Biology subjects

Aksimentiev, A.

Publications and source records attributed to Aksimentiev, A..

28 records · Page 2Linked to original sources

bioRxiv2021

Resolving isomeric posttranslational modifications using a nanopore

Posttranslational modifications (PTMs) of proteins are crucial for cellular function but pose analytical problems, especially in distinguishing chemically identical PTMs at different nearby locations within the same protein. Current methods, such as liquid chromatography-tandem mass spectrometry, are technically tantamount to de novo protein sequencing1. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems2-4. Here, we demonstrate the use of an engineered biological nanopore to differentiate positional isomers resulting from acetylation or methylation of histone protein H4, an important PTM target5,6. In contrast to strand sequencing, we differentiate positional isomers by recording ionic current modulations resulting from the stochastic entrapment of entire peptides in the pores sensing zone, with all residues simultaneously contributing to the electrical signal. Molecular dynamics simulations show that, in this whole-molecule sensing mode, the non-uniform distribution of the electric potential within the nanopore makes the added resistance contributed by a PTM dependent on its precise location on the peptide. Optimization of the pores sensitivity in combination with parallel recording and automated and standardized protein fragmentation may thus provide a simple, label-free, high-throughput analytical platform for identification and quantification of PTMs.

biophysics↗

bioRxiv2021

Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing

DNA is a promising next-generation data storage medium, but the recording latency and synthesis cost of oligos using the four natural nucleotides remain high. Here, we describe an improved DNA-based storage system that uses an extended 11-letter molecular alphabet combining natural and chemically modified nucleotides. Our extended-alphabet molecular storage paradigm offers a nearly two-fold increase in storage density and potentially the same order of reduction in the recording time. Experimental results involving a library of 77 custom-designed hybrid sequences reveal that one can readily detect and discriminate different combinations and orders of monomers via MspA nanopores. Furthermore, a neural network architecture designed to classify raw current signals generated by Oxford Nanopore Technologies sequencing ensures an average accuracy exceeding 60%, which is 39 times higher than that of random guessing. Molecular dynamics simulations reveal that the majority of modified nucleotides do not induce dramatic disruption of the DNA double helix, making the extended alphabet system potentially compatible with PCR-based random access data retrieval. The methodologies proposed provide a forward path for new implementations of molecular recorders.

synthetic biology↗

bioRxiv2021

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

Nanopore sensors could revolutionize single-molecule proteomics by providing a means for identification of known proteins through fingerprinting or by de novo sequencing. However, the complex chemical and physical properties of proteins present multiple challenges to the conventional nanopore sensing method, predominantly, the single-file threading of a protein chain into a nanopore and its transport through it. Herein we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore platform and a high concentration guanidinium chloride buffer enables protein unfolding and unidirectional transport through a pore, propelled by an electroosmotic effect that largely owes to the guanidinium chloride presence. The uniform and slow (~10 {micro}s/amino acid) single-file transport, when combined with supervised machine learning of the electrical current signatures obtained, allows us to use to discern the protein threading orientation and identity. In conjunction with a method for tail-modification of native proteins and higher-resolution nanopores, our approach could offer a path towards direct single-molecule protein fingerprinting without the requirement of a motor enzyme.

biophysics↗

bioRxiv2021

DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches including optical voltage-sensing dyes and genetically encoded voltage indicators have evolved. Here, we present a DNA nanotechnology-based system. Using DNA origami, we incorporate and optimize different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we use a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA connecting the DNA origami and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit is read out by changes of Fluorescence Resonance Energy Transfer (FRET) of single sensors attached to liposomes. They show a FRET change of [~]5% for {Delta}{Psi}=100 mV and allow adapting the potential range of highest sensitivity. Further, the working mechanism is rationalized by molecular dynamics simulations. Our approach holds potential for the application as non-genetically encoded sensors at membranes.

biophysics↗

bioRxiv2021

Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to rewind peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10-6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer. One-sentence summaryThis paper presents proof-of-concept experiments and simulations of a nanopore-based approach to sequencing individual proteins.

biophysics↗

bioRxiv2021

Membrane activity of a DNA-based ion channel depends on the stability of its double-stranded structure.

Structural 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 ion channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA-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, when introduced at an end of a DNA strand, are likely to cause fraying of the terminal base pairs as the DNA nanostructure adopts its energy-minimum configuration in the membrane. We also find that fraying of base pairs distorts nicked DNA constructs when embedded in a lipid bilayer. Here, we show that DNA nanostructures that do not have discontinuities (nicks) in their DNA backbones form considerably more stable DNA-induced conductive pores and insert into lipid membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows to design and maintain membrane-spanning helices in a tilted orientation within lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

synthetic biology↗

bioRxiv2021

A nanoscale reciprocating rotary mechanism with coordinated mobility control

Biological molecular motors transform chemical energy into mechanical work by coupling a cycle of catalytic reactions to large scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the celebrated example of F1Fo ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in the structure of the surrounding stator orchestrated by mechanical deformation. We designed the mechanism using DNA origami, characterized the structure of the components and the entire mechanism using cryo-electron microscopy, and examined the mechanisms dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. The data indicates that, while the camshaft can rotate inside the stator by diffusion, mechanical deformation of the stator makes the camshaft pause at a set of preferred orientations. By changing the mechanical stiffness of the stator, we could accelerate or suppress the Brownian rotation within the mechanism, thereby demonstrating an allosteric coupling between the movement of the camshaft and of the stator, and the ability to tailor the free energy landscape that governs the rotary motion. Our mechanism provides a framework for the manufacture of artificial nanomachines that, just like the man-made machines in the macroscopic world, function because of coordinated movements of their components.

biophysics↗

bioRxiv2021

Electrical Unfolding of Cytochrome c During Translocation Through a Nanopore Constriction

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 c (cyt c) through ultrathin silicon nitride (SiNx) 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 [~]30-100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5 nm 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 new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability. Significance StatementCan localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field-driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.

biophysics↗

bioRxiv2019

MrDNA: A multi-resolution model for predicting the structure and dynamics of nanoscale DNA objects

Although the field of structural DNA nanotechnology has been advancing with an astonishing pace, de novo design of complex 3D nanostructures 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 minutes or less, can produce an atomistic-resolution structure of an arbitrary DNA nanostructure with accuracy on par with that of a cryo-electron microscopy (cryo-EM) reconstruction. We demonstrate fidelity of our mrdna framework through direct comparison of the simulation results with the results of 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 a self-assembly principle other than origami, 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.

synthetic biology↗

bioRxiv2017

Outperforming nature: synthetic enzyme built from DNA flips lipids of biological membranes at record rates

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 membranes 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 single-molecule experiments we find the lipid transport rate catalyzed by the DNA nanostructure to exceed 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.

biophysics↗