Maßgeschneiderte Protein Inkapsulierung in einen DNA Käfig mittels geometrisch angeordneter supramolekularer Wechselwirkung

Im Rahmen dieser Dissertation wurden verschiedene Aspekte der DNA-Nanotechnologie, Biologie und der supramolekularen Chemie miteinander verknüpft und zur Anwendung gebracht. Die Arbeit kann in drei Teilbereiche unterteilt werden: (i) Das Designen eines geeigneten hexagonalen turbularen DNA-Käfigs mittels der Software CaDNAno2, dessen räumliche Dimensionen zur Immobilisierung des Proteins DegP in seinen unterschiedlichen Oligomerisierungs-Zuständen entsprechen musste. (ii) (ii) Die Synthese eines Oligonukleotid-Heptapeptid-Konjugates, welches supramolekular an die PDZ1 Domäne des Zielproteins bindet. (iii) (iii) Das Laden des Proteins in den Käfig mit anschließendem Versuch der gerichteten Freisetzung des Proteins. Der DNA-Origami-Käfig wurde mittels der Software CaDNAno2 als einlagiges, turbuläres und hexagonales DNA-Röhrchen mit einem inneren Radius von 20 nm und einem äußeren Radius von 23 nm entworfen und erfolgreich realisiert. Die einzelnen Seitenwände des Käfigs wurden durch jeweils speziell entworfene Oligonukleotide miteinander verknüpft, so dass zum einen die Orientierung der Seitenflächen zum Zentrum der Kavität (6p120 und 6p240) determiniert werden konnte, (vgl. Kapitel 3.2.1 und 3.2.2) und zum anderen durch eine flexible Verbindung der Seitenflächen mittels drei Thyminen eine zufällige Orientierung (6p180) der Seitenflächen zum Zentrum der Kavität vorlag. Zusätzlich wurde jede Seitenfläche mit der Option entworfen, mit null, ein, zwei oder drei (insgesamt 0cA1, 6cA1 oder 18cA1) orthogonal herausragenden Oligonukleotiden (Protruding-Arme), die als Verlängerung von zentral liegenden Oligonukleotiden zu sehen sind, ausgestattet zu werden. Die Protruding-Arme dienen als Träger für die mit Peptiden modifizierten komplementären Oligonukleotid-Liganden (Kapitel 3,3), welche wahlweise mit Fluorescein (Flc-A1-DPMFKLV) oder TAMRA (TAMRA-A1-DPMFKLV) ausgestattet waren. Mittels eines Biotin-modifizierten Liganden konnte durch Zugabe von Streptavidin die korrekte Formation der Konstrukte 6p120, 6p180 und 6p240 durch Gel-Elektrophorese und Raster-Kraft-Mikroskopie (AFM) bestätigt werden. Messungen durch das Transmissions-Elektronen-Mikroskop (durchgeführt von Pascal Lill am MPI in Dortmund im Rahmen seiner Masterarbeit) und der dynamischer Lichtstreuung bestätigten ebenfalls die korrekte Formation in Lösung. Unter Berücksichtigung des antiproportionalen Verhältnisses der Diffusionsrate durch den Käfig zur Größe des Proteins wurde der DNA-Origami-Komplex so geplant, dass das 12-mer des Proteins DegP bevorzugt binden sollte. Die Synthese des Peptidfragmentes der Liganden erfolgte mittels Festphasenpeptidsynthese nach Standardbedingungen. Die N-terminale Aminogruppe der Peptidsequenz konnte direkt in eine Maleimide Funktion überführt werden (Kapitel 6.5.2.8.2). Ohne die Verwendung eines üblichen Crosslinkers konnten Thiol-modifizierte Oligonukleotide über eine kovalente Bindung an das Maleimid gebunden werden. Dieser erfolgreich etablierte Weg stellt somit eine universelle Methode dar, N-terminale Peptide direkt an Thiol-modifizierte Oligonukleotide zu binden. Eine Charakterisierung der Konjugate erfolgte anschließend mittels MALDI-TOF. Das Laden diverser DegP Protein-Varianten (vgl. Kapitel 3.1, Tabelle 1) erfolgte nur in Anwesenheit des Peptid-Liganden (vgl. Kapitel 3.4.3), was die Selektivität der Methode erfolgreich demonstrierte und unspezifische Wechselwirkungen ausschloß. Das spezifische und erfolgreiche Binden an die DNA-Nano-Käfige konnte mittels interner Totalreflexionsfluoreszenzmikroskopie durch Überlagerung der Fluoreszenzsignale in Einzelmolekül-Experimenten bestätigt werden (durchgeführt durch die AG Birkedal, Aarhus Universität). Nach statistischer