Biology subjects

Tinnefeld, P.

Publications and source records attributed to Tinnefeld, P..

At least 19 recordsLinked to original sources

bioRxiv2026

A generalizable interface-seeded framework for de novo design of functional oligomers

Protein oligomers are ubiquitous in biological systems and essential for function. However, the de novo design of oligomers that controllably assemble in response to exogenous stimuli remains challenging. Here, we present an AI-based generative approach that leverages an interface-seeded strategy for designing responsive homo-oligomers from isolated interaction modules. Experimentally validated designs are highly accurate and explore new-to-nature topologies. We show that designs effectively respond to their chemical triggers with conditional oligomerization or to phosphorylation-driven conformational changes with reversible oligomerization. We further functionalized our responsive assemblies to build ligand-dependent membrane binding systems and phosphorylation-controlled gene regulatory switches. Our framework enables the generalizable design of responsive protein complexes, opening novel possibilities for the engineering of biosynthetic systems with sophisticated regulatory mechanisms.

biochemistry↗

bioRxiv2026

Resolving Sub-Microsecond Conformational Dynamics of Vertical Nucleic Acids on Graphene

The function of nucleic acids is governed not only by their structure but also by their dynamics. At the molecular scale, transitions between functional structural states are superimposed on rapid thermal fluctuations, resulting in an intricate interplay that is challenging to resolve experimentally, particularly at the single-molecule level. Here, we introduce a novel approach for unraveling sub-microsecond dynamics in oligonucleotides, enabling direct observation of fluctuations in single DNA molecules. By immobilizing nucleic acids vertically on graphene and exploiting distance-dependent graphene energy transfer of fluorescent molecules attached to the DNA, we relate fluctuations in fluorescence intensity to biomolecular dynamics. We show that ionic strength modulates the fluctuations and that structural defects in DNA, such as nucleotide gaps or mismatches, alter the measured dynamics. The experimental findings are complemented by atomistic molecular dynamics simulations and kinetic Monte Carlo simulations, establishing a direct link between theoretical predictions of structure and dynamics and experimentally accessible fluctuation timescales. Overall, our findings advance the understanding of how thermal fluctuations affect oligonucleotides and are modulated by both external and internal stimuli.

biophysics↗

bioRxiv2025

Brownian DNA Computing

Conventional silicon computing is constrained by energy demands, limited parallelism, and poor compatibility with living systems, motivating the exploration of molecular alternatives. Here, we realize Brownian DNA computing, a hitherto theoretical framework, for energy efficient computation in which coupled molecular balances on a DNA origami scaffold form Brownian Logic Elements (BLEs) that harnesses thermal fluctuations for computation. By encoding multiple logic gates into a programmed energy landscape, these BLEs execute all fundamental Boolean logic gates, complex circuits such as half-adders, and multi-input operations, while also enabling non-Boolean logic with multi-valued outputs in a single compact gate. Computation arises through thermally driven exploration of a near-flat configurational energy landscape, without reliance on consumable fuel strands for cascading signal propagation, making the process fast, resettable, and compatible with operation near reversible thermodynamic limits. Transient-input protocols further allow the BLE energy landscape to be controlled quasistatically, providing a route toward reversible Brownian computation. In combination with single-molecule readout, Brownian DNA computing establishes a new foundation for molecular information processing with potential applications in biocomputing, soft robotics, and energy-efficient nanodevices.

biophysics↗

bioRxiv2025

Localisation of Apicomplexa motor Myosin A with axial nanometric precision using graphene energy transfer

