessawy, Nada and Song, Woochul and Behera, Harekrushna and Dhiman, Raman and Boorla, Veda Sheersh and Kher, Kartik and Lin, Yi-Chih and Maranas, Costas and Aksimentiev, Aleksei and D. Freeman, Benny and Kumar, Manish} } @article {387, title = {DNA double helix, a tiny electromotor}, journal = {Nature Nanotechnology}, volume = {18}, year = {2023}, month = {12/2022}, pages = {238{\textendash}242}, chapter = {238}, abstract = {

Flowing fluid past chiral objects has been used for centuries to power rotary motion in man-made machines. By contrast, rotary motion in nanoscale biological or chemical systems is produced by biasing Brownian motion through cyclic chemical reactions. Here we show that a chiral biological molecule, a DNA or RNA duplex rotates unidirectionally at billions of revolutions per minute when an electric field is applied along the duplex, with the rotation direction being determined by the chirality of the duplex. The rotation is found to be powered by the drag force of the electro-osmotic flow, realizing the operating principle of a macroscopic turbine at the nanoscale. The resulting torques are sufficient to power rotation of nanoscale beads and rods, offering an engineering principle for constructing nanoscale systems powered by electric field.

}, issn = {1748-3387}, doi = {10.1038/s41565-022-01285-z}, url = {https://www.nature.com/articles/s41565-022-01285-z}, author = {Maffeo, Christopher and Quednau, Lauren and Wilson, James and Aksimentiev, Aleksei} } @article {395, title = {A Marcus-Type Inverted Region in the Translocation Kinetics of a Knotted Protein}, journal = {The Journal of Physical Chemistry Letters}, volume = {14}, year = {2023}, month = {11/2023}, pages = {10719-10726}, abstract = {

Knotted proteins are rare but important species, yet how their complex topologies affect their physical properties is not fully understood. Here we combine single molecule nanopore experiments and all-atom MD simulations to study the electric-field-driven unfolding during the translocation through a model pore of individual protein knots important for methylating tRNA. One of these knots shows an unusual behavior that resembles the behavior of electrons hopping between two potential surfaces: as the electric potential driving the translocation reaction is increased, the rate eventually plateaus or slows back down in the {\textquotedblleft}Marcus inverted regime{\textquotedblright}. Our results shed light on the influence of topology in knotted proteins on their forced translocation through a pore connecting two electrostatic potential wells.

}, doi = {10.1021/acs.jpclett.3c02183}, url = {https://doi.org/10.1021/acs.jpclett.3c02183}, author = {Tripathi, Prabhat and Mehrafrooz, Behzad and Aksimentiev, Aleksei and Jackson, Sophie E. and Gruebele, Martin and Wanunu, Meni} } @article {393, title = {Long-Range Conductivity in Proteins Mediated by Aromatic Residues}, journal = {ACS Physical Chemistry Au}, volume = {3}, year = {2023}, month = {09}, pages = {444{\textendash}455}, abstract = {

Single-molecule measurements show that many proteins, lacking any redox cofactors, nonetheless exhibit electrical conductance on the order of a nanosiemen over 10 nm distances, implying that electrons can transit an entire protein in less than a nanosecond when subject to a potential difference of less than 1 V. This is puzzling because, for fast transport (i.e., a free energy barrier of zero), the hopping rate is determined by the reorganization energy of approximately 0.8 eV, and this sets the time scale of a single hop to at least 1 μs. Furthermore, the Fermi energies of typical metal electrodes are far removed from the energies required for sequential oxidation and reduction of the aromatic residues of the protein, which should further reduce the hopping current. Here, we combine all-atom molecular dynamics (MD) simulations of non-redox-active proteins (consensus tetratricopeptide repeats) with an electron transfer theory to demonstrate a molecular mechanism that can account for the unexpectedly fast electron transport. According to our MD simulations, the reorganization energy produced by the energy shift on charging (the Stokes shift) is close to the conventional value of 0.8 eV. However, the non-ergodic sampling of molecular configurations by the protein results in reaction-reorganization energies, extracted directly from the distribution of the electrostatic energy fluctuations, that are only \~{}0.2 eV, which is small enough to enable long-range conductivity, without invoking quantum coherent transport. Using the MD values of the reorganization energies, we calculate a current decay with distance that is in agreement with experiment.

}, doi = {10.1021/acsphyschemau.3c00017}, url = {https://doi.org/10.1021/acsphyschemau.3c00017}, author = {Krishnan, Siddharth and Aksimentiev, Aleksei and Lindsay, Stuart and Matyushov, Dmitry} } @article {388, title = {Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates}, journal = {Journal of Chemical Theory and Computation}, volume = {19}, year = {2023}, month = {05/2023}, pages = {3721--3740}, abstract = {

Proteins containing intrinsically disordered regions are integral parts of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While the all-atom molecular dynamics method can, in principle, elucidate the conformational changes that arise from point mutations, the applications of this method to protein condensate systems is conditioned upon the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area, and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force fields that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA{\textendash}FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA{\textendash}protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

}, doi = {10.1021/acs.jctc.3c00148}, url = {https://pubs.acs.org/doi/10.1021/acs.jctc.3c00148}, author = {Kumar Sarthak and David Winogradoff and Yingda Ge and Sua Myong and Aleksei Aksimentiev} } @article {384, title = {Unidirectional single-file transport of full-length proteins through a nanopore}, journal = {Nature Biotechnology}, volume = {41}, year = {2023}, month = {01/2023}, pages = {1130--1139}, abstract = {

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 \textasciitilde1.4\thinspacenm 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.

}, issn = {1546-1696}, doi = {10.1038/s41587-022-01598-3}, url = {https://doi.org/10.1038/s41587-022-01598-3}, author = {Yu, Luning and Kang, Xinqi and Li, Fanjun and Mehrafrooz, Behzad and Makhamreh, Amr and Fallahi, Ali and Foster, Joshua C. and Aksimentiev, Aleksei and Chen, Min and Wanunu, Meni} } @article {386, title = {Super-Resolution Detection of DNA Nanostructures Using a Nanopore}, journal = {Advanced Materials}, year = {2023}, month = {01/2023}, pages = {2207434}, abstract = {

Abstract High-resolution analysis of biomolecules has brought unprecedented insights into fundamental biological processes and dramatically advanced biosensing. Notwithstanding the ongoing resolution revolution in electron microscopy and optical imaging, only a few methods are presently available for high-resolution analysis of unlabelled single molecules in their native states. Here, we demonstrate label-free electrical sensing of structured single molecules with a spatial resolution down to single-digit nanometres. Using a narrow solid-state nanopore, we detect the passage of a series of nanostructures attached to a freely translocating DNA molecule, resolving individual nanostructures placed as close as 6\ nm apart and with a surface-to-surface gap distance of only 2\ nm. We attribute such super-resolution ability to the nanostructure-induced enhancement of the electric field at the tip of the nanopore. Our work demonstrates a general approach to improving the resolution of single-molecule nanopore sensing and presents a critical advance towards label-free, high-resolution DNA sequence mapping and digital information storage independent of molecular motors.

}, keywords = {biosensing, DNA nanostructures, Nanopores, single molecule, super resolution}, doi = {https://doi.org/10.1002/adma.202207434}, author = {Chen, Kaikai and Choudhary, Adnan and Sandler, Sarah E. and Maffeo, Christopher and Ducati, Caterina and Aksimentiev, Aleksei and Keyser, Ulrich F.} } @article {377, title = {Multi-resolution simulation of DNA transport through large synthetic nanostructures.}, journal = {Phys Chem Chem Phys}, volume = {24}, year = {2022}, month = {2022 Feb 02}, pages = {2706-2716}, abstract = {

Modeling and simulation has become an invaluable partner in development of nanopore sensing systems. The key advantage of the nanopore sensing method - the ability to rapidly detect individual biomolecules as a transient reduction of the ionic current flowing through the nanopore - is also its key deficiency, as the current signal itself rarely provides direct information about the chemical structure of the biomolecule. Complementing experimental calibration of the nanopore sensor readout, coarse-grained and all-atom molecular dynamics simulations have been used extensively to characterize the nanopore translocation process and to connect the microscopic events taking place inside the nanopore to the experimentally measured ionic current blockades. Traditional coarse-grained simulations, however, lack the precision needed to predict ionic current blockades with atomic resolution whereas traditional all-atom simulations are limited by the length and time scales amenable to the method. Here, we describe a multi-resolution framework for modeling electric field-driven passage of DNA molecules and nanostructures through to-scale models of synthetic nanopore systems. We illustrate the method by simulating translocation of double-stranded DNA through a solid-state nanopore and a micron-scale slit, capture and translocation of single-stranded DNA in a double nanopore system, and modeling ionic current readout from a DNA origami nanostructure passage through a nanocapillary. We expect our multi-resolution simulation framework to aid development of the nanopore field by providing accurate, to-scale modeling capability to research laboratories that do not have access to leadership supercomputer facilities.

}, keywords = {Biological Transport, DNA, Electricity, Molecular Dynamics Simulation, Nanopores}, issn = {1463-9084}, doi = {10.1039/d1cp04589j}, author = {Choudhary, Adnan and Maffeo, Christopher and Aleksei Aksimentiev} } @article {385, title = {Trapping of protein cargo molecules inside DNA origami nanocages}, journal = {Nanoscale}, volume = {14}, year = {2022}, month = {11/2022}, pages = {18041{\textendash}18050}, abstract = {

The development of the DNA origami technique has directly inspired the idea of using three-dimensional DNA cages for the encapsulation and targeted delivery of drug or cargo molecules. The cages would be filled with molecules that would be released at a site of interest upon cage opening triggered by an external stimulus. Though different cage variants have been developed, efficient loading of DNA cages with freely-diffusing cargo molecules that are not attached to the DNA nanostructure and their efficient retention within the cages has not been presented. Here we address these challenges using DNA origami nanotubes formed by a double-layer of DNA helices that can be sealed with tight DNA lids at their ends. In a first step we attach DNA-conjugated cargo proteins to complementary target strands inside the DNA tubes. After tube sealing, the cargo molecules are released inside the cavity using toehold-mediated strand displacement by externally added invader strands. We show that DNA invaders are rapidly entering the cages through their DNA walls. Retention of \~{}70 kDa protein cargo molecules inside the cages was, however, poor. Guided by coarse-grained simulations of the DNA cage dynamics, a tighter sealing of the DNA tubes was developed which greatly reduced the undesired escape of cargo proteins. These improved DNA nanocages allow for efficient encapsulation of medium-sized cargo molecules while remaining accessible to small molecules that can be used to trigger reactions, including a controlled release of the cargo via nanocage opening.