Auswertung der AFM-Bilder konnte gezeigt werden, dass eine Präferenz zur Immobilisierung von 12-meren im Verhältnis 1 : 2.2 : 1.3 für DegP6, DegP12 und DegP24 vorlag, was der Zielsetzung bezüglich der Selektivtät entsprach. Durch das Assemblieren eines 6p120 Käfigs mit einer unterschiedlichen Anzahl an Protruding-Armen (vgl. Kapitel 3.4.3.5) und dementsprechend mit einer unterschiedlichen Anzahl von Liganden konnte gezeigt werden, dass zum einen ein Ligand ausreicht, um ein Protein (DegP6) erfolgreich zu immobilisieren und zum anderen ein proportionales Verhältnis zwischen Ladungseffiziens und der Anzahl der Protruding-Arme besteht. Um den positiven Effekt einer hohen lokalen Konzentration der Peptid-Liganden auf die Bindeeffiziens der Proteine zu zeigen, wurden die DNA-Käfige mit den jeweiligen Ausrichtungen der Protruding-Arme (6p120, 6p180 und 6p240, vgl. Kapitel 3.4.3.4) und gleicher Anzahl der Liganden mit DegP12/24A488SA versetzt und mittels eines Vergleiches der Intensität des Fluorescein-Signals die höchste Bindeeffiziens im Verhältnis 8 : 1.4 : 1 (6p120: 6p180 : 6p240) für die Käfige mit nach innen-orientierten Liganden bestimmt. Nach dem erfolgreichen Immobilisieren des Proteins innerhalb der Kavität des Käfigs wurde anschließend eine Freisetzung des Proteins durch einen Austausch des zum Protruding-Arm nicht vollständig komplementären, mit Fluorophoren markierten Peptid-Liganden durch ein vollständig komplementäres Oligonukleotid versucht. Der erfolgreiche Austausch der markierten Liganden konnte mittels Gel-Elektrophorese gezeigt werden, jedoch nicht unter vollständiger Freisetzung des Proteins. Eine verbesserte Freisetzung konnte trotz Änderung der Nettoladung des Proteins ebenfalls nicht erreicht werden (vgl. Kapitel 3.4.3.8). Zusammenfassend kann festgehalten werden, dass zum ersten Mal ein Protein durch schwache supramolekulare Interaktionen innerhalb einer DNA-Origamistruktur immobilisiert werden konnte. Diese Proteine konnten ohne chemische Veränderung nur aufgrund der räumlichen Nähe der Peptid-Liganden, die an die PDZ1-Domänen der Proteine bindeten, und der hohen lokalen Konzentration innerhalb der Kavität durch multivalente Wechselwirkungen mit geringer Reichweite im Käfig gehalten werden. Die Bedeutung der Oberflächenladung der Proteine für die Immobilisierung innerhalb der Kavität bedarf noch weiterer Untersuchungen. Ein Indiz für diese Bedeutung liefern die Versuche mit dem molekularen Tweezer, welche an die Lysine an der Oberfläche der Proteine bindet und deren positive Ladung abschirmt. Folglich führt dies zur Abschwächung der unspezifischen Wechselwirkungen zwischen den Proteinen und dem Käfig, so dass diese erfolgreich geladen werden konnten. Die analogen Ergebnisse der Experimente mit einer weiteren Struktur eines offenen Prismas im HoneyComb-Design (vgl. Kapitel 1.2.3) werden in Kapitel 8 gezeigt, da diese keine neuen, bzw. nur die bisherigen Ergebnisse bestätigende Resultate erbracht haben.During this PhD project, aspects of DNA nanotechnology, biology and supramolecular chemistry have been merged. This work can be divided into three main parts: (i) the design of a suitable tubular DNA cage, which is able to encapsulate all different oligomers of DegP; (ii) the synthesis of a DNA-hepta-peptide conjugate, which binds non-covalently to the PDZ1 domain of the target protein and (iii) the loading of the protein. The DNA origami cage was successfully designed and realized as a single-layer hexagonal DNA prism with an inner radius of 20 nm and an outer radius of 23 nm. Using special spatial staples strands for the face-to-face connections, the orientation of the faces towards the cavity could be programmed (6p120 and 6p240). Alternatively, stochastically oriented faces (6p180) were obtained using three thymines as flexible hinges (see chapter 