Homogeneous fluorescence-based aptamer assays: Evaluation and development of established and novel detection mechanisms for simplified aptamer Integration

In vitro assays are a reliable and convenient method of detecting disease biomarkers, conducting environmental and food safety screenings and controlling bioprocesses. These assays primarily rely on antibodies as recognition elements, exhibiting high affinity and specificity. Although antibodies used in immunoassays are viewed as the gold standard in assay development, aptamers are promising alternatives as recognition molecules for the integration into in vitro assays. Aptamers are short single stranded DNA or RNA sequences that bind the target molecules with high specificity and affinity due to their characteristic three-dimensional structure. With regard to the limitations of antibodies, aptamers have the advantage of having no immunogenicity, being more stable under extreme pH and heat, being chemically synthesized and additionally having the ability to refold after denaturation. These properties lead to assays, which have a longer shelf-life, are less expensive and are more expeditious to manufacture. In recent years, numerous high-affinity aptamers with exceptional specificity were selected in a process called systematic evolution of ligands by exponential enrichment (SELEX). Their application encompasses diverse matrices, target molecules, signal generation mechanisms, and a variety of detection methods. For instance, in heterogeneous assays, such as sandwich or competitive ELISAs, in which the recognition element or target molecule is immobilized, antibodies can be easily substituted by aptamers. However, these heterogeneous assays are often time-consuming and labor-intensive, which is why homogeneous assays are often a preferred alternative. Aptamers can also be applied in homogeneous assays, wherein commonly structure switching is employed as the signaling mechanism. During structure switching, the aptamers change their native tertiary structure induced by target binding. This is exploited in detection strategies like strand displacement and molecular aptamer beacon (MAB) assays, whereby mainly the fluorescence signal of fluorophore-labeled aptamers affected by quenching either induced or suppressed during target binding in measured. The phenomenon of structure switching has been described in literature as a generalizable effect for all aptamers that possess a distinct target-binding-induced tertiary structure. Yet not all aptamers have this property or there is a lack of sufficient information about their target binding regions and kinetics. In this thesis, the strand displacement assay and the MAB assay were therefore evaluated by integrating two model aptamers form literature to develop homogeneous assays without any prior knowledge about their ability to switch structure. Two literature aptamers were selected for this purpose, one against human urokinase plasminogen activator (huPA), a potential biomarker for cancer, and second against His-tag, which has potential use in recombinant protein quality control. For the literature aptamers, complementary strands were designed, and the aptamers were modified to form hairpin structures and tested for their binding to the target and functionality in the assays. For the assays described in literature, no efficient target detection could be determined with both tested aptamers without designing and evaluating further complementary primers and aptamer hairpin structures. For this reason a new kind of aptamer assay was developed by implementing free complementary primers to the hairpin aptamers in one mechanism. This Target Induced Primer Obstruction (TIPO) is used for aptamers which do not change their native tertiary structure when binding to a target molecule. The hairpin of the aptamer is configured to open only when primers hybridize to the aptamer, leading to an increase in fluorescence. This increase is inhibited by the presence of the target. In the homogeneous format, this assay achieved a calculated limit of detection (LOD) of 2 nM for the target huPA, 7 nM for the His-tagged mouse programmed cell death protein 1 and 4 nM for the His-tag small molecule. The TIPO mechanism was successfully established with components free in solution but also with aptamers immobilized in microtiter plates. This assay has the potential to serve as a substitute for established aptamer sandwich assays in cases where targets have only a single binding epitope or when aptamers devoid of structure-switching capabilities are utilized. Due to the seamless integration of various literature aptamers into the assay, the TIPO assay does not necessitate extensive establishment experiments. Consequently, this feature renders the assay a remarkably effective instrument in expediting the development of highly sensitive assays for the detection of disease biomarkers and recombinant protein quality control.