Добірка наукової літератури з теми "DNA self-assembling"

Hierarchical self-assembly is a process in which molecular building blocks form intermediate structures that self organise at macroscopic level. Remarkable examples can be found in nature, like, for instance, DNAs or viruses. Self-assembly offers interesting strategies to build new complex materials: Therefore, it is very important to understand its mechanism to design and control molecular architectures and to build structures with desired properties and morphologies. A major question is how the shape of the building blocks influences self- assembly. In this context, chirality plays a crucial role: It is extremely sensitive to subtleties on the molecular scale and can guide self-assembly; furthermore, chirality can act as an amplifier of changes that occur at the molecular level. From the theoretical point of view, the difficulty derives from the need of multiscale methods and models, able to connect the different length scales. To take into account the relationship among the building blocks, their supramolecular organization, and the properties of the aggregates, a detailed representation of intermolecular interactions is needed: This description has to be integrated into a suitable modeling of the system behaviour on a much longer length scale. This thesis deals with the development and implementation of models for chirality propagation from the molecular to the meso- and macroscopic levels in self-assembling systems. In particular, the research has been carried out along three lines. The first deals with self-assembly of hard helices, leading to the formation of anisotropic phases of vari- ous symmetry. The second topic is the linear aggregation and formation of liquid crystal phases by double-stranded nucleic acid oligomers (dsNA): The relationship between the sequence of oligonucleotides, their self-assembly and the properties of their cholesteric phase is investigated. The last topic is the aggregation of porphyrin-polypeptide conjugates in water. Depending on the problem and the length scale, we used different theoretical and com- putational methods, in particular: Statistical theories of liquids and molecular dynamics simulations (both atomistic and coarse-grained). The first and third topics have been carried out in collaboration with experimentalists, while for the second other groups of theoreticians have been involved. This thesis is organized in three parts. In Chapter 1, the concepts of self-assembling, chi- rality propagation and multiscale modeling are introduced. Moreover this Chapter presents an outline of the main properties of liquid crystals. The first Part, from Chapter 2 to Chapter 4, presents the study on the anisotropic phases formed by hard helices. Chapter 2 presents a study of the nematic phase, using an Onsager- like theory. The theoretical results are compared with Monte Carlo simulations. In Chapter 3, a theoretical model for the cholesteric phase is presented and used to investigate the relationship between the helical shape and the properties of the cholesteric phase. In Chapter 4, the complete phase diagram of hard helices is presented, together with the characterization of a novel chiral nematic phase. The second Part deals with liquid crystal phases formed by dsNA. Chapter 5 focuses on the relation between the sequence of oligonucleotides and their organization in the cholesteric phase, using a molecular theory and coarse-grained modelling based on sequence dependent structural data. Chapter 6 describes the theoretical model for the cholesteric phase formed by self-assembling oligomers, which integrates the theory for cholesteric order presented in Chapter 3 with that for linear aggregation in the nematic phase. The last Part, from Chapter 7 to Chapter 9, deals with the aggregation of porphyrin- peptide conjugates in water. In Chapter 7 the main concepts of circular dichroism are introduced and the state-of-the-art of self-assembly of porphyrins is reviewed. Chapter 8 describes atomistic molecular dynamics simulation of aggregates of porphyrin-peptide conjugates. Chapter 9 presents a study of the same systems by coarse-grained molecular dynamics simulations, using the MARTINI model. Finally, Chapter 10 presents a summary, which highlights the relevant results obtained in this Thesis, and three Appendices follow.
