xvii, 203 p. / Gold nanoparticles have garnered recent attention due to their intriguing size- and shape-dependent properties. Routine access to well-defined gold nanoparticle samples in terms of core diameter, shape, peripheral functionality and purity is required in order to carry out fundamental studies of their properties and to utilize these properties in future applications. For this reason, the development of methods for preparing well-defined gold nanoparticle samples remains an area of active research in materials science. In this dissertation, two methods, diafiltration and aptamer mediated synthesis, are explored as possible routes towards well-defined gold nanoparticle samples. It is shown that diafiltration has considerable potential for the efficient and convenient purification and size separation of water-soluble nanoparticles. The suitability of diafiltration for (i) the purification of water-soluble gold nanoparticles, (ii) the separation of a bimodal distribution of nanoparticles into fractions, (iii) the fractionation of a polydisperse sample and (iv) the isolation of [rimers from monomers and aggregates is studied. NMR, thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS) measurements demonstrate that diafiltration produces highly pure nanoparticles. UV-visible spectroscopic and transmission electron microscopic analyses show that diafiltration offers the ability to separate nanoparticles of disparate core size, including linked nanoparticles. These results demonstrate the applicability of diafiltration for the rapid and green preparation of high-purity gold nanoparticle samples and the size separation of heterogeneous nanoparticle samples. In the second half of the dissertation, the identification of materials specific aptamers and their use to synthesize shaped gold nanoparticles is explored. The use of in vitro selection for identifying materials specific peptide and oligonucleotide aptamers is reviewed, outlining the specific requirements of in vitro selection for materials and the ways in which the field can be advanced. A promising new technique, in vitro selection on surfaces (ISOS), is developed and the discovery using ISOS of RNA aptamers that bind to evaporated gold is discussed. Analysis of the isolated gold binding RNA aptamers indicates that they are highly structured with single-stranded polyadenosine binding motifs. These aptamers, and similarly isolated peptide aptamers, are briefly explored for their ability to synthesize gold nanoparticles. This dissertation contains both previously published and unpublished co-authored material. / Adviser: James E. Hutchison

abstract: The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution. / Dissertation/Thesis / Ph.D. Biochemistry 2013

abstract: Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this line, my Ph.D. dissertation focuses on the in vitro selection of two important biomolecules, deoxynucleotide acid (DNA) and protein with binding properties. Chapter two focuses on in vitro selection of DNA. Aptamers are single-stranded nucleic acids that generated from a random pool and fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers. By employing negative selection step to eliminate aptamers that bind with off-target through charge unselectively, an aptamer that binds with histone H4 protein with high specificity (>100 fold)was generated. Chapter four focuses on another functional molecule: protein. It is long believed that complex molecules with different function originated from simple progenitor proteins, but very little is known about this process. By employing a previously selected protein that binds and catalyzes ATP, which is the first and only protein that was evolved completely from random pool and has a unique α/β-fold protein scaffold, I fused random library to the C-terminus of this protein and evolved a multi-domain protein with decent properties. Also, in chapter 3, a unique bivalent molecule was generated by conjugating peptides that bind different sites on the protein with nucleic acids. By using the ligand interactions by nucleotide conjugates technique, off-the shelf peptide was transferred into high affinity protein capture reagents that mimic the recognition properties of natural antibodies. The designer synthetic antibody amplifies the binding affinity of the individual peptides by ∼1000-fold to bind Grb2 with a Kd of 2 nM, and functions with high selectivity in conventional pull-down assays from HeLa cell lysates. / Dissertation/Thesis / Ph.D. Biochemistry 2013

