Transmembrane molecular machines

Transmembrane molecular machines are ubiquitous in nature. These evolved systems demonstrate superlative elegance and efficiency of operation. Imitation and hijacking of biological components such as proteins and DNA has emerged as a means of imparting desirable characteristics to rationally designed synthetic molecular machines. This thesis presents work towards various synthetic transmembrane molecular machines based on alpha-haemolysin (α-HL). Chapter One reviews progress towards synthetic transmembrane machines, introducing natural examples, defining criteria for being ‘a molecular machine’ cataloguing examples and trends in synthetic molecular machines in solution, on surfaces and in membranes. Examples are evaluated in terms of their machine like behaviour and α-HL emerges as a particularly promising component in the development of synthetic transmembrane molecular machines. Chapter Two examines solvent isotope effects resulting from substitution of hydrogen by deuterium in water at the nanoscale – on the rates of transmembrane ion transport and transmembrane translocation of ssDNA through α-HL, both of which are of concern in the context of building molecular machines which use α-HL as a component. Chapters Three to Six look at different machine applications of related transmembrane architectures based on individual transmembrane rotaxanes constructed in α-HL from DNA/PEG copolymer ‘thread’ strands and DNA ‘primer’ strands. Chapter Three uses this approach to observe translational motion of the thread strand in both directions along the z-axis due to nucleotide incorporation and pyrophosphorolysis in real-time with single-nucleotide resolution. Chapter Four provides the first demonstration of asymmetrical, hysteretic cyclical behaviour in the translational motion of the thread strand by incorporation of a nicking site which resets the system after nucleotide incorporations have occurred. Chapter Five introduces a novel variant of the rotaxane architecture using a circularised primer strand which allows real time observation of rolling circle amplification at the single molecule level by coupling the process to the unidirectional translocation of the thread strand. Chapter Six considers the use of the vestibule of α-HL as a transmembrane DNA ligase mimic with the DNA thread/primer complex as substrate

Engineering a multi-functional DNA origami nanorod for the control of nanoscale processes

Processes that occur at the nanoscale are the foundational building blocks of our world. As such, there is considerable interest in ways to study and manipulate matter at this scale, with applications in biomedicine and other fields. DNA origami has emerged over the past decade as a promising technology for nanofabrication, offering the capacity for precise and tunable nanoscale synthesis while maintaining the ease and scale of bottom-up self-assembly. The goal of this work is to develop novel ways in which DNA origami can be used to manipulate nanoscale processes. To this end, I developed a single DNA origami nanorod which is used in two distinct studies, highlighting the multifunctionality of this structure. I first investigated the effect of iron oxide nanoparticle clustering on MRI contrast generation by organizing particles in precise patterns on the nanorod. I found that small changes in the number of attached iron oxide nanoparticles lead to significant enhancement in T2 relaxivity, while inter-particle spacing has a minimal effect. In the second part of thesis, I developed the first DNA origami molecular motor, which converts chemical energy into mechanical activity and demonstrates autonomous directed motion over micron distances. By leveraging the unique addressability of DNA origami, I found that these motors predominately exhibit a rolling motion and that this behavior can be tuned via small alterations to the nanorod. Combined, this work demonstrates two novel applications for DNA origami nanostructures. We expect this work will serve as an initial platform for further studies and open up a range of new possibilities for the use of DNA origami as MRI contrast agents and molecular motors.Ph.D

