Dr. Barbara Saccà
University of Duisburg-Essen
- A switchable DNA origami/plasmonic hybrid device with a precisely tuneable DNA-free interparticle gap
Erkelenz, M. and Kosinski, R. and Giesler, H. and Sritharan, O. and Jose, J. and Saccà, B. and Schlücker, S.
Chemical Communications 58 (2022)
view abstract 10.1039/d2cc05324a
- Design, Mechanical Properties, and Dynamics of Synthetic DNA Filaments
Stenke, L.J. and Saccà, B.
Bioconjugate Chemistry (2022)Over the past 40 years, structural and dynamic DNA nanotechnologies have undoubtedly demonstrated to be effective means for organizing matter at the nanoscale and reconfiguring equilibrium structures, in a predictable fashion and with an accuracy of a few nanometers. Recently, novel concepts and methodologies have been developed to integrate nonequilibrium dynamics into DNA nanostructures, opening the way to the construction of synthetic materials that can adapt to environmental changes and thus acquire new properties. In this Review, we summarize the strategies currently applied for the construction of synthetic DNA filaments and conclude by reporting some recent and most relevant examples of DNA filaments that can emulate typical structural and dynamic features of the cytoskeleton, such as compartmentalization in cell-like vesicles, support for active transport of cargos, sustained or transient growth, and responsiveness to external stimuli. ©
view abstract 10.1021/acs.bioconjchem.2c00312
- Growth Rate and Thermal Properties of DNA Origami Filaments
Stenke, L.J. and Saccà, B.
Nano Letters (2022)Synthetic DNA filaments exploit the programmability of the individual units and their predictable self-association to mimic the structural and dynamic features of natural protein filaments. Among them, DNA origami filamentous structures are of particular interest, due to the versatility of morphologies, mechanical properties, and functionalities attainable. We here explore the thermodynamic and kinetic properties of linear structures grown from a ditopic DNA origami unit, i.e., a monomer with two distinct interfaces, and employ either base-hybridization or base-stacking interactions to trigger the dimerization and polymerization process. By observing the temporal evolution of the system toward equilibrium, we reveal kinetic aspects of filament growth that cannot be easily captured by postassembly studies. Our work thus provides insights into the thermodynamics and kinetics of hierarchical DNA origami assembly and shows how it can be mastered by the anisotropy of the building unit and its self-association mode. © 2022 The Authors. Published by American Chemical Society.
view abstract 10.1021/acs.nanolett.2c02255
- Self-Assembled Artificial DNA Nanocompartments and Their Bioapplications
Huang, J. and Gambietz, S. and Saccà, B.
Small (2022)Compartmentalization is the strategy evolved by nature to control reactions in space and time. The ability to emulate this strategy through synthetic compartmentalization systems has rapidly evolved in the past years, accompanied by an increasing understanding of the effects of spatial confinement on the thermodynamic and kinetic properties of the guest molecules. DNA nanotechnology has played a pivotal role in this scientific endeavor and is still one of the most promising approaches for the construction of nanocompartments with programmable structural features and nanometer-scaled addressability. In this review, the design approaches, bioapplications, and theoretical frameworks of self-assembled DNA nanocompartments are surveyed. From DNA polyhedral cages to virus-like capsules, the construction principles of such intriguing architectures are illustrated. Various applications of DNA nanocompartments, including their use for programmable enzyme scaffolding, single-molecule studies, biosensing, and as artificial nanofactories, ending with an ample description of DNA nanocages for biomedical purposes, are then reported. Finally, the theoretical hypotheses that make DNA nanocompartments, and nanosystems in general, a topic of great interest in modern science, are described and the progresses that have been done until now in the comprehension of the peculiar phenomena that occur within nanosized environments are summarized. © 2022 The Authors. Small published by Wiley-VCH GmbH.
view abstract 10.1002/smll.202202253
- The role of DNA nanostructures in the catalytic properties of an allosterically regulated protease
Kosinski, R. and Perez, J.M. and Schöneweiß, E.-C. and Ruiz-Blanco, Y.B. and Ponzo, I. and Bravo-Rodriguez, K. and Erkelenz, M. and Schlücker, S. and Uhlenbrock, G. and Sanchez-Garcia, E. and Saccà, B.