}, doi = {10.1039/d2nr05356j}, author = {Merle Scherf and Florian Scheffler and Christopher Maffeo and Ulrich Kemper and Jingjing Ye and Aleksei Aksimentiev and Ralf Seidel and Uta Reibetanz} } @article {372, title = {Percolation transition prescribes protein size-specific barrier to passive transport through the nuclear pore complex.}, journal = {Nature Communications}, volume = {13}, year = {2022}, month = {09/2022}, pages = {5138}, chapter = {5138}, abstract = {

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex{\textquoteright}s pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of experimentally derived NPC structures with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein transport reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistics of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

}, keywords = {NPC, nuclear pore, passive transport}, issn = {2041-1723}, doi = {10.1038/s41467-022-32857-1}, author = {Winogradoff, David and Chou, Han-Yi and Maffeo, Christopher and Aleksei Aksimentiev} } @article {380, title = {Leakless end-to-end transport of small molecules through micron-length DNA nanochannels}, journal = {Science Advances}, volume = {8}, year = {2022}, month = {09/09/2022}, abstract = {
Designed and engineered protein and DNA nanopores can be used to sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between nonadjacent compartments or cells. Here, we design micrometer-long, 7-nm-diameter DNA nanochannels that small molecules can traverse according to the laws of continuum diffusion. Binding DNA origami caps to channel ends eliminates transport and demonstrates that molecules diffuse from one channel end to the other rather than permeating through channel walls. These micrometer-length nanochannels can also grow, form interconnects, and interface with living cells. This work thus shows how to construct multifunctional, dynamic agents that control molecular transport, opening ways of studying intercellular signaling and modulating molecular transport between synthetic and living cells.
}, doi = {10.1126/sciadv.abq4834}, url = {https://www.science.org/doi/10.1126/sciadv.abq4834}, author = {Li, Yi and Maffeo, Christopher and Joshi, Himanshu and Aleksei Aksimentiev and M{\'e}nard, Brice and Schulman, Rebecca} } @article {374, title = {Discrimination of RNA fiber structures using solid-state nanopores}, journal = {Nanoscale}, volume = {14}, year = {2022}, month = {05/2023}, pages = {6866-6875}, abstract = {

RNA fibers are a class of biomaterials that can be assembled using HIV-like kissing loop interactions. Because of the programmability of molecular design and low immunorecognition, these structures present an interesting opportunity to solve problems in nanobiotechnology and synthetic biology. However, the experimental tools to fully characterize and discriminate among different fiber structures in solution are limited. Herein, we utilize solid-state nanopore experiments and Brownian dynamics simulations to characterize and distinguish several RNA fiber structures that differ in their degrees of branching. We found that, regardless of the electrolyte type and concentration, fiber structures that have more branches produce longer and deeper ionic current blockades in comparison to the unbranched fibers. Experiments carried out at temperatures ranging from 20{\textendash}60 {\textdegree}C revealed almost identical distributions of current blockade amplitudes, suggesting that the kissing loop interactions in fibers are resistant to heating within this range.

}, issn = {2040-3364}, doi = {10.1039/D1NR08002D}, url = {http://pubs.rsc.org/en/content/articlepdf/2022/NR/D1NR08002D}, author = {Tripathi, Prabhat and Chandler, Morgan and Maffeo, Christopher Michael and Fallahi, Ali and Makhamreh, Amr and Halman, Justin and Aleksei Aksimentiev and Afonin, Kirill A. and Wanunu, Meni} } @article {373, title = {Single-molecule biophysics experiments in silico: Toward a physical model of a replisome}, journal = {iScience}, volume = {25}, year = {2022}, month = {05/2022}, pages = {104264}, abstract = {

The interpretation of single-molecule experiments is frequently aided by computational modeling of biomolecular dynamics. The growth of computing power and ongoing validation of computational models suggest that it soon may be possible to replace some experiments outright with computational mimics. Here, we offer a blueprint for performing single-molecule studies in silico using a DNA-binding protein as a test bed. We demonstrate how atomistic simulations, typically limited to sub-millisecond durations and zeptoliter volumes, can guide development of a coarse-grained model for use in simulations that mimic single-molecule experiments. We apply the model to recapitulate, in silico, force-extension characterization of protein binding to single-stranded DNA and protein and DNA replacement assays, providing a detailed portrait of the underlying mechanics. Finally, we use the model to simulate the trombone loop of a replication fork, a large complex of proteins and DNA.

}, doi = {10.1016/j.isci.2022.104264}, url = {https://api.elsevier.com/content/article/PII:S258900422200534}, author = {Maffeo, Christopher and Chou, Han-Yi and Aleksei Aksimentiev} } @article {366, title = {Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing}, journal = {Nano Letters}, year = {2022}, month = {02/2022}, abstract = {

DNA is a promising next-generation data storage medium, but challenges remain with synthesis costs and recording latency. Here, we describe a prototype of a DNA data storage system that uses an extended molecular alphabet combining natural and chemically modified nucleotides. Our results show that MspA nanopores can discriminate different combinations and ordered sequences of natural and chemically modified nucleotides in custom-designed oligomers. We further demonstrate singlemolecule sequencing of the extended alphabet using a neural network architecture that classifies raw current signals generated by Oxford Nanopore sequencers with an average accuracy exceeding 60\% (39{\texttimes} larger than random guessing). Molecular dynamics simulations show that the majority of modified nucleotides lead to only minor perturbations of the DNA double helix. Overall, the extended molecular alphabet may potentially offer a nearly 2-fold increase in storage density and potentially the same order of reduction in the recording latency, thereby enabling new implementations of molecular recorders.

}, doi = {10.1021/acs.nanolett.1c04203}, author = {Tabatabaei, S Kasra and Pham, Bach and Pan, Chao and Liu, Jingqian and Chandak, Shubham and Shorkey, Spencer A and Hernandez, Alvaro G and Aleksei Aksimentiev and Chen, Min and Schroeder, Charles M and Milenkovic, Olgica} } @article {364, title = {A nanoscale reciprocating rotary mechanism with coordinated mobility control.}, journal = {Nature Communications}, volume = {12}, year = {2021}, month = {12/2021}, pages = {7138}, chapter = {7138}, abstract = {

Biological molecular motors transform chemical energy into mechanical work by coupling cyclic catalytic reactions to large-scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the FoF1 ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in a surrounding stator orchestrated by mechanical deformation. We design the mechanism using DNA origami, characterize its structure via cryo-electron microscopy, and examine its dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. While the camshaft can rotate inside the stator by diffusion, the stator{\textquoteright}s mechanics makes the camshaft pause at preferred orientations. By changing the stator{\textquoteright}s mechanical stiffness, we accelerate or suppress the Brownian rotation, demonstrating an allosteric coupling between the camshaft and the stator. Our mechanism provides a framework for manufacturing artificial nanomachines that function because of coordinated movements of their components.

}, keywords = {Cryoelectron Microscopy, DNA, Molecular Conformation, Molecular Dynamics Simulation, Nanostructures, Nanotechnology, Rotation}, issn = {2041-1723}, doi = {10.1038/s41467-021-27230-7}, author = {Bertosin, Eva and Maffeo, Christopher M and Drexler, Thomas and Honemann, Maximilian N and Aleksei Aksimentiev and Dietz, Hendrik} } @article {363, title = {Membrane Activity of a DNA-Based Ion Channel Depends on the Stability of Its Double-Stranded Structure}, journal = {Nano Letters}, volume = {21}, year = {2021}, month = {11/2021}, pages = {9789{\textendash}9796}, chapter = {9789}, abstract = {

DNA nanotechnology has emerged as a promising method for designing spontaneously inserting and fully controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based membrane channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA{\textendash}lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications can cause fraying of terminal base pairs of nicked DNA constructs, distorting them when embedded in a lipid bilayer. Importantly, we show that DNA nanostructures with no backbone discontinuities form more stable conductive pores and insert into membranes with higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows the design and maintenance of membrane-spanning helices in a tilted orientation within the lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

}, keywords = {DNA structures, lipid membranes, nicks, protein-mimicking, synthetic ion channel, tilt}, doi = {10.1021/acs.nanolett.1c03791}, url = {https://pubs.acs.org/doi/10.1021/acs.nanolett.1c03791}, author = {Morzy, Diana and Joshi, Himanshu and Sandler, Sarah E. and Aleksei Aksimentiev and Keyser, Ulrich F.} } @article {369, title = {DNA sequence and methylation prescribe the inside-out conformational dynamics and bending energetics of DNA minicircles.}, journal = {Nucleic Acids Res}, volume = {49}, year = {2021}, month = {11/2021}, pages = {11459-11475}, abstract = {

Eukaryotic genome and methylome encode DNA fragments{\textquoteright} propensity to form nucleosome particles. Although the mechanical properties of DNA possibly orchestrate such encoding, the definite link between {\textquoteright}omics{\textquoteright} and DNA energetics has remained elusive. Here, we bridge the divide by examining the sequence-dependent energetics of highly bent DNA. Molecular dynamics simulations of 42 intact DNA minicircles reveal that each DNA minicircle undergoes inside-out conformational transitions with the most likely configuration uniquely prescribed by the nucleotide sequence and methylation of DNA. The minicircles{\textquoteright} local geometry consists of straight segments connected by sharp bends compressing the DNA{\textquoteright}s inward-facing major groove. Such an uneven distribution of the bending stress favors minimum free energy configurations that avoid stiff base pair sequences at inward-facing major grooves. Analysis of the minicircles{\textquoteright} inside-out free energy landscapes yields a discrete worm-like chain model of bent DNA energetics that accurately account for its nucleotide sequence and methylation. Experimentally measuring the dependence of the DNA looping time on the DNA sequence validates the model. When applied to a nucleosome-like DNA configuration, the model quantitatively reproduces yeast and human genomes{\textquoteright} nucleosome occupancy. Further analyses of the genome-wide chromatin structure data suggest that DNA bending energetics is a fundamental determinant of genome architecture.

}, keywords = {DNA Methylation, DNA, Circular, Molecular Dynamics Simulation, Nucleic Acid Conformation}, issn = {1362-4962}, doi = {10.1093/nar/gkab967}, author = {Yoo, Jejoong and Park, Sangwoo and Maffeo, Christopher and Ha, Taekjip and Aleksei Aksimentiev} } @article {355, title = {The Manipulation of the Internal Hydrophobicity of FraC Nanopores Augments Peptide Capture and Recognition}, journal = {ACS Nano}, year = {2021}, month = {06/2021}, abstract = {

The detection of analytes and the sequencing of DNA using biological nanopores have seen major advances over recent years. The analysis of proteins and peptides with nanopores, however, is complicated by the complex physicochemical structure of polypeptides and the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. In this work, we show that introducing aromatic amino acids at precise positions within the lumen of α-helical fragaceatoxin C (FraC) nanopores increased the capture frequency of peptides and largely improved the discrimination among peptides of similar size. Molecular dynamics simulations determined the sensing region of the nanopore, elucidated the microscopic mechanism enabling accurate characterization of the peptides\ via\ ionic current blockades in FraC, and characterized the effect of the pore modification on peptide discrimination. This work provides insights to improve the recognition and to augment the capture of peptides by nanopores, which is important for developing a real-time and single-molecule size analyzer for peptide recognition and identification.

}, doi = {10.1021/acsnano.0c09958}, url = {https://doi.org/10.1021/acsnano.0c09958}, author = {Florian Leonardus Rudolfus Lucas and Kumar Sarthak and Erica Mariska Lenting and David Coltan and Nieck Jordy van der Heide and Roderick Corstiaan Abraham Versloot and Aleksei Aksimentiev and Giovanni Maglia} } @article {356, title = {The emerging landscape of single-molecule protein sequencing technologies}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {06/2021}, chapter = {604-617}, abstract = {

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

}, doi = {10.1038/s41592-021-01143-1}, url = {https://doi.org/10.1038/s41592-021-01143-1}, author = {Javier Antonio Alfaro and Peggy Bohl{\"a}nder and Mingjie Dai and Mike Filius and Cecil J. Howard and Xander F. van Kooten and Shilo Ohayon and Adam Pomorski and Sonja Schmid and Aleksei Aksimentiev and Eric V. Anslyn and Georges Bedran and Chan Cao and Mauro Chinappi and Etienne Coyaud and Cees Dekker and Gunnar Dittmar and Nicholas Drachman and Rienk Eelkema and David Goodlett and S{\'e}bastien Hentz and Umesh Kalathiya and Neil L. Kelleher and Ryan T. Kelly and Zvi Kelman and Sung Hyun Kim and Bernhard Kuster and David Rodriguez-Larrea and Stuart Lindsay and Giovanni Maglia and Edward M. Marcotte and John P. Marino and Christophe Masselon and Michael Mayer and Patroklos Samaras and Kumar Sarthak and Lusia Sepiashvili and Derek Stein and Meni Wanunu and Mathias Wilhelm and Peng Yin and Amit Meller and Chirlmin Joo} } @article {350, title = {Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures}, journal = {Journal of the American Chemical Society}, volume = {143}, year = {2021}, month = {05/2021}, pages = {7358-7367}, chapter = {7358}, abstract = {

The interplay between nucleic acids and lipids\ underpins several key processes in molecular biology, synthetic biotechnology, vaccine technology, and nanomedicine. These interactions are often electrostatic in nature, and much of their rich phenomenology remains unexplored in view of the chemical diversity of lipids, the heterogeneity of their phases, and the broad range of relevant solvent conditions. Here we unravel the electrostatic interactions between zwitterionic lipid membranes and DNA nanostructures in the presence of physiologically relevant\ cations, with the purpose of identifying new routes to program DNA-lipid complexation and membrane-active nanodevices. We demonstrate that this interplay is influenced by both the phase of the lipid membranes and the valency of the ions and observe divalent cation bridging between nucleic acids and gel-phase bilayers. Furthermore, even in the presence of hydrophobic modifications on the DNA, we find that cations are still required to enable DNA adhesion to liquid-phase membranes. We show that the latter mechanism can be exploited to control the degree of attachment of cholesterol-modified DNA nanostructures by modifying their overall hydrophobicity and charge. Besides their biological relevance, the interaction mechanisms we explored hold great practical potential in the design of biomimetic nanodevices, as we show by constructing an ion-regulated DNA-based synthetic enzyme.