3.2.1 and 3.2.2). Additionally, each face was equipped with zero, one, two or three orthogonal protruding arms (for a total of 0cA1, 6cA1 or 18cA1 arms, respectively). These arms were used for hybridization with DNA-peptide conjugates equipped with optional fluorophores (chapter 3.3). Correct formation of the different designs (6p120, 6p180 and 6p240) was proven by AFM and gel electrophoresis, after adding streptavidin to the biotinylated ligands. TEM characterization (performed by Pascal Lill at the MPI in Dortmund) and dynamic light scattering confirmed the correct dimensions of the DNA structures in solution. The peptide sequence DPMFKLV was synthesized via solid phase peptide synthesis under standard coupling conditions. Transforming the N-terminal amino group of the peptide directly into a maleimide function allowed reaction with the thiol group of an oligonucleotide without the use of any crosslinking agents. This successful method represents a general method to link the terminal amino-group of a peptide to a thiol bearing oligonucleotide. Purification via HPLC allowed characterization of the DNA-DPMFKLV ligands per MALDI-TOF. Loading of diverse DegP proteins (compare chapter 3.1, table 1) only took place in the presence of the A1-DPMFKLV ligands, demonstrating the validity of the encapsulation strategy (compare chapter 3.4.3). Successful and specific binding was shown at the single-molecule level using total internal reflection fluorescence (TIRF) microscopy, (performed by AG Birkedal, Aarhus University). Statistical evaluation of AFM images revealed preferential encapsulation of the DegP12 protein, with a ratio of 1.3 : 2.2 : 1 for the DegP6, DegP12 and DegP24, respectively. Loading experiments performed with DegP6A633SA and cages with a different number of PAs (see chapter 3.4.3.5) and a correspondingly different number of A1-peptide ligands, showed that one A1-DPMFKLV ligand is sufficient for encapsulation of the protein, with a loading efficiency proportional to the number of ligands. DNA origami cages with identical number of ligands but different orientations of the PAs (6p120, 6p180 and 6p240, see chapter 3.4.3.4) were loaded with DegP12/24A488SA. Gel electrophoresis analysis showed a highest binding efficiency for the 6p120 design in a ratio of 8 : 1.4 : 1 for the 6p120-, 6p180- and 6p240-designs, respectively, thus indicating the importance of a high local concentration of peptide ligands. After successful encapsulation of the protein, experiments to release the protein from the cage were performed, using single strand displacement reactions. Disappearance of the fluorophore signals from the cage sample showed successful displacement of the ligands; however, without releasing the protein. Variation of the pH of the solution and its ionic strength did not result in any beneficial effects (see chapter 3.4.3.8). To sum up, it could be shown that a protein can be encapsulated within a DNA origami cage by weak non-covalent supramolecular interactions without any previous chemical treatment of the protein. The arrangement of a distinct number of peptide ligands in the vicinity of the corresponding binding sites on the protein surface allowed modulation of local concentration effects and multivalent short-range interactions in a single system. The importance of the net charge of the protein for encapsulation within the cavity of the DNA nanochamber and the different binding efficiencies observed for distinct fluorophores, require further investigation. Evidence for the important role of the net charge is the successful loading of the DegP12 protein in presence of molecular tweezers, targeting the lysine residues on the surface of the protein. Results of analogue experiments performed with a second DNA cage, (HoneyComb design, see chapter 1.2.3) are shown in chapter 8

