University thesis:
Dissertation, Universität Freiburg, 2021
Footnote:
Description:
Abstract: Aptamers are short single-stranded DNA or RNA oligonucleotides with the ability to bind a molecular target with high affinity and specificity. Other positive properties are their high thermal and chemical stability and that their denaturation is reversible, so they can be easily reused. Therefore, they have the potential to replace antibodies in many diagnostic and therapeutic applications in the future.<br>However, a major problem in aptamer research is to find the right aptamer sequence with the desired properties. The current selection method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) consists of alternating selection and amplification cycles, with the aim of evolutionary enriching the “best/fittest” aptamers. In reality, however, SELEX is severely limited due to many biases. Sequences that perform best in PCR amplification have the greatest selection advantage, but often displace better binders that amplify worse. In addition, SELEX is very time-consuming and cost-intensive, as the best of all sequences in the final pool have to be characterised individually.<br>Therefore, a completely novel selection method called AptaSwift was developed, and a first proof-of-concept was partly realised within this dissertation. AptaSwift aims to decouple selection and amplification by limiting each amplification and also providing optimal monitoring at each amplification step. This is enabled by the combination of a novel digital solid-phase PCR method which limits the amount of generated DNA per sequence with a label-free measurement system called SCORE (Single COlor REflectometry), enabling to monitor the binding properties of each sequence in parallel. In the context of this thesis, the digital solid-phase PCR and SCORE analysing step is tested, as well as additionally combined with a photocleavage process, in order to enable a later release of DNA sequences according to demand. Using the SCORE system, all potential aptamers on a microarray generated by digital solid-phase PCR can be analysed and characterised simultaneously. Subsequently, the best binders are regained in the photocleavage process by cleaving the photocleavable groups of the solid-phase primer using a microscope and short-wave light.<br>In this thesis, microarrays were generated from a pool of three aptamer species via the solid-phase PCR process using two different approaches, and the aptamers were successfully analysed for their binding affinity to thrombin using SCORE. In addition, the photocleavage of a thrombin aptamer was demonstrated with sequencing data. To evaluate the kinetic analytical power of the SCORE system, aptamer screenings were performed with synthetic aptamer arrays binding thrombin and ICAM-1 (intercellular adhesion molecule 1), and DNA binders against Min proteins. A systematic investigation of thrombin aptamer sequences could be performed, showing especially that the height of the binding signal nearly linearly depends on the number of Thymine (T-) spacer between aptamer and surface.<br>5<br>In contrast to thrombin aptamers, DNA interactions with ICAM-1 were found to be nearly completely non-specific, as positive and negative controls also showed binding. Also contrary to current models and theories, the investigated Min proteins bind much better to single-stranded than to double-stranded DNA. The validation of the SCORE system was concluded with a comparative study of six different biosensing systems. In addition, the influence of the analysis software and the user on the determination of the binding constants was investigated. It could be shown that the software has only minor impact, but the quite often manual selection of the fitting windows can impact the KD value by a factor of up to 10. But this selection is already impacted by the definition of the assay itself, as, for example, the off-kinetic time may be determined to be 5 min instead of 10 min, which can have an impact of an order of magnitude for the KD analysis. Therefore “the human factor” may be a major impact to many kinetic analysis publications