L’autoassemblaggio gerarchico è un processo nel quale "building block" molecolari formano strutture intermedie che si auto-organizzano a livello macroscopico. Molti esempi possono essere trovati in natura, come il DNA o i virus. L’autoassemblaggio offre interessanti strategie per costruire nuovi materiali complessi: di conseguenza, risulta molto importante capirne i meccanismi per disegnare e controllare le architetture molecolari e per costruire strutture con proprietà e morfologie desiderate. Uno dei principali quesiti cui dare risposta è come la forma del building block influenzi l’autoassemblaggio. In questo contesto, la chiarità svolge un ruolo cruciale: è estremamente sensibile ai dettagli molecolari e può guidare l’autoassemblaggio; inoltre, essa può amplificare le differenze che avvengono su scala molecolare. Dal punto di vista teorico, la difficoltà deriva dalla necessità di metodi e modelli mul- tiscala, capaci di connettere le differenti scale di lunghezza. Per tenere in considerazione la relazione tra i building blocks, la loro organizzazione supramolecolare e le proprietà degli aggregati, si rende necessaria una rappresentazione dettagliata delle interazioni inter- molecolari: questa descrizione deve poi essere integrata in una modellizazione opportuna del comportamento del sistema su scale di lunghezza più grandi. Il tema di questa tesi è lo sviluppo e l’implementazione di modelli per la propagazione di chiralità dalla scala molecolare alla scala meso e macroscopica in sistemi autoassem- blati. Tre diverse linee di ricerca sono state portate avanti. La prima si è concentrata sull’autoassemblaggio di eliche dure, ed in particolare sulla formazione di fasi anisotrope di diversa simmetria. Il secondo argomento riguarda l’aggregazione lineare e la formazione di fasi liquido-cristalline a partire da oligomeri di acidi nucleici a doppio filamento prendendo in considerazione le relazioni tra la sequenza di oligonucleotidi, l’autoassemblaggio e le proprietà della loro fase colesterica. L’ultimo argomento è dedicato all’autoassemblaggio di coniugati porfirina-peptide in acqua. In base al problema e alla scala di lunghezza, sono stati utilizzati diversi metodi teorici e computazionali, in particolare: teorie statistiche dei liquidi e simulazioni di dinamica molecolare (sia atomistica che a grana grossa). La prima e la terza linea di ricerca sono stati condotte in collaborazione con sperimentali, mentre la seconda ha coinvolto altri gruppi teorici. La tesi è organizzata in tre parti. Nel Capitolo 1, il processo di autoassemblaggio, la propagazione di chiralità e il concetto di modellizzazione multiscala vengono descritti. Inoltre in questo Capitolo si presentano le principali proprietà dei cristalli liquidi. Nella prima Parte, dal Capitolo 2 al Capitolo 4, viene presentato il lavoro svolto sulle fasi anisotrope di eliche dure. Il Capitolo 2 presenta lo studio della fase nematica usando una teoria Onsager-like. I risultati teorici sono confrontati con simulazioni Monte Carlo. Nel Capitolo 3, viene presentato un modello teorico per la fase colesterica utilizzato poi per studiare l’effetto della forma elicoidale sulle proprietà della fase colesterica. Nel Capitolo 4 viene presentato l’intero diagramma di fase delle eliche dure, assieme alla caratterizzazione di una nuova fase nematica chirale. La seconda Parte concerne le fasi liquido-cristalline formate da dsNA. Il Capitolo 5 si focalizza sulla relazione tra la sequenza di oligonucleotidi e la loro organizzazione nella fase colesterica utilizzando una teoria molecolare e la modellizzazione a grana grossa, basata su dati strutturali dipendenti dalla sequenza. Nel Capitolo 6, viene descritto il modello teorico per la fase colesterica formata da oligomeri autoassemblati, che mette assieme la teoria per l’ordine colesterico presentata nel Capitolo 3 con quella dell’aggregazione lineare in fase nematica. L’ultima Parte, dal Capitolo 7 al Capitolo 9, si concentra sull’aggregazione di coniugati porfirina-peptide in acqua. Nel Capitolo 7, vengono introdotti i principali concetti relativi al dicroismo circolare e viene commentato lo stato dell’arte dell’autoassemblaggio di porfirine. Il Capitolo 8 descrive le simulazioni atomistiche di dinamica molecolare di aggregati porfirina- peptide . Capitolo 9 presente uno studio degli stessi sistemi condotto attraverso simulazioni di dinamica molecolare a grana grossa, che utilizzano il modello MARTINI. Infine, il Capitolo 10 presenta un sommario delle tre linee di ricerca, mettendo in evidenza i risultati notevoli ottenuti in questa tesi. Seguono poi tre Appendici.