}, doi = {10.1021/jacs.1c00166}, url = {https://doi.org/10.1021/jacs.1c00166}, author = {Morzy, Diana and Rubio-S{\'a}nchez, Roger and Joshi, Himanshu and Aleksei Aksimentiev and Di Michele, Lorenzo and Keyser, Ulrich F.} } @article {346, title = {Electrical unfolding of cytochrome during translocation through a nanopore constriction.}, journal = {Proceedings of the National Academy of Sciences}, volume = {118}, year = {2021}, month = {04/2021}, pages = {e2016262118}, abstract = {

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome (cyt ) through ultrathin silicon nitride (SiN) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of \~{}30 to 100 MV/m, cyt is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (\~{}ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.

}, issn = {1091-6490}, doi = {10.1073/pnas.2016262118}, author = {Tripathi, Prabhat and Benabbas, Abdelkrim and Mehrafrooz, Behzad and Yamazaki, Hirohito and Aleksei Aksimentiev and Champion, Paul M and Wanunu, Meni} } @article {341, title = {Translocation of DNA through Ultrathin Nanoslits}, journal = {Advanced Materials}, volume = {33}, year = {2021}, month = {02/2021}, pages = {2007682}, chapter = {2007682}, abstract = {

2D nanoslit devices, where two crystals with atomically flat surfaces are separated by only a few nanometers, have attracted considerable attention because their tunable control over the confinement allows for the discovery of unusual transport behavior of gas, water, and ions. Here, the passage of double-stranded DNA molecules is studied through nanoslits fabricated from exfoliated 2D materials, such as graphene or hexagonal boron nitride, and the DNA polymer behavior is examined in this tight confinement. Two types of events are observed in the ionic current: long current blockades that signal DNA translocation and short spikes where DNA enters the slits but withdraws. DNA translocation events exhibit three distinct phases in their current-blockade traces{\textemdash}loading, translation, and exit. Coarse-grained molecular dynamics simulation allows the different polymer configurations of these phases to be identified. DNA molecules, including folds and knots in their polymer structure, are observed to slide through the slits with near-uniform velocity without noticeable frictional interactions of DNA with the confining graphene surfaces. It is anticipated that this new class of 2D-nanoslit devices will provide unique ways to study polymer physics and enable lab-on-a-chip biotechnology.

}, keywords = {2D nanoslits, biopolymers, DNA translocation, graphene, Nanofluidics}, doi = {https://doi.org/10.1002/adma.202007682}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/adma.202007682}, author = {Yang, Wayne and Radha, Boya and Choudhary, Adnan and You, Yi and Mettela, Gangaiah and Geim, Andre K. and Aleksei Aksimentiev and Keerthi, Ashok and Dekker, Cees} } @article {340, title = {Chiral Systems Made from DNA}, journal = {Advanced Science}, volume = {8}, year = {2021}, month = {01/2021}, pages = {2003113}, type = {Progress Report}, chapter = {2003113}, abstract = {

The very chemical structure of DNA that enables biological heredity and evolution has non-trivial implications for the self-organization of DNA molecules into larger assemblies and provides limitless opportunities for building functional nanostructures. This progress report discusses the natural organization of DNA into chiral structures and recent advances in creating synthetic chiral systems using DNA as a building material. How nucleic acid chirality naturally comes into play in a diverse array of situations is considered first, at length scales ranging from an individual nucleotide to entire chromosomes. Thereafter, chiral liquid crystal phases formed by dense DNA mixtures are discussed, including the ongoing efforts to understand their origins. The report then summarizes recent efforts directed toward building chiral structures, and other structures of complex topology, using the principle of DNA self-assembly. Discussed last are existing and proposed functional man-made nanostructures designed to either probe or harness DNA{\textquoteright}s chirality, from plasmonics and spintronics to\ biosensing.

}, doi = {10.1002/advs.202003113}, url = {https://doi.org/10.1002/advs.202003113}, author = {D Winogradoff and Pin-Yi Li and H Joshi and L Quednau and C Maffeo and Aleksei Aksimentiev} } @article {357, title = {A tetrahedral DNA nanorobot with conformational change in response to molecular trigger}, journal = {Nanoscale}, year = {2021}, month = {01/2021}, abstract = {

Dynamic DNA origami nanostructures that respond to external stimuli are promising platforms for cargo delivery and nanoscale sensing. However, the low stability of such nanostructures under physiological conditions presents a major obstacle for their use in biomedical applications. This article describes a stable tetrahedral DNA nanorobot (TDN) programmed to undergo a controlled conformational change in response to epithelial cell adhesion molecule (EpCAM), a molecular biomarker specifically expressed on the circulating tumor cells. Multiresolution molecular dynamics simulations verified the overall stability of the folded TDN design and characterized local distortions in the folded structure. Atomic force microscopy and gel electrophoresis results showed that tetragonal structures are more stable than unfolded DNA origami sheets. Live cell experiments demonstrated the low cytotoxicity and target specificity of TDN. In summary, the proposed TDN can not only effectively resist nuclease catalysis but also has the potential to monitor EpCAM-positive cells precisely.

}, issn = {2040-3364}, doi = {10.1039/D1NR02757C}, url = {http://xlink.rsc.org/?DOI=D1NR02757Chttp://pubs.rsc.org/en/content/articlepdf/2021/NR/D1NR02757Chttp://pubs.rsc.org/en/content/articlepdf/2021/NR/D1NR02757C}, author = {Liu, Fengyu and Liu, Xiaoming and Shi, Qing and Maffeo, Christopher and Kojima, Masaru and Dong, Lixin and Aleksei Aksimentiev and Huang, Qiang and Fukuda, Toshio and Arai, Tatsuo} } @article {338, title = {High-Fidelity Capture, Threading, and Infinite-Depth Sequencing of Single DNA Molecules with a Double-Nanopore System}, journal = {ACS Nano}, volume = {14}, year = {2020}, month = {11/2020}, pages = {15566-15576}, chapter = {15566}, abstract = {

Nanopore sequencing of nucleic acids has an illustrious history of innovations that eventually made commercial nanopore sequencing possible. Nevertheless, the present nanopore sequencing technology leaves much room for improvement, especially with respect to accuracy of raw reads and detection of nucleotide modifications. Double-nanopore sequencing{\textemdash}an approach where a DNA molecule is pulled back and forth by a tug-of-war of two nanopores{\textemdash}could potentially improve single-molecule read accuracy and modification detection by offering multiple reads of the same DNA fragment. One principle difficulty in realizing such a technology is threading single-stranded DNA through both nanopores. Here, we describe and demonstrate through simulations a nanofluidic system for loading and threading DNA strands through a double-nanopore setup with nearly 100\% fidelity. The high-efficiency loading is realized by using hourglass-shaped side channels that not only deliver the molecules to the nanopore but also retain molecules that missed the nanopore at the first passage to attempt the nanopore capture again. The second nanopore capture is facilitated by an orthogonal microfluidic flow that unravels the molecule captured by the first nanopore and delivers it to the capture volume of the second nanopore. We demonstrate the potential utility of our double-nanopore system for DNA sequencing by simulating repeat back-and-forth motion{\textemdash}flossing{\textemdash}of a DNA strand through the double-nanopore system. We show that repeat exposure of the same DNA fragments to the nanopore sensing volume considerably increases accuracy of the nucleotide sequence determination and that correlated displacement of ssDNA through the two nanopores may facilitate recognition of homopolymer fragments.

}, keywords = {Gene delivery, Genetics, Molecules, Monomers, Nanopores}, doi = {10.1021/acsnano.0c06191}, url = {https://doi.org/10.1021/acsnano.0c06191}, author = {Choudhary, Adnan and Joshi, Himanshu and Chou, Han-Yi and Sarthak, Kumar and Wilson, James and Maffeo, Christopher and Aleksei Aksimentiev} } @article {335, title = {Scalable molecular dynamics on CPU and GPU architectures with NAMD}, journal = {J. Chem. Phys.}, volume = {153}, year = {2020}, month = {07/2020}, chapter = {044130}, abstract = {

NAMD is a molecular dynamics program designed for high-performance simulations of very large biological objects on CPU- and GPU- based architectures. NAMD offers scalable performance on petascale parallel supercomputers consisting of hundreds of thousands of cores, as well as on inexpensive commodity clusters commonly found in academic environments. It is written in C++ and leans on Charm++ parallel objects for optimal performance on low-latency architectures. NAMD is a versatile, multipurpose code that gathers state-of-the-art algorithms to carry out simulations in apt thermodynamic ensembles, using the widely popular CHARMM, AMBER, OPLS, and GROMOS biomolec- ular force fields. Here, we review the main features of NAMD that allow both equilibrium and enhanced-sampling molecular dynamics\ simulations with numerical efficiency. We describe the underlying concepts utilized by NAMD and their implementation, most notably for handling long-range electrostatics; controlling the temperature, pressure, and pH; applying external potentials on tailored grids; leveraging massively parallel resources in multiple-copy simulations; and hybrid quantum-mechanical/molecular-mechanical descriptions. We detail the variety of options offered by NAMD for enhanced-sampling simulations aimed at determining free-energy differences of either alchem- ical or geometrical transformations and outline their applicability to specific problems. Last, we discuss the roadmap for the development of NAMD and our current efforts toward achieving optimal performance on GPU-based architectures, for pushing back the limitations that have prevented biologically realistic billion-atom objects to be fruitfully simulated, and for making large-scale simulations less expensive and easier to set up, run, and analyze. NAMD is distributed free of charge with its source code at www.ks.uiuc.edu.

