An aptamer-based sensing platform for luteinising hormone pulsatility measurement

Normal fertility in human involves highly orchestrated communication across the hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile release of Luteinising Hormone (LH) is a critical element for downstream regulation of sex steroid hormone synthesis and the production of mature eggs. Changes in LH pulsatile pattern have been linked to hypothalamic dysfunction, resulting in multiple reproductive and growth disorders including Polycystic Ovary Syndrome (PCOS), Hypothalamic Amenorrhea (HA), and delayed/precocious puberty. Therefore, assessing the pulsatility of LH is important not only for academic investigation of infertility, but also for clinical decisions and monitoring of treatment. However, there is currently no clinically available tool for measuring human LH pulsatility. The immunoassay system is expensive and requires large volumes of patient blood, limiting its application for LH pulsatility monitoring. In this thesis, I propose a novel method using aptamer-enabled sensing technology to develop a device platform to measure LH pulsatility. I first generated a novel aptamer binding molecule against LH by a nitrocellulose membrane-based in vitro selection then characterised its high affinity and specific binding properties by multiple biophysical/chemical methods. I then developed a sensitive electrochemical-based detection method using this aptamer. The principal mechanism is that structure switching upon binding is associated with the electron transfer rate changes of the MB redox label. I then customised this assay to numerous device platforms under our rapid prototyping strategy including 96 well automated platform, continuous sensing platform and chip-based multiple electrode platform. The best-performing device was found to be the AELECAP (Automated ELEctroChemical Aptamer Platform) – a 96-well plate based automatic micro-wire sensing platform capable of measuring a series of low volume luteinising hormone within a short time. Clinical samples were evaluated using AELECAP. A series of clinical samples were measured including LH pulsatility profile of menopause female (high LH amplitude), normal female/male (normal LH amplitude) and female with hypothalamic amenorrhea (no LH pulsatility). Total patient numbers were 12 of each type, with 50 blood samples collected every 10 mins in 8 hours. Results showed that the system can distinguish LH pulsatile pattern among the cohorts and pulsatility profiles were consistent with the result measured by clinical assays. AELECAP shows high potential as a novel approach for clinical aptamer-based sensing. AELECAP competes with current automated immunometric assays system with lower costs, lower reagent use, and a simpler setup. There is potential for this approach to be further developed as a tool for infertility research and to assist clinicians in personalised treatment with hormonal therapy.Open Acces

Generating aptamers towards human sperm cells using massively parallel sequencing

Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection. [Abstract copyright: © 2021. The Author(s).

Preparation and Evaluation of Single-Stranded DNA Aptamer-Based Immunological Adjuvant in Broiler Chickens

Mineral oils and metal salts are commonly used as adjuvants to enhance acquired immunity. Recently, monoclonal antibodies (MAbs) and recombinant peptides agonist CD40 receptor have shown remarkable promise for induction of rapid and robust immune responses. Limitations of this approach MAb production costs and multiple administrations due to anti-MAb immune responses. Here we demonstrate the development of a unique and sophisticated DNA aptamer-based alternative for CD40-directed delivery of universal antigens as an alternative in chickens, and potentially other vertebrate species. This receptor, expressed by antigen-presenting cells, acts as a costimulatory molecule for activated T helper lymphocytes. After initially selecting for high affinity aptamers of independent sequence, we utilized a polymerization process where multiple aptamers were simultaneously and sequentially arranged through rolling circle amplification products (RCA-p), potentially capable of binding multiple recognition sites and causing receptor clustering. Selected sequences were demonstrated to effectively activate chicken macrophage HD11 cell line through CD40 receptors, demonstrating proof of concept in vitro. Additionally, using limited proteolysis liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), we deduced the actual amino acid sequences targeted by these aptamer products, which are essential for activation. In chapter III, we produced a biotinylated version of an effective CD40-activating RCA-p, and streptavidin-conjugated this RCA-p to a biotinylated peptide of the highly conserved type-A influenza peptide M2e. This conjugate was administered subcutaneously at either (25μg/bird) or (50μg/bird) at age 7 and 21 days. Anti-M2e IgG responses were determined at 14, 21, 28 and 35 days of age by ELISA. The RCA-p-M2e complex, at 50μg/bird, was able to induce robust seroconversion as early as 7 days post-immunization, with high antibody titer consistent through the end of the experiment. The lower dose, however, showed delayed responses and required the second administration. Taken together, these results constitute proof of concept for RCA-p-directed antigen delivery for peptide antigens and a potential tool for rapid generation of new vaccines against animal diseases. In chapter IV we discuss PCR artifacts that develop during traditional DNA aptamers selection and proposed a repetitive thermal correction cycles [(65°C/10min, 20°C/1min)x6] of the PCR. This may improve future selection of aptamers for similar purposes