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 Biology

Computational protein design (CPD) is a burgeoning field that uses a physical-chemical or knowledge-based scoring function to create protein variants with new or improved properties. This exciting approach has recently been used to generate proteins with entirely new functions, ones that are not observed in naturally occurring proteins. For example, several enzymes were designed to catalyze reactions that are not in the repertoire of any known natural enzyme. In these designs, novel catalytic activity was built de novo (from scratch) into a previously inert protein scaffold. In addition to de novo enzyme design, the computational design of protein-protein interactions can also be used to create novel functionality, such as neutralization of influenza. Our goal here was to design a protein that can self-assemble with DNA into nanowires. We used computational tools to homodimerize a transcription factor that binds a specific sequence of double-stranded DNA. We arranged the protein-protein and protein-DNA binding sites so that the self-assembly could occur in a linear fashion to generate nanowires. Upon mixing our designed protein homodimer with the double-stranded DNA, the molecules immediately self-assembled into nanowires. This nanowire topology was confirmed using atomic force microscopy. Co-crystal structure showed that the nanowire is assembled via the desired interactions. To the best of our knowledge, this is the first example of a protein-DNA self-assembly that does not rely on covalent interactions. We anticipate that this new material will stimulate further interest in the development of advanced biomaterials.

The field of DNA nanotechnology offers a wide range of design strategies with which nanometer-sized structures with a desired shape, size and aspect ratio can be built. The most established techniques in the field rely on close-packed 'solid' DNA nanostructures produced with either the DNA origami or the single-stranded tile techniques. These structures depend on high-salt buffer solutions and require more material than comparable size hollow wireframe structures. This dissertation explores the construction of hollow wireframe DNA nanostructures composed of equilateral triangles. To achieve maximal material efficiency the design is restricted to use a single DNA double helix per triangle edge. As a proof of principle, the DNA origami technique is extended to produce a series of truss structures including the flat, tetrahedral, octahedral, or irregular dodecahedral truss designs. In contrast to close packed DNA origami designs these structures fold at low-salt buffer conditions. These structures have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. The design process of these structures is simplified by a custom design software. Next, the triangulated construction motif is extended to the single-stranded DNA tile technique. A collection of finite structures, as well as one-dimensional crystalline assemblies is explored. The ideal assembly conditions are determined experimentally and using molecular dynamics simulations. A custom design software is presented to simplify the design and handling of these structures. At last, the cost-effective prototyping of triangulated wireframe DNA origami structures is explored. This is achieved through the introduction of single-stranded “gap” regions along the triangle edges. These gap regions are then filled using a DNA polymerase rather than by synthetic oligonucleotides. This technique also allows the mechanical transformation of these structures, which is exemplified by the transition of a bent into a straight structure upon completion of the gap filling.:Abstract v Publications vii Acknowledgements ix Contents xi Chapter 1 A short introduction into DNA nanotechnology 1 1.1 Nanotechnology 1 1.1.1 Top down 1 1.1.2 Bottom up 3 1.2 Deoxyribonucleic acid (DNA) 4 1.3 DNA Nanotechnology 6 1.3.1 Tile based assembly 9 1.3.2 DNA origami and single-stranded tiles 10 1.3.3 Some applications of DNA nanotechnology 12 1.3.4 Wireframe structures 15 1.3.5 Computational tools and DNA nanotechnology. 17 Chapter 2 Motivation and objectives 19 Chapter 3 Design and Synthesis of Triangulated DNA Origami Trusses 20 3.1 Introduction 20 3.2 Results and Discussion 21 3.2.1 Design 21 3.2.2 Nomenclature and parameters of the tube structures 23 3.2.3 Gel electrophoreses analysis 25 3.2.4 Imaging of the purified structures 26 3.2.5 Optimizing the folding conditions 28 3.2.6 Comparison to vHelix 29 3.3 Conclusions 29 3.4 Methods 30 3.4.1 Standard DNA origami assembly reaction. 30 3.4.2 Gel purification. 30 3.4.3 AFM sample preparation. 31 3.4.4 TEM sample preparation. 31 3.4.5 Instructions for mixing the staple sets. 31 Chapter 4 Triangulated wireframe structures assembled using single-stranded DNA tiles 33 4.1 Introduction 33 4.2 Results and Discussion 35 4.2.1 Designing the structures 35 4.2.2 Synthesis of test structures 37 4.2.3 Molecular dynamics simulations of 6-arm junctions 38 4.2.4 Assembly of the finite structures 40 4.2.5 Influence of salt concentration and folding times 42 4.2.6 Molecular dynamics simulations of the rhombus structure 43 4.2.7 1D SST crystals 44 4.2.8 Controlling the crystal growth 46 4.3 Conclusions 48 4.4 Methods 49 4.4.1 SST Folding 49 4.4.2 Agarose Gel Electrophoresis 49 4.4.3 tSEM Characterization 49 4.4.4 AFM Imaging 49 4.4.5 AGE-Based Folding-Yield Estimation 49 4.4.6 Molecular Dynamics Simulations 50 Chapter 5 Structural transformation of wireframe DNA origami via DNA polymerase assisted gap-filling 52 5.1 Introduction 52 5.2 Results and Discussion 54 5.2.1 Design of the Structures 54 5.2.2 Folding of Gap-Structures 56 5.2.3 Inactivation of Polymerase. 57 5.2.4 Secondary Structures. 58 5.2.5 Folding Kinetics of Gap Origami. 60 5.3 Conclusions 61 5.4 Methods 62 5.4.1 DNA origami folding 62 5.4.2 Gap filling of the wireframe DNA origami structures 63 5.4.3 Agarose gel electrophoresis 63 5.4.4 PAGE gel analysis 63 5.4.5 tSEM characterization 64 5.4.6 AFM imaging 64 5.4.7 AGE based folding-yield estimation 64 5.4.8 Gibbs free energy simulation using mfold 65 5.4.9 List of sequence for folding the DNA origami triangulated structures 65 Chapter 6 Summary and outlook 67 Appendix 69 A.1 Additional figures from chapter 369 A.2 Additional figures from chapter 4 77 A.3 Additional figures from chapter 5 111 Bibliography 127 Erklärung 138