It is now widely known that some nucleic acid molecules, either DNA or RNA, are capable of forming intricate three-dimensional structures and carrying out functions of molecular recognition and catalysis. Most of known functional nucleic acids are isolated from DNA or RNA pools with random sequences using the technique of in vitro selection. With intensive research for the past three decades, a variety of functional nucleic acids have been discovered and examined for potential applications. The general objective of this thesis is to expand the repertoire of functional nucleic acids via new in vitro selection experiments and pursue their biosensing applications. I started by asking the question of whether it is possible to develop a new kind of functional nucleic acids: chimeric RNA/DNA substrates that have high activity for ribonuclease H2 from the important bacterial pathogen Clostridium difficile but much reduced activity towards the same enzymes from other bacterial species. The key rationale behind pursuing these special functional nucleic acids is my hypothesis that these molecules can eventually be developed into useful biosensors for diagnosing Clostridium difficile infection. For this reason, in my first project, I applied the in vitro selection technique to a random-sequence DNA pool, obtained several highly selective chimeric RNA/DNA substrates, and carried out in-depth analysis of their reactivities and their structural properties. During this study, I accidentally discovered a family of highly guanine-rich DNA molecules that are able to form an unusual guanine-quadruplex structure in 7 molar urea, a strong denaturing condition for nucleic acid structures. This discovery constitutes a novel observation and therefore, in my second project, I fully characterized the sequence and structural properties of these special DNA molecules and established the conditions that allow these molecules to create stable structures in 7 molar urea. I then got interested in devising a unique application to take advantage of the urea-resistant property exhibited by these molecules. Towards this end, in my third project, I used one such DNA molecule to set up a DNA detection method capable of detecting single nucleotide polymorphism in very long DNA sequences, a desired application that has never been demonstrated before. The findings made in these projects contribute to the ever-growing appreciation of functional capability and practical utility of nucleic acids. / Thesis / Doctor of Philosophy (PhD)

Peptides are a promising class of therapeutic candidates due to their high specificity and affinity for cellular protein targets. However, peptides are susceptible to protease degradation and are typically not cell-permeable. In efforts to design more effective peptide drug discovery systems, investigators have discovered that incorporation of non-canonical amino acids (ncAAs) and macrocyclization overcome these limitations, making peptides more drug-like. In this work, we exploit the promiscuity of wild-type aminoacyl-tRNA synthetases (aaRSs) to ‘mischarge’ ncAAs onto tRNA and ribosomally incorporate them into peptides using a cell-free translation system. We have demonstrated the ability to incorporate five ncAAs into a single peptide with near-wild type yield and fidelity. We also demonstrated the in situ incorporation of ncAAs containing azide and alkyne functionalities, enabling the use of CuAAC (click chemistry) to generate triazole-bridged cyclic peptides. When combined with bisalkylation of peptides containing two cysteines via an α,α’-dibromo-m-xylene linker, we created bicyclic peptides which are structurally similar to the highly bioactive knotted peptide natural products. Biological display methods, such as mRNA display, are powerful peptide discovery tools based on their ability to generate libraries of >1014 unique peptides. We combined our ability to incorporate ncAAs with our bicyclization technique adapted for use with mRNA display to create knotted peptide library scaffolds. We performed side-by-side monocyclic and bicyclic in vitro selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nM affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance. We used a new library that enables the generation of a diverse collection of linear, monocyclic and bicyclic scaffolds in one pot, increasing the likelihood of target-ligand conformational alignment. We performed a second selection against streptavidin and revealed a nearly unanimous preference for linear peptides containing an HPQ motif, a known streptavidin-binding sequence. However, when we used these libraries for in vitro selection against a biological target, DNA repair protein XRCC4, we did not observe convergence. In summary, we have developed a novel technique for production of bicyclic peptide libraries. These highly-constrained protease-stable scaffolds can be used as platforms to identify high affinity, drug-like ligands using mRNA display.