Migration in Apicomplexan parasites, a phylum that includes Plasmodium spp. responsible for malaria and the zoonotic Toxoplasma, is explained by the linear migration motor model. This model predicts that Myosin A is confined beneath the plasma membrane and above the inner membrane complex (IMC), a membranous barrier separating the cell membrane and the cytoplasm. Cumulative data, using mutant cell lines and biochemical data support this model. Paradoxically, a proof of the precise localization of Myosin A is still lacking due to limitations in resolving the motor with nanometric precision within the space comprising the membrane and the IMC. Here, we implement graphene energy transfer (GET), a novel axial nanometric ruler with a resolution of [~]1 nm, to determine the relative axial position of Myosin A and IMC1 (a reference protein defining IMC position). Using GET, we estimate the IMC dimensions with precision matching electron microscopy (EM) data and the added advantage of identifying specific bauplan proteins. We complement these measurements with 2D STED microscopy in MIRA confiners, uExMIC, and cryo-immunolabeling. Our data present the first direct localization of Myosin A populations with nanometric resolution in the third dimension, which is compatible with the linear motor model. One sentence summaryUsing the novel GET method, this study presents the first direct localization of Myosin A populations in Toxoplasma gondii with 1-4 nm axial precision, supporting the linear model.

biophysics↗

bioRxiv2025

A silicon rhodamine-fused glibenclamide to label and detect malaria-infected red blood cells

The malaria parasite Plasmodium falciparum affects the lives of millions of people worldwide every year. The detection of replicating parasites within human red blood cells is of paramount importance, requiring appropriate diagnostic tools. Herein, we design and apply a silicon rhodamine-fused glibenclamide (SiR-glib). We first test this far-red fluorescent, fluorogenic and endoplasmic reticulum-targeting sulfonylurea in mammalian cells and pancreatic tissues, before characterizing its labeling performance in red blood cells infected with the asexual developmental stages of Plasmodium falciparum. We further combine SiR-glib with a portable smartphone-based microscope to easily and rapidly identify parasitized red blood cells, providing proof of principle for diagnostic use in rural endemic areas without major healthcare facilities.

biochemistry↗

bioRxiv2025

Bayesian Inference of Binding Kinetics from Fluorescence Time Series

The study of binding kinetics via the analysis of fluorescence time traces is often con-founded by measurement noise and photophysics. Although photoblinking can be mitigated by using labels less likely to photoswitch, photobleaching generally cannot be eliminated. Current methods for measuring binding and unbinding rates are therefore limited by concurrent photobleaching events. Here, we propose a method to infer binding and unbinding rates alongside photobleaching rates using fluorescence intensity traces. Our approach is a two-stage process involving analyzing individual regions of interest (ROIs) with a Hidden Markov Model to infer the fluorescence intensity levels of each trace. We then use the inferred intensity level state trajectory from all ROIs to infer kinetic rates. Our method has several advantages, including the ability to analyze noisy traces, account for the presence of photobleaching events, and provide uncertainties associated with the inferred binding kinetics. We demonstrate the effectiveness and reliability of our method through simulations and data from DNA origami binding experiments.

biochemistry↗

bioRxiv2025

Minimally Invasive DNA-Mediated Photostabilization for Extended Single-Molecule and Super-resolution Imaging

Photobleaching of fluorescence labels poses a major limitation in single-molecule and super-resolution microscopy. Conventional photostabilization methods, such as oxygen removal and addition of high concentrations of photostabilization additives, often require careful fluorophore selection and can disrupt the biological environment. To address these limitations, we developed a modular and minimally invasive photostabilization approach that utilizes DNA-mediated delivery of a photostabilizer directly to the imaging site. Under lower excitation intensities, the DNA-mediated strategy outperformed solution-based approaches, achieving efficient photostabilization at significantly lower additive concentrations. However, at higher excitation intensities, the stability of a single photostabilizer molecule became the limiting factor. To overcome this and reduce the loss of localizations in DNA-PAINT experiments we have also implemented a recovery scheme where the photostabilizer is continuously replenished at the imaging site. We further extended the approach to cell imaging, demonstrating improved localization rate and precision in 3D-DNA PAINT measurements. DNA-mediated photostabilization offers a promising solution for imaging applications where high additive concentrations are prohibited. Its modularity enables adaptation to various imaging schemes and ultimately expands the repertoire of fluorophores suitable for single-molecule and super-resolution imaging.