}, doi = {10.1063/5.0014475}, author = {James C. Phillips and David J. Hardy and Julio D. C. Maia and John E. Stone and Jo{\~a}o V. Ribeiro and Rafael C. Bernardi and Ronak Buch and Giacomo Fiorin and J{\'e}r{\^o}me H{\'e}nin and Wei Jiang and Ryan McGreevy and Marcelo C. R. Melo and Brian K. Radak and Robert D. Skeel and Abhishek Singharoy and Yi Wang and Beno{\^\i}t Roux and Aleksei Aksimentiev and Zaida Luthey-Schulten and Laxmikant V. Kal{\'e} and Klaus Schulten and Christophe Chipot and Emad Tajkhorshid} } @article {317, title = {Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore}, journal = {Nature Biotechnology}, volume = {38}, year = {2020}, month = {02/2020}, pages = {176-181}, chapter = {176}, abstract = {

Efforts to sequence single protein molecules in nanopores have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

Research Highlights

1.\ Tang, Lei. "Sensing proteinogenic amino acids." Nature Methods 17(2):126 (2020) DOI:\ 10.1038/s41592-020-0741-z

2.\ Howorka, Stefan, and Zuzanna S. Siwy. "Reading amino acids in a nanopore." Nature Biotechnology 38(2):159 (2020) DOI:\ 10.1038/s41587-019-0401-y

3.\ Nanopores can identify the amino acids in proteins, the first step to sequencing, Highlight by Illinois news bureau.\ \ 

}, isbn = {1546-1696}, doi = {10.1038/s41587-019-0345-2}, url = {https://doi.org/10.1038/s41587-019-0345-2}, author = {Ouldali, Hadjer and Sarthak, Kumar and Ensslen, Tobias and Piguet, Fabien and Manivet, Philippe and Pelta, Juan and Behrends, Jan C. and Aleksei Aksimentiev and Oukhaled, Abdelghani} } @article {318, title = {Artificial Water Channels Enable Fast and Selective Water Permeation Through Water-wire Networks}, journal = {Nature Nanotechnology.}, volume = {15,}, year = {2020}, month = {01/2020}, pages = {73-79}, chapter = {73}, abstract = {

Artificial water channels are synthetic molecules that aim to mimic the structural and functional features of biological water channels (aquaporins). Here we report on a cluster-forming organic nanoarchitecture, peptide-appended hybrid[4]arene (PAH[4]), as a new class of artificial water channels. Fluorescence experiments and simulations demonstrated that PAH[4]s can form, through lateral diffusion, clusters in lipid membranes that provide synergistic membrane-spanning paths for rapid and selective water permeation through water-wire networks. Quantitative transport studies revealed that PAH[4]s can transport \>109\ water molecules per second per molecule, which is comparable to aquaporin water channels. The performance of these channels exceeds the upper bound limit of current desalination membranes by a factor of ~104, as illustrated by the water/NaCl permeability{\textendash}selectivity trade-off curve. PAH[4]{\textquoteright}s unique properties of a high water/solute permselectivity via cooperative water-wire formation could usher in an alternative design paradigm for permeable membrane materials in separations, energy production, and barrier applications.

}, doi = {https://doi.org/10.1038/s41565-019-0586-8}, author = {Woochul Song and Himanshu Joshi and Ratul Chowdhury and Joseph S. Najem and Yue-xiao Shen and Chao Lang and Codey B. Henderson and Yu-Ming Tu and Megan Farell and Megan E. Pitz and Costas D. Maranas and Paul S. Cremer and Robert J. Hickey and Stephen A. Sarles and Jun-li Hou and Aleksei Aksimentiev and Manish Kumar} } @article {325, title = {MrDNA: a multi-resolution model for predicting the structure and dynamics of DNA systems}, journal = {Nucleic Acids Research}, year = {2020}, abstract = {

Although the field of structural DNA nanotechnology has been advancing with an astonishing pace, de novo design of complex 3D nanostructures and functional devices remains a laborious and time-consuming process. One reason for that is the need for multiple cycles of experimental characterization to elucidate the effect of design choices on the actual shape and function of the self-assembled objects. Here, we demonstrate a multi-resolution simulation framework, mrdna, that, in 30 min\ or less, can produce an atomistic-resolution structure of a self-assembled DNA nanosystem. We demonstrate fidelity of our mrdna framework through direct comparison of the simulation results with the results of cryo-electron microscopy (cryo-EM) reconstruction of multiple 3D DNA origami objects. Furthermore, we show that our approach can characterize an ensemble of conformations adopted by dynamic DNA nanostructures, the equilibrium structure and dynamics of DNA objects constructed using off-lattice self-assembly principles, i.e.\ wireframe DNA objects, and to study the properties of DNA objects under a variety of environmental conditions, such as applied electric field. Implemented as an open source Python package, our framework can be extended by the community and integrated with DNA design and molecular graphics tools.

}, doi = {10.1093/nar/gkaa200}, url = {https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa200/5814051}, author = {Maffeo, Christopher and Aleksei Aksimentiev} } @article {308, title = {Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism}, journal = {Cell}, volume = {179}, year = {2019}, month = {11/2019}, pages = {1098 - 1111.e23}, abstract = {

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore{\textquoteright}s structural integrity and robust energy conversion. Parallels are drawn with the more universal\ mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

}, keywords = {bioenergetics, biological membranes, charge transport, chromatophore, integrative model, MD, mitochondria, Molecular Dynamics Simulation, optical spectroscopy, photosynthesis}, issn = {0092-8674}, doi = {10.1016/j.cell.2019.10.021}, url = {http://www.sciencedirect.com/science/article/pii/S0092867419311717}, author = {Abhishek Singharoy and Christopher Maffeo and Karelia H. Delgado-Magnero and David J.K. Swainsbury and Melih Sener and Ulrich Kleinekath{\"o}fer and John W. Vant and Jonathan Nguyen and Andrew Hitchcock and Barry Isralewitz and Ivan Teo and Danielle E. Chandler and John E. Stone and James C. Phillips and Taras V. Pogorelov and M. Ilaria Mallus and Christophe Chipot and Zaida Luthey-Schulten and D. Peter Tieleman and C. Neil Hunter and Emad Tajkhorshid and Aleksei Aksimentiev and Klaus Schulten} } @article {307, title = {Single molecule analysis of structural fluctuations in DNA nanostructures}, journal = {Nanoscale}, volume = {11}, year = {2019}, month = {10/2019}, pages = {18475 - 18482}, abstract = {

DNA origami is an excellent tool for building complex artificial nanostructures. Functionalization of these structures provides the possibility of precise organization of matter at the nanoscale. In practice, efforts in this endeavour can be impeded by electrostatic repulsion or other dynamics at the molecular scale, resulting in uncompliant local structures. Using single molecule FRET microscopy combined with coarse-grained Brownian dynamics simulations, we investigated here the local structure around the lid of a DNA origami box, which can be opened by specific DNA keys. We found that FRET signals for the closed box depend on buffer ion concentrations and small changes to the DNA structure design. Simulations provided a view of the global and local structure and showed that the distance between the box wall and lid undergoes fluctuations. These results provide methods to vizualise and improve the local structure of three-dimensional DNA origami assemblies and offer guidance for exercising control over placement of chemical groups and ligands.

}, issn = {2040-3364}, doi = {10.1039/C9NR03826D}, url = {http://xlink.rsc.org/?DOI=C9NR03826Dhttp://pubs.rsc.org/en/content/articlepdf/2019/NR/C9NR03826Dhttp://pubs.rsc.org/en/content/articlepdf/2019/NR/C9NR03826D}, author = {Jepsen, Mette D. E. and S{\o}rensen, Rasmus Sch{\o}ler and Maffeo, Christopher and Aleksei Aksimentiev and Kjems, J{\o}rgen and Birkedal, Victoria} } @article {295, title = {Electro-Mechanical Conductance Modulation of a Nanopore Using a Removable Gate}, journal = {ACS Nano}, volume = {13}, year = {2019}, month = {02/2019}, pages = {2398--2409}, chapter = {2398}, abstract = {

Ion channels form the basis of information processing in livings cells by facilitating exchange of electrical signals across and along cellular membranes. Applying the same principles to man-made systems requires development of synthetic ion channels that can alter their conductance in response to a variety of external manipulations. By combining single-molecule electrical recordings with all-atom molecular dynamics simulations, we here demonstrate a new hybrid nanopore system that allows for both a stepwise change of its conductance and a non-linear current-voltage dependence. The conductance modulation is realized by using a short flexible peptide gate that carries opposite electric charge at its ends. We show that a constant transmembrane bias can position, and in a later stage remove, the peptide gate right at the most sensitive sensing region of a biological nanopore FraC, thus partially blocking its channel and producing a stepwise change in the conductance. Increasing or decreasing the bias while having the peptide gate trapped in the pore stretches or compresses the peptide within the nanopore, thus modulating its conductance in a non-linear but reproducible manner. We envision a range of applications of this removable-gate nanopore system, e.g. from an element of biological computing circuits to a test bed for probing the elasticity of intrinsically disordered proteins.

}, doi = {10.1021/acsnano.8b09266}, url = {https://doi.org/10.1021/acsnano.8b09266}, author = {Shidi Zhao and Restrepo-Perez, Laura and Maglia, Giovanni and Misha Soskine and Chirlmin Joo and Cees Dekker and Aleksei Aksimentiev} } @article {296, title = {Molecular Mechanisms of DNA Replication and Repair Machinery: Insights from Microscopic Simulations}, journal = {Advanced Theory and Simulations}, volume = {2}, year = {2019}, month = {02/2019}, pages = {1800191}, abstract = {

Reproduction, the hallmark of biological activity, requires making an accurate copy of the genetic material to allow the progeny to inherit parental traits. In all living cells, the process of DNA replication is carried out by a concerted action of multiple protein species forming a loose protein{\textendash}nucleic acid complex, the replisome. Proofreading and error correction generally accompany replication but also occur independently, safeguarding genetic information through all phases of the cell cycle. Advances in biochemical characterization of intracellular processes, proteomics, and the advent of single-molecule biophysics have brought about a treasure trove of information awaiting to be assembled into an accurate mechanistic model of the DNA replication process. This review describes recent efforts to model elements of DNA replication and repair processes using computer simulations, an approach that has gained immense popularity in many areas of molecular biophysics but has yet to become mainstream in the DNA metabolism community. It highlights the use of diverse computational methods to address specific problems of the fields and discusses unexplored possibilities that lie ahead for the computational approaches in these areas.

}, keywords = {coarse-grained simulation, DNA damage, DNA repair, homologous recombination, Molecular dynamics, nucleic acids, QM/MM, replisome}, doi = {10.1002/adts.201800191}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/adts.201800191}, author = {Maffeo, Christopher and Chou, Han-Yi and Aleksei Aksimentiev} } @article {292, title = {Sequence-Dependent DNA Condensation as a Driving Force of DNA Phase Separation}, journal = {Nucleic Acids Research}, volume = {46}, year = {2018}, month = {10/2018}, pages = {9401--9413}, abstract = {

The physical properties of DNA have been suggested to play a central role in spatio-temporal organization of eukaryotic chromosomes. Experimental correlations have been established between the local nucleotide content of DNA and the frequency of inter- and intra-chromosomal contacts but the underlying physical mechanism remains unknown. Here, we combine fluorescence resonance energy transfer (FRET) measurements, precipitation assays, and molecular dynamics simulations to characterize the effect of DNA nucleotide content, sequence, and methylation on inter-DNA association and its correlation with DNA looping. First, we show that the strength of DNA condensation mediated by poly-lysine peptides as a reduced model of histone tails depends on the DNA{\textquoteright}s global nucleotide content but also on the local nucleotide sequence, which turns out to be qualitatively same as the condensation by spermine. Next, we show that the presence and spatial arrangement of C5 methyl groups determines the strength of inter-DNA attraction, partially explaining why RNA resists condensation. Interestingly, multi-color single molecule FRET measurements reveal strong anti-correlation between DNA looping and DNA{\textendash}DNA association, suggesting that a common biophysical mechanism underlies them. We propose that the differential affinity between DNA regions of varying sequence pattern may drive the phase separation of chromatin into chromosomal subdomains.

