Local PAINT superresolution microscopy for multiplexed RNA detection
Aim of this project is the improvement of imaging speed and suppression of background in PAINT super-resolution microscopy applied to DNA:RNA:protein targets. The DC will develop local PAINT (L-PAINT) with amino acids probe design that exhibits two binding sites to the target structure. The stronger binding site upconcentrates the probe at the target structure while the weaker binding site equipped with a fluorescent dye is continuously localised while it samples the binding sites in the local environment. The doctoral candidate will study the “phasespace” for optimal L-PAINT probe design using DNA origami model structures with defined patterns of binding sites and a landscape of interactions strengths. What should be the interactions strengths of strong and weak binding site? What is the optimal length of the linker? How does the binding kinetics scale with distance to the strong binding site and how does it depend on the mechanical properties of the linker (single-stranded vs double-stranded or “nunchucks” design with alternating single- and double-stranded regions). The doctoral candidate will then use L-PAINT for cell receptor imaging and for single-molecule imaging of aptamers. Most importantly, a new approach for multiplexed detection of RNA and RNA:DNA nanostructures will be developed in cooperation with the Keyser lab.
Principal Investigator: Prof. Philip Tinnefeld
Center for NanoScience and Faculty of Chemistry & Pharmacy, LMU Munich
Doctoral Candidate:
Decoding and Engineering Immunological Synapses: Super-Resolution Mapping and Patterned Therapeutics
This project focuses on two aspects related to the molecular architecture and dynamics of immunological synapses (IS). First, we will use state-of-the-art super-resolution fluorescence microscopy, including DNA-PAINT, to unravel the molecular architecture of immunological synapses (IS) at an unprecedented level of detail. The project will establish advanced labeling toolkits, incorporating nanobody- and single-chain variable fragment (scFv)-based probes, for precise targeting of proteins involved in IS formation, including T cell receptors (TCR), major histocompatibility complexes (MHC), co-stimulatory molecules (CD28, CD80), and inhibitory checkpoint regulators (PD-1, PD-L1). These probes will enable multiplexed imaging of molecular assemblies within the IS, elucidating interactions and nanoscale spatial arrangements critical to immune response modulation. Next, we will use DNA origami to arrange therapeutic agents such as monoclonal antibodies, T cell bispecifics (TCBs), immune checkpoint modulators, and engineered chimeric antigen receptors (CARs) to influence the spatial organization and functional dynamics of key IS components. It will be a key question how the distribution of the imaged surface proteins correlates with the influence of the arrangements of the therapeutic agents on top of the DNA origami. The approach aims at understanding the mechanistic impact of “patterned therapeutic” agents on IS organization, focusing on how changes in protein clustering, interaction affinities, and molecular dynamics influence immune cell activation or suppression. These insights will guide the rational design of next-generation immunotherapeutics with optimized efficacy and reduced off-target effects. Ultimately, this knowledge will enhance Roche’s capacity to model and design immune-modulatory drugs across oncology, autoimmunity, and inflammatory diseases.
Principal Investigator: Prof. Ralf Jungmann
Max Planck Institute of Biochemistry and LMU Munich, Germany
Doctoral Candidate:
Computational de novo protein design of specific NA-binders
Aim of this project is the development of a computational pipeline to design biohybrid materials consisting of protein binders for predefined sequences in nucleic acids (NA), enabling the development of tools for cargo encapsulation and delivery as well as diagnostic tests including viral infections. Previously we have developed a pipeline for target peptide sensing by adjusting the binding pockets of natural membrane receptor systems comprised of proteins whose dimerization and signalling relies on extracellular peptide binding. The doctoral candidate will combine state-of-the-art physics- and artificial intelligence-based prediction and design methods to develop an analogous pipeline for specific targeting of DNA and RNA secondary structure motifs, using as inspiration natural NA-binding proteins. Through rounds of design-build-test-learn, the doctoral candidate will establish a library of building blocks for NA binders in complex environments.