biophysics↗

bioRxiv2024

Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor

The high functionality of DNA nanostructures makes them a promising tool for biomedical applications, their intrinsic instability under application-relevant conditions, still remains challenging. Protective coating of DNA nanostructures with materials like silica or cationic polymers has evolved as a simple, yet powerful strategy to improve their stability even under extreme conditions. While over time, various materials and protocols have been developed, the characterization and quality assessment of the coating is either time consuming, highly invasive or lacks detailed insights on single nanostructures. Here, we introduce a cyanine dye based molecular sensor designed to non-invasively probe the coating of DNA origami by either a cationic polymer or by silica, in real-time and on a single nanostructure level. The cyanine dye reports changes in its local environment upon coating via increased fluorescence lifetime induced by steric restriction and water exclusion. Exploiting the addressability of DNA origami, the molecular sensor can be placed at selected positions to probe the coating layer with nanometer precision. We demonstrate the reversibility of the sensor and use it to study the stability of the different coatings in degrading conditions. To showcase the potential for correlative studies, we combine the molecular fluorescence lifetime sensor with DNA PAINT super-resolution imaging to investigate coating and structural integrity as well as preserved addressability of DNA nanostructures. The reported sensor presents a valuable tool to probe the coating of DNA nanodevices in complex biochemical environments in real-time and at the single nanosensor level and aids the development of novel stabilization strategies.

biophysics↗

bioRxiv2024

Bringing Attomolar Detection to the Point-of-Care with Nanopatterned DNA Origami Nanoantennas

Creating increasingly sensitive and cost-effective nucleic acid detection methods is critical for enhancing point-of-care (POC) applications. This involves capturing all desired biomarkers in a sample with high specificity and transducing the capture events to a detector. However, the signal from biomarkers present at extremely low amounts often falls below the detection limit of typical fluorescence-based methods, making molecular amplification a necessary step. Here, we present a nucleic acid detection assay of a 151-nucleotide sequence specific to antibiotics-resistant Klebsiella pneumoniae, based on single-molecule fluorescence detection of non-amplified DNA down to the attomolar level, using Trident NanoAntennas with Cleared HOtSpots (NACHOS). Our NACHOS-diagnostics assay leverages a compact microscope with a large field-of-view and cost-efficient components, including microfluidic flow to enhance capturing efficiency. Fluorescence enhancement is provided by DNA origami NanoAntennas, arranged in a dense array using a combination of nanosphere lithography and site-specific DNA origami placement. Our method can detect 200 {+/-} 50 out of 600 molecules in a 100 {micro}L sample volume within an hour. This represents typical number of pathogens in clinical samples commonly detected by Polymerase Chain Reaction but without the need for molecular amplification. We achieve similar sensitivity in untreated human blood plasma, enhancing the practical applicability of the system. Our platform can be adapted to detect shorter nucleic acid fragments that are not compatible with traditional amplification-based technologies. This broadens its potential for diverse diagnostic and healthcare applications, providing a robust and scalable solution for sensitive nucleic acid detection in various clinical settings.

biophysics↗

bioRxiv2024

Spring-loaded DNA origami arrays as energy-supplied hardware for modular nanorobots

DNA origami nanodevices allow us to mimic cellular functions in a rationally controlled manner. They describe machineries which respond to environmental stimuli by conducting different tasks. To date, this mostly is achieved by constructing conformational two-state switches which upon activation by stimuli change their conformation resulting in the performance of a priorly programmed task. Their applicability however is often limited to a single, specific stimuli - output combination due to their intrinsic properties as two-state systems only. This makes expanding them further to include multiple stimuli/ outputs challenging. Here, we address this problem by introducing reconfigurable DNA origami arrays as a coupled network of two-state systems. We use this network to create a universal design strategy in which different operational units can be incorporated into any two-state system of our nanorobot. The resulting nanorobot is capable of receiving different stimuli, computing the response to the received stimuli using multi-level Boolean logic gating and yielding multiple programmed output operations with controlled order, timing and spatial position. We expect that this strategy will be a crucial step towards further developing DNA origami nanorobots for applications in various technological fields.

