Nanopore Translocation Reveals Electrophoretic Force on Noncanonical RNA:DNA Double Helix.

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.

Electro-osmotic Flow Generation via a Sticky Ion Action.

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.

Single molecule delivery into living cells.

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

Dehydrated Biomimetic Membranes with Skinlike Structure and Function.

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'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.

The structure and physical properties of a packaged bacteriophage particle.

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 species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, 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 mechanism7 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-capsid interface.

Electric field of DNA in solution: who is in charge?

In solution, DNA is a highly charged macromolecule which bears a unit of negative charge on each phosphate of its sugar-phosphate backbone. Although partially compensated by counterions adsorbed at or condensed near it, DNA still produces a substantial electric field in its vicinity, which is screened by buffer electrolyte at longer distances from the DNA. Such field has been explored so far predominantly within the scope of a primitive model of the electrolytic solution, not considering more complicated structural effects of the water solvent. In this paper we investigate the distribution of electric field around DNA using linear response nonlocal electrostatic theory, applied here for helix-specific charge distributions, and compare the predictions of such theory with specially performed fully atomistic large scale molecular dynamics simulations. The main finding of this study is that oscillations in the electrostatic potential distribution are present around DNA, caused by the overscreening effect of structured water. Surprisingly, electrolyte ions at physiological concentrations do not strongly disrupt these oscillations, and rather distribute according to these oscillating patterns, indicating that water structural effects dominate the short-range electrostatics. We also show that (i) structured water adsorbed in the grooves of DNA lead to a positive electrostatic potential core, (ii) the Debye length some 10 {\AA} away from the DNA is reduced, effectively renormalised by the helical pitch of the DNA, and (iii) Lorentzian contributions to the nonlocal dielectric function of water, effectively reducing the dielectric constant close to the DNA, enhances the overall electric field. The impressive agreement between the atomistic simulations and the developed theory substantiates the use of nonlocal electrostatics when considering solvent effects in molecular processes in biology.

Molecular Determinants of Current Blockade Produced by Peptide Transport Through a Nanopore.

The nanopore sensing method holds the promise of delivering a single molecule technology for identification of biological proteins, direct detection of post-translational modifications, and perhaps de novo determination of a protein's amino acid sequence. The key quantity measured in such nanopore sensing experiments is the magnitude of the ionic current passing through a nanopore blocked by a polypeptide chain. Establishing a relationship between the amino acid sequence of a peptide fragment confined within a nanopore and the blockade current flowing through the nanopore remains a major challenge for realizing the nanopore protein sequencing. Using the results of all-atom molecular dynamics simulations, here we compare nanopore sequencing of DNA with nanopore sequencing of proteins. We then delineate the factors affecting the blockade current modulation by the peptide sequence, showing that the current can be determined by (i) the steric footprint of an amino acid, (ii) its interactions with the pore wall, (iii) the local stretching of a polypeptide chain, and (iv) the local enhancement of the ion concentration at the nanopore constriction. We conclude with a brief discussion of the prospects for purely computational prediction of the blockade currents.

RNA regulates cohesiveness and porosity of a biological condensate.

Biological condensates have emerged as key elements of a biological cell function, concentrating disparate biomolecules to accomplish specific biological tasks. RNA was identified as a key ingredient of such condensates, however, its effect on the physical properties of the condensate was found to depend on the condensate's composition while its effect on the microstructure has remained elusive. Here, we characterize the physical properties and the microstructure of a protein-RNA condensate by means of large-scale coarse-grained (CG) molecular dynamics simulations. By developing a custom CG model of RNA compatible with a popular CG model of proteins, we systematically investigate the structural, thermodynamic, and kinetic properties of condensate droplets containing thousands of individual protein and RNA molecules over a range of temperatures. While we find RNA to increase the condensate's cohesiveness, its effect on the condensate's fluidity is more nuanced with longer molecules compacting the condensate and making it less fluid. We show that a biological condensate has a sponge-like morphology of interconnected channels of size that increases with temperature and decreases in the presence of RNA. Our results suggest that longer RNA form a dynamic scaffold within a condensate, regulating not only its fluidity but also permeability to intruder molecules.

