The question about the origins of life often appears as a difficult question to answer. A more reliable candidate molecule for the chemical origins of life would be a molecule that is capable of making copies of itself from simple precursors. With the finding of the catalytic activities of RNA molecules by Thomas Cech and Sid Altman in late 1980s, the term ribozyme was introduced to define an RNA molecule with catalytic activity. The RNA World is a conceptual period in the early stages in the development of life because RNA simultaneously possesses evolvability and catalytic function. An RNA molecule that could evolve in such a fashion is likely to have been one of the Earth's first life forms. The most important problem facing the RNA World is the difficulty of prebiotic synthesis of RNA. Different prebiotic environments could provide the right reaction conditions for synthesis of catalytically active RNA molecules. Most importantly, these environments can support new ways to assemble monomers into polymers. In order to understand and demonstrate how small inactive RNA oligomers can self-assemble into an autocatalytic ribozyme molecule, here I have used the Azoarcus Group I intron. First, the fragmentation and the self-assembly reactions were done using the natural IGS-tag combination of the Azoarcus group I intron, which is GUG/CAU. The main purpose was to experimentally demonstrate that the ribozyme can be broken down into five or more shorter RNA fragments and these fragments can self-assemble into a catalytically active covalent full-length molecule. Then, with the successful demonstration that five inactive RNA fragments can self-assemble, the next step to test the other possible breaking locations with mutated IGS/tag combinations. A new IGS/tag pair GCG-CGU also successfully demonstrated the five-piece self-assembly reaction. Finally, we tested these reactions in different Mg2+ concentrations to optimize the self-assembly reactions. By focusing not only one single reaction but on a collection of different sequence requirement combinations and with the development of evaporation and subsequent rehydration by spinning down methods this study successfully illustrates that covalent self-assembly from inactive RNA oligomers is possible. Therefore, this thesis work focuses on a more broader aspect of intermolecular interactions in the study of the RNA World, as illustrated in following chapters.