Principal Investigator: Prof. Alena Khmelinskaia
Group of Protein Design and Self Assembly, LMU Munich, Germany
Doctoral Candidate:
DNA and RNA strand displacement in complex environments
The aim of this project is to design and operate DNA- and RNA-based strand displacement circuits with predictable kinetics in complex environments. These environments involve competing molecular interactions, crowding effects, and the presence of nucleic acid-binding proteins. Achieving precise control over such reactions will enable the use of such circuits for biosensing in analyte samples, synthetic cell models, and within living cells. We will design a range of nucleic acid circuits that respond to biologically relevant inputs such as RNA, proteins, small biomolecules, or combinations thereof. These circuits will span from simple sensors to logic functions and multilayered networks. Potential application scenarios include:
– complex analyte mixtures for point-of-care biosensing,
– implementation of logic or analog circuits as controllers in synthetic cells, and
– in vivo operation targeting endogenous RNA or proteins.
Each of these environments presents distinct challenges in terms of molecular concentrations, competing interactions, crowding, and degradation. To tackle these challenges, strategies such as molecular amplification (feedback circuits), protection against degradation through secondary structures, protein binding, and spatial control via compartmentalization or co-localization will be pursued.
Principal Investigator: Prof. Friedrich Simmel
TUM School of Natural Sciences, Department of Bioscience, TU Munich, Germany
Doctoral Candidate:
Probing silicification as a means to achieve impermeable or selectively permeable DNA origami
The aim of this project is to explore new methods to overcome the limitations of DNA origami and biohybrid systems due to their vulnerability in complex environments. The project will use the method of silicification of DNA origami nanostructures, recently demonstrated by the Heuer-Jungemann lab. Silicified structures will be modified with different proteins for biosensing applications. How silicification can reduce the inherent porosity of DNA origami structures and prevent the leaking of cargo from a DNA origami cage will be reported. Differently-sized enzymes placed inside the silicified DNA origami cage, as well as DNA strand displacement reactions inside the cage will be studied to determine leakage rates via fluorescence. Additionally, strand displacement reactions in these confined environments will be studied. By carefully designing sections of ssDNA within the DNA origami structure, controllable pores in the silicified DNA origami will be created, which can react to sense external input such as hybridization or protein binding. The size of these pores and the ability to translocate molecules through them will be investigated by different means, including the influx or efflux of enzyme-specific substrates, fluorescently-labelled DNA strands, intra-cage strand displacement reactions, DNA PAINT and will, in collaboration, be supplemented by predictive modelling.
Principal Investigator: Prof. Amelie Heuer-Jungemann
Faculty of Chemistry and Chemical Biology, TU Dortmund, Germany
Doctoral Candidate:
Nanopore screening of nucleic acid:protein hybrids for antibacterial functions
Aim of this project is to set up a screening process for the selection of aptamers that can act as antimicrobials. The successful candidate will collaborate with the teams’ specialists in nanopores sensing (Keyser) and in the antimicrobial activity of DNA nanostructures (Mela). The candidate will test the affinity of both RNA and DNA aptamer binding to pharmacologically relevant target proteins (Mela) in a single nanopore experiment. The role holder will focus on selection of nucleic acid structures that can form non-covalent hybrids with proteins that are crucial for bacterial function. The relative ease in varying the aptamer sequences enabled by the design of both RNA and DNA hybrid nanostructures for nanopore sensing will rapidly yield quantitative data. The binding studies will guide a deeper understanding of the design rules for selective aptamer binding. The project will provide data for the collaborators in the BioHYBRITE project who will input and suggest possible improvements of the aptamer designs and analysis of the nanopore translocation data. In the second part of the project, the selected aptamers will be chemically modified to enable covalent binding to improve the dissociation constant compared to conventional antibody designs. The project will concentrate on antibacterial aptamers (Mela) and include the characterization of off-target effects and toxicity of the designed RNA and DNA aptamers for human cells.
Principal Investigator: Prof. Ulrich Keyser
Cavendish Laboratory, University of Cambridge, UK
Principal Investigator: Dr. Ioanna Mela
Department of Pharmacology, University of Cambridge, UK
Doctoral Candidate:
RNA-producing antimicrobial synthetic cells made from nucleic acid:enzyme hybrids
Aim of this project is to design and construct synthetic cells that can conditionally produce, store and release antimicrobial RNA constructs for future applications in the targeted treatment of infections. The synthetic cells will consist of Giant Unilamellar lipid Vesicles that can enzymatically produce multivalent, branched RNA nanostructures via in-vitro transcription, as already demonstrated by the UCAM team. The DC will design new branched nanostructures comprising one or more bacteria-targeting DNA or RNA aptamers and linking to moieties with antimicrobial properties, e.g. siRNA, antibiotics or photosensitisers. The antimicrobial moieties will either be synthesised within the synthetic cells or pre-encapsulated. For initial experiments, the synthesis and release of the antimicrobial constructs will be externally induced using light or temperature.
