Biology subjects

Bertosin, E.

Publications and source records attributed to Bertosin, E..

5 recordsLinked to original sources

bioRxiv2026

A modular DNA toolkit for single-molecule biophysics

Long, customizable DNA molecules are an essential tool for in vitro single-molecule biophysics. Most studies still rely on the {lambda}-phage genome, which has a fixed sequence, a non-uniform GC distribution, and limited options for modification. To overcome these limitations, we developed a modular DNA (ModDNA) toolkit that integrates in silico sequence design with cloning, to create long (up to 100 kb) DNA molecules with entirely user-defined sequences. We used a computational workflow with curated databases to design a motif-depleted sequence backbone, into which any desired sequence can be inserted at any position. Golden Gate assembly then builds these sequences into constructs, which we extend iteratively in defined increments up to 100 kb. We show precise placement of local features, e.g. a fluorophore, a bound protein, and a DNA-origami structure, and illustrate the construct's utility in single-molecule visualization assays, including DNA loop extrusion by human condensin I. ModDNA offers a scalable, sequence-programmable alternative to {lambda}-DNA and other restrictive substrates. More broadly, the motif-aware feature-free design connects single-molecule biophysics to emerging efforts in machine-guided regulatory-sequence design and synthetic-genome engineering, where controlled programmable sequence context is increasingly essential.

biophysics↗

bioRxiv2026

Dilatable DNA origami nanopores as nuclear pore mimics

The nuclear pore complex (NPC) is a massive protein system that controls all nucleocytoplasmic transport via a dynamic network of intrinsically disordered proteins called FG-Nups. We developed an NPC-mimicking DNA origami nanostructure that self-assembles into a tetrameric, octagonal nanopore with an inner diameter that, through addition of DNA oligonucleotides, can be tuned in a user-defined fashion from 57 to 66 nm - mimicking the contracted and dilated states of NPCs. We demonstrated that these origami nanopores can dilate within minutes and recontract at slower timescales. Both contracted and dilated pores can be inserted into lipid bilayers, where they maintain their conformation and allow transmembrane transport of fluorescent proteins. With outer diameters up to 87 nm, these are, to our knowledge, the largest DNA origami nanopores inserted into lipid membranes. Functionalization of the pores with the FG-Nup Nsp1 reduces non-specific cargo diffusion across the lipid membrane while facilitating translocation of the transport receptor Kap95, demonstrating transport selectivity much like biological NPCs. The work establishes a size-tunable platform to dissect nuclear transport mechanisms, with potential applications for engineering programmable artificial channels.

biophysics↗

bioRxiv2024

Compliant DNA Origami Nanoactuators as Size-Selective Nanopores

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, we harness the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between three stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behaviour with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug-delivery, biomolecule sorting and sensing, as well as bottom-up synthetic biology.

biophysics↗

bioRxiv2022

Orientation-locked DNA origami for stable trapping of small proteins in the NEOtrap

Nanopores are versatile single-molecule sensors that offer a simple label-free readout with great sensitivity. We recently introduced the Nanopore Electro-Osmotic trap (NEOtrap) which can trap and sense single unmodified proteins for long times. The trapping is achieved by the electro-osmotic flow (EOF) generated from a DNA-origami sphere docked onto the pore, but thermal fluctuations of the origami limited the trapping of small proteins. Here, we use site-specific cholesterol functionalization of the origami sphere to firmly link it to the lipid-coated nanopore. We can lock the origami in either a vertical or horizontal orientation which strongly modulates the EOF. The optimized EOF greatly enhances the trapping capacity, yielding reduced noise, reduced measurement heterogeneity, an increased capture rate, and 100-fold extended observation times. We demonstrate the trapping of a variety of single proteins, including small ones down to a molecular mass of 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.

biophysics↗

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↗