Geometric modeling, simulation, and visualization methods for plasmid DNA molecules

Plasmid DNA molecules are a special type of DNA molecules that are used, among other applications, in DNA vaccination and gene therapy. These molecules are characterized by, when in their natural state, presenting a closed-circular conformation and by being supercoiled. The production of plasmid DNA using bacteria as hosts implies a purification step where the plasmid DNA molecules are separated from the DNA of the host and other contaminants. This purification process, and all the physical and chemical variations involved, such as temperature changes, may affect the plasmid DNA molecules conformation by uncoiling or even by open them, which makes them useless for therapeutic applications. Because of that, researchers are always searching for new purification techniques that maximize the amount of supercoiled plasmid DNA that is produced. Computer simulations and 3D visualization of plasmid DNA can bring many advantages because they allow researchers to actually see what can happen to the molecules under certain conditions. In this sense, it was necessary to develop reliable and accurate geometric models specific for plasmid DNA simulations. This dissertation presents a new assembling algorithm for B-DNA specifically developed for plasmid DNA assembling. This new assembling algorithm is completely adaptive in the sense that it allows researchers to assemble any plasmid DNA base-pair sequence along any arbitrary conformation that fits the length of the plasmid DNA molecule. This is specially suitable for plasmid DNA simulations, where conformations are generated by simulation procedures and there is the need to assemble the given base-pair sequence over that conformation, what can not be done by conventional predictive DNA assembling methods. Unlike traditional molecular visualization methods that are based on the atomic structure, this new assembling algorithm uses color coded 3D molecular surfaces of the nucleotides as the building blocks for DNA assembling. This new approach, not only reduces the amount of graphical objects and, consequently, makes the rendering faster, but also makes it easier to visually identify the nucleotides in the DNA strands. The algorithm used to triangulate the molecular surfaces of the nucleotides building blocks is also a novelty presented as part of this dissertation. This new triangulation algorithm for Gaussian molecular surfaces introduces a new mechanism that divides the atomic structure of molecules into boxes and spheres. This new space division method is faster because it confines the local calculation of the molecular surface to a specific region of influence of the atomic structure, not taking into account atoms that do not influence the triangulation of the molecular surface in that region. This new method also guarantees the continuity of the molecular surface. Having in mind that the aim of this dissertation is to present a complete set of methods for plasmid DNA visualization and simulation, it is also proposed a new deformation algorithm to be used for plasmid DNA Monte Carlo simulations. This new deformation algorithm uses a 3D polyline to represent the plasmid DNA conformation and performs small deformations on that polyline, keeping the segments length and connectivity. Experiments have been performed in order to compare this new deformation method with deformation methods traditionally used by Monte Carlo plasmid DNA simulations These experiments shown that the new method is more efficient in the sense that its trial acceptance ratio is higher and it converges sooner and faster to the elastic energy equilibrium state of the plasmid DNA molecule. In sum, this dissertation successfully presents an end-to-end set of models and algorithms for plasmid DNA geometric modelling, visualization and simulation