Science Advances 8 (2022)DNA-scaffolded enzymes typically show altered kinetic properties; however, the mechanism behind this phenomenon is still poorly understood. We address this question using thrombin, a model of allosterically regulated serine proteases, encaged into DNA origami cavities with distinct structural and electrostatic features. We compare the hydrolysis of substrates that differ only in their net charge due to a terminal residue far from the cleavage site and presumably involved in the allosteric activation of thrombin. Our data show that the reaction rate is affected by DNA/substrate electrostatic interactions, proportionally to the degree of DNA/enzyme tethering. For substrates of opposite net charge, this leads to an inversion of the catalytic response of the DNA-scaffolded thrombin when compared to its freely diffusing counterpart. Hence, by altering the electrostatic environment nearby the encaged enzyme, DNA nanostructures interfere with charge-dependent mechanisms of enzyme-substrate recognition and may offer an alternative tool to regulate allosteric processes through spatial confinement. © 2022 The Authors.
view abstract 10.1126/sciadv.abk0425
- DNA origami
Dey, S. and Fan, C. and Gothelf, K.V. and Li, J. and Lin, C. and Liu, L. and Liu, N. and Nijenhuis, M.A.D. and Saccà, B. and Simmel, F.C. and Yan, H. and Zhan, P.
Nature Reviews Methods Primers 1 (2021)Biological materials are self-assembled with near-atomic precision in living cells, whereas synthetic 3D structures generally lack such precision and controllability. Recently, DNA nanotechnology, especially DNA origami technology, has been useful in the bottom-up fabrication of well-defined nanostructures ranging from tens of nanometres to sub-micrometres. In this Primer, we summarize the methodologies of DNA origami technology, including origami design, synthesis, functionalization and characterization. We highlight applications of origami structures in nanofabrication, nanophotonics and nanoelectronics, catalysis, computation, molecular machines, bioimaging, drug delivery and biophysics. We identify challenges for the field, including size limits, stability issues and the scale of production, and discuss their possible solutions. We further provide an outlook on next-generation DNA origami techniques that will allow in vivo synthesis and multiscale manufacturing. © 2021, Springer Nature Limited.
view abstract 10.1038/s43586-020-00009-8
- DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity
Ochmann, S.E. and Joshi, H. and Büber, E. and Franquelim, H.G. and Stegemann, P. and Saccà, B. and Keyser, U.F. and Aksimentiev, A. and Tinnefeld, P.
Nano Letters 21 (2021)Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches, including optical voltage-sensing dyes and genetically encoded voltage indicators, have evolved. Here, we present a DNA nanotechnology-based system and demonstrated its functionality on liposomes. Using DNA origami, we incorporated and optimized different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we used a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA to connect the nanostructure and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit was read out by fluorescence resonance energy transfer (FRET) changes of single sensors attached to liposomes. A FRET change of ∼5% for Δψ = 100 mV was observed. The working mechanism of the sensor was rationalized by molecular dynamics simulations. Our approach holds potential for an application as nongenetically encoded membrane sensors. © 2021 American Chemical Society.
view abstract 10.1021/acs.nanolett.1c02584
- Pumilio2 promotes growth of mature neurons
Schieweck, R. and Schöneweiss, E.-C. and Harner, M. and Rieger, D. and Illig, C. and Saccà, B. and Popper, B. and Kiebler, M.A.
