title: Development of RNA aptamers binding environmental and food contaminants
Creators: name: Kramat, Janice
Publisher: name: UNSPECIFIED
Year: 2023

description: The pollution of the environment by various substances is a central issue of our time. Humans introduce a wide variety of pollutants into nature through industrial processes, the use of certain substances in agriculture and the application of pharmaceuticals in factory farming. These contaminants can exert negative effects on the environment and living organisms due to their presence or specific effect and return to humans through various pathways. The detection of such substances in environmental samples or food is therefore essential for uncovering their entry routes and distribution. Analytical methods such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS) are already used for this purpose. Both methods offer precise analysis and can detect even the smallest traces of various substances. However, this requires specially qualified staff, extensive laboratory equipment and very expensive instruments. In addition, the analyses take a certain amount of time and cannot be used flexibly or portably. Detection methods that do not have these disadvantages are based on sensor technology using biological elements. They represent rapid, simple, comparatively inexpensive analysis platforms that can be used on site. These are so-called biosensors and are based on a harmonised interplay of different components. The basis for the recognition of an analyte is provided by the bioreceptors. These can consist of whole microorganisms, isolated proteins such as enzymes or antibodies, or even short DNA or RNA molecules. They are responsible for the sensing of the analyte and enable the generation of a signal. This is subsequently converted into a visible or measurable signal by a transducer. Finally, a third component can be used to amplify and process this signal. Thus, biosensors provide not only a qualitative but also a quantitative evaluation of a wide variety of analytes. Based on the problems caused by environmental as well as food contamination, the aim of this study has been the development of short, single-stranded RNA sequences as suitable bioreceptors. These are also called aptamers and their application as recognition elements in biosensors make them aptasensors. Aptamers are able to bind a variety of different ligands. These include whole cells, proteins, but also ions or small compounds. In this work, the latter were selected as target molecules for RNA aptamers. The substance classes of artificial sweeteners as sugar substitutes, bisphenols, which are necessary to produce synthetic materials, as well as antibiotics used in human medicine and factory farming were chosen in particular. The individual representatives of these classes were acesulfame K, cyclamate, saccharin and sucralose as sweeteners, the bisphenols A, F, S and 4-hydroxyacetophenone, a degradation product of bisphenol A, as well as the antibiotics kanamycin A and levofloxacin. Some of these substances can already be detected in the environment and partially cause damage to nature and living organisms or are suspected of doing so. Therefore, they represent interesting analytes for aptamer-based biosensors. The origin of the development of aptamers involved different in vitro selections of ligand-binding RNA molecules from an extensive sequence library. The method used for this was derived from SELEX (systematic evolution of ligands by exponential enrichment), which was developed in 1990. It is called Capture-SELEX and is based on an iterative process. This always includes the immobilisation of RNA molecules, elution of these through interaction with the chosen ligand, amplification of the selected sequences and their preparation to be used as a new RNA pool for the next selection round. This led to an enrichment of potential aptamers for the target molecules bisphenol A, kanamycin A as well as levofloxacin. The selection against bisphenol A revealed that binding did not occur to the ligand itself but to ethanol, which was required for the solubility of the bisphenol in aqueous solution. Based on this discovery, further steps in the development of an RNA aptamer were focused on monohydric alcohols as ligands. In addition to ethanol, isopropanol and methanol were also examined as target molecules in Capture-SELEX experiments. No binding between RNA and the ligand could be detected for methanol. Six different RNA sequences could be identified for ethanol and isopropanol using cloning and sequencing techniques. These sequences were evaluated for their ability to distinguish between the two alcohols, which led to the ‘aptamer I’ that preferentially binds isopropanol. Based on the prediction of a secondary structure, various truncations of aptamer I were made subsequently. These were again assayed for their ability to distinguish ethanol and isopropanol as ligands. Certain truncations led to a loss of the binding ability of the RNA to the applied ligands. Others still exhibited binding but did not show any improvement in specificity. Attention has to be paid to the possibility that the monohydric alcohols ethanol and isopropanol are ligands that are capable of forming non-specific interactions with RNA. Therefore, further research is needed to determine whether it is a 'classical' aptamer-ligand binding. The in vitro selection against levofloxacin resulted in an enrichment of target-binding aptamers. Subsequently, the entire selection was analysed using Next Generation Sequencing. The resulting data were bioinformatically evaluated in cooperation with the research group ‘Self-Organising Systems’ of the Department of Electrical Engineering and Information Technology. In addition to assessing the enrichment process of levofloxacin-binding RNA molecules, eight different sequences of potential aptamers were identified. These were tested for their ability to distinguish between two closely related ligands. In addition to the intended target molecule, the structurally very similar ciprofloxacin was used. This resulted in the aptamer LXC, which had the best distinguishing ability and a dissociation constant in the low micromolar range for binding to levofloxacin. Subsequently, based on the predicted secondary structure of LXC, truncations as well as mutations of the sequence were carried out. These were evaluated by isothermal titration calorimetry (ITC). It resulted in the levofloxacin-binding aptamer trLXC. Based on the ITC mutation studies and subsequent in-line probing experiments, the predicted secondary structure could be confirmed. Furthermore, it allowed the identification of regions involved in ligand binding. Thus, a levofloxacin-binding aptamer ready to be used as a receptor in a biosensor was successfully developed and characterised.