RNA AS A UNIQUE POLYMER TO BUILD CONTROLLABLE NANOSTRUCTURES FOR NANOMEDICINE AND NANOTECHNOLOGY

RNA nanotechnology is an emerging field that involves the design, construction and functionalization of nanostructures composed mainly of RNA for applications in biomedical and material sciences. RNA is a unique polymer with structural simplicity like DNA and functional diversity like proteins. A variety of RNA nanostructures have been reported with different geometrical structures and functionalities. This dissertation describes the design and construction of novel two-dimensional and three-dimensional self-assembled RNA nanostructures with applications in therapeutics delivery, cancer targeting and immunomodulation. Firstly, by using the ultra-stable pRNA three-way junction motif with controllable angles and arm lengths, tetrahedral architectures composed purely of RNA were successfully assembled via one-pot bottom-up assembly with high efficiency and thermal stability. By introducing arm sizes of 22 bp and 55 bp, two RNA tetrahedrons with similar global contour structure but with different sizes of 8 nm and 17 nm were successfully assembled. The RNA tetrahedrons were also highly amenable to functionalization. Fluorogenic RNA aptamers, ribozyme, siRNA, and protein-binding RNA aptamers were integrated into the tetrahedrons by simply fusing the respective sequences with the tetrahedral core modules. Secondly, I reported the design and construction of molecularly defined RNA cages with cube and dodecahedron shapes based on the stable pRNA 3WJ. The RNA cages can be easily self-assembled by single-step annealing. The RNA cages were further characterized by gel electrophoresis, cryo-electron microscopy and atomic force microscopy, confirming the spontaneous formation of the RNA cages. I also demonstrated that the constructed RNA cages could be used to deliver model drugs such as immunomodulatory CpG DNA into cells and elicit enhanced immune responses. Thirdly, by using the modular multi-domain strategy, molecular defined RNA nanowires can be successfully self-assembled via a bottom-up approach. Only four different 44-nucleotide single-stranded RNAs were used to assemble the RNA nanowire. The reported RNA nanowire has the potential to be explored in the future as the carrier for drug delivery or matrix for tissue engineering. Fourthly, the construction of RNA polygons for delivering immunoactive CpG oligonucleotides will be presented. When CpG oligonucleotides were incorporated into the RNA polygons, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced, while RNA polygon controls induced unnoticeable cytokine induction. Moreover, the RNA polygons were delivered to macrophages specifically and the degree of immunostimulation greatly depended on the size, shape, and the number of payload per RNA polygon. Collectively, these findings demonstrated RNA nanotechnology can produce controllable nanostructures with different functionalities and result in potential applications in nanomedicine and nanotechnology

Accurate phase diagram of tetravalent DNA nanostars

We evaluate, by means of molecular dynamics simulations employing a realistic DNA coarse-grained model, the phase behaviour and the structural and dynamic properties of tetravalent DNA nanostars, i.e. nanoconstructs completely made of DNA. We find that, as the system is cooled down, tetramers undergo a gas--liquid phase separation in a region of concentrations which, if the difference in salt concentration is taken into account, is comparable with the recently measured experimental phase diagram [S. Biffi \textit{et al}, Proc. Natl. Acad. Sci, \textbf{110}, 15633 (2013)]. We also present a mean-field free energy for modelling the phase diagram based on the bonding contribution, derived by Wertheim in its studies of associating liquids, combined with mass action law expressions appropriate for DNA binding and a numerically evaluated reference free energy. The resulting free energy qualitatively reproduces the numerical data. Finally, we report information on the nanostar structure, e.g. geometry and flexibility of the single tetramer and of the collective behaviour, providing a useful reference for future small angle scattering experiments, for all investigated temperatures and concentrations.Comment: 10 pages, 11 figure

Coarse-Grained Simulations of the Self-Assembly of DNA-Linked Gold Nanoparticle Building Blocks

The self-assembly of nanoparticles (NPs) of varying shape, size, and composition for the purpose of constructing useful nanoassemblies with tailored properties remains challenging. Although progress has been made to design anisotropic building blocks that exhibit the required control for the precise placement of various NPs within a defined arrangement, there still exists obstacles in the technology to maximize the programmability in the self-assembly of NP building blocks. Currently, the self-assembly of nanostructures involves much experimental trial and error. Computational modeling is a possible approach that could be utilized to facilitate the purposeful design of the self-assembly of NP building blocks into a desired nanostructure. In this report, a coarse-grained model of NP building blocks based on an effective anisotropic mono-functionalization approach, which has shown the ability to construct six building block configurations, was used to simulate various nanoassemblies. The purpose of the study was to validate the model’s ability to simulate the self-assembly of the NP building blocks into nanostructures previously produced experimentally. The model can be programmed to designate up to six oligonucleotides attached to the surface of a Au NP building block, with a modifiable length and nucleotide sequence. The model successfully simulated the self-assembly of Au NP building blocks into a number of previously produced nanostructures and demonstrated the ability to produce visualizations of self-assembly as well as calculate interparticle distances and angles to be used for the comparison with the previous experimental data for validation of the model. Also, the model was used to simulate nanoassemblies which had not been produced experimentally for its further validation. The simulations showed the capability of the model to use specific NP building blocks and self-assemble. The coarse-grained NP building block model shows promise as a tool to complement the purposeful experimental design of functional nanostructures