A DNA turbine powered by a transmembrane potential across a nanopore.

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 24-27 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.

Length-dependent Intramolecular Coil-to-Globule Transition in Poly(ADP-ribose) Induced by Cations.

Poly(ADP-ribose) (PAR), as part of a post-translational modification, serves as a flexible scaffold for noncovalent protein binding. Such binding is influenced by PAR chain length through a mechanism yet to be elucidated. Structural insights have been elusive, partly due to the difficulties associated with synthesizing PAR chains of defined lengths. Here, we employ an integrated approach combining molecular dynamics (MD) simulations with small-angle X-ray scattering (SAXS) experiments, enabling us to identify highly heterogeneous ensembles of PAR conformers at two different, physiologically relevant lengths: PAR 15 and PAR 22 . Our findings reveal that numerous factors including backbone conformation, base stacking, and chain length contribute to determining the structural ensembles. We also observe length-dependent compaction of PAR upon the addition of small amounts of Mg 2+ ions, with the 22-mer exhibiting ADP-ribose bundles formed through local intramolecular coil-to-globule transitions. This study illuminates how such bundling could be instrumental in deciphering the length-dependent action of PAR.

A Marcus-Type Inverted Region in the Translocation Kinetics of a Knotted Protein.

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 "Marcus inverted regime". Our results shed light on the influence of topology in knotted proteins on their forced translocation through a pore connecting two electrostatic potential wells.

Long-Range Conductivity in Proteins Mediated by Aromatic Residues.

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.

Sulfur-Containing Foldamer-Based Artificial Lithium Channels.

Unlike many other biologically relevant ions (Na+ , K+ , Ca2+ , Cl- , etc) and protons, whose cellular concentrations are closely regulated by highly selective channel proteins, Li+ ion is unusual in that its concentration is well tolerated over many orders of magnitude and that no lithium-specific channel proteins have so far been identified. While one naturally evolved primary pathway for Li+ ions to traverse across the cell membrane is through sodium channels by competing with Na+ ions, highly sought-after artificial lithium-transporting channels remain a major challenge to develop. Here we show that sulfur-containing organic nanotubes derived from intramolecularly H-bonded helically folded aromatic foldamers of 3.6 Šin hollow cavity diameter could facilitate highly selective and efficient transmembrane transport of Li+ ions, with high transport selectivity factors of 15.3 and 19.9 over Na+ and K+ ions, respectively.

Engineering Biological Nanopore Approaches toward Protein Sequencing.

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

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-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-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.

All-Atom Molecular Dynamics Simulations of Membrane-Spanning DNA Origami Nanopores.

Building on the recent technological advances, all-atom molecular dynamics (MD) simulations have become an indispensable tool to study the molecular behavior at nanoscale. Molecular simulations have been used to characterize the structure, dynamics, and mechanical and electrical properties of DNA origami objects. In this chapter we describe a method to build all-atom model of lipid-spanning DNA origami nanopores and perform molecular dynamics simulations in explicit electrolyte solutions.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral components 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 all-atom molecular dynamics method can, in principle, elucidate the conformational changes responsible for the aging of the condensate, the applications of this method to protein condensate systems is conditioned by 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 field 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-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-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.

Super-Resolution Detection of DNA Nanostructures Using a Nanopore.

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 unlabeled single molecules in their native states. Here, label-free electrical sensing of structured single molecules with a spatial resolution down to single-digit nanometers is demonstrated. Using a narrow solid-state nanopore, the passage of a series of nanostructures attached to a freely translocating DNA molecule is detected, resolving individual nanostructures placed as close as 6 nm apart and with a surface-to-surface gap distance of only 2 nm. Such super-resolution ability is attributed to the nanostructure-induced enhancement of the electric field at the tip of the nanopore. This 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.

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

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