(A) Constructing a Three-Dimensional DNA Nanomachine to Achieve Rapid Isothermal Signal Amplification for Nucleic Acid Detection (B) Regulation of DNA Strand Displacement Using an Allosteric DNA Toehold

(A) We developed a new strategy to achieve rapid isothermal signal amplification through the construction of DNA nanomachine. DNA nanomachine built from a DNA functionalized gold nanoparticle (DNA−AuNP), which moves a DNA walker along a three-dimensional (3-D) DNA−AuNP track and executes the task of releasing signal reporters (SRs) to generate fluorescence. The movement of the DNA walker is powered by a nicking endonuclease that cleaves specific DNA substrates on the track. During the movement, each DNA walker cleaves multiple substrates, resulting in the rapid release of SRs to achieve signal amplification at a constant temperature. The 3-D DNA nanomachine is highly efficient due to the high local effective concentrations of all DNA components that have been co-conjugated on the same AuNP. Moreover, the activity of the 3-D DNA nanomachine can be controlled by introducing a protecting DNA probe that can hybridize to or dehybridize from the DNA walker in a target-specific manner. This property allows us to tailor the DNA nanomachine into a DNA nanosensor that is able to achieve rapid, isothermal, and homogeneous signal amplification for detection of nucleic acids in both buffer and a complicated biomatrix. (B) Toehold-mediated DNA strand displacement has proven extremely powerful in the construction and operation of DNA devices, including reconfigurable structures, DNA circuits, and amplifications. To achieve the construction of such DNA devices, toeholds are required for controllable activation and regulation. Usually, the complicated strand displacement behaviors and functions are achieved by combining conventional toehold-mediated strand displacement, associative toehold-mediated strand displacement, and remote toehold strand displacement toehold activation mechanisms. We still need to enrich the toolbox of strand displacement techniques with alternative approaches for toehold activation to construct devices of higher complexity. Here we introduce an allosteric DNA toehold (A-toehold) design that allows flexible activation or regulation of DNA strand displacement

Self-Assembly from Milli- to Nanoscales: Methods and Applications

The design and fabrication techniques for microelectromechanical systems (MEMS) and nanodevices are progressing rapidly. However, due to material and process flow incompatibilities in the fabrication of sensors, actuators and electronic circuitry, a final packaging step is often necessary to integrate all components of a heterogeneous microsystem on a common substrate. Robotic pick-and-place, although accurate and reliable at larger scales, is a serial process that downscales unfavorably due to stiction problems, fragility and sheer number of components. Self-assembly, on the other hand, is parallel and can be used for device sizes ranging from millimeters to nanometers. In this review, the state-of-the-art in methods and applications for self-assembly is reviewed. Methods for assembling three-dimensional (3D) MEMS structures out of two-dimensional (2D) ones are described. The use of capillary forces for folding 2D plates into 3D structures, as well as assembling parts onto a common substrate or aggregating parts to each other into 2D or 3D structures, is discussed. Shape matching and guided assembly by magnetic forces and electric fields are also reviewed. Finally, colloidal self-assembly and DNA-based self-assembly, mainly used at the nanoscale, are surveyed, and aspects of theoretical modeling of stochastic assembly processes are discussed

Multivalent Programmable Interactions Between Lipid Vesicles: Towards Responsive Soft Materials

