Biology subjects

Simmel, F. C.

Publications and source records attributed to Simmel, F. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Experimental umbrella sampling of a rotary nanomachine

The free-energy landscape of a molecular system, which describes its free energy as a function of a few collective coordinates, governs its dynamics and function, but is challenging to determine experimentally. In molecular simulations, enhanced-sampling methods such as umbrella sampling use bias potentials to access regions of this landscape that are rarely visited by equilibrium fluctuations. Here, we transfer this concept to a single-molecule experiment: we combine electric-field biasing, in situ calibration of the bias potentials, and statistical reweighting to reconstruct the angular free-energy landscapes of rotary DNA nanomachines. The method resolves barriers exceeding 10 kBT and detects changes in local landscape shape caused by small modifications of the stator design. A systematic analysis of bias calibration, sampling, and finite-exposure averaging establishes the conditions required for accurate reconstruction. Experimental umbrella sampling thus relates nanoscale structure to the underlying free-energy landscape and supports the physics-informed design of artificial molecular machinery.

biophysics↗

bioRxiv2026

DNA Origami Nanomechanical Amplifiers for Resolving Single-Molecule Binding Events

Mechanical amplification of minute length changes enables precise measurements across many orders of magnitude, from macroscopic metrology to optical instrumentation. Extending this principle to molecular systems could provide a route to monitoring nanoscale structural changes without relying on analyte labeling or fluorescence-based distance measurements. Here we present a DNA origami nanomechanical amplifier that converts subnanometre-scale molecular conformational changes into amplified mechanical displacements that can be tracked in real time at the single-molecule level. The platform resolves geometric changes associated with DNA hybridization, secondary-structure formation, DNA strand-exchange dynamics, and ligand-induced aptamer folding, enabling quantitative analysis of molecular kinetics and direct observation of transient intermediates and heterogeneous conformational ensembles. By translating molecular recognition events into mechanically amplified signals, our approach establishes a general framework for monitoring binding-coupled conformational dynamics and extends the scope of single-molecule measurements beyond conventional optical readouts.

biophysics↗

bioRxiv2026

Magnetic DNA Origami Nanorotors

Self-assembled DNA nanostructures show great promise as functional devices, highly configurable materials, and in nanorobotics. Magnetic control can provide a powerful actuation mechanism in a broad range of contexts, since it affords a high-level of external control, it is biocompatible, and orthogonal to chemical or electrical stimuli. Here we demonstrate magnetic molecular nanoactuators by leveraging the unique site-specificity of DNA origami to assemble highly anisotropic magnetic nanocubes on high-aspect ratio DNA origami bundles. We traced and controlled 100s of our DNA origami nanorotors at the single-rotor level and demonstrated their programmable magnetic clamping and controlled rotation under uniform and rotating magnetic fields. By varying the population and inter-particle spacing of the nanocubes, magnetic torque values in the order of 10-100 pN nm are achieved at field strengths < 10 mT. Monte Carlo simulations reveal that assembly of nanocubes on DNA origami rotors leads to collective magnetic properties, with numerically estimated torque values in good agreement with the experiments. Our magnetic nanorotors offer a foundation for biocompatible nanorobotics, as well as high-throughput magnetic force and torque tweezers.

biophysics↗

bioRxiv2025

Vesicle-templated self-assembly of freestanding multi-μm DNA shells

In the quest to create increasingly complex synthetic cell-mimicking systems, a wide range of DNA nanostructures have been developed to coat, permeabilize, sculpt, or otherwise functionalize lipid vesicles. In a complementary strategy, DNA architectures have been used as scaffolds to direct the growth of lipid membrane vesicles. Here we introduce a simple and broadly applicable method to realize freestanding, membrane-mimicking DNA shells: DNA shells are first assembled on the outer surface of giant unilamellar vesicles and then liberated by surfactant-mediated liposome removal. The resulting structures faithfully retain the geometry of their membrane template. We demonstrate the approach with two distinct classes of DNA tectons: a complex barrel-shaped DNA origami with programmable inter-subunit interactions, and a simple nanostar-inspired motif composed of only eleven oligonucleotides. The site-specific addressability of the former enable the rational design of binding interfaces, as demonstrated by controlled multilayer formation. The success of both strategies underscores the generality of our approach and the feasibility of creating shell-like compartments from different DNA architectures. This method enables the construction of tunable, DNA-only containers spanning the size range of eukaryotic cells, offering a fundamentally new type of compartmentalization for bottom-up synthetic biology.

