Biology subjects

Scheckenbach, M.

Publications and source records attributed to Scheckenbach, M..

5 recordsLinked to original sources

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↗

bioRxiv2022

Full site-specific addressability in DNA origami-templated silica nanostructures

DNA nanotechnology allows for the fabrication of nano-meter-sized objects with high precision and selective addressability as a result of the programmable hybridization of complementary DNA strands. Such structures can template the formation of other materials, including metals and complex silica nanostructures, where the silica shell simultaneously acts to protect the DNA from external detrimental factors. However, the formation of silica nanostructures with site-specific addressability has thus far not been explored. Here we show that silica nanostructures templated by DNA origami remain addressable for post silicification modification with guest molecules even if the silica shell measures several nm in thickness. We used the conjugation of fluorescently labelled oligonucleotides to different silicified DNA origami structures carrying a complementary ssDNA handle as well as DNA PAINT super-resolution imaging to show that ssDNA handles remain unsilicified and thus ensure retained addressability. We also demonstrate that not only handles, but also ssDNA scaffold segments within a DNA origami nanostructure remain accessible, allowing for the formation of dynamic silica nanostructures. Finally we demonstrate the power of this approach by forming 3D DNA origami crystals from silicified monomers. Our results thus present a fully site-specifically addressable silica nanostructure with complete control over size and shape.

biophysics↗