Selección in vitro de aptámeros de DNA y RNA frente a la proteína Core del virus de la hepatitis C: aplicaciones en diagnóstico y terapia

Los aptámeros son ácidos nucleicos de cadena sencilla (RNA o ssDNA) que, gracias a su estructura tridimensional en solución, pueden unirse a un amplio rango de dianas (desde moléculas pequeñas hasta células y tejidos) con alta afinidad y especificidad. Se obtienen mediante selección in vitro utilizando un procedimiento llamado SELEX y constituyen herramientas moleculares prometedoras en biotecnología y biomedicina (por ejemplo, para el diagnóstico y terapia de enfermedades infecciosas). El virus de la hepatitis C (VHC) causa hepatitis crónica, que puede progresar hacia fibrosis, cirrosis e incluso carcinoma hepatocelular. Los tests de diagnóstico actuales se basan principalmente en ensayos serológicos que detectan anticuerpos anti-VHC producidos por los pacientes infectados, así como en ensayos moleculares que cuantifican el RNA genómico viral en plasma o suero. Sin embargo, se requieren herramientas analíticas más rápidas, baratas y sensibles para el diagnóstico y terapia. La proteína core del VHC es una diana de gran interés: es la proteína más conservada del virus, es la primera que se sintetiza durante la traducción del genoma viral y ejerce funciones importantes en el ciclo de vida del virus, entre ellas la encapsidación y protección de su genoma. En esta Tesis se han seleccionado in vitro aptámeros de ssDNA y RNA frente a diferentes variantes de la proteína core del VHC que pertenecen a los genotipos 1 a 4. Tras realizar de 9 a 14 rondas de SELEX, los aptámeros individuales presentes en las poblaciones enriquecidas fueron analizados por secuenciación Sanger y secuenciación masiva (UDS), y las constantes de afinidad (Kd) fueron cuantificadas mediante ELONA colorimétrico y ELONA-qPCR (para aptámeros de DNA) o ELONA-RTqPCR (para aptámeros de RNA). El análisis bioinformático mostró que el aptámero de DNA AptD-1312 estaba altamente representado en todos los procesos de selección y su secuencia compartía motivos de secuencia con otras que habían sido seleccionadas solamente frente a algunas variantes de la proteína core del VHC. Además, tanto AptD-1312 como AptD-1932 presentaron la mayor afinidad (con Kds en el rango nanomolar bajo) y especificidad por la proteína core del VHC. El aptámero AptD-1312 se utilizó como sonda de bio-reconocimiento en un aptasensor basado en grafeno que detecta la proteína core de VHC en plasma en el rango attomolar, convirtiéndose en un biosensor ultrasensible de gran utilidad para el diagnóstico clínico. Además, los dos aptámeros de mayor prevalencia y alta afinidad (AptD-1312 y AptD-1932) fueron analizados en cultivo celular humano infectado con VHC, produciendo una inhibición de la producción de progenie viral, un descenso de los niveles de RNA extracelular y una acumulación del RNA viral intracelular. Tales resultados revelan su capacidad para unirse a la proteína core e inhibir el ensamblaje de la cápsida del VHC en cultivo celular, impidiendo así la formación y liberación de viriones infecciosos. Esto sugiere una futura aplicación clínica como fármacos antivirales, complementarios o alternativos los utilizados actualmente en el tratamiento de la hepatitis C

Click-SELEX enables the selection of Δ9-tetrahydrocannabinol-binding nucleic acids