Durch die Spleißreaktion werden nicht-kodierende Sequenzelemente (Introns) aus eukaryotischen Vorläufer-mRNAs entfernt und die kodierenden Sequenzelemente (Exons) miteinander zu einem offenen Leserahmen verbunden. Dieser zentrale Prozessierungsschritt während der eukaryotischen Genexpression wird durch das Spleißosom katalysiert, das aus den vier kleinen nukleären Ribonucleoproteinpartikeln (snRNPs) U1, U2, U4/U6 und U5, sowie einer Vielzahl weiterer Proteinfaktoren gebildet wird. Alle snRNPs besitzen eine gemeinsame ringförmige Kernstruktur, die aus sieben gemeinsamen Sm-Proteinen (SmB/B‘-D1-D2-D3-E-F-G) besteht, die ein einzelsträngiges Sequenzmotiv auf der snRNAs binden. Während sich diese, als Sm-Core-Domäne bezeichnete Struktur in vitro spontan ausbilden kann, erfolgt die Zusammenlagerung in vivo in einem assistierten und hochregulierten Prozess. Dieser ist abhängig von insgesamt mindestens 12 trans-agierenden Faktoren, die in den PRMT5- und SMN-Komplexen organisiert sind. Der PRMT5-Komplex agiert in der frühen Phase der Zusammenlagerung, indem er die Sm-Proteine durch die Untereinheit pICln rekrutiert und die symmetrische Methylierung von Argininresten in den C terminalen Schwänzen von SmB/B‘, SmD1 und SmD3 katalysiert. Als Resultat dieser frühen Phase befinden sich die Sm-Proteine SmD1-D2-E-F-G und SmB/B‘-D3 in zwei getrennten und durch pICln organisierten Komplexen. Während SmB/B‘-D3-pICln am PRMT5-Komplex gebunden bleibt, existiert der zweite Komplex als freies Intermediat mit einem Sedimentationskoeffizienten von 6S. Diese Intermediate können nicht mit RNA assoziieren, sodass für die Fortsetzung des Zusammenlagerungsprozesses die Interaktion der Sm-Proteine mit pICln aufgelöst werden muss. Dies geschieht in der späten Phase der Sm-Core-Zusammenlagerung, in der die Sm-Proteine vom SMN-Komplex (bestehend aus SMN, Gemin2-8 und unrip) übernommen werden und pICln dissoziiert wird. Dadurch werden die Sm-Proteine für ihre Interaktion mit der snRNA aktiviert und können auf die Sm-Bindestelle transferiert werden, wodurch die Formierung des Sm-Core abgeschlossen wird. Im Rahmen dieser Arbeit konnten mit Hilfe einer Kombination röntgenkristallographischer und elektronenmikroskopischer Methoden zwei wichtige Intermediate dieses Zusammenlagerungs-prozesses strukturbiologisch charakterisiert werden. Bei diesen Intermediaten handelt es sich um den 6S-Komplex, sowie um ein Sm-Protein-Transferintermediat mit einem Sedimentations-koeffizienten von 8S. In diesem ist der 6S-Komplex an zwei zentrale Untereinheiten des SMN-Komplexes (SMN und Gemin2) gebunden, während pICln den Komplex noch nicht verlassen hat. Der 8S-Komplex stellt daher ein „gefangenes“ Intermediat zwischen der frühen und späten Phase der Zusammenlagerung dar. Zunächst gelang es eine erste Kristallform des rekombinant hergestellten 8S-Komplexes zu erhalten, die jedoch keine Strukturlösung erlaubte. Durch eine kombinierte Optimierung der Kristallisationsbedingung und der verwendeten Proteine wurde eine weitere ähnliche Kristallform erhalten, mit der die Kristallstruktur des 8S-Komplexes gelöst werden konnte. Die Kristallisation des 6S-Komplexes gelang im Anschluss auf Basis der Hypothese, dass Kristalle beider Komplexe aufgrund der kompositionellen Verwandtschaft zwischen 6S und 8S auch Ähnlichkeiten in der Architektur ihrer Kristallgitter aufweisen könnten. Daher wurden innerhalb von pICln gezielt Aminosäuren substituiert, die sich innerhalb von Kristallkontakten der 8S-Kristalle befanden und konformationell eingeschränkt waren. Mit entsprechend rekonstituierten 6S-Präparationen konnten dann zwei Kristallformen erzeugt werden, die eine Strukturlösung des 6S-Komplexes ermöglichten. Durch die Kristallstruktur des 6S-Komplexes konnte für pICln eine strukturelle Mimikry der Sm-Proteine identifiziert werden. Diese ermöglicht eine Bindung der Sm-Proteine und eine frühzeitige topologische Organisation des Sm-Pentamers D1-D2-F-E-G in einer geschlossenen hexameren Ringstruktur. Die Kristallstruktur des 8S-Komplexes zeigt, wie der SMN-Komplex über Gemin2 an das Sm-Pentamer bindet. In Kombination mit einer EM-Struktur des 8S-Komplexes gelang es weiterhin, einen plausiblen Mechanismus für die Elimination von pICln und die Aktivierung der Sm-Proteine für die snRNA-Bindung zu formulieren. Somit konnten diese Arbeiten zu einem besseren Verständnis der Funktionen von trans-agierenden Faktoren bei Zusammenlagerung von RNA-Protein-Komplexen in vivo beitragen. / Splicing is the process in which non-coding sequence elements (introns) are removed from eukaryotic pre-mRNAs and coding sequence elements (exons) are linked to an open reading frame. This central step in eukaryotic gene expression is catalyzed by the spliceosome, which is composed of the four small nuclear Ribonucleoproteins (snRNPs) U1, U2, U4/U6, U5 and a large number of additional protein factors. The snRNPs possess a common ring-shaped core structure that is formed by the seven Sm proteins (SmB/B’-D1-D2-D3-E-F-G) around a single-stranded sequence (Sm site) of the snRNAs. While this so-called Sm core domain forms spontaneously in vitro, its assembly is a highly regulated and assisted process in vivo. It is dependent on the action of at least 12 trans-acting factors which are organized in the PRMT5 and SMN complexes. The PRMT5 is active in the early phase of assembly and recruits the Sm proteins via its pICln subunit and catalyzes the symmetrical di methylation of arginine residues in the C-terminal tails of SmB/B’, SmD1 and SmD3. As a result of the early phase the Sm proteins SmD1-D2-E-F-G and SmB/B’-D3 are organized by pICln in two distinct complexes. While SmB/B’-D3 remains bound to the PRMT5 complex, the second complex exists as a free intermediate with a sedimentation coefficient of 6S. These intermediates cannot associate with RNA and the interaction of the Sm proteins with pICln has to be resolved for the assembly process to be continued. This happens in the late phase of Sm core assembly in which the Sm proteins are taken over by the SMN complex and pICln is dissociated. Afterwards the Sm proteins can be transferred onto the Sm site of the snRNA and the Sm core is formed. As part of this thesis two key intermediates of this assembly process could structurally be characterized by a combination of crystallographic and electron microscopic methods. These intermediates comprise the 6S complex and an Sm protein transfer-intermediate with a sedimentation coefficient of 8S. In this 8S complex the 6S complex is bound to two central subunits of the SMN complex (SMN and Gemin2) while pICln is still associated with the Sm proteins. Hence, this complex represents a trapped intermediate between the early and late phase of assembly. In the beginning a first crystal form of a recombinantly prepared 8S complex was obtained that did not allow the solution of the structure. By a combined optimization of the crystallization condition and the proteins a further similar crystal form was obtained that allowed for the solution of the 8S crystal structure. The crystallization of the 6S complex could successfully be accomplished based on the hypothesis that the lattices of crystals of both complexes might show an architectural similarity because of the similar composition of the complexes. Hence, amino acids of pICln that were conformationally restricted within crystal contacts of the 8S crystals were targeted for substitution to alanine. 6S preparations reconstituted with these proteins yielded two new crystal forms that allowed for the structure solution of the 6S complex. Based on the crystal structure of the 6S complex a structural mimicry of Sm proteins by pICln was revealed. This enables binding of the Sm proteins by pICln which is the basis for an early topological organisation of the Sm Pentamer D1-D2-F-E-G within a closed hexameric ring structure. The crystal structure of the 8S complex revealed how the SMN complex binds to the Sm Pentamer via its Gemin2 subunit. In combination with an EM structure of the 8S complex both structures revealed a plausible mechanism for the elimination of pICln and the activation of the Sm proteins for snRNA binding. The solution of both structures helps to better understand the function of trans-acting factors during the in vivo assembly of RNA-protein complexes.