Design of an environment-indipendent, tunable 3D DNA-origami plasmonic sensor

DNA origami nanotechnology engineers DNA as the building blocks of newly conceived self-assembled materials and devices. Due to its high degree of customization and its precise spatial addressability, DNA origami provides an unmatched platform for nanoscale structures and devices design. Gold nanoparticles (AuNP) have been largely investigated because of their peculiar optical properties and in particular their localized surface plasmon resonance (LSPR) that modifies significantly the electromagnetic environment in a thin shell around them, and provides a tool with unrivalled potential to tune the local optical properties. The combination of DNA origami frameworks and AuNP into DNA based-plasmonic nanostructures offers a concrete approach for optical properties engineering. It has been successfully applied to design biosensor and to enhance Raman scattering or fluorescence emission. Moreover, it has been exploited to design molecular ruler in which the inter-particle gap is controlled with nanometric precision through the transduction of the conformational changes into univocally detectable optical signals. In this thesis I present my PhD work which aims at the design of an environment-independent AuNP decorated-DNA origami. A tetrahedral DNA shape structure has been selected for its three dimensional robustness and thus a DNA origami prototype has been assembled, characterized with SEM, TEM and AFM to verify the proper folding of the structure. The origami was equipped with an actuator probe which recognizes a specific target oligonucleotide inducing a structural reconfiguration of the tetrahedron. To detect the conformational change triggered by the hybridization event, I functionalized the origami with two gold nanoparticles placed in two opposite facets at a known distance of 10 nm: the change of the interparticle gap is effectively transduced in a LSPR shift. This working principle has been verified with optical extinction measurements and the interparticle distance reduction has been confirmed by SEM imaging and SAXS analysis performed in the SAXS beamline of Elettra Synchrotron, thus confirming that the operation of the device and its transduction mechanism are the same no matter of the external conditions, being them dry, liquid or solid

Structure-function analysis of a multifunctional enzyme using the atomic force microscope

The type I R-M enzyme EcoR124I is a multifunctional, multisubunit molecular motor with the ability to self-assemble. In the presence of hydrophobic compounds, subunit disassembly has been observed leading to the possibility of using the enzyme as a nanoactuator in toxicity biosensors. A better understanding of single molecule interactions between the subunits has been investigated using atomic force microscopy (AFM), a powerful tool for measuring forces and dynamics between single molecules with a picoNewton sensitivity. AFM imaging of DNA fragments with a single recognition binding site for EcoR124I positioned in the middle or at 1/3 of the length of DNA, was used to study the assembled holoenzyme. Reproducible DNA imaging was investigated using divalent cations (Mg2+, Ca2+, Ni2+). The presence of only one EcoR124I holoenzyme bound to DNA was observed, confirming the specificity of binding. Molecular volume (Vm) measurements were used to identify subunits and complexes. The effect of ATP analogues (ATP-γ-S and AMP-pnp) on enzyme stability was also investigated. The addition of ATP, although not novel, confirmed the enzyme activity by showing the ability of the enzyme to translocate. Biotin-avidin interactions were studied using AFM force curves as a model to probe the novel HsdR-MTase system. AFM tips were functionalised using both glutaraldehyde and a PEG linker. In the former, many multiple event force curves were seen, although the final “pull-off” event yielded information on single-molecule or near single-molecule interactions: a single biotin-avidin interaction at 56 ± 13 pN was measured, with further periodic force maxima at 98 ± 15 and 161 ± 3 pN (two and three interactions, respectively). The use of a PEG linker allowed more sensitive measurements to be made, with a single biotin-avidin interaction at 47 ± 9.5 pN and, again, periodic maxima were seen at 93 ± 7 and 143 ± 4 pN. The PEG linker method allowed more single molecules interactions to be measured (ca. 70% of analysed forcedistance curves). Forces between a GST-HsdR(PrrI) motor subunit attached to an AFM tip using a PEG linker and MTase on poly-L-lysine pre-treated mica were studied using dynamic force spectroscopy (DFS). A single barrier in the energy landscape of the complex was found in the dissociation pathway (xdiss) to be located 13.5 Å from the bound state. The value kdiss for the GST-HsdR(PrrI)-MTase complex was calculated to be 0.16 s-1 and the lifetime t(0) of the GST-HsdR(PrrI)-MTase bond was found to be 6.25 s. GST – anti GST antibody interactions and HsdR – anti-GST antibody interactions suggest that forces measured between HsdR and MTase were realistic for the GSTHsdR(PrrI)-MTase complex.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Dna studies: Latest spectroscopic and structural approaches

This review looks at the different approaches, techniques, and materials devoted to DNA studies. In the past few decades, DNA nanotechnology, micro-fabrication, imaging, and spectro-scopies have been tailored and combined for a broad range of medical-oriented applications. The continuous advancements in miniaturization of the devices, as well as the continuous need to study biological material structures and interactions, down to single molecules, have increase the interdisciplinarity of emerging technologies. In the following paragraphs, we will focus on recent sensing approaches, with a particular effort attributed to cutting-edge techniques for structural and mechanical studies of nucleic acids

The architectonics of programmable RNA and DNA nanostructures

The past several years have witnessed the emergence of a new world of nucleic-acid-based architectures with highly predictable and programmable self-assembly properties. For almost two decades, DNA has been the primary material for nucleic acid nanoconstruction. More recently, the dramatic increase in RNA structural information led to the development of RNA architectonics, the scientific study of the principles of RNA architecture with the aim of constructing RNA nanostructures of any arbitrary size and shape. The remarkable modularity and the distinct but complementary nature of RNA and DNA nanomaterials are revealed by the various selfassembly strategies that aim to achieve control of the arrangement of matter at a nanoscale level. Introduction The complex supramolecular (see glossary) structures that emerged in living organisms through billions of years of evolution rely on two basic self-assembly processes: the spontaneous folding of one polymer chain into a stable well-defined 3D structure; and the assembly of multiple subunits into defined, modular supramolecular architectures. Key characteristics are hierarchical organization, modular components, and stereochemically specific and selective interactions. Programmable assembly (see glossary) results from the application of folding and assembly principles gleaned from biological structures to design molecules that will, in a predictable manner, fold into specific shapes and subsequently assemble with one another into supramolecular architectures according to the structural information encoded within their primary structure. Although programmable self-assembly is at the core of supramolecular chemistry Proteins are the material of choice for building the structural, catalytic and regulatory components of cells, but their folding and assembly remain challenging to predict and design because of the inherent complexity of their 3D structures (see the reviews from Ranganathan, Waters, Kuhlman and Chin in this issue). By contrast, DNA, as the carrier of the genetic information in cells, has only four deoxynucleotide chemical building blocks, a high chemical stability, and predictable folding and assembly properties that are readily amenable to the rational design and construction of 3D nanostructures by programmable self-assembly [2,3,4 -6 ]. RNA has recently emerged as a challenger to DNA, interesting in its own right as a medium for programmable nanoconstruction (e.g. [7,8,9 ,10,11 ,12 ]). Despite a chemical structure very similar to that of DNA, RNA is chemically more labile than DNA, but is also more prone to fold into complex tertiary structures with recognition and catalytic properties reminiscent of those of proteins. Natural RNAs are the working components of biologically important molecular machines that are capable of using cellular energy in the form of ATP or GTP to perform mechanical work and to carry out complex tasks of information processing, such as template-directed protein synthesis and multiplexed gene regulation Basic structural properties and modularity of RNA and DNA nanostructures RNA and DNA modularity is hierarchically expressed at a chemical, structural and supramolecular level Despite a limited number of known DNA tertiary (38) structure motifs (see glossary; At a 48 structure level, RNA and DNA modular units assemble further into complex and highly modular supramolecular architectures in a predictable manner using base-pair rules as organizational instructions. The dimensionality of these nanostructures is directly related to the number, shape, geometry and orientation of cohesive, assembling interfaces formed between constitutive RNA or DNA tiles (see glossary) [6 ] ( DNA architectonics: variations on the same structural theme Because of the lack of stable natural 38 structure motifs, much effort has been expended designing robust and rigid DNA self-assembling building blocks A subtle balance of flexibility and stress is required for building good self-assembling tiles The monolithic structure of most DNA tiles imposes strong geometrical constraints on the positioning of their cohesive interfaces In the future, the use of triple helices RNA architectonics: sculpting new RNA structures The concept of RNA tectonics (see glossary) was initially defined as referring to the modular character of RNA structures that can be decomposed and reassembled to create new modular RNA units, called tectoRNAs (see glossary), which are able to self-assemble into nanoscale and mesoscale architectures of any desired size and shape b The persistence lengths of RNA and DNA were determined experimentally by single-molecule analysis (e.g. Current Opinion in Structural Biology The characterization of tectoRNA folding and self-assembly properties is typically performed by biochemical and biophysical methods, and visualization techniques, such as atomic force microscopy (AFM) [9 ,47] and transmission electron microscopy (TEM) Although still a new field of investigation, RNA architectonics has already generated a great variety of tectoRNA units able to assemble into highly modular supramolecular architectures of arbitrary shapes ( Nanoparticles, filaments and 2D RNA architectures The first tectoRNAs to be generated by RNA architectonics self-assemble through loop-receptor interfaces to form dimeric nanoparticles Collinear kissing loop interactions can generate strong 48 intermolecular interfaces to promote the formation of RNA particles of different sizes [50] The high modularity and hierarchical supramolecular structure of tectosquares makes it possible to construct a large number of them from a limited set of tectoRNAs that assemble through strong 48 interaction loop-loop interfaces Strategies for programmable nucleic acid self-assembly Two main approaches can be distinguished for programmable self-assembly of nucleic acid architectures ( Figure 4 Programmable supramolecular RNA architectures. (a) 0D loop-receptor (RL) dimeric tectoRNA particle: the original 38 structure model (left) Stepwise assembly can be used to generate programmable architectures of finite size, with the position of each of the constitutive molecules known and therefore addressable within the final architecture. The first demonstration of this approach led to the fabrication of RNA nanogrids of finite size Each of these approaches can make use of additional nonmutually exclusive self-assembly strategies, such as algorithmic self-assembly, directed nucleation (or templated) self-assembly and scaffolded self-assembly. In algorithmic self-assembly, a set of nucleic acid tiles, defined as Wang tiles (see glossary), is viewed as the algorithm for a particular computational task leading to the formation of 1D, 2D and 3D patterns. This strategy was used to compute the formation of aperiodic fractal 2D patterns based on the Sierpinski triangle pattern Nucleic acid architectonics Jaeger and Chworos 539 The main strategies for programmable self-assembly. (a) Single-step self-assembly: all the molecules are mixed together and assembled through a slow cool annealing procedure (most DNA architectures are formed this way). (b) Stepwise hierarchical self-assembly [9 ,58]: specific sets of molecules are first separately assembled into small supramolecular entities that are then mixed in a stepwise fashion to form the final architecture. Hierarchical assembly is favored by the use of 48 interactions with different stabilities and magnesium requirements. (c) Scaffolded self-assembly or scaffolded DNA origami: a long singlestranded molecule is folded into an arbitrary shape with small oligonucleotides acting as staples Additional principles of nucleic acid architectonics Principle of orientational compensation The inherent asymmetric nature of RNA and DNA tiles can have a dramatic effect on the larger nanostructures that they form by introducing various degrees of curvature. By using the principle of orientational compensation, whereby two adjacent units are related by a local twofold pseudo-rotational axis of symmetry, one source of asymmetry can be locally eliminated, so that asymmetric tiles that are not perfectly flat can still assemble in a plane instead of forming nanotube