International Journal of Molecular Sciences 22 (2021)RNA-binding proteins (RBPs) are essential regulators controlling both the cellular transcriptome and translatome. These processes enable cellular plasticity, an important prerequisite for growth. Cellular growth is a complex, tightly controlled process. Using cancer cells as model, we looked for RBPs displaying strong expression in published transcriptome datasets. Interestingly, we found the Pumilio (Pum) protein family to be highly expressed in all these cells. Moreover, we observed that Pum2 is regulated by basic fibroblast growth factor (bFGF). bFGF selectively enhances protein levels of Pum2 and the eukaryotic initiation factor 4E (eIF4E). Exploiting atomic force microscopy and in vitro pulldown assays, we show that Pum2 selects for eIF4E mRNA binding. Loss of Pum2 reduces eIF4E translation. Accordingly, depletion of Pum2 led to decreased soma size and dendritic branching of mature neurons, which was accompanied by a reduction in essential growth factors. In conclusion, we identify Pum2 as an important growth factor for mature neurons. Consequently, it is tempting to speculate that Pum2 may promote cancer growth. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
view abstract 10.3390/ijms22168998
- Site-specific facet protection of gold nanoparticles inside a 3D DNA origami box: a tool for molecular plasmonics
Erkelenz, M. and Kosinski, R. and Sritharan, O. and Giesler, H. and Saccà, B. and Schlücker, S.
Chemical Communications 57 (2021)Bare gold nanocubes and nanospheres with different sizes are incorporated into a rationally designed 3D DNA origami box. The encaged particles expose a gold surface accessible for subsequent site-specific functionalization, for example, for applications in molecular plasmonics such as SERS or SEF. © The Royal Society of Chemistry 2021.
view abstract 10.1039/d0cc07712g
- Insights into the Structure and Energy of DNA Nanoassemblies
Jaekel, A. and Lill, P. and Whitelam, S. and Saccà, B.
Molecules (Basel, Switzerland) 25 (2020)Since the pioneering work of Ned Seeman in the early 1980s, the use of the DNA molecule as a construction material experienced a rapid growth and led to the establishment of a new field of science, nowadays called structural DNA nanotechnology. Here, the self-recognition properties of DNA are employed to build micrometer-large molecular objects with nanometer-sized features, thus bridging the nano- to the microscopic world in a programmable fashion. Distinct design strategies and experimental procedures have been developed over the years, enabling the realization of extremely sophisticated structures with a level of control that approaches that of natural macromolecular assemblies. Nevertheless, our understanding of the building process, i.e., what defines the route that goes from the initial mixture of DNA strands to the final intertwined superstructure, is, in some cases, still limited. In this review, we describe the main structural and energetic features of DNA nanoconstructs, from the simple Holliday junction to more complicated DNA architectures, and present the theoretical frameworks that have been formulated until now to explain their self-assembly. Deeper insights into the underlying principles of DNA self-assembly may certainly help us to overcome current experimental challenges and foster the development of original strategies inspired to dissipative and evolutive assembly processes occurring in nature.
view abstract 10.3390/molecules25235466
- The primordial life of DNA dynamic networks
Winat, L.J. and Saccà, B.
Nature Catalysis 3 (2020)
view abstract 10.1038/s41929-020-00536-3
- Manipulating enzymes properties with DNA nanostructures
Jaekel, A. and Stegemann, P. and Saccà, B.
Molecules 24 (2019)Nucleic acids and proteins are two major classes of biopolymers in living systems. Whereas nucleic acids are characterized by robust molecular recognition properties, essential for the reliable storage and transmission of the genetic information, the variability of structures displayed by proteins and their adaptability to the environment make them ideal functional materials. One of the major goals of DNA nanotechnology—and indeed its initial motivation—is to bridge these two worlds in a rational fashion. Combining the predictable base-pairing rule of DNA with chemical conjugation strategies and modern protein engineering methods has enabled the realization of complex DNA-protein architectures with programmable structural features and intriguing functionalities. In this review, we will focus on a special class of biohybrid structures, characterized by one or many enzyme molecules linked to a DNA scaffold with nanometer-scale precision. After an initial survey of the most important methods for coupling DNA oligomers to proteins, we will report the strategies adopted until now for organizing these conjugates in a predictable spatial arrangement. The major focus of this review will be on the consequences of such manipulations on the binding and kinetic properties of single enzymes and enzyme complexes: an interesting aspect of artificial DNA-enzyme hybrids, often reported in the literature, however, not yet entirely understood and whose full comprehension may open the way to new opportunities in protein science. © 2019 by the authors.
view abstract 10.3390/molecules24203694
- Sites of high local frustration in DNA origami
Kosinski, R. and Mukhortava, A. and Pfeifer, W. and Candelli, A. and Rauch, P. and Saccà, B.
