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

Associative Pattern Recognition for Biological Regulation Data

In the last decade, bioinformatics data has been accumulated at an unprecedented rate, thanks to the advancement in sequencing technologies. Such rapid development poses both challenges and promising research topics. In this dissertation, we propose a series of associative pattern recognition algorithms in biological regulation studies. In particular, we emphasize efficiently recognizing associative patterns between genes, transcription factors, histone modifications and functional labels using heterogeneous data sources (numeric, sequences, time series data and textual labels). In protein-DNA associative pattern recognition, we introduce an efficient algorithm for affinity test by searching for over-represented DNA sequences using a hash function and modulo addition calculation. This substantially improves the efficiency of \textit{next generation sequencing} data analysis. In gene regulatory network inference, we propose a framework for refining weak networks based on transcription factor binding sites, thus improved the precision of predicted edges by up to 52%. In histone modification code analysis, we propose an approach to genome-wide combinatorial pattern recognition for histone code to function associative pattern recognition, and achieved improvement by up to $38.1\%$. We also propose a novel shape based modification pattern analysis approach, using this to successfully predict sub-classes of genes in flowering-time category. We also propose a combination to combination associative pattern recognition, and achieved better performance compared against multi-label classification and bidirectional associative memory methods. Our proposed approaches recognize associative patterns from different types of data efficiently, and provides a useful toolbox for biological regulation analysis. This dissertation presents a road-map to associative patterns recognition at genome wide level

Interferon-γ aptamers for the diagnosis of extra-pulmonary tuberculosis

Tuberculosis (TB) is a major global health problem. About 15-20% of the global population who are HIV negative have extra-pulmonary TB (EPTB) such as pleural TB. This increases to 50-70% in HIV positive people. The diagnosis of EPTB is challenging because of the low bacillary burden. Interferon gamma (IFN-γ) has been identified as a promising biomarker for the diagnosis of EPTB. The development of rapid and accurate point-of-care (POC) diagnostic technologies becomes crucial in controlling EPTB. Aptamers referred to as "synthetic antibodies" have been recently explored as a replacement for antibodies in diagnostic platforms. These single-stranded nucleic acid molecules have high affinity and specificity comparable, and in some instances even superior, to those of antibodies; in addition to their relatively low cost and simple method of production they have the potential to reduce assay turnaround time. The aim of this thesis was to develop aptamers to IFN-γ, a biomarker specific for EPTB, thus facilitating the development of aptamer-based POC tests for the diagnosis of EPTB

DNA Sequencing

This book illustrates methods of DNA sequencing and its application in plant, animal and medical sciences. It has two distinct sections. The one includes 2 chapters devoted to the DNA sequencing methods and the second includes 6 chapters focusing on various applications of this technology. The content of the articles presented in the book is guided by the knowledge and experience of the contributing authors. This book is intended to serve as an important resource and review to the researchers in the field of DNA sequencing

Development of fluorogenic RNA aptamers for cellular imaging of RNA and genomic loci

In recent years, there has been an explosion of SELEX-evolved fluorescent RNA aptamers, such as Spinach, Broccoli, Corn and Mango. Fluorogenic RNA aptamers have sparked a lot of interest and hold great potential to enable background-free visualisation of RNA molecules in cellular environments. However, their application has been limited by relatively inefficient folding in vivo and fluorescent stability. Therefore, evolving new RNA aptamers with improved brightness and stability should better their use in cellular imaging. Three new Mango-based aptamers have recently been selected from the original Mango RNA SELEX pool using microfluidic- assisted in vitro compartmentalization and fluorescence-activated sorting. This thesis demonstrates the use of these new aptamer variants to image small non-coding RNAs (such as 5S rRNA, U6 snRNA and mgU2-47 scaRNA) in both fixed and live human cells with improved sensitivity and resolution. Upon expression the modified RNAs subcellular localisation pattern is conserved, as validated using immunofluoresence. Recent work with tandem Mango arrays shows increased sensitivity, which enables the visualization of single mRNA molecules in live and fixed cells. Furthermore, it is shown that the tandem Mango arrays don’t affect the expected localization of a cytoplasmic mRNA (β-actin) and the nuclear long non- coding RNA (NEAT-1). Furthermore, these RNA aptamers can also be used to label genomic loci via CRISPR/Cas9 mediated genome targeting with improved contrast. This allows for the targeting of short genomic repeats in a less invasive manner with regards to current methodologies. Taken together this data shows that new Mango aptamers are vastly improved for cellular imaging over previous RNA aptamers, and can in principle be incorporated into a wide range of coding and non-coding RNAs.Open Acces