}, doi = {10.1093/nar/gky639 }, author = {Hyunju Kang and Jejoong Yoo and Hong Soo Lee and Byeong-Kwon Sohn and Seung-Won Lee and Wenjie Ma and Jung-Min Kee and Aleksei Aksimentiev and Hajin Kim} } @article {290, title = {PoreDesigner for tuning solute selectivity in a robust and highly permeable outer membrane pore}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {09/2018}, pages = {3661}, abstract = {

Monodispersed angstrom-size pores embedded in a suitable matrix are promising for highly selective membrane-based separations. They can provide substantial energy savings in water treatment and small molecule bioseparations. Such pores present as membrane proteins (chiefly aquaporin-based) are commonplace in biological membranes but difficult to implement in synthetic industrial membranes and have modest selectivity without tunable selectivity. Here we present PoreDesigner, a design workflow to redesign the robust beta-barrel Outer Membrane Protein F as a scaffold to access three specific pore designs that exclude solutes larger than sucrose (\>360 Da), glucose (\>180 Da), and salt (\>58 Da) respectively. PoreDesigner also enables us to design any specified pore size (spanning 3{\textendash}10 {\r A}), engineer its pore profile, and chemistry. These redesigned pores may be ideal for conducting sub-nm aqueous separations with permeabilities exceeding those of classical biological water channels,\ aquaporins, by more than an order of magnitude at over 10 billion water molecules per channel per second.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-06097-1}, url = {https://doi.org/10.1038/s41467-018-06097-1}, author = {Chowdhury, Ratul and Ren, Tingwei and Shankla, Manish and Decker, Karl and Grisewood, Matthew and Prabhakar, Jeevan and Baker, Carol and Golbeck, John H and Aleksei Aksimentiev and Kumar, Manish and Maranas, Costas D} } @article {286, title = {A synthetic enzyme built from DNA flips 10$^{7}$ lipids per second in biological membranes}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {07/2018}, pages = {2426}, abstract = {

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane{\textquoteright}s inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

}, keywords = {Computational biophysics, DNA nanostructures, Membrane biophysics, Nanobiotechnology}, doi = {10.1038/s41467-018-04821-5}, url = {http://www.nature.com/articles/s41467-018-04821-5}, author = {Ohmann, Alexander and Li, Chen-Yu and Maffeo, Christopher and Al Nahas, Kareem and Baumann, Kevin N. and G{\"o}pfrich, Kerstin and Yoo, Jejoong and Keyser, Ulrich F. and Aleksei Aksimentiev} } @article {285, title = {Inchworm movement of two rings switching onto a thread by biased Brownian diffusion represent a three-body problem}, journal = {Proceedings of the National Academy of Sciences}, year = {2018}, month = {05/2018}, abstract = {

This article describes a molecular realization of the classical three-body problem, where the motion of three or more bodies is directed by a set of pairwise forces. Surprisingly, motion of the components of the three-body molecular systems is found to be highly choreographed by differences in strength of intercomponent interactions, promoting a rare inchworm-like loading of molecular rings onto a molecular thread. Our work demonstrates the utility of an integrative approach to design and develop functional molecular machines.The coordinated motion of many individual components underpins the operation of all machines. However, despite generations of experience in engineering, understanding the motion of three or more coupled components remains a challenge, known since the time of Newton as the {\textquotedblleft}three-body problem.{\textquotedblright} Here, we describe, quantify, and simulate a molecular three-body problem of threading two molecular rings onto a linear molecular thread. Specifically, we use voltage-triggered reduction of a tetrazine-based thread to capture two cyanostar macrocycles and form a [3]pseudorotaxane product. As a consequence of the noncovalent coupling between the cyanostar rings, we find the threading occurs by an unexpected and rare inchworm-like motion where one ring follows the other. The mechanism was derived from controls, analysis of cyclic voltammetry (CV) traces, and Brownian dynamics simulations. CVs from two noncovalently interacting rings match that of two covalently linked rings designed to thread via the inchworm pathway, and they deviate considerably from the CV of a macrocycle designed to thread via a stepwise pathway. Time-dependent electrochemistry provides estimates of rate constants for threading. Experimentally derived parameters (energy wells, barriers, diffusion coefficients) helped determine likely pathways of motion with rate-kinetics and Brownian dynamics simulations. Simulations verified intercomponent coupling could be separated into ring{\textendash}thread interactions for kinetics, and ring{\textendash}ring interactions for thermodynamics to reduce the three-body problem to a two-body one. Our findings provide a basis for high-throughput design of molecular machinery with multiple components undergoing coupled motion.

}, issn = {0027-8424}, doi = {10.1073/pnas.1719539115}, url = {http://www.pnas.org/content/early/2018/05/03/1719539115}, author = {Benson, Christopher R. and Maffeo, Christopher and Fatila, Elisabeth M. and Liu, Yun and Sheetz, Edward G. and Aleksei Aksimentiev and Singharoy, Abhishek and Flood, Amar H.} } @article {281, title = {Optical voltage sensing using DNA origami}, journal = {Nano Letters}, volume = {18}, year = {2018}, pages = {1962-1971}, abstract = {

We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nano-devices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labelled with a single pair of FRET dyes. The DNA structures were reversibly immobilised on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterised voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures, and highlights potential applications of dynamic DNA nanostructures as voltage sensors.

}, doi = {10.1021/acs.nanolett.7b05354}, url = {https://doi.org/10.1021/acs.nanolett.7b05354}, author = {Hemmig, Elisa Alina and Fitzgerald, Clare and Maffeo, Christopher and Hecker, Lisa and Ochmann, Sarah Elisabeth and Aleksei Aksimentiev and Tinnefeld, Philip and Keyser, Ulrich F} } @inbook {277, title = {A Practical Guide to Molecular Dynamics Simulations of DNA Origami Systems}, booktitle = {DNA Nanotechology: Methods and Protocols}, series = {Methods in Molecular Biology}, volume = {1811}, year = {2018}, pages = {209-229}, publisher = {Humana Press}, organization = {Humana Press}, edition = {2}, chapter = {A Practical Guide to Molecular Dynamics Simulations of DNA Origami Systems}, address = {New York}, abstract = {

The DNA origami method exploits the self-assembly property of nucleic acids to build diverse nanoscale systems. The all-atom molecular dynamics (MD) method has emerged as a powerful computational tool for atomic-resolution characterization of the in situ structure and physical properties of DNA origami objects. This chapter provides step-by-step instructions for building atomic-scale models of DNA origami systems, using the MD method to simulate the models, and performing basic analyses of the resulting MD trajectories.\ 

}, issn = {978-1-4939-8581-4}, doi = {10.1007/978-1-4939-8582-1_15}, url = {https://link.springer.com/protocol/10.1007/978-1-4939-8582-1_15}, author = {Jejoong Yoo and Chen-Yu Li and Scott Michael Slone and Christopher Maffeo and Aleksei Aksimentiev}, editor = {Giampaolo Zuccheri} } @article {279, title = {Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization}, journal = {ACS Nano}, volume = {11}, year = {2017}, month = {12/2017}, pages = {11931-11945}, abstract = {

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein{\textquoteright}s insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal{\textquoteright}s DNA-ratchet function.

}, doi = {10.1021/acsnano.7b06980}, url = {http://pubs.acs.org/doi/abs/10.1021/acsnano.7b06980}, author = {Cressiot*, Benjamin and Greive*, Sandra J. and Si*, Wei and Pascoa, Tomas and Mojtabavi, Mehrnaz and Chechik, Maria and Jenkins, Huw Thomas and Lu, Xueguang and Zhang, Ke and Aleksei Aksimentiev and Antson, Alfred A. and Wanunu, Meni} } @article {273, title = {Molecular mechanism of DNA association with single-stranded DNA binding protein}, journal = {Nucleic Acids Research}, volume = {45}, year = {2017}, month = {12/2017}, pages = {12125{\textendash}12139}, chapter = {12125}, abstract = {

During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA{\textendash}SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB{\textendash}ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-{\textmu}s-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion.

}, doi = {10.1093/nar/gkx917}, url = {http://dx.doi.org/10.1093/nar/gkx917}, author = {Maffeo, Christopher and Aleksei Aksimentiev} } @article {274, title = {Quantification of Membrane Protein-Detergent Complex Interactions}, journal = {The Journal of Physical Chemistry B}, volume = {121}, year = {2017}, month = {11/2017}, pages = {10228-10241}, chapter = {10228}, abstract = {

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semi-quantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

}, doi = {10.1021/acs.jpcb.7b08045}, url = {http://pubs.acs.org/doi/abs/10.1021/acs.jpcb.7b08045}, author = {Wolfe, Aaron John and Si, Wei and Zhang, Zhengqi and Blanden, Adam R and Hsueh, Yi-Ching and Gugel, Jack Fredrick and Pham, Bach and Chen, Min and Loh, Stewart N and Rozovsky, Sharon and Aleksei Aksimentiev and Movileanu, Liviu} } @article {251, title = {Selective Permeability of Truncated Aquaporin 1 in Silico}, journal = {ACS Biomaterials Science \& Engineering}, volume = {3}, year = {2017}, month = {02/2017}, pages = {342-348}, chapter = {342}, abstract = {

Aquaporin (AQP) proteins function as highly efficient water transport channels that support homeostasis in many types of living cells. Their structure-function relationships have been characterized extensively in fundamental and applied research, primarily via structural analysis, mutational studies, and computational approaches. The present study evaluates the effects of progressive truncations on the permeability and ionic conductivity of AQP-1 (bovine). The use of truncations to determine critical features has not been considered previously, as physical truncation of AQP is likely not technically feasible due to the ornate arrangement of six interwoven alpha helices in a single pore structure. However, structures not obtainable through protein assembly can be realized via synthetic chemistry approaches and studied using molecular dynamics (MD) simulations. Here, we apply the MD method to characterize the permeability of AQP variants truncated along the pore axis from both cytoplasmic and extracellular sides of the channel. The simulation results suggest that AQP-1 can retain its function even after deletion of up to 50\% of the channel{\textquoteright}s length, representing 50\% of proteins{\textquoteright}s molecular mass. Deletions such as these are expected to greatly simplify future biomimicry efforts of reproducing the AQP functionality using synthetic macromolecules. This study demonstrates the potential of in silico approaches to support the creation of streamlined functional analogs of biological molecular machines.

}, doi = {10.1021/acsbiomaterials.6b00583 }, author = {Karl Decker and Martin Page and Ashley Boyd and Irene MacAllister and Mark Ginsberg and Aleksei Aksimentiev} } @article {236, title = {Ion Channels Made from a Single Membrane-Spanning DNA Duplex}, journal = {Nano Letters}, volume = {16}, year = {2016}, month = {06/2016}, pages = {4665--4669}, abstract = {

Due to their hollow interior, transmembrane channels are capable of opening up pathways for ions across lipid membranes of living cells. Here, we demonstrate ion conduction induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, our duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, we elucidate the microscopic conductance pathway\ (see\ water channel\ trajectory, require login to nanoHUB). Ions flow (see ion transport\ trajectory)\ at the DNA-lipid interface as the lipid head groups tilt towards the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism realized here, with the smallest possible DNA-based ion channel, offers a route to design a new class of synthetic ion channels with maximum simplicity.

}, doi = {10.1021/acs.nanolett.6b02039}, author = {Kerstin G{\"o}pfrich and Chen-Yu Li and Iwona Mames and Satya Prathyusha Bhamidimarri and Maria Ricci and Jejoong Yoo and Adam Mames and Alexander Ohmann and Mathias Winterhalter and Eugen Stulz and Aleksei Aksimentiev and Ulrich F Keyser} } @article {214, title = {De Novo Reconstruction of DNA Origami Structures through Atomistic Molecular Dynamics Simulation}, journal = {Nucleic Acids Research}, volume = {44}, year = {2016}, month = {04/2016}, pages = {3013-3019}, chapter = {3013}, abstract = {

The DNA origami method has brought nanometer-precision fabrication to molecular biology labs, offering myriads of potential applications in the fields of synthetic biology, biomolecular medicine, molecular computation, etc. Advancing the method further requires controlling self-assembly down to the atomic scale. Here we demonstrate a computational method that allows the equilibrium structure of a large, complex DNA origami object to be determined to atomic resolution. Through direct comparison with the results of cryo-electron microscopy, we demonstrate de novo reconstruction of a 4.7 megadalton pointer structure by means of fully atomistic molecular dynamics simulations. Furthermore, we show that elastic network-guided simulations performed without solvent can yield similar accuracy at a fraction of the computational cost, making this method an attractive approach for prototyping and validation of self-assembled DNA nanostructures.