synthetic biology↗

bioRxiv2025

A Nanoscale Jitterbug Transformer from DNA

Many viruses have intricate polyhedral shells capable of symmetric transformations in response to external stimuli to initiate payload release. Such deployable auxetic nanostructures are not available in the synthetic realm. Here we present a nanoscale Jitterbug transformer using DNA origami that can reconfigure its structure upon chemical and optical signals while maintaining a Poissons ratio of -1. By leveraging molecular dynamics simulations, we design the Jitterbug DNA to form a compact octahedron by storing elastic energy and spontaneously transition into an expanded cuboctahedron by releasing it. DNA transformers are explored like viruses that can create nanopores on lipid membranes and regulate payload release into vesicles. This work integrates programmable DNA self-assembly with free-energy-guided mechanical design, providing a pathway toward adaptive nanomaterials with potential in synthetic organelles and stimuli-responsive nanodevices.

biophysics↗

bioRxiv2025

Operating CRISPR/Cas12a in a complex nucleic acid sequence background

Since their discovery, CRISPR-Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a - a Class 2, Type V effector - and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR-Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the guide RNA seed region significantly affect kinetic inhibition by background polynucleotides. Guide RNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.

biophysics↗

bioRxiv2025

Genetic Manipulation of Mammalian Cells in Microphysiological Hydrogels

Engineering functional 3D tissue constructs is essential for developing advanced organ-like systems, with applications ranging from fundamental biological research to drug testing. The generation of complex multicellular structures requires the integration of external geometric and mechanical cues with the ability to activate genetic programs that regulate and stimulate cellular self-organization. Here, we demonstrate that gelatin methacryloyl (GelMA) hydrogels serve as effective matrices for 3D cell culture, supporting both in situ genetic manipulation and cell growth. HEK293T cells embedded in GelMA remained viable and proliferated over 16 days, forming clusters within the matrix. We achieved efficient gene delivery in the 3D hydrogel environment using both plasmid DNA and mRNA as gene vectors. Furthermore, we applied in situ prime editing to induce permanent genetic modifications in embedded cells. To achieve spatially confined gene expression, we introduced gel-embedded channels that allowed localized stimulation via doxycycline perfusion through a Tet-On system. Our findings establish GelMA hydrogel matrices as a versatile platform for generating spheroidal cell cultures while enabling precise genetic control and spatially resolved cellular manipulation through diffusible cues.

bioengineering↗

bioRxiv2025

Digital Phage Biology in Droplets

Since their discovery, bacteriophages - viruses that infect bacteria - have been an invaluable source of insight for molecular biology and have provided a plethora of molecular components for biotechnology. Driven by the imminent global antibiotic crisis, interest in using bacteriophages as antimicrobial agents has strongly increased in the past few years. While standard quantification of phage counts and infectivity by double-layer plaque assays (DLA) have provided foundational insights, they are limited by their inability to monitor infection dynamics over time and the inflexibility in experimental setups. Here, we introduce a novel high-throughput method using droplet microfluidics to quantify individual phage infection events, enhancing the understanding of phage-host dynamics at the single-event level. By co-encapsulating individual phages and bacteria in microfluidic droplets, we can control key experimental parameters such as exposure time and the ratio of phages to bacteria. Our system allows for precise quantification of lysis events and the direct observation of lysis kinetics unaffected by the continuous release of progeny phages that is typical in bulk cultures. Moreover, our method is potentially applicable to any phage-host pair. Our findings suggest that droplet microfluidics can provide a more accurate and dynamic understanding of phage biology. This approach could be particularly valuable in the development of phage-based antimicrobial strategies in the face of rising antibiotic resistance.

biophysics↗

bioRxiv2024

Flow-Induced Vascular Remodeling On-Chip: Implications for Anti-VEGF Therapy

Impaired vascular remodeling is linked to tumor progression, impacting the efficacy of anti-vascular therapies. However, the interplay between blood flow and vascular endothelial growth factor (VEGF) in driving vascular remodeling remains unclear. We optimized a human vasculature-on-chip system for long-term perfusion to quantify vascular remodeling under physiologically low and tumor-mimicking high VEGF conditions. Live imaging shows that at low VEGF levels, flow-conditioned vascular networks remodeled like animal models, en-hancing flow velocities. Conversely, static networks lacking flow exhibited continuous growth, loss of network hierarchy, and decreased flow velocities. At tumor-mimicking high VEGF levels, flow-conditioned vessels showed aberrant overgrowth, which was blocked by the anti-VEGF tumor drug bevacizumab, restoring flow-induced remodeling. Without flow, however, high VEGF slowed vessel growth compared to low VEGF, while anti-VEGF treatment restored continuous growth. These findings provide valuable insights into optimizing anti-angiogenic therapies by exploiting VEGF modulation and flow conditions for vascular remodeling and improved tumor treatment.