Nature Communications 10 (2019)The self-assembly of a DNA origami structure, although mostly feasible, represents indeed a rather complex folding problem. Entropy-driven folding and nucleation seeds formation may provide possible solutions; however, until now, a unified view of the energetic factors in play is missing. Here, by analyzing the self-assembly of origami domains with identical structure but different nucleobase composition, in function of variable design and experimental parameters, we identify the role played by sequence-dependent forces at the edges of the structure, where topological constraint is higher. Our data show that the degree of mechanical stress experienced by these regions during initial folding reshapes the energy landscape profile, defining the ratio between two possible global conformations. We thus propose a dynamic model of DNA origami assembly that relies on the capability of the system to escape high structural frustration at nucleation sites, eventually resulting in the emergence of a more favorable but previously hidden state. © 2019, The Author(s).
view abstract 10.1038/s41467-019-09002-6
- Hierarchical Assembly of DNA Filaments with Designer Elastic Properties
Pfeifer, W. and Lill, P. and Gatsogiannis, C. and Saccà, B.
ACS Nano 12 (2018)The elastic features of protein filaments are encoded in their component units and in the way they are connected, thus defining a biunivocal relationship between the monomer and the result of its self-assembly. Using DNA origami approaches, we constructed a reconfigurable module, composed of two quasi-independent domains and four possible interfaces, capable of facial and lateral growing through specific recognition patterns. Whereas the flexibility of the intra-domains region can be regulated by switchable DNA motifs, the inter-domain interfaces feature mutually and self-complementary shapes, whose pairwise association leads to filaments of programmable periodicity and variable persistence length. Thus, we show here that the assembly pathway leading to oligomeric chains can be finely tuned and fully controlled, enabling the emulation of protein-like filaments using a single construction principle. Our approach results in artificial materials with a large variety of ultrastructures and bending strengths comparable, or even superior, to their natural counterparts. © 2017 American Chemical Society.
view abstract 10.1021/acsnano.7b06012
- Synthetic DNA filaments: From design to applications
Pfeifer, W. and Saccà, B.
Biological Chemistry 399 (2018)Natural filaments, such as microtubules and actin filaments, are fundamental components of the cell. Despite their relatively simple linear structure, filaments play a number of crucial roles in living organisms, from scaffolding to cellular adhesion and motility. The mechanical properties of natural filaments mostly rely on the structural features of the component units and on the way they are connected together, thus providing an ideal molecular model for emulation purposes. In this review, we describe the progresses done in this field using DNA for the rational design of synthetic filamentous-like materials with tailored structural and physical characteristics. We firstly survey the strategies that have been adopted until now for the construction of individual DNA building components and their programmable self-assembly into linear oligomeric structures. We then describe the theoretical models of polymer elasticity applied to calculate the bending strength of DNA filaments, expressed in terms of persistence length. Finally, we report some of the most exciting examples of truly biomimetic DNA filaments, which are capable of mimicking not only the sophisticated structural features of their natural counterparts but also their responsiveness to external stimuli, thus resulting in active motion and growing networks between distant loci. © 2018 Walter de Gruyter GmbH, Berlin/Boston.
view abstract 10.1515/hsz-2018-0110
- Three-Dimensional DNA Origami as Programmable Anchoring Points for Bioreceptors in Fiber Optic Surface Plasmon Resonance Biosensing
Daems, D. and Pfeifer, W. and Rutten, I. and Saccà, B. and Spasic, D. and Lammertyn, J.
