In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides and aptamers, as therapeutic and diagnostic entities. Therapeutic Bio-Oligomers; A series of peptoid analogs have been designed and synthesised using solid phase synthesis. These peptoids have been subjected to biological evaluation to determine structure-activity relationships that define their antimicrobial activity. In total 13 peptoids were synthesised. Out of 13 different peptoids, only one peptoid called Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal activity. TOP kills bacteria under non-dividing and dividing conditions. The Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM. The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the 10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for TOP as 100. This TI is well above previously reported peptidomimetics TI of around 20. TOP demonstrates selective bacterial killing in co-culture systems and intracellular bacterial killing activity. Diagnostic Bio-Oligomers; In the second part of my thesis, I investigated aptamer and peptide-based molecular probes to detect MRSA. As well as screening aptamers and peptide probes against whole MRSA, I over-expressed and purified PBP2A protein. This purified protein was used as a target for aptamer and peptide probes to detect MRSA. Two different aptamer libraries were initially screened for utility. In-vitro conditions for SELEX were optimised. Biopanning with a phage derived peptides was also performed. Target sequences for both methods were identified and chemically synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and confocal imaging showed no specificity for MRSA detection with either method. The Bio-Oligomers and the in-vitro selection methodology require further refinement to improve diagnostic utility.

Philosophiae Doctor - PhD / Tetrodotoxin (TTX) is a nonpeptidic neurotoxin with a high rate of food poisoning mortality (60%) that has been associated with the consumption of diets from puffer fish and mud snails harbouring TTX-producing bacteria. As this neurotoxin has no known antidote and could not be mitigated by cooking, the only way for safety appears to be the detection of TTX-contaminated fishes at the points of harvest and control. The overall aim of this study was to develop amperometric and impedimetric sensors for TTX based on ionophores and aptamer immobilised on the modified conducting electroactive polyaniline (PANI)/electrode. The undoped polyaniline and poly(4-styrenesulfonic acid) (PSSA) doped electroactive polyanilines were prepared in perchloric acid/acetonitrile and phosphoric acid respectively by electrochemical oxidative polymerisation. Two types of electropolymerisation were applied to prepare the neutral and p-doped PANI−PSSA films composites. The dynamic electroinactivity of TTX was studied which revealed that TTX is not electrochemically active on bare Au, GC, Pt, PG, Ni, Ti and BDD (Boron dopeddiamond) electrodes in acetate buffer pH 4.8. Using ion transfer voltammetry and UV-Vis analysis, the complexation of TTX with two neutral ionophores (sodium ionophore X (NaX) and dibenzo-18-crown6 (B18C6)) was investigated. The cyclic voltammograms (CVs) recorded from ion transfer voltammetry presented no redox peak and no increasing/decreasing current was observed which indicates that no TTX ions transfer from the liquid to the organic phase. In addition, the absorption spectra of the mixture of TTX/NaX and TTX/B18C6 presented the same absorption bands recorded for NaX and B18C6 respectively. Three absorptions bands at 250.4, 278.3, and 370.6 nm for NaX and two at 222.03 and 274.10 nm for B18C6 were observed before and after mixing TTX with NaX and TTX with B18C6 separately. No chemical reaction occurred between the TTX and both ionophores, therefore, sodium ionophore X and dibenzo-18-crown-6 did not form a complex with TTX. Thus, TTX ion sensor cannot be developed based on these two neutral compounds. The electrodynamics of the PANI and PANI−PSSA films electropolymerised on the bare precious metal electrodes were also investigated through various electrochemical techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies in sodium phosphate (SPB) and acetate (OAc) buffer revealed that both neutral and p-doped films synthesized were thin (thickness L < 5 nm in acetate buffer and L < 10 nm in sodium phosphate buffer) film polymers.