Lipid membranes and lipid vesicles have been studied extensively in the last 50 years in order to characterise their biological, chemical and physical properties. Such work is of interest from a fundamental biological perspective, but also due to the applications that their biocompatibility affords: in biotechnological, pharmaceutical, food science and cosmetic applications. From this work, it is clear that lipid membranes display a large number of remarkable traits: they can form a wide range of sizes and morphologies, are deformable and can be functionalised with a variety of structures. More recently, multivalent interactions have been exploited to drive self-assembly of nanoparticles, hard colloids and compliant units including emulsion droplets and lipid vesicles. By applying this to deformable lipid vesicles, formation of links between two membranes produces morphological changes unachievable in hard colloidal systems, and the liquid interface of liquid-phase bilayers allows for the diffusion of the multivalent constructs across the membrane of the lipid vesicle. Against this background of membrane science and multivalent interactions, this thesis develops new experimental approaches to exploit these extra degrees of freedom to develop novel lipid-based soft responsive materials with potential ’real-world’ applications, such as in molecular sensing. In Chapter 1, the motivations for this work are introduced, before introducing the requisite background literature and general experimental techniques in Chapters 2 and 3 respectively. In Chapter 4 we show a system of single lipid vesicles adhering to a flat supported lipid bilayer through multimeric multivalent interactions, which we study to characterise the morphological and mechanical changes of the vesicles in response to external ligands. We show that the mechanical properties of the vesicles, in particular their membrane tension, change dramatically on adhesion, and that the number of adhering vesicles is dependent on the concentration of the external ligand due to combinatorial entropy, which we confirm through consideration of a simple statistical mechanical model. In Chapter 5 we use Differential Dynamic Microscopy to study the dynamics of a thermoreversible gel consisting of diffusive attractive soft colloids (large unimlamellar vesicles functionalised with complementary DNA constructs), and fit the dynamics with a stretched/compressed exponential model. From the fit parameters, we observe differing levels of spatial heterogeniety of the dynamics of the sample within different regimes below, above and around the gel/melting points, as well as differing length scales of the dynamics, which differ between quenching and melting experiments. From the statics and dynamics, we see evidence for multiple phenomena, including coarsening as well as ballistic events corresponding to strand breakages. In Chapter 6 we propose a method for high-throughput vesicle production. We characterise the method and the vesicles produced, as well as demonstrating novel applications, most notably the high-throughput production of vesicles encapsulating responsive DNA circuitry, highlighting the potential of this method in bottom-up synthetic biology and the design of programmable materials. Furthermore, we demonstrate the possibility of on-chip functionalisation of membrane constructs into the lipid membranes, in this case cholesterol anchored DNA constructs. In Chapter 7 we study dense packings of vesicles assembled using multivalent complementary DNA interactions, through passive tracking of diffusive colloidal particles and active microrheology using magnetic tweezers. We observe changes in the structure in response to increased temperature, DNA concentration and aging leading to reduced pore sizes. From a rheological standpoint, we observe strain hardening of the material through repeated creep tests, with the ability to reset the material by increasing the temperature above the melting point of the system. The material stiffens and becomes more viscous, which we observe through the application of a constitutive and fractional rheological model respectively. In this thesis we demonstrate the responsiveness of these multivalent construct functionalised lipid vesicle based soft materials by showing the ability to tune the structure, rheology and dynamics of such materials, as well as proposing a method for high throughput, monodisperse production of functionalised lipid vesicles. These results lead to further potential avenues of research, and demonstrate suitability for and preliminary steps towards applications of these responsive materials in fields such as molecular sensing.EPSRC CDT for Sensor Technologies and Application