ACS Applied Materials and Interfaces 10 (2018)Many challenges in biosensing originate from the fact that the all-important nanoarchitecture of the biosensor surface, including precise density and orientation of bioreceptors, is not entirely comprehended. Here, we introduced a three-dimensional DNA origami as a bioreceptor carrier to functionalize the fiber optic surface plasmon resonance (FO-SPR) sensor with nanoscale precision. Starting from a 24-helix bundle, two distinct DNA origami structures were designed to position thrombin-specific aptamers with different densities and distances (27 and 113 nm) from the FO-SPR surface. The origami-based biosensors not only proved to be capable of reproducible, label-free thrombin detection but revealed also valuable innovative features: (1) a significantly better performance in the absence of backfilling, known as essential in the biosensing field, suggesting improved bioreceptor orientation and accessibility, and (2) a wider linear range compared to previously reported thrombin biosensors. We envisage that our method will be beneficial for both scientists and clinicians looking for new surface (bio)chemistry and improved diagnostics. Copyright © 2018 American Chemical Society.
view abstract 10.1021/acsami.8b04757
- Enzyme-functionalized DNA nanostructures as tools for organizing and controlling enzymatic reactions
Grossi, G. and Jaekel, A. and Andersen, E.S. and Saccà, B.
MRS Bulletin 42 (2017)Enzyme sequestration and compartmentalization are key factors in cell signaling and metabolism, evolved to solve the challenges of slow turnover rates, undesired pathway intermediates, and competing reactions. Inspired by nature, DNA nanoengineers have developed organizational systems to confine enzymes in two- and three-dimensional environments and to actuate them in response to precise external stimuli. DNA-scaffolded enzymes have applications for not only the in vitro reconstitution of proteins, peptides, and other molecular assemblies, but also to enable the generation of advanced functional nanomaterials for the development of, for example, fuel cells, biosensors, and drug delivery systems. Despite several challenges that still remain unsolved, the use of DNA scaffolds to arrange enzymes in space and time will help to realize biochemical nanofactories, where multiple components work together to produce novel and improved functional materials, rivaling the efficiency of biological systems. © Copyright Materials Research Society 2017.
view abstract 10.1557/mrs.2017.269
- Irregular model DNA particles self-assemble into a regular structure
Preisler, Z. and Saccà, B. and Whitelam, S.
Soft Matter 13 (2017)DNA nanoparticles with three-fold coordination have been observed to self-assemble in experiment into a network equivalent to the hexagonal (6.6.6) tiling, and a network equivalent to the 4.8.8 Archimedean tiling. Both networks are built from a single type of vertex. Here we use analytic theory and equilibrium and dynamic simulation to show that a model particle, whose rotational properties lie between those of the vertices of the 6.6.6 and 4.8.8 networks, can self-assemble into a network built from three types of vertex. Important in forming this network is the ability of the particle to rotate when bound, thereby allowing the formation of more than one type of binding motif. The network in question is equivalent to a false tiling, a periodic structure built from irregular polygons, and possesses 40 particles in its unit cell. The emergence of this complex structure, whose symmetry properties are not obviously related to those of its constituent particles, highlights the potential for creating new structures from simple variants of existing nanoparticles. © 2017 The Royal Society of Chemistry.
view abstract 10.1039/c7sm01627a
- Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions
Sprengel, A. and Lill, P. and Stegemann, P. and Bravo-Rodriguez, K. and Schöneweiß, E.-C. and Merdanovic, M. and Gudnason, D. and Aznauryan, M. and Gamrad, L. and Barcikowski, S. and Sanchez-Garcia, E. and Birkedal, V. and Gatsogiannis, C. and Ehrmann, M. and Saccà, B.
Nature Communications 8 (2017)The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging. © 2017 The Author(s).
view abstract 10.1038/ncomms14472
- The collective behavior of spring-like motifs tethered to a DNA origami nanostructure
Schöneweiß, E.-C. and Saccà, B.