In recent years, there has been an increasing demand for newer, more accurate, technologies that can detect and identify biomolecules or biological entities related to health, agriculture or the environment. With the discovery of new properties of nucleic acids beyond the storage and transfer of genetic information, a new class of nucleic acid-based biosensors is emerging, using DNA and RNA as target recognition elements with the advantage of being simpler and more cost-effective compared to antibodies-based biosensor. Two sequences, TrG14MC and TrG10SC, with evidence to suggest that they are capable of inhibit the metalloenzyme CIP, were isolated from a selection conducted by Dr. Razvan Nutiu. Here we study the inhibitory properties of these two aptamer candidates and measure the IC50 value, determined as 94 nM for TrG14MC and 83 nM for TrG10SC. Different bivalent constructs, designed to increase the inhibitory effect of the isolated sequences, are studied showing a pronounce influence of the linker length improving the inhibitory effect over CIP. Modulating the interaction of the isolated sequences and the CIP is of key importance in order to develop a successful biosensor. Therefore, we try to recover CIP from the inhibition effect by using antisense sequences complementary to different segments of the construct. The maximum recovery, 75%, was achieved by an antisense sequence fully complemented to the inhibitory bivalent construct. We also study here the use of a linker in the bivalent construct that forms a secondary hairpin structure, and the effect of linearizing that structure with an antisense sequence complementary to the linker. This resulted in as 12% of the inhibitory effect. The purpose of this investigation was to establish the first steps toward the development of a new class of biosensors capable of disinhibiting CIP upon the recognition of a specific target, taking advantage of the suggested CIP-inhibitory properties of the isolated sequences TrG14MC and TrG10SC. / Thesis / Master of Applied Science (MASc)

No description available.

The development of the in vitro selection technique permits the creation of synthetic DNA molecules with ligand-binding capabilities (DNA aptamers), or abilities to catalyze chemical reactions (DNAzymes), or both (aptazymes). Significant research efforts in this field over the past two decades have led to the creation of a large array of DNA aptamers and DNAzymes and ever-increasing interests in taking advantage of these molecular species for diverse applications. One area of remarkable potential and development is the exploration of functional DNA molecules for bioanalytical applications. The work described in this dissertation aims to pursue innovative concepts and technologies that expand utility of functional DNA molecules for biosensing applications. I have focused on two functional DNA species: RNA-cleaving DNAzymes and protein-binding DNA aptamers. My key interest is to develop simple but effective colorimetric assays that employ these functional DNA molecules and to establish an effective strategy that makes functional DNA biosensors highly functional in biological samples. / Thesis / Doctor of Philosophy (PhD)

<p>The coagulation system is initiated when subendothelial tissue factor is exposed to circulating blood, and culminates in the production in the enzyme thrombin, which catalyzes clot formation. However, the failure of built-in controls to limit thrombin generation to sites of vascular damage, leads to pathological clot formation, termed thrombosis. Anticoagulants, such as heparin and warfarin, are used to prevent thrombosis; however, these can cause bleeding. As a result, there is a need for novel anticoagulants that limit clot formation without the risk of bleeding. DNA aptamers are small oligonucleotides designed to bind a specific target with high affinity. HD1 is a DNA aptamer that was developed by Griffin and Leung in 1993 to bind thrombin. The focus of this work was to better characterize the anticoagulant properties of HD1.</p><p>We demonstrate that HD1 binds a subdomain of exosite 1 that is fully developed in prothrombin, unlike other exosite 1-directed ligands, such as Hir⁵⁴⁻⁶⁵(SO₃⁻) and fibrin, which do not bind prothrombin. As a result, HD1 binds prothrombin with high affinity and inhibits prothrombin activation by prothrombinase with an IC₅₀ value of 115 nM. HD1 competes with tV a for binding to prothrombin. Based on its capacity to inhibit both thrombin and prothrombin, HD1 is more potent than Hir⁵⁴⁻⁶⁵(SO₃⁻) at inhibiting standard plasma clotting assays, but not in the thrombin clotting time. Furthermore, HDl inhibits thrombin generation by prolonging the lag time, reducing peak thrombin, and reducing the ETP to a greater extent than Hir⁵⁴⁻⁶⁵(SO₃⁻). Based on thrombin generation assays conducted in cofactor-deficient plasma, we demonstrate that HD 1 inhibits thrombin feedback activation of fV, but not fVIII, and that inhibition of prothrombinase accounts for the reduction in both peak thrombin and prolongation of the lag time. HDI is neutralized in buffer-based and plasma-based coagulation assays by an antisense oligonucleotide, antiHD1. AntiHD1 preferentially targets free HD1 because HD1 bound to either thrombin or prothrombin is protected from neutralization by antiHD1. Based on our data, HD1 may be a useful anticoagulant due to its capacity to inhibit both prothrombin and thrombin. Positioned at the junction of the intrinsic and extrinsic pathways prothrombin could be a useful target for aptamer development into anticoagulant applications.</p> / Thesis / Doctor of Philosophy (PhD)