biophysics↗

bioRxiv2023

Single-Molecule FRET at 10 MHz Count Rates

A bottleneck in many studies utilizing single-molecule Forster Resonance Energy Transfer (smFRET) is the attainable photon count rate as it determines the temporal resolution of the experiment. As many biologically relevant processes occur on timescales that are hardly accessible with currently achievable photon count rates, there has been considerable effort to find strategies to increase the stability and brightness of fluorescent dyes. Here, we use DNA nanoantennas to drastically increase the achievable photon count rates and to observe fast biomolecular dynamics in the small volume between two plasmonic nanoparticles. As a proof of concept, we observe the coupled folding and binding of two intrinsically disordered proteins which form transient encounter complexes with lifetimes on the order of 100 s. To test the limits of our approach, we also investigated the hybridization of a short single-stranded DNA to its complementary counterpart, revealing a transition path time of 17 s at photon count rates of around 10 MHz, which is an order-of-magnitude improvement when compared to the state of the art. Concomitantly, the photostability was increased, enabling many seconds long megahertz fluorescence time traces. Due to the modular nature of the DNA origami method, this platform can be adapted to a broad range of biomolecules, providing a promising approach to study previously unobservable ultrafast biophysical processes.

biophysics↗

bioRxiv2023

Real-Time Structural Biology of DNA and DNA-Protein Complexes on an Optical Microscope

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription, and repair. To better understand these interactions, it is crucial to develop tools to study DNA-protein complexes with high spatial and temporal resolution. Here, we use the vertical orientation that DNA adopts on graphene and investigate its interactions with proteins via energy transfer from a probe dye to graphene, achieving spatial resolution down to the [A]ngstrom scale. We measured the bending angle of DNA induced by adenine tracts, bulges, abasic sites and the binding of Escherichia coli endonuclease IV with unprecedented precision and millisecond time resolution. Additionally, we observed the translocation of the O6-alkylguanine DNA alkyltransferase along double-stranded DNA, reaching single-base pair resolution and detecting an affinity for adenine tracts. Overall, we foresee that this method will become a widespread tool for the dynamical study of nucleic acid and nucleic acid-protein interactions.

biophysics↗

bioRxiv2023

Engineering Modular and Tunable Single Molecule Sensors by Decoupling Sensing from Signal Output

Biosensors play key roles in medical research and diagnostics, but there currently is a lack of sensing platforms that combine easy adaptation to new targets, strategies to tune the response window to relevant analyte concentration ranges and allow for the incorporation of multiple sensing elements to benefit from multivalency. Utilizing a DNA origami nanostructure as a scaffold for arranging the different sensor components, we here propose an approach for the development of modular and tunable single-molecule sensors capable of detecting a variety of biomolecular targets such as nucleic acids, antibodies and restriction enzymes while offering mechanisms to tune the dynamic window, the specificity, and the cooperativity of the sensor.

biophysics↗

bioRxiv2023

DNA Origami Vesicle Sensors with Triggered Cargo Transfer

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Leveraging the programmable nature of DNA origami, we engineered a biosensor harnessing single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a rectangular DNA origami. In a vesicle-free environment, the ssDNA adopts a coiled stance, ensuring high FRET efficiency. However, upon lipid vesicle binding to cholesterol anchors on the DNA origami, the hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, leading to reduced FRET efficiency. The strategic placement of cholesterol anchors further modulates this interaction, affecting the observed FRET populations. Beyond its role as a vesicle sensor, we show targeted cargo transport of the acceptor dye unit to the vesicle. The cargo transport is initiated by vesicle bound DNA and a strand displacement reaction. Our interaction platform opens pathways for innovative interaction such as biosensing and molecular transport with complex biosystems.