Nanoscale 9 (2017)Dynamic DNA nanotechnology relies on the integration of small switchable motifs at suitable positions of DNA nanostructures, thus enabling the manipulation of matter with nanometer spatial accuracy in a trigger-dependent fashion. Typical examples of such motifs are hairpins, whose elongation into duplexes can be used to perform long-range, translational movements. In this work, we used temperature-dependent FRET spectroscopy to determine the thermal stabilities of distinct sets of hairpins integrated into the central seam of a DNA origami structure. We then developed a hybrid spring model to describe the energy landscape of the tethered hairpins, combining the thermodynamic nearest-neighbor energy of duplex DNA with the entropic free energy of single-stranded DNA estimated using a worm-like chain approximation. We show that the organized scaffolding of multiple hairpins enhances the thermal stability of the device and that the coordinated action of the tethered motors can be used to mechanically unfold a G-quadruplex motif bound to the inner cavity of the origami structure, thus surpassing the operational capabilities of freely diffusing motors. Finally, we increased the complexity of device functionality through the insertion of two sets of parallel hairpins, resulting in four distinct states and in the reversible localization of desired molecules within the reconfigurable regions of the origami architecture. © The Royal Society of Chemistry.
view abstract 10.1039/c6nr08314e
- Characterizing the Effect of Multivalent Conjugates Composed of Aβ-Specific Ligands and Metal Nanoparticles on Neurotoxic Fibrillar Aggregation
Streich, C. and Akkari, L. and Decker, C. and Bormann, J. and Rehbock, C. and Müller-Schiffmann, A. and Niemeyer, F.C. and Nagel-Steger, L. and Willbold, D. and Saccà, B. and Korth, C. and Schrader, T. and Barcikowski, S.
ACS Nano 10 (2016)Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody-antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aβ-specific small molecules to inhibit Aβ peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aβ are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for β-sheet content, and inhibition of cellular Aβ excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer's disease. © 2016 American Chemical Society.
view abstract 10.1021/acsnano.6b02627
- From Nano to Macro through Hierarchical Self-Assembly: The DNA Paradigm
Pfeifer, W. and Saccà, B.
ChemBioChem (2016)From atoms to molecules and bio-macromolecules, from organelles to cells, tissues, to the whole living system, nature shows us that the formation of complex systems with emergent properties originates from the hierarchical self-assembly of single components in guided bottom-up processes. By using DNA as a fundamental building block with well-known self-recognition properties, scientists have developed design rules and physical-chemical approaches for the fully programmable construction of highly organized structures with nanosized features. This review highlights the basic principles of hierarchical self-assembly in terms of type and number of distinguishable components and their interaction energies. Such general concepts are then applied to DNA-based systems. After a brief overview of the strategies used until now for the construction of individual DNA units, such as DNA tile motifs and origami structures, their self-association into assemblies of higher order is discussed. Particular emphasis is given to the forces involved in the self-assembly process, understanding and rational combination of which might help to coordinate the single elements of hierarchical structures both in space and time, thus advancing our efforts towards the creation of devices that mimic the complexity and functionality of natural systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract 10.1002/cbic.201600034
- Determinants of amyloid fibril degradation by the PDZ protease HTRA1
Poepsel, S. and Sprengel, A. and Saccà, B. and Kaschani, F. and Kaiser, M. and Gatsogiannis, C. and Raunser, S. and Clausen, T. and Ehrmann, M.
Nature Chemical Biology 11 (2015)Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved serine protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed. © 2015 Nature America, Inc. All rights reserved.
view abstract 10.1038/nchembio.1931
- Reversible reconfiguration of DNA origami nanochambers monitored by single-molecule FRET
Saccà, B. and Ishitsuka, Y. and Meyer, R. and Sprengel, A. and Schöneweiß, E.-C. and Nienhaus, G.U. and Niemeyer, C.M.
Angewandte Chemie - International Edition 54 (2015)Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance-dependent energy transfer between two preloaded fluorophores. Interestingly, single-molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine-tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/anie.201408941
- Site-Directed, On-Surface Assembly of DNA Nanostructures
Meyer, R. and Saccà, B. and Niemeyer, C.M.