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.

The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy is a topical microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal small inhibitory RNA (siRNA) application. To overcome the challenges of using siRNAs to knock down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in both the genital and rectal tracts of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity, provide durable target gene silencing for up to three weeks in vitro, and maintain effectiveness in a hydroxyethyl cellulose (HEC) gel formulation. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in cervicovaginal explants. When applied intravaginally to humanized mice, CD4-AsiCs provided durable protection against transmission of the virus. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent the sexual transmission of HIV.

No description available.

Aptamere besitzen als nahezu universelle Binder ein großes Anwendungspotential in Biotechnologie und Medizin; ihre Selektion aus zufälligen Sequenzbibliotheken liefert jedoch nur suboptimale Ergebnisse. Während der Selektion schlagen sich in der Bibliothek Bindungsinformationen nieder, die zur Optimierung des Verfahrens eingesetzt werden können. Die vorliegende Arbeit widmet sich der bioinformatischen Erschließung dieser Informationen. Für die initiale Auswertung der gewonnenen Aptamersequenzdaten erwiesen sich unter Zuhilfenahme von n-Gramm-Deskriptoren und Affinitäten die Regressionsanalyse und auf statistisch-symbolischer Ebene die Mustersuche als wirkungsvolle Verfahren. Für die Aufklärung der Komplexstruktur aus Aptamer und Zielprotein wurde eine Bewertungsfunktion gefunden, die die Identifikation sogenannter nahe-nativer Bindungskonformationen unter den Ergebnissen einer Dockingsimulation erlaubt. Nach dieser methodischen Evaluation erfolgte die Selektion eines Aptamers gegen das Kapsid des humanen Norovirus. Auf Basis der erhobenen Sequenzdaten wurde die Bindegeometrie zwischen Aptamer und Zielprotein durch Anwendung der im Verfahrensprotokoll festgehaltenen Kombination der Analysemethoden aufgeklärt. Das dabei als bindungsrelevant identifizierte Sequenzmotiv kann bei der Erzeugung targetspezifisch optimierter Selektionsbibliotheken als a priori-Information einfließen.:Inhaltsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Formelverzeichnis Abkürzungsverzeichnis Vorwort 1 Thematische Einleitung 1.1 Hypothesen und Fragestellungen 1.2 Aufbau der Arbeit 2 Allgemeine Grundlagen 2.1 Protein-Nukleinsäure-Komplexe 2.1.1 Aufbau von Proteinen 2.1.2 Aufbau von Nukleinsäuren 2.1.3 Interaktionen zwischen Proteinen und Nukleinsäuren 2.2 Aptamere und deren Gewinnung 2.2.1 Aptamere als universelle Binder 2.2.2 Das Grundverfahren der Aptamerselektion 2.2.3 Methodische Modifikationen des Selektionsverfahrens 3 Auswertung der Primär- und Sekundärstruktur von Nukleinsäuren 3.1 Numerische Beschreibung von Nukleinsäuren 3.1.1 Nukleobasendeskriptoren 3.1.2 