Development of photo-responsive synthetic RNA devices

The different functions of RNA have led to great and increasing interest in the field of synthetic biology. One such example of versatile RNA molecules used in synthetic biology is aptamers. Aptamers are able to bind to a wide range of target molecules with a high affinity and specificity, which is why they are often compared to antibodies. Using the SELEX method, they can be generated in vitro against target molecules for a broad range of possible applications. In nature, they work as the sensing domain of riboswitches and regulate gene expression by binding their cognate target molecule. Riboswitches can also be generated synthetically from in vitro generated aptamers. In the course of this work, a novel RNA aptamer which selectively binds to one of two light-induced isoforms of a specific small molecule ligand (azoCm) was developed. The potential function of such an aptamer as a riboswitch was evaluated. A previously carried out SELEX experiment against azoCm yielded a specifically binding aptamer (aptamer 42). At the beginning of this work, its secondary structure was analyzed using in-line probing. As it was previously shown aptamer 42 could not work as a riboswitch regulating GFP expression in the model organisms Saccharomyces cerevisiae, other aptamers from the same SELEX were tested under the same conditions using in vivo screening. As no functional riboswitch could be identified, a new SELEX experiment using a new aptamer library was started. The new affinity SELEX yielded aptamers specifically binding to azoCm. However, in vivo screening of aptamers from this SELEX did not result in an azoCm dependent riboswitch. Therefore, a novel light SELEX method was developed and established. This light SELEX protocol enriched isoform selective aptamers by a light-induced conformational change of azoCm during the SELEX process. As in vivo screening of light SELEX aptamers did not yield a riboswitch again, in vitro binding studies of aptamers from the light SELEX were performed. Comparing three aptamers which showed the highest discrimination between the two isoforms of azoCm to each other led to the discovery of a 13 nt sequence motif that only these aptamers shared. A next-generation sequencing experiment performed with the SELEX and light SELEX rounds revealed that this sequence motif (“light motif”) was specifically enriched during light SELEX rounds, but was gradually depleted during the later, more stringent affinity SELEX. Based on this discovery, a new library for SELEX was designed, containing the partially randomized light motif, as well as entirely randomized flanking regions. SELEX performed with this light motif doped library led to a fast enrichment within five rounds, and aptamers from this SELEX were tested regarding their in vivo functionality. From the aptamers tested in vivo, four showed gene regulatory function, however with a low regulatory factor of 1.25- to 1.35-fold. Based on the best functional aptamer B2, partially randomized aptamer libraries were generated for further in vivo screenings. While the regulatory factor of B2 could not be improved using this approach, a modified version of it called B2-1 could be shown to regulate gene expression in yeast cells in an azoCm dose-dependent manner. To learn more about the aptamer B2-1, its binding affinity was analyzed using isothermal calorimetry. The kD of B2-1 was determined to be 23 nM, depending on the calculation model used. A truncated version of B2-1 showed a low micromolar binding to azoCm, indicating that structurally relevant parts of B2-1 had been deleted during the truncation. However, both B2-1 and its truncated version did selectively bind to only one isoform of azoCm, while binding to the other isoform could not be detected. The aptamer developed in this work shows a much stronger discrimination between the two isoforms of its light-switchable ligand than previously reported isoform-selective aptamers. It also shows the highest binding affinity to its ligand compared to the isoform-selective aptamers in literature to date. While a riboswitch based on this aptamer shows only slight regulatory function, dose dependent regulation of gene expression could be shown. This work therefore constitutes the first steps towards the generation of a light dependent riboswitch