Biology subjects

Dietz, H.

Publications and source records attributed to Dietz, H..

10 recordsLinked to original sources

bioRxiv2024

Alphafold2 refinement improves designability of large de novo proteins

Recent advances in computational protein design have enabled the creation of novel proteins for a variety of purposes. The capability for producing custom-shape high-quality backbones for very large proteins would further expand the scope of protein design. To this end, here we introduce the AlphaFold2 (AF2) cycler (af2cycler) design pipeline. AF2cycler is used to refine draft protein backbone geometries of arbitrary shape created by the Chroma diffusion model or other methods, using a combination of AF2 and ProteinMPNN to achieve high designability with minimal deviations from the initial target structure. In silico testing on multiple protein designs (100-1000 amino acids) demonstrated improvements in structural integrity and designability after af2cycling confirmed by enhanced ESMFold repredictions. Experimental wet lab validation through the design of a variety of test proteins with distinct shapes, each comprising 1000 amino acids, showed structural agreement between in silico predictions and transmission electron microscopy (TEM) imaging, establishing the af2cyclers efficacy in translating designs into real-world results. Af2cycler provides a convenient and reliable protein design workflow, particularly for large proteins, with potential for expanding to applications in areas such as design of higher-order protein complexes and multi-state backbone optimization.

bioinformatics↗

bioRxiv2024

Programmable DNA shell scaffolds for directional membrane budding

In the pursuit of replicating biological processes at the nanoscale, controlling cellular membrane dynamics has emerged as a key area of interest. Here, we report a system mimicking virus assembly to control directional membrane budding. We employ three-dimensional DNA origami techniques to construct cholesterol-modified triangles that self-assemble into polyhedral shells on lipid vesicles, resulting in gradual curvature induction, bud formation and spontaneous neck scission. Strategic positioning of cholesterols on the triangle surface provides control over the directionality of bud growth and yields daughter vesicles with DNA endo- or exoskeletons reminiscent of clathrin-coated vesicles. This process occurs with rapid kinetics and across various lipid compositions. When combined into a two-step process, nested bivesicular objects with DNA shells encapsulated between lipid vesicles could be produced. Our work replicates key aspects of natural endocytic and exocytic pathways, opening new avenues for exploring membrane mechanics and applications in targeted drug delivery and synthetic biology.

biophysics↗

bioRxiv2023

Hepatitis B virus neutralization with DNA origami nanoshells

We demonstrate the use of DNA origami to create virus-trapping nanoshells that efficiently neutralize hepatitis B virus (HBV) in cell culture. By modifying the shells with a synthetic monoclonal antibody that binds to the HBV envelope, the effective neutralization potency per antibody is increased by approximately 100 times compared to using free antibodies. The improvements in neutralizing the virus are attributed to two factors: first, the shells act as a physical barrier that block the virus from interacting with host cells; second, the multivalent binding of the antibodies inside the shells lead to stronger attachment to the trapped virus, a phenomenon known as avidity. Pre-incubation of shells with HBV and simultaneous addition of both components separately to cells lead to comparable levels of neutralization, indicating rapid trapping of the virions by the shells. Our study highlights the potential of the DNA shell system to rationally create novel antivirals using components that, when used individually, show little to no antiviral effectiveness.

biophysics↗

bioRxiv2023

Single-cell precision nanotechnology in vivo

Targeting nanoparticle therapeutics with cellular accuracy in whole organisms could open breakthrough opportunities in precision medicine. However, evaluating and fine-tuning the biodistribution of such systems in the whole organism at the cellular level remains a major obstacle. Here, we constructed targetable DNA origami, and analyzed biodistribution in transparent mice, in addition to studying tolerability, clearance kinetics, and immune response parameters. Untargeted DNA origami primarily accumulated in the spleen and the liver, while an immune cell-targeting variant successfully attached to immune cells throughout the body. A cancer cell-targeting mimetic co-localized on solid-tumor metastasis in the liver and the lung. These findings indicate that DNA origami can be directed in vivo, providing an important proof-of-concept and highlights the potential of high-resolution tissue-clearing imaging technologies in their development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/550304v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@ce216borg.highwire.dtl.DTLVardef@f40bb4org.highwire.dtl.DTLVardef@2c6a66org.highwire.dtl.DTLVardef@1fd7fde_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThis study demonstrates the potential of DNA origami-based drug delivery systems as versatile tool for for targeted delivery, which could be used to treat a range of diseases with applications. C_LIO_LIThe immune compatibility, half-life, targeting efficiency, and the biodistribution evaluation of DNA origami indicate its potential for systemic drug delivery. C_LIO_LIOur approach enables the assessment of biodistribution of nanoparticles in the intact body with a sensitivity to the single-cell level, highlighting the high-resolution tissue clearing technoloies in revealing DNA origamis feasibility for drug targeting. C_LI

