Frankfurt (Main), Univ., Diss., 2001.

Universiẗat, Diss., 2002--Frankfurt am Main.

Univ., Diss., 2006--Frankfurt (Main) / Zsfassung in engl. und dt. Sprache

Universiẗat, Diss., 2005--Hamburg. / Erscheinungsjahr an der Haupttitelstelle: 2004.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 103-108).

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical analysis Candidate Michaela Beranová Supervisor doc. PharmDr. Miroslav Miletín, Ph.D. Title of thesis Preparation of fluorescent azaphthalocyanines for labeling of oligonucleotide probes I. Azaphthalocyanines (AzaPc) are macrocyclic compounds with large system of conjugated bonds and are therefore used as dyes and pigments. Their potential as diagnostic and therapeutic means is also under intensive examination. The aim of this thesis is a synthesis of precursors for cyclotetramerization of asymmetric fluorescent tetrapyrazinoporphyrazines (TPyzPz) via solid phase method bearing two various moieties, one suitable for binding to solid phase and the other intended for binding of the final TPyzPz to oligonucleotides. At the beginning of the thesis, basic terms related to AzaPc are defined. Also the main principles of synthesis of TPyzPz as well as possible modifications of their final structure are discussed. An important part of the thesis is the description of the typical properties of TPyzPz, the influence of the structure on the individual parameters and the fields of their use. Afterwards the preparation of each precursor with two different substituents for cyclotetramerization is...

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The increasing number of nucleic acid-based therapeutics demonstrates the potential to treat diseases at the genetic level. Although oligonucleotides show clinical potential, challenges remain including nuclease degradation, rapid clearance when administered systemically, low cell permeability, and limited distribution to tissues of interest. This is largely imparted by the polyanionic phosphate backbone, which produces unfavourable electrostatic interactions at cell membranes. As a result, their clinical translation is dependent on delivery technologies that improve stability, facilitate cell entry, and increase target affinity. Spherical nucleic acids (SNAs) consist of radially orienting linear nucleic acids onto a nanoparticle core, conferring them a three-dimensional, spherical architecture. These structures enter cells readily and display distinct properties that are independent of their nanoparticle core. Accordingly, we decided to replace the intrinsically anionic phosphodiester linkage of DNA with a phosphoramidate linkage (P-N), allowing us to incorporate new functionality at the phosphate backbone. With this handle, we inserted cationic and hydrophobically modified functional groups that were compatible with nanoscale architectures, giving rise to new properties relevant in biological contexts. Specifically, amine and guanidinium derivatized functional groups provided SNAs with a ~10-fold increase in cell uptake at early incubation times compared with unmodified SNAs. This demonstrates that we can tune the behaviour of SNAs with phosphate backbone modifications in a highly controlled manner. We hypothesize that the stringent control over location and placement of functional groups within the SNA framework will afford them favourable interactions at cell membranes, not only increasing their cell uptake, but also access to alternative uptake mechanisms and potency as therapeutics. / Thesis / Master of Science (MSc) / Oligonucleotides are short synthetic sequences of DNA or RNA that have the capacity to treat diseases at the genetic level. However, they face challenges such as degradation, low cell uptake, and poor tissue distribution. To overcome this issue, we plan to incorporate chemical modifications at the phosphate backbone of oligonucleotides to make them more stable and facilitate more favourable interactions at cell membranes. Conferring oligonucleotides into a 3D arrangement further enhances their stability and cell uptake relative to linear oligonucleotides. By densely functionalizing them onto a nanoparticle core, we can create spherical nucleic acids (SNAs). We hypothesize that the modifications imparted onto the phosphate backbone of linear oligonucleotides will translate their properties into SNAs. The new properties afforded to the SNAs will provide increased cell uptake, alternative uptake mechanisms, and access to cytosolic and nuclear targets, highlighting their potency and therapeutic potential.

<p> Attempts to prepare silicone oligonucleotide copolymers are complicated by the large difference in hydrophobicity in the two materials. Two approaches were followed to overcome this challenge. Initially, highly sterically hindered tetraisopropyldisiloxanes were used to bind 5'-0-(4,4'-dimethoxytrityl)-thyrnidine at the 5'-0H. These compounds proved to be hydrolytically more stable than the analogous dimethylsiloxane compounds, which were also prepared. Alternatively, Si-C bonds, which are hydrolytically stable, can be used to bind the two species together. Introduction of allyl ether by traditional Williamson conditions was followed by hydrosilylation with hydride terminated (Si-H) silicone, catalyzed by using platinum complexes, to give the nucleoside-functionalized silicone. We also introduced an epoxy group to one end of a silicone chain and found it to be stable to hydrolysis. Once the epoxy group binds nucleoside-functionalized silicone to solid phase, it is expected that the nucleoside-functionalized silicone via a trimethylene spacer linkage might be a starter for preparation of oligonucleotide-functionalized silicones in future work. </p> / Thesis / Master of Science (MSc)

Thesis advisor: Tim van Opijnen / Thesis advisor: Michelle Meyer / Molecules that have high affinity and specificity for their target are critical for functioning biosensors and effective therapeutics. Aptamers, or single-stranded oligonucleotides, are one type of molecule capable of both high affinity and specificity. Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the iterative in vitro process for identifying aptamers with high affinity and specificity from an initial pool of approximately 1015 randomized nucleotide molecules. There have been a multitude of SELEX variations developed over the years to include incorporation of machine learning algorithms to address the limited success (~30%), cost, and time required to identify high affinity and specific aptamers. While some SELEX variations have been more successful than others in addressing some of the challenges, issues remain. To confront these challenges, the digitalSELEX platform introduces a novel de novo design approach. The platform has two main components. The first component analyzes the target molecule identifying clusters of amino acids along the molecule’s surface based on their accessibility and proximity of atoms relevant to target-aptamer binding. The platform then proposes aptamers built from sequences of nucleotides that paired to the amino acids in the clusters. The second component improves these aptamers sequentially. This is done via simulation-based optimization procedure which uses molecular docking and stochastic optimization techniques. It explores small adjustments made on the starting aptamer that increase the affinity and specificity that is calculated extracting binding related features from the output of the docker. Once in silico counter-selection is complete, the best possible sequences are extracted for in vitro validation. To validate digitalSELEX, aptamers were designed for four different target molecules of varying size ranging from 18 – 140 kDa. Some of the aptamers were designed with specific counter-targets while others did not have counter-target molecules. In total, 19 oligonucleotides were chemically synthesized, and their affinity and specificity tested for five explicit validation problems. All 19 aptamers demonstrated high affinity for their respective target molecules. Sixteen of the 19 oligonucleotides were tested for specificity with nine meeting the 4-times Kd-value difference specificity criteria. Depending on the computational capacity being employed for each problem, the approximate time required from initiating the de novo design to the point of validation was 170 hours. The cost of in silico oligonucleotide design is negligible while validation of a few aptamers is few hundred dollars. The digitalSELEX platform was comprehensively tested examining the initial de novo design through affinity and specificity determination. The digtalSELEX platform is a prototype that has the opportunity for further development such as employing different molecular simulators. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.