Principal Investigator: Lorenzo di Michele
Department of Chemical Engineering and Nanotechnology, University of Cambridge, UK
Doctoral Candidate:
Designing programmable biohybrid molecular machines
This project aims to investigate how DNA nanostructures with novel functional geometries can replicate key biochemical and mechanical features that support adaptability in living systems. Building on recent advances in DNA origami and computational design softwares, the project will explore novel nanoscale architectures capable of controlled shape transformation and mechanical actuation. The goal is to build robot-like DNA assemblies with emphasis on shape morphing and mechanically responsive behavior. These robot-like DNA nanostructures will be designed to geometrically detect perturbations by incorporating, within the molecular system, proteins, or RNA to propose platforms to probe mechanobiological processes with molecular precision. In collaboration with the Ouldridge group, the project will combine oxDNA simulations for structural reshaping via strand-displacement reactions to facilitate the programming of dynamic structural features within 3D DNA origami. Structural and dynamic properties of the constructs will be characterized using cryo-electron microscopy and fluorescence-based approaches, including single-molecule FRET providing real-time insights into conformational changes.
Principal Investigator: Dr. Emmanuel Margeat
Centre of Structural Biology, CNRS, University Montpellier, France
Principal Investigator: Dr. Gaëtan Bellot
Centre of Structural Biology, CNRS, University Montpellier, France
Doctoral Candidate:
Mechanics of DNA–RNA Hybrids and Their Interaction with Proteins
This project explores the mechanical and structural behavior of DNA–RNA hybrids and long noncoding RNAs (lncRNAs) through multidisciplinary biophysical approaches. We will follow three lines of research. First, we will focus on the mechanics of DNA-RNA hybrids using magnetic and optical tweezers to perform force-extension measurement on single-stranded RNA (RNA) and ssRNA complexed with complementary ssRNA or ssDNA molecules. Atomic force microscopy (AFM) will also be used to analyze hybrid structures and dynamics both in air and liquid environments. Second, we will investigate two lncRNAs—NIHCOLE and CONCR—involved in DNA repair and chromatid cohesion, respectively. Their structures and protein interactions (with Ku70/Ku80 and DDX11) will be studied using nanopore techniques and the OT-Curtains single-molecule assay, allowing real-time observation of RNA–protein binding and structural changes. The third research line will employ super-resolution microscopy to visualize the spatial organization of these lncRNAs. By labeling specific RNA regions or immobilizing them on DNA origami scaffolds, this research line aims to correlate RNA folding, Mg²⁺-induced tertiary structures, and protein interactions, combining advanced microscopy and molecular manipulation methods.
Principal Investigator: Prof. Fernando Moreno-Herrero
National Center of Biotechnology (CNB-CSIC), Department of Macromolecular Structure, Madrid, Spain
Doctoral Candidate:
DNA-protein hybrids for tracking and sensing biochemical cues
During this PhD project the doctoral candidate will use DNA-protein conjugates to build DNA-based structures and assemblies that can have different applications in sensing and drug-delivery. In one example, enzyme-conjugated DNA-based assemblies will be designed to self-propel in fluid in the presence of specific chemical cues. The DNA-enzyme hybrid nanostructures will be also decorated with sensing elements (i.e. recognition elements such as antigens, aptamers, etc) so that they will be able to sense their environment during their propulsion movement and provide a measurable optical signal (fluorescence). During the project the doctoral candidate will investigate different geometries for the nanostructures ranging from simple tubular structures to more complex 3D shapes. The possibility to use different enzymes and to control in a programmable way the surface enzyme density will also be studied as a way to optimise propulsion. The use of strand displacement reactions to control the movement of the structure on the fly will also be studied.