}, doi = {10.1093/nar/gkw155}, url = {http://nar.oxfordjournals.org/content/early/2016/03/14/nar.gkw155}, author = {Christopher Maffeo and Jejoong Yoo and Aleksei Aksimentiev} } @article {216, title = {The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study}, journal = {PLoS ONE}, volume = {11}, year = {2016}, month = {02/2016}, pages = {e0152002}, abstract = {

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50\% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.

}, doi = {10.1371/journal.pone.0152002}, url = {http://journals.plos.org/plosone/article?id=10.1371\%2Fjournal.pone.0152002}, author = {Lili Sun and Marcin Tabaka and Sen Hou and Lin Li and Krzysztof Burdzy and Aleksei Aksimentiev and Christopher Maffeo and Xuzhu Zhang and Robert Holyst} } @article {202, title = {Highly permeable artificial water channels that self-assemble into two-dimensional arrays}, journal = {Proceedings of the National Academy of Science}, volume = {112}, year = {2015}, month = {07/2015}, pages = {9810-9815}, chapter = {9810}, abstract = {

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. We characterized a new architecture of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0({\textpm} 0.3){\texttimes}10-14\ cm3/s or 3.5({\textpm} 1.0){\texttimes}108\ water molecules/s, which is in the range of AQPs (3.4~40.3{\texttimes}108\ water molecules/s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0{\texttimes}108\ water molecules/s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ~107\ water molecules/s). Furthermore, within lipid bilayers, PAP channels can self-assemble into two-dimensional arrays. Importantly for permeable membrane design, the pore density of PAP channel arrays (~2.6{\texttimes}105\ pores/μm2) is two orders of magnitude higher than CNT membranes (0.1~2.5{\texttimes}103\ pores/μm2). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability and propensity to form arrays.

}, keywords = {membrane, nanochannel, permeation}, doi = {10.1073/pnas.1508575112}, author = {Yue-xiao Shen and Wen Si and Mustafa Erbakan and Karl Decker and Rita De Zorzi and Patrick O. Saboe and You Jung Kang and Sheereen Majd and Peter J. Butler and Thomas Walz and Aleksei Aksimentiev and Jun-li Hou and Manish Kumar} } @article {139, title = {Close encounters with DNA}, journal = {J Phys Condens Matter}, volume = {26}, year = {2014}, month = {10/2014}, pages = {413101}, abstract = {

Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena. We also discuss the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field.

}, keywords = {Review}, issn = {1361-648X}, doi = {10.1088/0953-8984/26/41/413101}, author = {Christopher Maffeo and Yoo, J and Jeffrey Comer and Wells, D B and Luan, B and Aleksei Aksimentiev} } @article {123, title = {A Coarse-Grained Model of Unstructured Single-Stranded DNA Derived from Atomistic Simulation and Single-Molecule Experiment}, journal = {Journal of Chemical Theory and Computation}, volume = {10}, year = {2014}, month = {08/2014}, pages = {2891{\textendash}2896}, abstract = {

A simple coarse-grained model of single-stranded DNA (ssDNA) was developed, featuring only two sites per nucleotide that represent the centers of mass of the backbone and sugar/base groups. In the model, the interactions between sites are described using tabulated bonded potentials optimized to reproduce the solution structure of DNA observed in atomistic molecular dynamics simulations. Isotropic potentials describe nonbonded interactions, implicitly taking into account the solvent conditions to match the experimentally determined radius of gyration of ssDNA. The model reproduces experimentally measured force{\textendash}extension dependence of an unstructured DNA strand across 2 orders of magnitude of the applied force. The accuracy of the model was confirmed by measuring the end-to-end distance of a dT14 fragment via FRET while stretching the molecules using optical tweezers. The model offers straightforward generalization to systems containing double-stranded DNA and DNA binding proteins.

}, doi = {10.1021/ct500193u}, url = {http://pubs.acs.org/doi/abs/10.1021/ct500193u}, author = {Christopher Maffeo and Ngo, Thuy T. M. and Ha, Taekjip and Aleksei Aksimentiev} } @article {119, title = {Two Structural Scenarios for Protein Stabilization by PEG.}, journal = {J Phys Chem B}, volume = {118}, year = {2014}, month = {05/2014}, pages = {8388{\textendash}8395}, abstract = {

PEGylation, or addition of poly(ethylene glycol) chains to proteins, is widely used to improve delivery in pharmaceutical applications. Recent studies suggest that stabilization of a protein by PEG, and hence its proteolytic degradability, are sequence-dependent and require only short PEG chains. Here we connect stabilization by short PEG chains directly to the structural dynamics of the protein and PEG chain. We measured the stability of human Pin1 WW domain with PEG-4 at asparagine 19 for a full mutant cycle at two positions thought to influence PEG-protein interaction: Ser16Ala and Tyr23Phe. We then performed explicit solvent molecular dynamics simulations on all PEGylated and PEG-free mutants. The mutant cycle yields a non-additive stabilization effect where the pseudo wild type and double mutant are more stabilized relative to unPEGylated proteins than are the two single mutants. The simulation reveals why: the double mutant suffers loss of beta sheet structure, which PEGylation restores even though the PEG extends as a coil into the solvent. In contrast, in one of the single mutants PEG preferentially interacts with the protein surface while disrupting the interactions of its asparagine host with a nearby methionine side chain. Thus PEG attachment can stabilize a protein differentially depending on the local sequence, and either by interacting with the surface, or by extending into the solvent. A simulation with PEG-45 attached to asparagine 19 shows that PEG even can do both in the same context.

}, issn = {1520-5207}, doi = {10.1021/jp502234s}, author = {Shu-Han Chao and Matthews, Sam S and Paxman, Ryan and Aleksei Aksimentiev and Martin Gruebele and Price, Joshua L} } @article {20, title = {Stretching and controlled motion of single-stranded DNA in locally heated solid-state nanopores}, journal = {ACS Nano}, volume = {7}, year = {2013}, month = {08/2013}, pages = {6816-24}, abstract = {

Practical applications of solid-state nanopores for DNA detection and sequencing require the electrophoretic motion of DNA through the nanopores to be precisely controlled. Controlling the motion of single-stranded DNA presents a particular challenge, in part because of the multitude of conformations that a DNA strand can adopt in a nanopore. Through continuum, coarse-grained and atomistic modeling, we demonstrate that local heating of the nanopore volume can be used to alter the electrophoretic mobility and conformation of single-stranded DNA. In the nanopore systems considered, the temperature near the nanopore is modulated via a nanometer-size heater element that can be radiatively switched on and off. The local enhancement of temperature produces considerable stretching of the DNA fragment confined within the nanopore. Such stretching is reversible, so that the conformation of DNA can be toggled between compact (local heating is off) and extended (local heating is on) states. The effective thermophoretic force acting on single-stranded DNA in the vicinity of the nanopore is found to be sufficiently large (4-8 pN) to affect such changes in the DNA conformation. The local heating of the nanopore volume is observed to promote single-file translocation of DNA strands at transmembrane biases as low as 10 mV, which opens new avenues for using solid-state nanopores for detection and sequencing of DNA.

}, issn = {1936-086X}, doi = {10.1021/nn403575n}, author = {Maxim Belkin and Christopher Maffeo and David B Wells and Aleksei Aksimentiev} } @article {15, title = {End-to-end attraction of duplex DNA}, journal = {Nucleic Acids Res}, volume = {40}, year = {2012}, month = {2012 May}, pages = {3812-21}, abstract = {

Recent experiments [Nakata, M. et al., End-to-end stacking and liquid crystal condensation of 6 to 20 basepair DNA duplexes. Science 2007; 318:1276-1279] have demonstrated spontaneous end-to-end association of short duplex DNA fragments into long rod-like structures. By means of extensive all-atom molecular dynamic simulations, we characterized end-to-end interactions of duplex DNA, quantitatively describing the forces, free energy and kinetics of the end-to-end association process. We found short DNA duplexes to spontaneously aggregate end-to-end when axially aligned in a small volume of monovalent electrolyte. It was observed that electrostatic repulsion of 5{\textquoteright}-phosphoryl groups promoted the formation of aggregates in a conformation similar to the B-form DNA double helix. Application of an external force revealed that rupture of the end-to-end assembly occurs by the shearing of the terminal base pairs. The standard binding free energy and the kinetic rates of end-to-end association and dissociation processes were estimated using two complementary methods: umbrella sampling simulations of two DNA fragments and direct observation of the aggregation process in a system containing 458 DNA fragments. We found the end-to-end force to be short range, attractive, hydrophobic and only weakly dependent on the ion concentration. The relation between the stacking free energy and end-to-end attraction is discussed as well as possible roles of the end-to-end interaction in biological and nanotechnological systems.

}, keywords = {DNA, Kinetics, Molecular Dynamics Simulation, Nucleic Acid Conformation}, issn = {1362-4962}, doi = {10.1093/nar/gkr1220}, author = {Christopher Maffeo and Luan, Binquan and Aleksei Aksimentiev} } @article {18, title = {Modeling and simulation of ion channels}, journal = {Chem Rev}, volume = {112}, year = {2012}, month = {2012 Dec 12}, pages = {6250-6284}, abstract = {

Transport of ions through pores in membranes is a process of fundamental importance to cell biology. In living organisms, such transport is facilitated by ion channels that utilize the ionic flux to perform diverse biological functions, such as cell{\textendash}cell communication and signaling, osmotic stress response, muscle contraction, etc. The action of ion channels is responsible for most of what we (humans) perceive as reality in the form of sound, smell, sight, taste, and touch and forms the physiological basis for thought. Biomimetic ion channels are ubiquitous in engineering, with application ranging from water desalination to fuel cells. Here, we review efforts to model and simulate ion channels that occurred within the past 10 years.

}, keywords = {Review}, issn = {1520-6890}, doi = {10.1021/cr3002609}, author = {Christopher Maffeo and Bhattacharya, Swati and Yoo, Jejoong and David B Wells and Aleksei Aksimentiev} } @article {30, title = {Optimization of the molecular dynamics method for simulations of DNA and ion transport through biological nanopores}, journal = {Methods Mol Biol}, volume = {870}, year = {2012}, month = {2012}, pages = {165-86}, abstract = {

Molecular dynamics (MD) simulations have become a standard method for the rational design and interpretation of experimental studies of DNA translocation through nanopores. The MD method, however, offers a multitude of algorithms, parameters, and other protocol choices that can affect the accuracy of the resulting data as well as computational efficiency. In this chapter, we examine the most popular choices offered by the MD method, seeking an optimal set of parameters that enable the most computationally efficient and accurate simulations of DNA and ion transport through biological nanopores. In particular, we examine the influence of short-range cutoff, integration timestep and force field parameters on the temperature and concentration dependence of bulk ion conductivity, ion pairing, ion solvation energy, DNA structure, DNA-ion interactions, and the ionic current through a nanopore.