Programmable and Multifunctional DNA-Based Materials for Biomedical Applications

DNA encodes the genetic information; recently, it has also become a key player in material science. Given the specific Watson–Crick base‐pairing interactions between only four types of nucleotides, well‐designed DNA self‐assembly can be programmable and predictable. Stem‐loops, sticky ends, Holliday junctions, DNA tiles, and lattices are typical motifs for forming DNA‐based structures. The oligonucleotides experience thermal annealing in a near‐neutral buffer containing a divalent cation (usually Mg2+) to produce a variety of DNA nanostructures. These structures not only show beautiful landscape, but can also be endowed with multifaceted functionalities. This Review begins with the fundamental characterization and evolutionary trajectory of DNA‐based artificial structures, but concentrates on their biomedical applications. The coverage spans from controlled drug delivery to high therapeutic profile and accurate diagnosis. A variety of DNA‐based materials, including aptamers, hydrogels, origamis, and tetrahedrons, are widely utilized in different biomedical fields. In addition, to achieve better performance and functionality, material hybridization is widely witnessed, and DNA nanostructure modification is also discussed. Although there are impressive advances and high expectations, the development of DNA‐based structures/technologies is still hindered by several commonly recognized challenges, such as nuclease instability, lack of pharmacokinetics data, and relatively high synthesis cost. </p

Probing DNA-Induced Colloidal Interactions and Dynamics with Scanning-Line Optical Tweezers

A promising route to forming novel nanoparticle-based materials is directed self-assembly, where the interactions among multiple species of suspended particles are intentionally designed to favor the self-assembly of a specific cluster arrangement or nanostructure. DNA provides a natural tool for directed particle assembly because DNA double helix formation is chemically specific — particles with short single-stranded DNA grafted on their surfaces will be bridged together only if those strands have complementary base sequences. Moreover, the temperature-dependent stability of such DNA bridges allows the resulting attraction to be modulated from negligibly weak to effectively irreversible over a convenient range of temperatures. Surprisingly, existing models for DNA-induced particle interactions are typically in error by more than an order of magnitude, which has hindered efforts to design complex temperature, sequence and time-dependent interactions needed for the most interesting applications. Here we report the first spatially resolved measurements of DNA-induced interactions between pairs of polystyrene microspheres at binding strengths comparable to those used in self-assembly experiments. The pair-interaction energies measured with our optical tweezers instrument can be modeled quantitatively with a conceptually straightforward and numerically tractable model, boding well for their application to direct self-assembly. In addition to understanding the equilibrium interactions between DNA-labeled particles, it is also important to consider the dynamics with which they bind to and unbind from one another. Here we demonstrate for the first time that carefully designed systems of DNA-functionalized particles exhibit effectively diffusion-limited binding, suggesting that these interactions are suitable to direct efficient self-assembly. We systematically explore the transition from diffusion-limited to reaction-limited binding by decreasing the DNA labeling density, and develop a simple dynamic model that is able to reproduce some of the anomalous kinetics observed in multivalent binding processes. Specifically, we find that when compounded, static disorder in the melting rate of single DNA duplexes gives rise to highly non-exponential lifetime distributions in multivalent binding. Together, our findings motivate a nanomaterial design approach where novel functional structures can be found computationally and then reliably realized in experiment

Analysis of extracellular vesicles as emerging theranostic nanoplatforms

Extracellular vesicles (EVs) are nanoscale lipid membrane–bound vesicles that are secreted by cells of both prokaryotes and eukaryotes and carry bioactive cargos including proteins, nucleic acid and lipids from source cells. Given their prominent ability in transporting bioactive components, EVs are regarded as promising biomarkers for disease diagnosis and emerging therapeutic nanoparticles. However, to exert their effect in clinical applications, effective isolation and sensitive analysis of EVs from complex biofluids is required. Recent advances in EV-related research has provided feasible approaches for developing emerging therapeutic nanoplatforms using EVs. With this review, we aim to provide a comprehensive and in-depth summary of recent advances in diverse assay methods for EVs including fluorescence, Raman/Surface-enhanced Raman Spectroscopy (SERS) analysis and other methods, as well as their clinical potential in constructing EV-based theranostic nanoplatforms towards various diseases. In particular, microfluidic-assisted analysis sytems, single EV detection and the main approaches of utilizing EVs for therapeutic purposes are highlighted. We anticipate this review will be inspirational for researchers in related fields and will provide a general introduction to scientists with various research backgrounds.</p