Principal Investigator: Prof. Francesco Ricci
Laboratory of Biosensors and Nanomachines, University of Rome Tor Vergata, Italy
Doctoral Candidate:
Intelligent design of artificial nucleic acid sequences (aptamers) for sensing
Aim of this project is optimising, validating and exploiting hybrid biosystems based on aptamers as biosensors. This will be based on the application of machine learning and molecular dynamics simulations for the design and analysis of aptamers (Stuber et al. ACS Nano 2023, Douaki et al. Chem. Comm. 2023, Douaki et al. Biosensors 2022). The PhD candidate will mainly work on the design and simulation of the nucleic acid sequences, considering applications towards highly selective and sensitive sensing. The results of the design will be then synthesised and integrated in hybrid systems where a solid- state device will be modified with the aptamers. Moreover, the model to be developed within this project will be used by the PhD student as a powerful tool to investigate the interaction between artificial nucleic acid sequences and DNA:RNA origami and hybrid biosystems comprising proteins of different size. The optimised aptamers developed by the PhD student will be also used in collaboration with other partners in the demonstration of the integration of optimised aptamers in silicified DNA origami and for the highly specific reaction in biohybrid cargo that can be modified with the aptamers.
Principal Investigator: Prof. Denis Garoli
Department of Science and Engineering, University of Modena and Reggio Emilia, Italy
Doctoral Candidate:
Predictive modelling of translocation properties of nucleic acids and protein assemblies
This project will develop and apply advanced theoretical and computational models for two purposes. First, it will characterize how the structure of nucleic acids, proteins, and their biohybrid assemblies affects their motion through nanopores. The results will then be used to design molecules with desired pore translocation properties. The theoretical/computational project will involve close secondments with experimental and theoretical partner groups of the BioHybrite network, through which the doctoral researcher will iteratively refine models by comparing them with experiments and, finally, formulate predictions.
Principal Investigator: Prof. Cristian Micheletti
Statistical and Biological Physics, International School for Advanced Studies SISSA Trieste, Italy
Doctoral Candidate:
DNA origami-directed liposome biohybrid assemblies
This project aims at training a doctoral candidate in DNA:RNA origami and liposome technologies, and apply them in programmable hybrid liposome assemblies / vesicular architectures that can be further adjusted for biomedical and bioengineering applications, including biocatalytic control, disease-specific drug delivery, or tissue-specific gene transfer. The expected results include the formation of DNA origami-modulated liposomal building blocks and the channelling of cargo materials between liposomal containers or between liposomes and cells using molecular gating systems.
Principal Investigator: Assoc. Prof. Veikko Linko
Institute of Technology, University of Tartu, Estonia
Doctoral Candidate:
Predictive modelling for nucleic acid strand displacement in hybrid settings
This project will develop predictive models of nucleic acid strand displacement rates to allow the rational design of complex networks of ever-increasing functionality. State of the art models mainly focus on highly-idealised systems that involve only carefully designed DNA strands at low concentrations in well-mixed solutions. In this project, the doctoral candidate will study strand displacement technology in more complex, hybrid environments to demonstrate predictive models that can account for these more complex settings. Using molecular-level simulations, basic theory and experimental data, the student will develop predictive models of strand displacement reactions in non-ideal settings, in collaboration with the other doctoral candidates within BIOHYBRITE. These areas include: structured environments, such as the synthetic cells; environments with competing interaction partners; and systems with hybrid RNA-DNA duplexes.
Principal Investigator: Prof. Thomas Ouldridge
Department of Bioengineering, Imperial College London, UK
Doctoral Candidate:
Functional proteins combined with DNA-Origami assisted fluorescence enhancement for next generation sequencing
The aim of this project is to develop DNA-origami–assisted fluorescence enhancement, combining the high signal amplification enabled by plasmonic DNA-origami nanoantennas with the functional capabilities of proteins and enzymes. Previous studies have optimized antenna geometry, dye selection, and achievable fluorescence enhancement factors. This project addresses the key challenge of precisely positioning functional proteins within the fluorescence hotspot while maintaining their full biological activity. Successfully doing so will unlock a broad range of applications. For example, localizing DNA polymerases at the hotspot could enable next-generation DNA sequencing, offering substantially higher throughput at significantly lower cost compared with current technologies. Moreover, incorporating DNA translocases within or near the hotspot can create synthetic nanopore systems, enabling ultra-sensitive sequencing of DNA – and potentially proteins – with unprecedented resolution and speed.
Principal Investigator: Prof. Philip Tinnefeld
Center for NanoScience and Faculty of Chemistry & Pharmacy, LMU Munich
Doctoral Candidate: