Research

Current Directions

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Research overview

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Molecular Caging

We are currently employing DNA origami technology for the construction of artificial DNA-based nanocontainers, which mimic the shape and function of natural compartmentalization systems.
We aim at using such artificial DNA devices for selective loading/release of proteins and envisage potential applications in biosensing as well as cargo transport and delivery.

Hierarchical Self-assembly

The complexity of natural macromolecular assemblies is normally achieved through a hierarchy paradigm, which mostly relies on step-wise interactions between shape-complementary subunits. One of our goals is to understand and reproduce this winning design principle to generate artificial systems with tailored properties.

Structural Reconfiguration

Another important aspect of biological self-assembling systems, to which we are inspired, is the capability to change their shape in response to an external event. Structural reconfiguration allows complex systems to perform different tasks depending on environmental conditions and therefore to switch on/off critical functions. Dynamic behavior is therefore a fundamental aspect of self-assembly. We use switchable DNA motifs and integrate them within static DNA nanostructures to achieve control of matter distribution both in space and time.

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Published Results

Travelling the energy landscape of DNA origami folding

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. 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.

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R. Kosinski, A. Mukhortava, W. Pfeifer, A. Candelli, P. Rauch and B. Saccà. Sites of high local frustration in DNA origami. Nat. Commun. 2019, 10, 1061.

DNA filaments with different ultrastructures and persistence lengths

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. In this work, we show that the assembly pathway leading to oligomeric chains can be finely tuned and fully controlled, enabling to emulate the formation of protein-like filaments using a single construction principle. This leads to artificial materials with a large variety of ultrastructures and bending strengths comparable, or even superior, to their natural counterparts.

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W. Pfeifer, P. Lill, C. Gatsogiannis and B. Saccà. Hierarchical assembly of DNA filaments with designer elastic ptoperties. ACS Nano. 2018, 12, 44-55.

A spring-like DNA origami device

In this work, we used temperature-dependent FRET spectroscopy to extract the thermal stabilities of distinct sets of hairpins integrated into the central seam of a DNA origami structure. We 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 into the inner cavity of the origami structure, thus surpassing the operational capabilities of freely diffusing motors.

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E.-C. Schöneweiß and B. Saccà. The collective behaviour of spring-like motifs tethered to a DNA origami nanostructure. Nanoscale 2017, 9, 4486-4496.

Rational design of DNA hosts for protein guests

Natural host-guest complexes are normally stabilized by non-covalent interactions between geometrically matching surfaces. Using this principle, we realized one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach relies on the decoration of the inner surface of a hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action.

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A. Sprengel, P. Lill, P. Stegemann, K. Bravo-Rodriguez, E.-C. Schöneweiß, M. Merdanovic, D. Gudnason, M. Aznauryan, L. Gamrad, S. Barcikowski, E. Sanchez-Garcia, V. Birkedal, C. Gatsogiannis, M. Ehrmann, B. Saccà. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions. Nat. Commun. 2017, 8, 14472.

Hierarchical self-assembly of DNA nanostructures

From atoms to molecules and biomacromolecules, from organelles to cells, tissues, till the whole living system: so nature shows us that the realization of complex systems with emergent properties originates from the hierarchical self-assembly of single components in a guided bottom-up process. 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.

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W. Pfeifer and B. Saccà. From nano to macro through hierarchical self-assembly: the DNA paradigm. Chembiochem. 2016, 17, 1063-1080.

Reversible reconfiguration of a planar DNA cage followed by single-molecule FRET

Reversible reconfiguration of a planar DNA cage followed by single-molecule FRET We use switchable DNA motifs and integrate them within the central seam of a DNA origami structure to reversibly enlarge and contract its inner cavity for modulation of fluorescence energy transfer between two internalized fluorophores and selective trapping of proteins.

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B. Saccà,* Y. Ishitsuka, R. Meyer, A. Sprengel, E. C. Shöneweiß, U. Nienhaus,* C. M. Niemeyer.* Reversible reconfiguration of DNA origami nanochambers monitored by single-molecule FRET. Angew Chem Int Ed Engl. 2015, 54, 3592-3597.
*corresponding authors

Planar lattices of switchable DNA motors

We used a DNA-based self-assembly method for the construction of switchable DNA devices based on G-quadruplex moieties, which are patterned on quasi-planar DNA arrays with nanoscale precision. The reversible switching of the devices was 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. Altogether, this study confirmed 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.

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B. Saccà and C.M. Niemeyer. Covalent tethering of protruding arms for addressable DNA nanostructures. Small 2011, 7, 2887-2898.
B. Saccà, B. Siebers, R. Meyer, M. Bayer and C.M. Niemeyer. Nanolattices of switchable DNA-based motors. Small 2012, 8, 3000-3008

Monitoring the self-assembly of DNA nanostructures in real time by FRET spectroscopy

We described a method for the real-time and high-throughput monitoring of the self-assembly and disassembly of complex DNA superstructures, using temperature-dependent Förster resonance energy transfer (FRET) spectroscopy. Compared with other spectroscopic approaches, such as UV-visible and circular dichroism, the method has advantages in terms of sensitivity, feasibility for high-throughput analysis and applicability to virtually any kind of supramolecular structure. To this end, two oligonucleotides out of the entire set building up the superstructure were labeled with a fluorescein and a tetramethylrhodamine, as FRET donor and acceptor, respectively. Correct assembly of the superstructure was monitored by temperature-dependent FRET efficiency. In case of reversible and cooperative assembly/disassembly of the DNA superstructure, application of the van't Hoff law allows for the determination of the thermodynamic parameters of the process. Monitoring the self-assembly of DNA nanostructures in real time by FRET spectroscopy

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B. Saccà*, R. Meyer, U. Feldkamp, H. Schroeder and C.M. Niemeyer*. High-throughput, realtime monitoring of the self-assembly of DNA nanostructures by FRET spectroscopy. Angew. Chem. Int. Ed. Engl. 2008, 47, 2135-2137.
B. Saccà*, R. Meyer and C.M. Niemeyer*. Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA nanostructures in real time. Nat. Protoc. 2009, 4, 271-285.
*corresponding authors