Transformationsstrategien 3.1.3 Direkt anwendbare Beschreibungskonzepte 3.2 Konzeption einer Strategie zur Evaluation der Beschreibungskonzepte 3.2.1 Beschreibung und Vorverarbeitung des Datensatzes 3.2.2 Zusammenstellung von Deskriptorensets 3.2.3 Eingesetzte Methoden 3.3 Ergebnisse der Evaluation 3.3.1 Gegenüberstellung der Beschreibungskonzepte 3.3.2 Überprüfung der Plausibilität 3.3.3 Verhältnismäßigkeit 3.3.4 Abschließende Betrachtung 4 Mustersuche in biologischen Sequenzen 4.1 Sequenzmuster 4.1.1 Definition der Sequenzmuster 4.1.2 Bewertung konkreter Musterfunde 4.1.3 Bewertung einzelner Musterinstanzen 4.1.4 Visualisierung 4.2 Algorithmus zur Mustersuche 4.2.1 Suche in Suffixbäumen 4.2.2 Durchsuchen des Musterraumes 4.2.3 Optimierung der Suchstrategie 4.2.4 Ordnung der Ergebnisse 4.2.5 Zusammenfassung 5 Auswertung der Tertiärstruktur von Protein-Nukleinsäure-Komplexen 5.1 Übersicht der Bewertungsmodelle für Protein-Nukleinsäure-Komplexe 5.1.1 Wissensbasierte Paarpotentiale 5.1.2 Molekularmechanische Bewertung 5.1.3 Auswahl der Konzepte für die weitere Betrachtung 5.2 Vorstellung und Herleitung der ausgewählten Bewertungsmodelle 5.2.1 Die SPA-PN-Potentiale 5.2.2 Die modifizierten SPA-PN Potentiale 5.2.3 Die ITScore-PR Potentiale 5.2.4 Molekularmechanische Bewertung 5.3 Konzeption des Vergleichs der Bewertungsmodelle 5.3.1 Auswahl und Vorstellung der Referenzkomplexe 5.3.2 Generierung von Decoy-Strukturen 5.3.3 Quantifizierung der strukturellen Abweichung 5.4 Ergebnisse des Vergleichs 5.4.1 Referenzkomplex 4PDB 5.4.2 Referenzkomplex 5CMX 5.4.3 Gewichtung der HADDOCK-Bewertung 5.4.4 Visualisierung der Bewertung 5.5 Zusammenfassung 6 Selektion und Analyse eines Norovirus-Aptamers 6.1 Der Norovirus als Zielstruktur der Aptamerselektion 6.1.1 Epidemiologische Aspekte 6.1.2 Aufbau des Norovirus 6.1.3 Nachweis des Norovirus 6.1.4 Eingesetzter Norovirusstamm 6.2 Experimentelle Durchführung und Auswertung 6.2.1 Aptamerselektion 6.2.2 Evaluation der Anreicherung von Aptamerkandidaten 6.2.3 Experimentelle Verifikation der Bindung 6.2.4 Entwurf eines bioinformatischen Analyseprotokolls 6.3 Bioinformatische Analysen auf Basis der Primär- und Sekundärstruktur 6.3.1 Vorhersage der Sekundärstrukturen für die Aptamerkandidaten 6.3.2 Untersuchung der Anreicherung über die Clusteranalyse 6.3.3 Untersuchung der n-Gramm-Zusammensetzung 6.3.4 Durchführung einer Mustersuche 6.3.5 Validierung der Musterfunde 6.4 Bioinformatische Analyse auf Basis der Tertiärstruktur 6.4.1 Bestimmung der Struktur des Zielproteins 6.4.2 Bestimmung der Aptamerstruktur 6.4.3 Bildung eines Komplexes aus Aptamer und Zielprotein 6.4.4 Einschätzung der Relevanz für die Epitope der Proteinoberfläche 6.5 Konzepte für die spezifische Anreicherung von Oligonukleotidbibliotheken 6.5.1 Ableitbare Unterräume des Sequenz- und Strukturraumes 6.5.2 Zusammensetzung einer Bibliothek 7 Abschließende Betrachtung 7.1 Zusammenfassung der Ergebnisse 7.2 Ausblick Literatur Versicherung