bioengineering↗

bioRxiv2023

Efficient and scalable de novo protein design using a relaxed sequence space

Deep learning techniques are being used to design new proteins by creating target backbone geometries and finding sequences that can fold into those shapes. While methods like ProteinMPNN provide an efficient algorithm for generating sequences for a given protein backbone, there is still room for improving the scope and computational efficiency of backbone generation. Here, we report a backbone hallucination protocol that uses a relaxed sequence representation. Our method enables protein backbone generation using a gradient descent driven hallucination approach and offers orders-of-magnitude efficiency enhancements over previous hallucination approaches. We designed and experimentally produced over 50 proteins, most of which expressed well in E. Coli, were soluble and adopted the desired oligomeric state along with the correct composition of secondary structure as measured by CD. Exemplarily, we determined 3D electron density maps using single-particle cryo EM analysis for three single-chain de-novo proteins comprising 600 AA which closely matched with the designed shape. These have no structural analogues in the protein data bank (PDB), representing potentially novel folds or arrangement of domains. Our approach broadens the scope of de novo protein design and contributes to accessibility to a wider community.

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

A synthetic tubular molecular transport system

We report the bottom-up construction of a macromolecular transport system in which molecular pistons diffusively move through micrometer-long, hollow filaments. The pistons can cover micrometer distances in fractions of seconds. We built the system using multi-layer DNA origami and analyzed the structures of the components using transmission electron microscopy. We studied the motion of the pistons along the tubes using single-molecule fluorescence microscopy and performed Langevin simulations to reveal details of the free energy surface that directs the motions of the pistons. The tubular transport system achieves diffusivities and displacement ranges known so far only from natural molecular motors and realizes mobility improvements over five orders of magnitude compared to previous artificial random walker designs. Electric fields can also be employed to actively pull the pistons along the filaments, thereby realizing a nanoscale electric rail system. Our system presents a platform for artificial motors that move autonomously driven by chemical fuels and for performing nanotribology studies, and it could form a basis for future molecular transportation networks.

biophysics↗

bioRxiv2021

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations

Many strategies have been pursued to trap and monitor single proteins over time in order to detect the molecular mechanisms of these essential nanomachines. Single protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection based on ion currents. Here we present the Nanopore Electro-Osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with sub-millisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtraps ability to sensitively distinguish proteins based on size and shape, and discriminate nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

biophysics↗

bioRxiv2021

Reconstitution of ultrawide DNA origami pores in liposomes for transmembrane transport of macromolecules

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed new methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective as dextran molecules with a diameter up to 22 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modelling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, synthetic biology, to drug delivery.

biophysics↗

bioRxiv2019

Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

Multi-colored gene reporters such as fluorescent proteins are indispensable for biomedical research, but equivalent tools for electron microscopy (EM), a gold standard for deciphering mechanistic details of cellular processes1,2 and uncovering the network architecture of cell-circuits3,4, are still sparse and not easily multiplexable. Semi-genetic EM reporters are based on the precipitation of exogenous chemicals5-9 which may limit spatial precision and tissue penetration and can affect ultrastructure due to fixation and permeabilization. The latter technical constraints also affect EM immunolabeling techniques10-13 which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core14-16, but its small size complicates differentiation of individual ferritin particles from cellular structures. To enable multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM), we here introduce the encapsulin system of Quasibacillus thermotolerans (Qt) as a fully genetic iron-biomineralizing nanocompartment. We reveal by cryo-electron reconstructions that the Qt monomers (QtEnc) self-assemble to nanospheres with T=4 icosahedral symmetry and an ~44 nm diameter harboring two putative pore regions at the fivefold and threefold axes. We furthermore show that the native cargo (QtlMEF) auto-targets to the inner surface of QtEnc and exhibits ferroxidase activity leading to efficient iron sequestration inside mammalian cells. We then demonstrate that QtEnc can be robustly differentiated from the non-intermixing encapsulin of Myxococcus xanthus17 (Mx, ~32 nm) via a deep-learning model, thus enabling automated multiplexed EM gene reporter imaging in mammalian cells.

synthetic biology↗