Synthetic Supramolecular Systems in Life-like Materials and Protocell Models

One of the biggest challenges in modern chemistry is the preparation of synthetic materials with life-like behavior for the assembly of artificial cells. In recent years, numerous artificial systems that mimic cellular components and functions have been developed. Supramolecular chemistry plays a key role in such cell mimics given that non-covalent interactions control the shape and function of many biomolecules, such as DNA base pairing, protein structure, ligand-receptor binding, and lipid membrane packing. However, the complexity of living cells constitutes a major challenge for their bottom-up assembly from pure synthetic materials. Inspired by the building blocks of nature, a wide range of supramolecular systems have been developed to reproduce cellular functions such as cell-cell communication, signaling cascades, and dynamic cytoskeleton assemblies. This review surveys a selection of key advances in synthetic derivatives of biomolecules with supramolecular organization and life-like behavior by addressing their non-covalent foundation and integration as increasingly complex protocell modelsThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI; SAF2017-89890-R), Xunta de Galicia (ED431C 2017/25, 2016-AD031, and ED431G/09), ISCIII (RD16/0008/003), and the European Commission (EC; European Regional Development Fund). I.I. thanks the EC and AEI for MSCA-IF (2018-843332) and JdC (FJCI-2017-31795) fellowships, respectively. J.M. received a Ramón y Cajal grant (RYC-2013-13784), an ERC-Stg grant (DYNAP-677786), and a Young Investigator Grant from the Human Frontier Science Program (RGY0066/2017)S

Rational Design and Development of Purely Peptidic Amphiphiles for Gene Delivery

Gene therapy depends on viral and non-viral delivery systems to ferry nucleic acids into target cells 1,2. In recent years, gene insertion and interference therapies have made a ground breaking impact in the treatment of rare inherited diseases, neurological disorders, cardiac diseases, and cancer 3. Several disadvantages associated with viral vectors, such as high toxicity and immunogenicity, limitation in size of transgenic DNA, and high manufacturing cost have triggered the rapid expansion of non-viral delivery systems including peptide-based vectors 4. The advantages of peptides are not only their biocompatibility and biodegradability, but sheer limitless possible combinations and modifications of amino acid residues that are able to promote the assembly of modular, multiplexed delivery systems 5. With the advantages of peptides in mind, we looked into the potential of peptide-based nanoassemblies in developing a non-viral gene delivery system. The thesis is structured to successively address (i) the design and development of purely amphiphilic peptides self-assembling into multicompartment micelles (MCMs), (ii) the efficient DNA cargo entrapment up to 100 nucleotides in length into self-assembled peptide MCMs and the delivery thereof, and (iii) targeting of oligonucleotides to the nucleus via a nuclear localization signal (NLS) integrated in the peptide-based carrier. The challenge was to rationally design the peptides and identify the proper conditions in which the DNA entrapment does not interfere with multicompartment micellar self-assembly. In addition, to fulfil the prerequisites of a successful gene delivery system that overcomes cellular barriers, we incorporated biologically active amino acids in our peptide sequences. A systematic characterization of the physicochemical features of the peptidic nanostructures was carried out to gain insight into the mechanism underlying self-assembly and to shed light on ways to tune these features for prospective biomedical applications. Taking into account our findings on how the size/type of genetic payload together with the peptide amphiphile’s charge and length impact the self-assembly process, we successfully established a non-toxic, purely peptidic delivery system that serves as a cornerstone for developing oligonucleotide therapy platforms

Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer‐scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self‐assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site‐specific (spatially addressable) DNA ‘staples’. This revolutionary approach has led to the creation of a multitude of two‐dimensional and three‐dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. WIREs Nanomed Nanobiotechnol 2012, 4:139–152. doi: 10.1002/wnan.170 For further resources related to this article, please visit the WIREs website .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90282/1/170_ftp.pd

Elektrostatisk självorganisation av DNA origami och guldnanopartiklar

Spatially well-ordered structures of gold nanoparticles(AuNPs) and other metal nanoparticles have unique electronic, magnetic and optical properties, and hence there is ever-increasing interest towards these kinds of nanomaterials. DNA and DNA nanostructures have successfully been used to direct the higher-ordered arrangement of AuNPs, but the programmable arrangement of them into larger, well-defined structures is still challenging. The objective of this thesis is to establish a self-assembly method based on electrostatic interactions in which DNA origami nanostructures can be used to guide the higher ordered arrangement of cationic AuNPs in a controlled and programmable manner. The AuNP binding properties of different DNA origami structures was studied with UV/Vis spectroscopy and agarose gel electrophoretic mobility shift assay. DNA origami-AuNP assemblies were formed during dialysis against decreasing ionic strength, and the formed assemblies were characterized using small-angle X-ray scattering, transmission electron microscopy and cryogenic electron tomography. Electrostatic self-assembly of DNA origami 6HB nanostructures and small AuNPs (D_core = 2.5 nm, D_hydrodynamic_diameter = 8.5 nm) yielded highly ordered superlattice structures with a 3D tetragonal lattice structure, whereas other studied combinations of DNA origami structures and AuNPs resulted in amorphous aggregates. These results suggest that both shape and charge complementarity between the building blocks are needed for well-ordered structures to be formed through electrostatic self-assembly. According to the results, electrostatic self-assembly guided by DNA origami structures seems promising for construction of novel, well-ordered structures with unique properties, such as lattice geometry, designed specifically for the chosen application.Guldnanopartiklar och andra metallnanopartiklar organiserade i välordnade strukturer har unika elektroniska, magnetiska och optiska egenskaper och därför finns det ett ständigt växande intresse för dessa typer av nanomaterial. DNA och nanostrukturer av DNA har framgångsrikt använts för att framställa välordnade, förutbestämda tredimensionella guldnanopartikelstrukturer, men det finns fortfarande utmaningar att tackla. Målet med detta diplomarbete är att utveckla en metod för självorganisation baserat på elektrostatiska interaktioner i vilken DNA-origaminanostrukturer på ett programmerbart och kontrollerat sätt kan användas för att styra hurudana strukturer som byggs upp av katjoniska guldnanopartiklar. De olika DNA-origamistrukturernas förmåga att binda guldnanopartiklar studerades med UV/Vis-spektroskopi och agarosgelelektrofores. DNA-origami-guldnanopartikelsystem byggdes upp genom dialys mot stegvis minskade jonkoncentrationer och de uppkomna strukturerna karaktäriserades med lågvinkelspridning, transmissionselektronmikroskopi och kryelektrontomografi. Elektrostatisk självorganisation av DNA-origami 6HB nanostrukturer och små guldnanopartiklar (D_kärna = 2.5 nm, D_hydrodynamisk_diameter = 8.5 nm) gav välordnade tredimensionella tetragonala kristallstrukturer, medan andra undersökta kombinationer av DNA origami strukturer och guldpartiklar endast resulterade i amorfa strukturer. Detta indikerar att de enskilda byggstenarna behöver kompletterande form och laddning för att välordnade strukturer skall kunna byggas upp genom elektrostatik självorganisation. Det förefaller dock finnas goda framtidsutsikter för elektrostatisk självorganisation som en metod att framställa välordnade strukturer med egenskaper, så som typ av kristallstruktur, lämpliga just för det önskade användningsområdet