Doctor of Philosophy (PhD) / Oligomeric surfactants, sometimes referred to as gemini surfactants, consist of two or more amphiphilic ‘monomer’ units linked together by spacer groups. The chemical identity of the spacer group is unconstrained, and it joins the individual units at or near the hydrophilic headgroups. Oligomeric surfactants display a range of interesting properties, including very low critical micelle concentrations, high surface activity and unusual rheology and self-assembly. Consequently they have many potential applications, both scientific and industrial. Until now, their use has been limited by the cost of their synthesis, which in some cases involve long and difficult procedures. This project developed from the idea that a synthesis based on polymerization could prove a useful and versatile method for producing these surfactants. The chemical starting point for this project was a series of polymerizable surfactants (‘surfmers’), upon which polymerization was performed. Two families of surfmers were investigated, both cationic and based on methacrylate and vinylpyridinium moieties respectively. The physical behaviour of these surfactants – a number of which are new – was investigated using standard techniques; these included the determination of the critical micelle concentration, characterization of phase behaviour, neutron scattering and surface adsorption. In producing oligomers, the initial focus was on free-radical polymerization, with control of molecular weight to be achieved by chain-transfer techniques. Due largely to analysis problems, this work proved unsuccessful. In its place a new reaction, not based on conventional polymerization methods, has been developed. The vinylpyridinium surfmers mentioned above readily undergo addition across the double bond to produce alkyl ring substituents. Under basic conditions, these alkylpyridiniums undergo an elimination/addition reaction in which they link together to form oligomers. This reaction can be started or stopped by raising or lowering the pH of the reaction solution, and has been performed in both organic and aqueous solutions. It is referred to in this thesis as LELA(Linkage by ELimination/Addition). The LELA reaction was used to produce mixtures of oligomers, the phase behaviour and surface adsorption of which were examined. Small-angle neutron scattering was used to monitor the reaction in real time and identify changes in self-assembly as the average oligomer length increased. Progress was also made towards a chromatographic protocol that would allow mixtures to be separated into their components and the pure oligomers to be studied. Finally, some of the compounds studied display interesting pH-dependent chromophoric properties which were also found to occur with other simple alkylpyridinium species. They are tentatively ascribed to inter- and intramolecular charge-transfer complexes, and evidence towards this conclusion was collected and is presented along with relevant calculations.