Laboratory directed evolution have enabled the discovery of numerous functional natural and synthetic macromolecules with tailor-made functions. However, approaches that use enzymes to effect the crucial translation from an information carrier molecule such as DNA or RNA to synthetic polymers are limited to producing close analogs of nucleic acids, either due to a strict requirement to hybridize with a nucleic acid template or as a consequence of the limited substrate scope of polymerase enzymes. / Chemistry and Chemical Biology

abstract: In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link between the enzymatic function being selected (phenotype) and the genetic information responsible for the function (genotype). Second, they must enable partitioning of active from inactive variants, often capturing only a small number of positive hits from a large population of variants. These principles have been applied to the selection of natural, modified, and even unnatural nucleic acids, peptides, and proteins. The ability to select for and characterize new functional molecules has significant implications for all aspects of research spanning the basic understanding of biomolecules to the development of new therapeutics. Presented here are four projects that highlight the ability to select for and characterize functional biomolecules through in vitro selection. Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents. Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells. Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis. Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

No description available.

CAR T-cellbehandling är en immunterapi som har visat lovande resultat vid behandling av cancer. Trots det riktade immunsvaret som kan uppnås, betonar komplexiteten i tillverkningsprocessen och behandlingsproceduren det utrymme somm finns för förbättringar. Omprogrammerade T-celler har illustrerat en hög persistens hos patienter, som utsätter dem för risken för systemisk toxicitet. In-vivo transienta CAR T-celler som använder självförstärkande mRNA leverade genom affinitetsproteinbelagda LNP, föreslås som ett standardiserat alternativ som möjligör dosering av terapin vid behov.  Med hjälp av fagdisplay utfördes ett urval av affibody molekyler mot de tre immunonkologiska målproteinerna CD5, CD8 och CD19, i fyra cykler. Monoklonal fag-ELISA och DNA-sekvensering identifierade sju förmodade kandidater mot CD5, en förmodad kandidat mot CD8 och tre mot CD19. SPR analys visade specifik binding från CD5 kandidaterna, medan binding till målprotein inte kunde påvisas för CD8- och CD19 kandidaterna. De identifierade CD5-bindarna kan konjugeras till LNP för T-cell inriktad leverans av själv-amplififerande mRNA, med genetisk kod för en valfri CAR. / Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy which has shown promising results in treating patients suffering from oncological malignancies. Despite the targeted immune response that can be achieved, elaborate manufacturing and procedure processes emphasise room for improvement. Engineered T-cells have illustrated a high persistence in patients, exposing them to the risk of systemic toxicity. In-vivo transient CAR T-cells using self-amplifying mRNA by delivery through affinity protein coated lipid nanoparticles (LNP) is proposed as a standardised and reversible alternative, allowing for dosing when needed. Using phage display technology, selection of affibody molecules toward the three immune oncology proteins CD5, CD8 and CD19 was performed in four cycles. Monoclonal phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing identified seven putative candidates toward CD5, one putative candidate was isolated toward CD8, and three toward CD19. Surface plasmon resonance analysis (SPR) showed specific target binding of the CD5 candidate binders, while target binding could not be demonstrated for the CD8 and CD19 candidates. The identified CD5 binders could be conjugated to LNP for T-cell targeted delivery of self-amplifying mRNA encoding any CAR of interest.

<p>Adaptation of nucleic acid <em>in vitro</em> selection for whole cell targets has been demonstrated to be an effective means of isolating useful sequences with applications in biomarker detection and therapeutics. The problem of efficient delivery of materials across cell membranes is common to a variety of research and medical fields. Existing aptamers isolated in surfacing binding selections have been successfully adapted for cell targeted therapies through complex modifications. However, better aptamers may be derived from a selection optimized to isolate internalized sequences directly. A cell selection experiment with the goal of identifying circular random-sequence DNA aptamers with the ability to facilitate their own internalization into MCF7 cells was conducted. Several classes of sequences isolated from this selection were shown to target cell nuclei at a rate significantly greater than control sequences as determined by qPCR relative recovery assays supported by <em>in situ</em> RCA fluorescence microscopy data. The localization of functional DNA sequences at the subcellular and intercellular levels suggests a receptor mediated mechanism. Techniques for the selection, purification and fluorescent detection of small circular DNAs were also developed for this study. Further work to characterize and identify targets should be pursued to better understand the mechanism of internalization and judge the suitability of G18d sequences as a delivery platform.</p> / Master of Science (MSc)