Design and synthesis of dynamically assembling DNA nanostructures

Kinetically controlled isothermal growth is fundamental to biological development, but it remains challenging to rationally design molecular systems that self-assemble isothermally into complex geometries via prescribed assembly and disassembly pathways. By exploiting the programmable chemistry of base pairing, sophisticated spatial and temporal control have both been demonstrated in DNA self-assembly, but largely as separate pursuits. This dissertation extends a new approach, called developmental self-assembly, that integrates temporal with spatial control by using a prescriptive molecular program to specify the kinetic pathways by which DNA molecules isothermally self-assemble into well-defined three-dimensional geometries.Chemistry and Chemical Biolog

DNA ORIGAMI ACTUATION AS A POWERFUL DYNAMIC AND TUNABLE ARCHITECTURE FOR PLASMONIC STRUCTURE

2013/2014In questa tesi presento il mio lavoro di ricerca di Dottorato in Nanotecnologie. Questo studio è concentrato sull'uso di nanotecnologia a DNA come strumento per la creazione di strutture nano-biologiche e funzionalizzazione di particelle d'oro. Le nano-particelle d'oro sono state largamente studiate e le loro proprietà sono state sfruttate per importanti applicazioni come la spettroscopia Raman, la rilevazione biologica e la terapia medica. Queste nano-particelle sono caratterizzate da una risonanza ottica plasmonica e proprietà di dispersione della luce ben definite ed esistono numerosi protocolli di provata efficienza per la loro funzionalizzazione chimica. Tra questi, il protocollo di auto-assemblaggio di DNA si è dimostrato eccellente nel comporre strutture di nano-particelle con dimensioni e forma controllate. Questo approccio è stato impiegato per l'ingegnerizzazione di proprietà ottiche, per la creazione di "hot spot” nel campo plasmonico in aggregati di nano-particelle e anche per la formazione di righelli plasmonici con dimeri di nano-particelle nei quali la loro spaziatura è controllata con precisione nanometrica. In questo studio confronto due strategie per la formazione di dimeri di nano-particelle d'oro usando l'ibridizzazione del DNA. Una di queste strategie mi ha permesso di raggiungere una al resa del 26% di formazioni di dimeri rispetto al totale delle AuNP, senza ulteriori procedure di filtrazione, dato che rappresenta il valore più alto riportato in letteratura; inoltre questo dato è stato replicato utilizzando sequenze di DNA molto corte, fino ad 11 nucleotidi, condizione che normalmente riduce l’efficienza del processo. Nella seconda parte della mia tesi, ho combinato le proprietà plasmoniche delle nano-particelle d'oro con strutture a DNA origami in modo da creare sistemi ibridi tra di loro. Questa combinazione mi ha permesso di esplorare architetture innovative per la il controllo della plasmonica con la prospettiva di essere un punto di partenza per lo sviluppo di biosensori. Ho sviluppato una strategia per un controllo innovativo, reversibile e continuo della risonanza plasmonica usando un'attuazione basata su DNA origami. Il meccanismo di attuazione è basato sull'ibridizzazione del DNA, in particolare si è visto uno spostamento del picco di risonanza fino a 6 nm utilizzando tre sequenza di DNA diverse. Il sistema proposto è potrà essere utilizzato per lo studio dei meccanismi di ibridazione di DNA in condizioni di stress controllato, oppure potrà essere usato come piattaforma per un controllo continuo della posizione della risonanza plasmonica o in spettroscopia Raman.XXVII Ciclo198

Hierarchical Assembly of DNA Nanostructures for Signal Transmission

In recent years scaffolded DNA origami has emerged as a novel technique for the construction of programmable nanostructures via molecular self-assembly. This technology provides unprecedented control over geometry and mechanical properties. These structures have demonstrated potential for a range of biomedical applications such as drug delivery, force measurement, and biomarker detection. Recent advancements have focused on the design of dynamic structures that can be triggered by DNA or other biological or environmental inputs to undergo actuated motion of the structure into different conformations. This work aims to exand on this foundation by developing of material systems where local conformational changes can be physically communicated to other parts of the material through propagated motion. We have designed a dynamic DNA nanostructure that can be assembled into arrays that can reach length scales ~10-100 times larger than the individual structure and can propagate conformational changes across the arrays. DNA strands specific to one end of the array initiate motion for the "trigger" structure at that end, which in turn propagates motion to subsequent structures in a sequential manner. This propagated motion is designed to transmit a signal across large distances. In the future, the ability to transmit a signal across micron-scale distances could lead to customizable molecular transport systems, programmable circuits, and long-range directional communication in biological environments.National Science FoundationInstitute for Materials ResearchUndergraduate Research Scholarship by the College of EngineeringA three-year embargo was granted for this item.Academic Major: Mechanical Engineerin