bioengineering↗

bioRxiv2023

Engineering an artificial catch bond using mechanical anisotropy

Catch bonds are a rare class of protein-protein interactions where the bond lifetime increases under an external pulling force. Here, we report how modification of anchor geometry generates catch bonding behavior for the mechanostable Dockerin G:Cohesin E (DocG:CohE) adhesion complex found on human gut bacteria. Using AFM single-molecule force spectroscopy in combination with bioorthogonal click chemistry, we mechanically dissociated the complex using five precisely controlled anchor geometries. When tension was applied through residue #13 on CohE and the N-terminus of DocG, the complex behaved as a two-state catch bond, while in all other tested pulling geometries, including the native configuration, it behaved as a slip bond. We used a kinetic Monte Carlo model with experimentally derived parameters to simulate rupture force and lifetime distributions, achieving strong agreement with experiments. Single-molecule FRET measurements further demonstrated that the complex does not exhibit dual binding mode behavior at equilibrium but unbinds along multiple pathways under force. Together, these results show how mechanical anisotropy and anchor point selection can be used to engineer artificial catch bonds.

biophysics↗

bioRxiv2023

Cooperative dynamics of DNA grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami

Magnetic nanoparticles (MNPs) enable unique capabilities for biosensing and actuation via coupling to DNA origami, yet how DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we demonstrate functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry conjugation with tunable grafting density. Several complementary methods show that particle translational and rotational dynamics exhibit a sigmoidal dependence on ssDNA grafting density. At low densities ssDNA strands are coiled and cause small changes to particle dynamics, while at high densities they form polymer brushes that cooperatively change particle dynamics. Intermediate ssDNA densities show the highest magnetic biosensing sensitivity for detection of target nucleic acids. Finally, we demonstrate that MNPs with high grafting densities are required to efficiently couple them to DNA origami. These results together establish ssDNA grafting density as a critical parameter for functionalization of MNPs for use in a broad range of applications.

biochemistry↗

bioRxiv2023

Super-Resolved FRET and Co-Tracking in pMINFLUX

Single-molecule FRET (smFRET) is widely used to investigate dynamic (bio) molecular interactions taking place over distances of up to 10 nm. With the advent of recent super-resolution methods such as MINFLUX, MINSTED or RASTMIN, the spatiotemporal resolution of these techniques advanced towards the smFRET regime. While these methods do not suffer from the spatial restriction of FRET, they only visualize one emitter at a time, thus rendering fast dynamics of interactions out of reach. Here, we describe two approaches to overcome this limitation in pMINFLUX using its intrinsic fluorescence lifetime information. First, we combined pMINFLUX with smFRET. This enabled us to track a FRET donor fluorophore and simultaneously colocalize its FRET acceptor with nanometer precision. To extend co-localized tracking beyond the FRET range, we developed pMINFLUX lifetime multiplexing, a method to simultaneously track two fluorophores with similar spectral properties but distinct fluorescence lifetimes. We demonstrated its application on both static and dynamic DNA origami systems with a precision better than 2 nm. Within the FRET range, pMINFLUX lifetime multiplexing additionally uses a novel combined phasor-microtimegating approach. This paves the way for nanometer precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, closing the resolution gap between smFRET and co-tracking. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/534096v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@11459forg.highwire.dtl.DTLVardef@1f1cee2org.highwire.dtl.DTLVardef@4592e6org.highwire.dtl.DTLVardef@1af3e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

Single-molecule experiments have changed the way we investigate the physical world but data analysis is typically time-consuming and prone to human bias. Here, we present Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software package consisting of an ensemble of deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, in particular from single-molecule Forster Resonance Energy Transfer (FRET) experiments. Deep-LASI automatically sorts single molecule traces, determines FRET correction factors and classifies the state transitions of dynamic traces, all in ~20-100 ms per trajectory. We thoroughly benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.

biophysics↗