Gold (Au) implantation in silicon (Si) has been a topic of great interest from both fundamental and applied perspectives. Ion implantation is a versatile technique due to its ability to form surface-embedded nanoparticles that provide better adhesion. Also, being an integral part of the substrate lattice, the nanoclusters produced by ion implantation are free from impurities and their size distribution can be controlled by carefully optimizing the beam parameters. During our experiments to produce nanoclusters of Au on Si for use as seeds for the growth of nanowires, we stumbled across an unusual pattern formation process under specific conditions. This unique self-assembly process is observed only within a critical threshold implantation fluence and above a threshold annealing temperature. Fabrication of ordered arrays of metal nanoparticles on Si substrates is of significance for both fundamental science associated with low-dimensional physics and technical app lications. The application of functional nanostructures strongly depends on their assembly in ordered one- or two- dimensional arrangements. These arrangements may play an important role in fabricating ordered arrays of semiconductor/oxide nanowires.This thesis discusses a systematic study performed to understand the temperature and time dependent nucleation, growth of Au nanoclusters and evolution of the self-assembled patterns. A growth model is proposed to show the re-crystallization behaviour of Au supersaturated amorphous silicon (a-Si) on Si substrate. The observed self-assembled periodic patterns of Au nanoclusters bear resemblance to the Liesegang ring structures prevalent in some chemical reaction-diffusion systems. Based on this systematic study of the growth and morphology of Au nanoclusters, a tentative growth mechanism has been proposed for the formation mechanism of this unusual self-assembled pattern. The pattern formation of this non-equilibrium process is expected to originate due to instabilities of the three scales of Au nanoclusters at elevated temperatures. The kinetics of pattern formation from a supersaturated solid solution (a-Si/Au alloy) is demonstrated using numerical solutions obtained by a two-dimensional growth model, which takes into account the nucleation, diffusion and the aggregation process. The numerical solution of the diffusion equations appear to be in good agreement with the experimental results.

The bottom-up assembly of functional devices requires novel building blocks to facilitate the incorporation of functional and structural hierarchy. Anisotropic building blocks can substantially broaden the creation of self-assembled devices with unique properties because of their morphological and/or chemical asymmetry. In this regard, we have created microspheres with one hemispherical face exposing silica and the other exposing gold. These microspheres were formed by the shadow deposition of gold onto silica microspheres. The two chemical surfaces allowed use of different surface reactions—silane chemistry for the silica side and thiol chemistry for the gold side—for immobilizing different oligonucleotide sequences on each of the two faces. These dual-functionalized microspheres were used in the selective orthogonal assembly of fluorophore-tagged target oligonucleotides. The DNA-directed assembly was confirmed by confocal microscopy of the microspheres. In essence, employing DNA as the linker molecule, these “Janus” particles can be assembled into various novel 1-D, 2-D, and 3-D structures, which are difficult to realize using symmetric building blocks. / Singapore-MIT Alliance (SMA)

The binding affinity of single stranded DNA (ssDNA) for gold nanoparticle surface is studied in this work. The data indicate that the strength of interaction between ssDNA and Au particle surface is closely related to the particle size, with smaller particles (5 nm) producing the most pronounced effects. From these experimental findings, a single stranded DNA (ssDNA) based method to assimilate 13 and 5 nm gold nanoparticles was developed, and verified by transmission electron microscopy (TEM). / Singapore-MIT Alliance (SMA)

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Dionisios G. Vlachos and Raul F. Lobo, Dept. of Chemical Engineering Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Mary E. Galvin-Donoghue, Dept. of Materials Science & Engineering. Includes bibliographical references.

This thesis describes the fabrication and characterization of integrated hollow Bragg waveguides fabricated by controlled thin film buckling. Hollow waveguides based on two different set of materials were studied. In the first case, thermal tuning of air-core dimensions was studied using waveguides, with chalcogenide glass and polymer claddings. Results showed that the change in air- core height as a function of small temperature variations was in good agreement with theory. Planar, silicon based, hollow core waveguides with Si/SiO2 Bragg reflector claddings are also described. Fabrication was accomplished by incorporating compressive stress in the sputtered Si and SiO2 layers and then heating samples to induce buckling along predefined areas of low adhesion. Several low adhesion layers were studied, but a fluorocarbon layer was deposited by CVD gave the best results. Optical experiments demonstrated optical confinement in the air-core, with loss in the ~5 dB/cm range at the 1550 nm wavelength. / Photonics and Plasmas

Thesis (Ph. D.)--University of Delaware, 2006. / Principal faculty advisor: Darrin J. Pochan, Dept. of Materials Science & Engineering. Includes bibliographical references.