biophysics↗

bioRxiv2024

Realizing Mechanical Frustration at the Nanoscale Using DNA Origami

Structural designs inspired by physical and biological systems have been previously utilized to develop advanced mechanical metamaterials. These are based on the clever geometric arrangement of their building blocks, resulting in enhanced mechanical properties such as shape morphing and auxetic behavior. Until now, the benefits from such designs have yet to be leveraged at the nanoscale. Here, we use the DNA origami method to realize a nanoscale metastructure exhibiting mechanical frustration, the mechanical counterpart of the well-known phenomenon of magnetic frustration. We show that this DNA metastructure can be precisely controlled to adopt either frustrated or non-frustrated mechanical states, each characterized by a distinct free energy profile. Switching among the states is achieved by engineering reconfigurable struts into the structure. Actuation of the struts causes a global deformation of the metastructures. In the non-frustrated state, strain can be distributed homogeneously throughout the structure, while in the frustrated state, strain is concentrated at a specific location. Molecular dynamics simulations reconcile the contrasting behaviors of the two modes and provide detailed insights into the mechanics. Our work demonstrates how combining programmable DNA self-assembly with mechanical design principles can overcome engineering limitations encountered at the macroscale, enabling the development of dynamic, deformable nanostructures with tunable responses. These may lay the foundation for mechanical energy storage elements, nanomechanical computation, and allosteric mechanisms in DNA-based nanomachinery.

biophysics↗

bioRxiv2024

Self-assembled cell-scale containers made from DNA origami membranes

DNA origami provides a methodology for the sequence-programmable generation of precisely defined molecular nanostructures with sizes of order 100 nm. A new frontier for the field is the generation of superstructures made from DNA origami subunits, which requires other self-assembly strategies than those used for DNA origami itself. Challenges faced by current approaches include the increasing complexity, cost and development time for the structures and off-target assembly. Here, we demonstrate how radially symmetric origami subunits that are inspired by the structure and interactions of lipids organize into giant DNA origami monolayer membranes that can be readily programmed to form vesicles or hollow tubes with diameters ranging from 100 nm to over 1 {micro}m. DNA origami membranes are an unprecedented approach for compartmentalization that opens up new possibilities for bottom-up biology and cell-scale soft robotics.

synthetic biology↗

bioRxiv2024

From Brownian to deterministic motor movement in a DNA-based molecular rotor

Molecular devices that have an anisotropic, periodic potential landscape can be operated as Brownian motors. When the potential landscape is cyclically switched with a chemical reaction or an external force, such devices can harness random Brownian fluctuations to generate directed motion. Recently, directed Brownian motor-like rotatory movement was demonstrated with an electrically switched DNA origami rotor with designed, ratchet-like obstacles. Here, we demonstrate that also the intrinsic anisotropy of DNA origami rotors that originally were not designed as Brownian motor devices is sufficient to result in motor movement. We show that for low amplitudes of an external switching field such devices operate as Brownian motors, while at higher amplitudes the movement is better described by the deterministic motion of an overdamped electrical motor. We characterize the amplitude and frequency dependence of the movements in both regimes, showing that after an initial steep rise the angular speed peaks and drops for excessive driving amplitudes and frequencies. The characteristics of the rotor movement are well described by a simple stochastic model of the system.

biophysics↗

bioRxiv2024

Single-Molecule Force Spectroscopy of Toehold-Mediated Strand Displacement

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and is the foundation for a wide range of DNA or RNA-based reaction circuits. Previous studies have typically relied on bulk fluorescence measurements to investigate the kinetics of TMSD, which only provide effective, bulk-averaged reaction rates and do not resolve the process on the level of individual molecules or even base pairs. In this work, we addressed this limitation by exploring the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy (SMFS) with an optical trap supported by state-of-the-art coarse-grained simulations. By probing the ends of the hairpin of a toehold structure, we can trigger and observe TMSD in real time with microsecond and nanometer resolution. Using a microfluidic assay where we expose the hairpin to a solution of trigger strands, we find that under load, TMSD proceeds very rapidly, with single step times of 1 s. Introducing mismatches into the invader sequence allows us to tune stability such that invasion and re-invasion occur in equilibrium, even under load. This allows us to study thousands of invasion/re-invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolating our findings to zero load, we find single step times for DNA invading DNA four times faster than for RNA invading RNA. Moreover, we used force to study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have importance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

biophysics↗

bioRxiv2022

Toehold-Mediated Strand Displacement in Random Sequence Pools

Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity between components can lead to cross-talk causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to one order of magnitude difference in reaction speed. Finally, we compare two established and a novel technique that speed up TMSD reactions in random sequence pools: a threeletter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.