Δ9-Tetrahydrocannabinol (THC) is the main psychoactive compound in the plant Cannabis sativa and the most widely used illegal drug in the world. Its consumption impairs driving skills such as keeping a vehicle on track and has been shown to increase the risk of accidents. Urine tests are available for the detection of THC in a roadside setting, but necessitate facilities and are prone to sample manipulation. In addition, the correlation between impairment and positive tests is poor as a THC-metabolite instead of the active THC is analysed. Oral fluid sampling has emerged as a simple and quick alternative that permits detection of THC itself and therefore evidence for driving under the acute influence of THC. Existing oral fluid tests that are based on antibodies fail to reach the sensitivity and specificity needed for roadside testing. We aimed to select a THC-binding aptamer for the development of new oral fluid tests for THC. Aptamers are single-stranded nucleic acids that specifically recognise their target molecule with high affinity. Numerous trials with both DNA and 2'F-modified RNA did not succeed in enriching such sequences for THC. Most likely, this is due to the high hydrophobicity of THC and its thereby constrained ability to interact with nucleic acids. Nucleobase-modified aptamers have been shown to dramatically increase the selection success for protein targets. We thus used a recently developed technique, called click-SELEX, that facilitates modification of DNA with an azide of choice. Emulating the interaction of THC with the human cannabinoid receptor 1, benzyl- and trifluoromethylbenzyl-residues were utilised as nucleobase-modifications. This enabled the immediate selection of sequences that bind THC immobilised on epoxy-sepharose. While none of the sequences could be shown to recognise THC in solution, this thesis established the synthesis of fluorescently-labelled THC-derivatives that can be utilised in fluorescence polarisation assays to ascertain affinity constants of future clickmers. In addition, THC-solubility in aqueous buffers with low amounts of organic solvent was thoroughly investigated. Altogether, the findings of this thesis lay the foundation for the selection of THC-binding clickmers that can be implemented in roadside tests in the future.Click-SELEX ermöglicht die Selektion Δ9-Tetrahydrocannabinol-bindender Nukleinsäuren Δ9-Tetrahydrocannabinol (THC) ist für den Großteil der psychoaktiven Wirkung der Pflanze Cannabis sativa verantwortlich und die meistgenutzte Droge der Welt. Der Konsum beeinträchtigt die Fahrfähigkeit: Fahrzeuge können nicht in der Spur gehalten werden und die Wahrscheinlichkeit von Unfällen steigt. Urintests werden von der Polizei verwendet, können aber verhältnismäßig leicht manipuliert werden und setzen das Vorhandensein von entsprechenden Anlagen voraus. Zusätzlich ist die Korrelation zwischen Einschränkung der Fahrfähigkeit und positiven Ergebnissen schlecht, da ein THC-Metabolit an Stelle des aktiven THCs analysiert wird. Speicheltests sind als eine schnelle und einfache Alternative bekannt geworden, die zudem die Detektion von THC selbst und entsprechend einen viel besseren Nachweis des Fahrens unter akutem Drogeneinfluss gestatten. Die bisher erhältlichen Tests sind Antikörper-basiert, erreichen jedoch nicht die benötigte Sensitivität und Spezifität. Wir wollten ein THC-bindendes Aptamer für die Entwicklung neuer Speicheltests für THC selektieren. Aptamere sind einzelsträngige Nukleinsäuren, die spezifisch und hochaffin ihr Zielmolekül erkennen. Zahlreiche Versuche mit sowohl DNA als auch 2'F-modifizierter RNA konnten keine THC-Aptamere anreichern. Vermutlich ist dies auf die hohe Hydrophobizität THCs und seine dadurch eingeschränkten Möglichkeiten zur Interaktion mit Nukleinsäuren zurückzuführen. Nukleobasen-modifizierte Aptamere konnten die Erfolgsrate von Selektionen für Protein-bindende Aptamere drastisch erhöhen. Entsprechend verwendeten wir eine vor kurzem entwickelte Technik, die sich Click-SELEX nennt, und die Modifikation der DNA mit einem frei wählbaren Azid ermöglicht. Um die Interaktion von THC mit dem humanen Cannabinoid Rezeptor 1 nachzuahmen, wurden Benzyl- und Trifluoromethylbenzyl-Reste als Nukleobasen-Modifikationen verwendet. Dies ermöglichte die sofortige Selektion von Sequenzen, die auf Epoxy-Sepharose immobilisiertes THC erkennen. Obgleich keine der Sequenzen THC in Lösung bindet, etablierte diese Arbeit die Synthese fluoreszenzmarkierter THC-Derivate, welche für die Bestimmung der Affinität zukünftiger Clickmere mit Hilfe von Fluoreszenz Polarisation genutzt werden können. Zusätzlich wurde die Löslichkeit von THC in wässrigen Puffersystemen mit geringen Mengen organischer Lösungsmittel sorgfältig untersucht. Alles in allem legen die in dieser Dissertation dargelegten Ergebnisse das Fundament für die Selektion THC-bindender Clickmere, die in zukünftige Speicheltests implementiert werden können