}, keywords = {Biological Transport, DNA, Electric Conductivity, Electroosmosis, Hemolysin Proteins, Ions, Lipid Bilayers, Molecular Dynamics Simulation, Nanopores, Porins, Solvents, Thermodynamics}, issn = {1940-6029}, doi = {10.1007/978-1-61779-773-6_10}, author = {David B Wells and Bhattacharya, Swati and Carr, Rogan and Christopher Maffeo and Anthony H. Ho and Jeffrey Comer and Aleksei Aksimentiev} } @article {195, title = {Rectification of the current in alpha-hemolysin pore depends on the cation type: the alkali series probed by MD simulations and experiments}, journal = {The Journal of Physical Chemistry C}, volume = {115}, year = {2011}, month = {2011 Feb 21}, pages = {4255-4264}, abstract = {

A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel{\textquoteright}s end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.

}, issn = {1932-7447}, doi = {10.1021/jp111441p}, author = {Bhattacharya, Swati and Muzard, L and Payet, Linda and Math{\'e}, J{\'e}r{\^o}me and Bockelmann, Ulrich and Aleksei Aksimentiev and Viasnoff, Virgile} } @inbook {192, title = {Third Generation DNA Sequencing with a Nanopore}, booktitle = {Nanopores: Sensing fundamental biological interactions at the single molecule level}, year = {2011}, pages = {287-311}, publisher = {Springer US}, organization = {Springer US}, chapter = {12}, abstract = {

With the advent of Next-Generation-Sequencing (NGS) technologies, an enormous volume of DNA sequencing data can be generated at low cost, placing genomic science within the grasp of everyday medicine. However, mired in this voluminous data, a new problem has emerged: the assembly of the genome from the short reads. In this chapter we examine the prospects for sequencing DNA using a synthetic nanopore. Nanopore sequencing has the potential for very long reads, reducing the computational burden posed by alignment and genome assembly, while at the same time eliminating logistically challenging and error-prone amplification and library formation due to its exquisite single molecule sensitivity. On the other hand, long high fidelity reads demand stringent control over both the DNA configuration in the pore and the translocation kinetics. We examine the prospects for satisfying these specifications with a synthetic nanopore.

}, keywords = {Comparison between biological and synthetic nanopores, Hydrodynamic focusing, Molecular dynamics simulations, Sequencing DNA, Trapping DNA, λ-DNA}, isbn = {978-1-4419-8251-3}, doi = {10.1007/978-1-4419-8252-0_12}, url = {http://dx.doi.org/10.1007/978-1-4419-8252-0_12}, author = {Gregory Timp and Mirsaidov, Utkur and Timp, Winston and Shim, Jiwook and Wang, Deqiang and Valentin Dimitrov and Scrimgeour, Jan and Lin, Chunchen and Jeffrey Comer and Anthony H. Ho and Zou, Xueqing and Aleksei Aksimentiev and Klaus Schulten}, editor = {Iqbal, Samir M. and Rashid Bashir} } @article {16, title = {Single molecule force measurements: insights from molecular simulations. Comment on "Biophysical characterization of DNA binding from single molecule force measurements" by Kathy R. Chaurasiya et al.}, journal = {Phys Life Rev}, volume = {7}, year = {2010}, month = {2010 Sep}, pages = {353-4; discussion 358-61}, keywords = {Biophysics, DNA, Microscopy, Atomic Force, Molecular Dynamics Simulation, Nucleic Acid Conformation, Thermodynamics}, issn = {1873-1457}, doi = {10.1016/j.plrev.2010.07.004}, author = {Christopher Maffeo and Aleksei Aksimentiev} } @article {17, title = {DNA-DNA interactions in tight supercoils are described by a small effective charge density}, journal = {Phys Rev Lett}, volume = {105}, year = {2010}, month = {2010 Oct 8}, pages = {158101}, abstract = {

DNA-DNA interactions are important for genome compaction and transcription regulation. In studies of such complex processes, DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation. Commonly, a charge adaptation factor is used to address limitations of this theoretical approach. Despite considerable theoretical and experimental efforts, a rigorous quantitative assessment of this parameter is lacking. Here, we comprehensively characterized DNA-DNA interactions in the presence of monovalent ions by analyzing the supercoiling behavior of single DNA molecules held under constant tension. Both a theoretical model and coarse-grained simulations of this process revealed a surprisingly small effective DNA charge of 40\% of the nominal charge density, which was additionally supported by all-atom molecular dynamics simulations.

}, keywords = {Biomechanical Phenomena, DNA, Superhelical, Molecular Dynamics Simulation, Monte Carlo Method, Salts, Static Electricity}, issn = {1079-7114}, doi = {10.1103/PhysRevLett.105.158101 }, url = {http://journals.aps.org/prl/abstract/10.1103/PhysRevLett.105.158101}, author = {Christopher Maffeo and Sch{\"o}pflin, Robert and Brutzer, Hergen and Stehr, Ren{\'e} and Aleksei Aksimentiev and Wedemann, Gero and Seidel, Ralf} } @article {41, title = {Slowing the translocation of double-stranded DNA using a nanopore smaller than the double helix}, journal = {Nanotechnology}, volume = {21}, year = {2010}, month = {2010 Oct 1}, pages = {395501}, abstract = {

It is now possible to slow and trap a single molecule of double-stranded DNA (dsDNA), by stretching it using a nanopore, smaller in diameter than the double helix, in a solid-state membrane. By applying an electric force larger than the threshold for stretching, dsDNA can be impelled through the pore. Once a current blockade associated with a translocating molecule is detected, the electric field in the pore is switched in an interval less than the translocation time to a value below the threshold for stretching. According to molecular dynamics (MD) simulations, this leaves the dsDNA stretched in the pore constriction with the base-pairs tilted, while the B-form canonical structure is preserved outside the pore. In this configuration, the translocation velocity is substantially reduced from 1 bp/10 ns to approximately 1 bp/2 ms in the extreme, potentially facilitating high fidelity reads for sequencing, precise sorting, and high resolution (force) spectroscopy.

}, keywords = {Bacteriophage lambda, DNA, DNA, Viral, Electrochemical Techniques, Microscopy, Electron, Transmission, Molecular Dynamics Simulation, Nanopores, Nanotechnology, Nucleic Acid Conformation, Particle Size}, issn = {1361-6528}, doi = {10.1088/0957-4484/21/39/395501}, author = {Mirsaidov, Utkur and Jeffrey Comer and Valentin Dimitrov and Aleksei Aksimentiev and Gregory Timp} } @article {36, title = {Nanopore Sequencing: Electrical Measurements of the Code of Life}, journal = {IEEE Trans Nanotechnol}, volume = {9}, year = {2010}, month = {2010 May 1}, pages = {281-294}, abstract = {

Sequencing a single molecule of deoxyribonucleic acid (DNA) using a nanopore is a revolutionary concept because it combines the potential for long read lengths (\>5 kbp) with high speed (1 bp/10 ns), while obviating the need for costly amplification procedures due to the exquisite single molecule sensitivity. The prospects for implementing this concept seem bright. The cost savings from the removal of required reagents, coupled with the speed of nanopore sequencing places the $1000 genome within grasp. However, challenges remain: high fidelity reads demand stringent control over both the molecular configuration in the pore and the translocation kinetics. The molecular configuration determines how the ions passing through the pore come into contact with the nucleotides, while the translocation kinetics affect the time interval in which the same nucleotides are held in the constriction as the data is acquired. Proteins like α-hemolysin and its mutants offer exquisitely precise self-assembled nanopores and have demonstrated the facility for discriminating individual nucleotides, but it is currently difficult to design protein structure ab initio, which frustrates tailoring a pore for sequencing genomic DNA. Nanopores in solid-state membranes have been proposed as an alternative because of the flexibility in fabrication and ease of integration into a sequencing platform. Preliminary results have shown that with careful control of the dimensions of the pore and the shape of the electric field, control of DNA translocation through the pore is possible. Furthermore, discrimination between different base pairs of DNA may be feasible. Thus, a nanopore promises inexpensive, reliable, high-throughput sequencing, which could thrust genomic science into personal medicine.

}, keywords = {Review}, issn = {1536-125X}, doi = {10.1109/TNANO.2010.2044418}, author = {Timp, Winston and Mirsaidov, Utkur M and Wang, Deqiang and Jeffrey Comer and Aleksei Aksimentiev and Gregory Timp} } @article {19, title = {Structure, dynamics, and ion conductance of the phospholamban pentamer}, journal = {Biophys J}, volume = {96}, year = {2009}, month = {2009 Jun 17}, pages = {4853-65}, abstract = {

A 52-residue membrane protein, phospholamban (PLN) is an inhibitor of an adenosine-5{\textquoteright}-triphosphate-driven calcium pump, the Ca2+-ATPase. Although the inhibition of Ca2+-ATPase involves PLN monomers, in a lipid bilayer membrane, PLN monomers form stable pentamers of unknown biological function. The recent NMR structure of a PLN pentamer depicts cytoplasmic helices extending normal to the bilayer in what is known as the bellflower conformation. The structure shows transmembrane helices forming a hydrophobic pore 4 {\r A} in diameter, which is reminiscent of earlier reports of possible ion conductance through PLN pentamers. However, recent FRET measurements suggested an alternative structure for the PLN pentamer, known as the pinwheel model, which features a narrower transmembrane pore and cytoplasmic helices that lie against the bilayer. Here, we report on structural dynamics and conductance properties of the PLN pentamers from all-atom (AA) and coarse-grained (CG) molecular dynamics simulations. Our AA simulations of the bellflower model demonstrate that in a lipid bilayer membrane or a detergent micelle, the cytoplasmic helices undergo large structural fluctuations, whereas the transmembrane pore shrinks and becomes asymmetric. Similar asymmetry of the transmembrane region was observed in the AA simulations of the pinwheel model; the cytoplasmic helices remained in contact with the bilayer. Using the CG approach, structural dynamics of both models were investigated on a microsecond timescale. The cytoplasmic helices of the CG bellflower model were observed to fall against the bilayer, whereas in the CG pinwheel model the conformation of the cytoplasmic helices remained stable. Using steered molecular dynamics simulations, we investigated the feasibility of ion conductance through the pore of the bellflower model. The resulting approximate potentials of mean force indicate that the PLN pentamer is unlikely to function as an ion channel.

}, keywords = {Calcium-Binding Proteins, Cell Membrane, Computer Simulation, Cytoplasm, Humans, Hydrophobic and Hydrophilic Interactions, Ions, Lipid Bilayers, Micelles, Models, Molecular, Protein Multimerization, Protein Structure, Quaternary}, issn = {1542-0086}, doi = {10.1016/j.bpj.2009.03.053}, author = {Christopher Maffeo and Aleksei Aksimentiev} } @article {49, title = {Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore}, journal = {Nucleic Acids Res}, volume = {37}, year = {2009}, month = {2009 Jul}, pages = {4170-9}, abstract = {

Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence--GAATTC--in the absence of a Mg(2+) ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI-DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments.

}, keywords = {Computer Simulation, Deoxyribonuclease EcoRI, DNA, DNA-Binding Proteins, Models, Molecular, Nanostructures, Protein Binding}, issn = {1362-4962}, doi = {10.1093/nar/gkp317}, author = {Dorvel, B and G Sigalov and Zhao, Q and Jeffrey Comer and Valentin Dimitrov and Mirsaidov, U and Aleksei Aksimentiev and Gregory Timp} } @article {58, title = {Synthetic ion channels via self-assembly: a route for embedding porous polyoxometalate nanocapsules in lipid bilayer membranes}, journal = {Nano Lett}, volume = {8}, year = {2008}, month = {2008 Nov}, pages = {3916-21}, abstract = {

Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels; however, their use as an artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this Letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na(+) cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane.