bioengineering↗

bioRxiv2022

Shape primed AC-electrophoretic microrobots

Second-order electrokinetic flow around colloidal particles caused by concentration polarization electro-osmosis (CPEO) can result in a phoretic motion of asymmetric particle dimers in a homogeneous AC electrical field, which we refer to as concentration polarization electro-phoresis (CPEP). To demonstrate this actuation mechanism, we created particle dimers from micron-sized silica spheres with sizes 1.0 {micro}m and 2.1 {micro}m by connecting them with DNA linker molecules. The dimers can be steered along arbitrarily chosen paths within a 2D plane by controlling the orientation of the AC electric field in a fluidic chamber with the joystick of a gamepad. Further utilizing induced dipole-dipole interactions, we demonstrate that particle dimers can be used to controllably pick up monomeric particles and release them at any desired position, and also to assemble several particles into groups. Systematic experiments exploring the dependence of the dimer migration speed on the electric field strength, frequency, and buffer composition align with the theoretical framework of CPEO and provide parameter ranges for the operation of our microrobots.

biophysics↗

bioRxiv2022

Algorithmic design of 3D wireframe RNA polyhedra

We address the problem of de novo design and synthesis of nucleic acid nanostructures, a challenge that has been considered in the area of DNA nanotechnology since the 1980s and more recently in the area of RNA nanotechnology. Towards this goal, we introduce a general algorithmic design process and software pipeline for rendering 3D wireframe polyhedral nanostructures in single-stranded RNA. To initiate the pipeline, the user creates a model of the desired polyhedron using standard 3D graphic design software. As its output, the pipeline produces an RNA nucleotide sequence whose corresponding RNA primary structure can be transcribed from a DNA template and folded in the laboratory. As case examples, we design and characterize experimentally three 3D RNA nanostructures: a tetrahedron, a bipyramid and a prism. The design software is openly available, and also provides an export of the targeted 3D structure into the oxRNA molecular dynamics simulator for easy simulation and visualization.

bioengineering↗

bioRxiv2021

Period Doubling Bifurcations in a Forced Cell-Free Genetic Oscillator

Complex non-linear dynamics such as period doubling and chaos have been previously found in computational models of the oscillatory gene networks of biological circadian clocks, but their experimental study is difficult. Here, we present experimental evidence of period doubling in a forced synthetic genetic oscillator operated in a cell-free gene expression system. To this end, an oscillatory negative feedback gene circuit is established in a microfluidic reactor, which allows continuous operation of the system over extended periods of time. We first thoroughly characterize the unperturbed oscillator and find good agreement with a four-species ODE model of the system. Guided by simulations, microfluidics is then used to periodically perturb the system by modulating the concentration of one of the oscillator components with a given amplitude and frequency. When the ratio of the external zeitgeber period and the intrinisic period is close to 1, we experimentally find period doubling and quadrupling in the oscillator dynamics, whereas for longer zeitgeber periods, we find stable entrainment. Our theoretical model suggests favorable conditions for which the oscillator can be utilized as an externally synchronized clock, but also demonstrates that related systems could, in principle, display chaotic dynamics.

synthetic biology↗

bioRxiv2021

Transcriptional interference in toehold switch-based RNA circuits

Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5 untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible for the ribosome. In the presence of a trigger RNA molecule protein production is turned on. Using antisense RNA against trigger molecules (anti-trigger RNA), gene expression can also be switched off again. We here study the utility and regulatory effect of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and anti-trigger transcription, whereas their cognate toehold switch, regulating expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is strongly influenced by transcriptional interference (TI) effects, leading to more than four-fold differences in expression levels. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. Based on this insight, we selected optimum promoter designs and arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers and two anti-triggers that function as a post-transcriptional RNA regulatory exclusive OR (XOR) gate.

synthetic biology↗