Angewandte Chemie - International Edition 54 (2015)Two-dimensional DNA lattices have been assembled from DNA double-crossover (DX) motifs on DNA-encoded surfaces in a site-specific manner. The lattices contained two types of single-stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface-bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA-tethered probe molecules on the opposite side of the lattices exposed to the solution. Site-specific lattice assembly and attachment of fluorophore-labeled oligonucleotides and DNA-protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/anie.201505553
- A facile method for preparation of tailored scaffolds for DNA-origami
Erkelenz, M. and Bauer, D.M. and Meyer, R. and Gatsogiannis, C. and Raunser, S. and Saccà, B. and Niemeyer, C.M.
Small 10 (2014)A convenient PCR cloning strategy allows one to prepare hundreds of picomoles of circular single-stranded DNA molecules, which are suitable as scaffolds for the assembly of DNA origami structures. The method is based on a combination of site-directed mutagenesis and site- and ligation-independent cloning protocols, with simultaneous insertion of a nicking endonuclease restriction site on a double-stranded plasmid of desired length and sequence. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/smll.201300701
- Nucleic acids nanotechnology
Methods 67 (2014)
view abstract 10.1016/j.ymeth.2014.04.018
- De novo design of nucleic acid structures
Saccà, B. and Sprengel, A. and Feldkamp, U.
De novo Molecular Design (2013)The molecular recognition properties of nucleic acids are at the basis of the storage and transmission of the genetic information of every living system. This fundamental biological property has been advantageously used by scientists to construct two- and three-dimensional materials made entirely of DNA or RNA, with nanoscaled features and impressive geometrical sophistication. In this chapter, we describe the design principles of the most important strategies currently used in structural DNA nanotechnology and illustrate how DNA objects can be realized through modern software tools. Although not treated here in detail, de novo design of nucleic acids extends also to DNA systems with programmed dynamic behavior and functional RNA nanoarchitectures. Clearly, the constant evolution of novel, and sometimes unexpectedly successful, design concepts shows us how limited is still our understanding of DNA and RNA self-assembly and how bright is the future of nucleic acids nanotechnology. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA. All rights reserved.
view abstract 10.1002/9783527677016.ch20
- DNA origami: The art of folding DNA
Saccà, B. and Niemeyer, C.M.
Angewandte Chemie - International Edition 51 (2012)The advent of DNA origami technology greatly simplified the design and construction of nanometer-sized DNA objects. The self-assembly of a DNA-origami structure is a straightforward process in which a long single-stranded scaffold (often from the phage M13mp18) is folded into basically any desired shape with the help of a multitude of short helper strands. This approach enables the ready generation of objects with an addressable surface area of a few thousand nm 2 and with a single "pixel" resolution of about 6 nm. The process is rapid, puts low demands on experimental conditions, and delivers target products in high yields. These features make DNA origami the method of choice in structural DNA nanotechnology when two- and three-dimensional objects are desired. This Minireview summarizes recent advances in the design of DNA origami nanostructures, which open the door to numerous exciting applications. Know when to fold ′em: As in the ancient art of paper folding, where a single sheet of paper is modeled into beautiful shapes, DNA origami technology allows nanoscale-addressable objects to be created from one single strand of DNA (see picture). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/anie.201105846
- Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates
Tennstaedt, A. and Pöpsel, S. and Truebestein, L. and Hauske, P. and Brockmann, A. and Schmidt, N. and Irle, I. and Saccà, B. and Niemeyer, C.M. and Brandt, R. and Ksiezak-Reding, H. and Tirniceriu, A.L. and Egensperger, R. and Baldi, A. and Dehmelt, L. and Kaiser, M. and Huber, R. and Clausen, T. and Ehrmanna, M.
Journal of Biological Chemistry 287 (2012)Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
view abstract 10.1074/jbc.M111.316232
- Nanolattices of switchable DNA-based motors
Saccà, B. and Siebers, B. and Meyer, R. and Bayer, M. and Niemeyer, C.M.