}, keywords = {Ion Channels, Kinetics, Lipid Bilayers, Liposomes, Models, Molecular, Molecular Conformation, Nanocapsules, Porosity, Tungsten Compounds}, issn = {1530-6984}, doi = {10.1021/nl802366k}, author = {Carr, Rogan and Weinstock, Ira A and Sivaprasadarao, Asipu and M{\"u}ller, Achim and Aleksei Aksimentiev} } @article {65, title = {Detecting SNPs using a synthetic nanopore}, journal = {Nano Lett}, volume = {7}, year = {2007}, month = {2007 Jun}, pages = {1680-5}, abstract = {

We have discovered a voltage threshold for permeation through a synthetic nanopore of dsDNA bound to a restriction enzyme that depends on the sequence. Molecular dynamic simulations reveal that the threshold is associated with a nanonewton force required to rupture the DNA-protein complex. A single mutation in the recognition site for the restriction enzyme, i.e., a single nucleotide polymorphism (SNP), can easily be detected as a change in the threshold voltage. Consequently, by measuring the threshold voltage in a synthetic nanopore, it may be possible to discriminate between two variants of the same gene (alleles) that differ in one base.

}, keywords = {Deoxyribonuclease EcoRI, DNA, DNA Mutational Analysis, Electromagnetic Fields, Macromolecular Substances, Materials Testing, Molecular Conformation, Nanostructures, Nanotechnology, Particle Size, Polymorphism, Single Nucleotide, Porosity, Sequence Analysis, DNA, Surface Properties}, issn = {1530-6984}, doi = {10.1021/nl070668c}, author = {Zhao, Q and G Sigalov and Valentin Dimitrov and Dorvel, B and Mirsaidov, U and S Sligar and Aleksei Aksimentiev and Gregory Timp} } @article {68, title = {The electromechanics of DNA in a synthetic nanopore}, journal = {Biophys J}, volume = {90}, year = {2006}, month = {2006 Feb 1}, pages = {1098-106}, abstract = {

We have explored the electromechanical properties of DNA on a nanometer-length scale using an electric field to force single molecules through synthetic nanopores in ultrathin silicon nitride membranes. At low electric fields, E < 200 mV/10 nm, we observed that single-stranded DNA can permeate pores with a diameter >/=1.0 nm, whereas double-stranded DNA only permeates pores with a diameter >/=3 nm. For pores <3.0 nm diameter, we find a threshold for permeation of double-stranded DNA that depends on the electric field and pH. For a 2 nm diameter pore, the electric field threshold is approximately 3.1 V/10 nm at pH = 8.5; the threshold decreases as pH becomes more acidic or the diameter increases. Molecular dynamics indicates that the field threshold originates from a stretching transition in DNA that occurs under the force gradient in a nanopore. Lowering pH destabilizes the double helix, facilitating DNA translocation at lower fields.

}, keywords = {Biophysics, Computer Simulation, DNA, Electrochemistry, Electromagnetic Fields, Electrophoresis, Electrophoresis, Agar Gel, Hydrogen-Ion Concentration, Ions, Membrane Potentials, Microscopy, Electron, Transmission, Models, Molecular, Nanostructures, Nanotechnology, Nucleic Acid Conformation, Polymerase Chain Reaction, Porosity, Silicon Compounds, Time Factors}, issn = {0006-3495}, doi = {10.1529/biophysj.105.070672}, author = {Jiunn B Heng and Aleksei Aksimentiev and Ho, C and Patrick Marks and Yelena V Grinkova and S Sligar and Klaus Schulten and Gregory Timp} } @article {69, title = {Stretching DNA using the electric field in a synthetic nanopore}, journal = {Nano Lett}, volume = {5}, year = {2005}, month = {2005 Oct}, pages = {1883-8}, abstract = {

The mechanical properties of DNA over segments comparable to the size of a protein-binding site (3-10 nm) are examined using an electric-field-induced translocation of single molecules through a nanometer diameter pore. DNA, immersed in an electrolyte, is forced through synthetic pores ranging from 0.5 to 1.5 nm in radius in a 10 nm thick Si(3)N(4) membrane using an electric field. To account for the stretching and bending, we use molecular dynamics to simulate the translocation. We have found a threshold for translocation that depends on both the dimensions of the pore and the applied transmembrane bias. The voltage threshold coincides with the stretching transition that occurs in double-stranded DNA near 60 pN.

}, keywords = {DNA, Nanotechnology, Static Electricity, Stress, Mechanical}, issn = {1530-6984}, doi = {10.1021/nl0510816}, author = {Jiunn B Heng and Aleksei Aksimentiev and Chuen Ho and Patrick Marks and Yelena V Grinkova and S Sligar and Klaus Schulten and Gregory Timp} } @article {70, title = {Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel}, journal = {Proc Natl Acad Sci U S A}, volume = {102}, year = {2005}, month = {2005 Aug 30}, pages = {12377-82}, abstract = {

We characterize the voltage-driven motion and the free motion of single-stranded DNA (ssDNA) molecules captured inside the approximately 1.5-nm alpha-hemolysin pore, and show that the DNA-channel interactions depend strongly on the orientation of the ssDNA molecules with respect to the pore. Remarkably, the voltage-free diffusion of the 3{\textquoteright}-threaded DNA (in the trans to cis direction) is two times slower than the corresponding 5{\textquoteright}-threaded DNA having the same poly(dA) sequence. Moreover, the ion currents flowing through the blocked pore with either a 3{\textquoteright}-threaded DNA or 5{\textquoteright} DNA differ by approximately 30\%. All-atom molecular dynamics simulations of our system reveal a microscopic mechanism for the asymmetric behavior. In a confining pore, the ssDNA straightens and its bases tilt toward the 5{\textquoteright} end, assuming an asymmetric conformation. As a result, the bases of a 5{\textquoteright}-threaded DNA experience larger effective friction and forced reorientation that favors co-passing of ions. Our results imply that the translocation process through a narrow pore is more complicated than previously believed and involves base tilting and stretching of ssDNA molecules inside the confining pore.

}, keywords = {Bacterial Toxins, Base Sequence, Biophysical Phenomena, Biophysics, DNA, Single-Stranded, Hemolysin Proteins, Ion Channels, Models, Molecular, Motion, Nucleic Acid Conformation, Poly A, Thermodynamics}, issn = {0027-8424}, doi = {10.1073/pnas.0502947102}, author = {Math{\'e}, J{\'e}r{\^o}me and Aleksei Aksimentiev and Nelson, David R and Klaus Schulten and Meller, Amit} } @article {59, title = {Beyond the Gene Chip}, journal = {Bell Labs Tech J}, volume = {10}, year = {2005}, month = {2005}, pages = {5-22}, abstract = {

We describe a prospective strategy for reading the encyclopedic information encoded in the genome: using a nanopore in a membrane formed from an MOS-capacitor to sense the charge in DNA. In principle, as DNA permeates the capacitor-membrane through the pore, the electrostatic charge distribution characteristic of the molecule should polarize the capacitor and induce a voltage on the electrodes that can be measured. Silicon nanofabrication and molecular dynamic simulations with atomic detail are technological linchpins in the development of this detector. The sub-nanometer precision available through silicon nanotechnology facilitates the fabrication of the detector, and molecular dynamics provides us with a means to design it and analyze the experimental outcomes.

}, issn = {1089-7089}, doi = {10.1002/bltj.20102}, author = {Jiunn B Heng and Aleksei Aksimentiev and Ho, C and Valentin Dimitrov and Sorsch, T and Miner, J and Mansfield, W and Klaus Schulten and Gregory Timp} } @inbook {189, title = {Toward understanding membrane channels}, booktitle = { Bacterial ion channels and their eukaryotic homologues}, year = {2005}, pages = {153{\textendash}190}, publisher = {ASM Press}, organization = {ASM Press}, address = {Washington, DC}, abstract = {

Membrane channels play key roles in all living cells. Owing to recent advances in protein crystallography, atomic-resolution structures for a representative set of membrane channels have become available. Transport of materials through these channels, however, is a dynamic process and cannot be fully understood from static structural information alone. Molecular dynamics simulations are in a unique position to describe the dynamics of channel transport in the cases in which the structures of channel proteins are known. However, simulation of channel permeability and gating in the native environment of lipid bilayer and water involves large systems with 100,000 - 300,000 atoms.

This review article focuses on four case studies that demonstrate the power of molecular dynamics simulations in unraveling the mechanisms underlying the function of membrane channels. Simulations of AQP water channels present a prime example of the effectiveness and usefulness of molecular dynamics in simulating conduction and explaining selectivity. A detailed energetic analysis of ion permeation through chloride channels proposes a two- ion permeation mechanism that reconciles naturally structural and physiological data. The study of ion and water permeation through hemolysin exemplifies how accurately one can simulate today even very large membrane channel systems. Finally, the study of mechanical gating of MscS shows that molecular dynamics can account even for complex gating motions of channels.

}, author = {Tajkhorshid, Emad and Cohen, Jordi and Aleksei Aksimentiev and Marcos Sotomayor and Klaus Schulten}, editor = {Boris Martinac and Andrzej Kubalski} } @article {181, title = {Scaling properties of the morphological measures at the early and intermediate stages of the spinodal decomposition in homopolymer blends}, journal = {The Journal of Chemical Physics}, volume = {112}, year = {2000}, pages = {6049{\textendash}6062}, abstract = {

The spinodal decomposition of the homopolymer blends has been studied by the numerical integration of the Cahn{\textendash}Hilliard{\textendash}Cook equation. We have investigated the time evolution of the morphological measures that characterize quantitatively the interface in the system. For symmetric blends we have found that the Euler characteristic of the interface is negative and increases with time as τ0.75 (connectivity of the domains decreases) regardless of the final quench temperature. The homogeneity index of the interface is constant in this case. This suggests that at the level of the integral geometry quantities (Minkowski functionals), the dynamic scaling hypothesis holds for the evolution of the interface morphology in quenched critical systems. The nonuniversal morphological evolution of the asymmetric blends have been studied. Also, we have shown that the thermal fluctuations can modify significantly the curvature distribution.

}, doi = {10.1063/1.481178}, author = {Aleksei Aksimentiev and Krzysztof Moorthi and Robert Ho{\l}yst} } @article {180, title = {Kinetics of the droplet formation at the early and intermediate stages of the spinodal decomposition in homopolymer blends}, journal = {Macromolecular Theory and Simulations}, volume = {9}, year = {2000}, pages = {661{\textendash}674}, abstract = {

The kinetics of the droplet formation during the spinodal decomposition (SD) of the homopolymer blends has been studied by numerical integration of the Cahn-Hilliard-Cook equation. We have found that the droplet formation and growth occurs when the minority phase volume fraction, fm , approaches the percolation threshold value, fthr = 0.3 {\textpm} 0.01. The time for the formation of the disperse droplet morphology (coarsening time) depends only on the equilibrium minority phase volume fraction, fm . fm approaches its equilibrium value logarithmically at the late SD stages, and, therefore, the coarsening time decreases exponentially as the average volume fraction or the quench depth decrease. Since the temporal evolution of the total interfacial area does not depend on the quench conditions and blend morphology, the average droplet size and the droplet number density is determined by the coarsening time. Within the time scale studied, the droplet number density decreases with time as t{\textendash}0.63{\textpm}0.03; the average mean curvature decreases as t{\textendash}0.35{\textpm}0.05; the average Gaussian curvature decreases as t{\textendash}0.42{\textpm}0.03, and the average droplet compactness \~{}V/S3/2 where S is the surface area and V is the volume) approaches a spherical limit logarithmically with time. The droplets with larger area have lower compactness and in the low compactness limit their area is a parabolic function of compactness. The size and shape distribution functions have been also investigated.

}, doi = {10.1002/1521-3919(20001101)9:8<661::AID-MATS661>3.0.CO;2-6}, author = {Aleksei Aksimentiev and Robert Ho{\l}yst and Krzysztof Moorthi} }