Phase specific transcriptional regulation of circadian clock and metabolism in mouse liver

The molecular clock has been conserved from cyanobacteria to mammals and is believed to align behavioral and biochemical processes with the diurnal cycle. This cellular mechanism has been an advantage to increase the fitness of organisms through the ability to anticipate food availability or predator presence. Accumulating evidence has revealed that the circadian clock is intimately interconnected with the metabolic cycle. But, the nature of these interconnections is yet not clear at the transcriptional level, and the contribution of cis-regulatory modules has not been elucidated. Accessible chromatin regions of the genome are implicated in diverse processes, such as gene regulation through enhancers and promoters, insulation of genomic domains or alternative splicing. They allow cell-type specific programs that are controlled by tissue-specific transcription factors and chromatin modifiers, although tissue-specific regulation of the circadian clock remains unclear. Therefore, we compared genome-wide BMAL1 binding and chromatin accessibility in the liver and in NIH3T3 fibroblasts. Moreover, for the first time, our study explores the dynamics of accessible regions, histone 3 lysine 27 acetylation (H3K27ac) and RNA polymerase II (Pol II) every 4 hours over one day in mouse liver. In this study, we show that a substantial fraction of these accessible sites are oscillating during a diurnal cycle with a circadian period. We observed that these accessible regions are enriched in the proximity of actively transcribed genes, and that they are dynamically affected by the binding of transcription factors such as BMAL1. We investigated wild-type (WT) and Bmal1-/- genotypes, in night restricted feeding regimen and in Light-Dark (LD) cycle, to study the circadian clock regulatory network underlying diurnal transcription. Therefore, we applied a penalized generalized linear model to infer the activity of transcription factor binding motifs in oscillating accessible sites using Pol II loadings at the transcription start sites of nearby genes. We were able to recapitulate the known regulatory elements of the circadian clock, notably E-box, D-Box, and ROR-responsive elements (RRE) in the WT genotype. On the other hand, we found that Forkhead box (FOX), glucocorticoids responsive elements (GRE), and C-AMP Response Element (CRE) were the main contributors of the regulation of oscillating genes in Bmal1-/-. Finally, using a mixture model to detect footprints with a base pair resolution, we studied the dynamics of the accessibility overlapping E-box. Our last analysis suggested that BMAL1/CLOCK is binding on double E-boxes with a spacer of 6 or 7 bp in a hetero-tetramer configuration. A 3D structure model further supported this binding mode. In Summary, we used DNase I-seq and ChIP-seq of Pol II and H3K27ac to study the circadian chromatin landscape in a 4h time-resolved experimental design. We uncovered the underlying circadian transcriptional regulatory network and, we dissected the chromatin accessibility around BMAL1 binding sites at a base pair resolution, which led to an unappreciated mode of binding of BMAL1/CLOCK in a hetero-tetramer conformation on double E-boxes. Lastly, we found tissue-specific factors that might contribute to tissue-specific binding of BMAL1/CLOCK

A Unified Framework for the Prioritization of Variants of Uncertain Significance in Hereditary Breast and Ovarian Cancer Patients

A significant proportion of hereditary breast and ovarian cancer (HBOC) patients receive uninformative genetic testing results, an issue exacerbated by the overwhelming quantity of variants of uncertain significance identified. This thesis describes a framework where, aside from protein coding changes, information theory (IT)-based sequence analysis identifies and prioritizes pathogenic variants occurring within sequence elements predicted to be recognized by proteins involved in mRNA splicing, transcription, and untranslated region binding and structure. To support the utilization of IT analysis, we established IT-based variant interpretation accuracy by performing a comprehensive review of mutations altering mRNA splicing in rare and common diseases. Custom probes targeting 20 complete HBOC genes for sequencing in 379 BRCA-uninformative patients identified 47,501 unique variants and we prioritized 429 variants in both BRCA and non-BRCA genes. Our approach focuses attention on a limited set of variants from a spectrum of functional mutation types for downstream functional and co-segregation analysis