DNA origami is a novel self-assembly technique that can be used to form various

2D and 3D objects, and to position matter with nanometer accuracy. It has been

used to coordinate the placement of nanoscale objects, both organic and inorganic, to make molecular motor and walkers; and to create optically active nanostructures. In this dissertation, DNA origami templates are used to assemble plasmonic structures. Specifically, engineered Surface Enhanced Raman Scattering (SERS) substrates were fabricated. Gold nanoparticles were selectively placed on the corners of rectangular origami and subsequently enlarged via solution-based metal deposition. The resulting assemblies exhibited "hot spots" of enhanced electromagnetic field between the nanoparticles. These hot spots significantly enhanced the Raman signal from Raman molecules covalently attached to the assemblies. Control samples with only one nanoparticle per DNA template, which therefore lacked inter-particle hot spots, did not exhibit strong enhancement. Furthermore, Raman molecules were used to map out the hot spots' distribution, as the molecules are photo-damaged when experiencing a threshold electric field. This method opens up the prospect of using DNA origami to rationally engineer and assemble plasmonic structures for molecular spectroscopy.

Dissertation

碩士
中山醫學大學
應用化學系碩士班
103
For effective gene delivery, structural degradation of synthetic carriers is crucial to nucleic acids releasing on the transfection time scale. In this study, we have synthesized the amphiphilic dendritic scaffolds with a photolabile o-nitrobenzyl (o-NB) group that can enable the structural decomposition and controlled release of nucleic acids under active light stimulation. The amphiphilic counterpart composed of a lipophilic cholesterol and hydrophilic poly(amido amine) (PAMAM) dendron allows the self-assembly into a core-shell-like pseudodendrimer above the critical aggregation concentration (CAC) of approximately 20 μM. On the basis of electrostatic interaction, the polycationic pseudodendrimers is capable of forming stable complexes with polyanionic cyclic reporter gene (pEGFP-C1) under low charge excess value, suggesting substantial binding affinity of the dednron assembly toward plasmid DNA. Because the o-NB group in the dendritic structure undergoes efficient photolytic cleavage, in vitro test shows that thus-formed “dendriplexes” are readily dissociated under 365-nm light irradiation, causing effective dendron degradation accompanied by DNA release. This photochemical strategy provides an opportunity to control over the gene binding and releasing in a spatiotemporal manner.

How can we engineer components to assemble into complex structures in a reliable manner? Experimental methods rely on interactions such as surface properties, magnetic dipoles, charge, and polarizability. Here pressure is shown to have the power to hold a system together, or to take it apart. Specific diagrams give visual clues to the nature of the pressure that is present. The ability to identify the properties of pressure will allow researchers to manipulate pressure’s power to their advantage. DNA base pairing, polynucleotide chain properties, and an enzyme-catalyzed reaction are used as examples of self-assembling structures conforming to laws of pressure.

An electronic microarray has been used to carry out directed self-assembly of higher order 3D structures from Biotin/Streptavidin and DNA derivatized nanoparticles. Structures with more than forty layers of alternating biotin and streptavidin and DNA nanoparticles were fabricated using a 400 site CMOS microarray system. In this process, reconfigurable electric fields produced by the microarray device have been used to rapidly transport, concentrate and accelerate the binding of 40 and 200 nanometer biotin, streptavidin, DNA and peroxidase derivatized nanoparticles to selected sites on the microarray. The nanoparticle layering process takes less than one minute per layer (10–20 seconds for addressing and binding nanoparticles, 40 seconds for washing). The nanoparticle addressing/binding process can be monitored by changes in fluorescence intensity as each nanoparticle layer is deposited. The final multilayered 3-D structures are about two microns in thickness and 50 microns in diameter. Work is now focused on assembling “micron size” biosensor devices from bio-molecule derivatized luminescent and fluorescent nanoparticles. The proposed structure for a nanolayered glucose sensor device includes a base layer of biotin/streptavidin nanoparticles, a layer of glucose oxidase derivatized nanoparticles, a layer of peroxidase derivatized nanoparticles, a layer of quantum dots, and a final layer of biotin/streptavidin nanoparticles. Such a device will serve as a prototype for a wide variety of applications which includes other biosensor devices, lab-on a-chip devices, in-vivo drug delivery systems and “micron size” dispersible bio/chem sensors for environmental, military and homeland security applications.