ZMB Member Barbara Saccà, Research
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.