Small 8 (2012)Miniaturization is an important aspect of device fabrication. Despite the advancements of modern top-down approaches, scaling-down to the sub-nanometer size is still a challenge. As an alternative, bottom-up approaches, such as the use of DNA as an engineering material, are therefore emerging, allowing control of matter at the single-molecule level. A DNA-based self-assembly method for the construction of switchable DNA devices is descrbied here based on G-quadruplex moieties, which are patterned on quasi-planar DNA arrays with nanoscale precision. The reversible switching of the devices is triggered by addition of DNA sequences ('fuels') and translated into linear extension/contractile movements. The conformational change of the devices was visualized by atomic force microscopy and FRET spectroscopy. Steady state fluorescence spectroscopy indicated that scaffolding of the G4 motors to either individual tiles or extended superlattices had no significant impact on the switching and optical performance of the system. However, time-resolved spectroscopy revealed that ordering in the microstructural environment enhances the fraction of molecules subject to FRET. Altogether, our study confirms that DNA superstructures are well-suited scaffolds for accommodation of mechanically switchable units and thus opens the door to the development of more sophisticated nanomechanical devices. Ordered planar nanoarrays bearing DNA-responsive devices are produced by DNA self-assembly procedures. The nanomotors display extension and contraction movements in response to addition of DNA fuels with efficient cycling operation even after repetitive cycles. This DNA-based assembly of nanomechanical units offers full control over the spatial arrangement of each single molecule and opens the door to the development of more sophisticated nanomechanical devices. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/smll.201200703
- Covalent tethering of protruding arms for addressable DNA nanostructures
Saccà, B. and Niemeyer, C.M.
Small 7 (2011)Functionalization of self-assembled DNA nanostructures is of fundamental importance for the realization of their application in nanotechnology and biosensing. Approaches reported so far suffer from lack of general applicability and usually require careful system design to avoid poor yields in the assembly of target structures. A novel approach well suited for fabrication of addressable DNA superstructures is reported here to generate DNA tile motifs. The method is based on the covalent linkage of a single-stranded protruding arm (covPA) to one of the oligomers forming the tile. Subsequent to assembly of tile motifs and superlattices, the covPA can be addressed by hybridization with complementary oligonucleotides or DNA-protein conjugates. The covPA can be located at arbitrary positions in a given tile motif without changing the general design and without compromising the structural integrity of the tile. The covPA strategy can also be readily extended to different PA sequences and multiple covPA arms can be linked to a tile. Superlattices obtained by self-assembly of covPA tiles reveal partial folding into double layers which possess an intrinsic order at the ultrastructural level. This phenomenon is likely associated with the increased flexibility of the covPA and might open up novel ways for DNA-based functionalization of solid surfaces and other applications of structural DNA nanotechnology. A novel class of DNA tile motifs bears a covalently linked, single-stranded protruding arm. The increased flexibility of this arm enables efficient modification of tiles and superlattices with complementary DNA-linked objects and induces formation of partially folded bilayers which posses an intrinsic order at the ultrastructural level. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/smll.201101010
- Functionalization of DNA nanostructures with proteins
Saccà, B. and Niemeyer, C.M.
Chemical Society Reviews 40 (2011)Proteins possess intrinsic functionalities, which have been optimized in billions of years of natural evolution. The conjugation of proteins with artificial nucleic acids allows one to further functionalize proteins with a synthetically accessible, physicochemically robust tag, which is addressable in a highly specific manner by Watson-Crick hybridization. The resulting DNA-protein conjugates can be advantageously used in a variety of applications, ranging from biomedical diagnostics to DNA-based nanofabrication. This critical review provides an overview on chemical approaches to the synthesis of DNA-protein conjugates and their applications in biomolecular nanosciences (96 references). © The Royal Society of Chemistry 2011.
view abstract 10.1039/c1cs15212b
- Orthogonal protein decoration of DNA origami
Saccà, B. and Meyer, R. and Erkelenz, M. and Kiko, K. and Arndt, A. and Schroeder, H. and Rabe, K.S. and Niemeyer, C.M.
Angewandte Chemie - International Edition 49 (2010)If the face fits: Self-labeling fusion proteins have been used for the site-specific decoration of DNA origami. This method even allows individual faces of the quasi-two-dimensional plane of the nanostructure to be specifically decorated (see picture), thereby enabling directional immobilization and thus control over the accessibility of distinct proteins presented on the structure. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/anie.201005931
atomic force microscopy