Clickmers and Aptamers as versatile tools for drug testing and fluorescence microscopy techniques

Abstract

Δ⁹-tetrahydrocannabinol (THC) is the main psychotropic compound of the plant cannabis sativa. THC consumption provokes several physical impairments, which in turn might reduce the capability of consumers, to drive vehicles. Hence, test-devices for the police are needed, that reliably detect small amounts of THC in biological matrices as, e.g., saliva. To date, most road-side test devices are based on antibodies and often lack the desired specificity and sensitivity. Therefore, chemically modified aptamers (clickmers) could serve as alternative biomolecules functioning as sensors in drug-test devices. <br /> Chemically modified aptamers are able to bind a multitude of targets with high affinity and specificity. Click-SELEX enables the modular modification of DNA libraries during the selection process. The incorporated building blocks ideally support the interaction of the oligonucleotides with the target molecule, which might increase the prospect of the selection-success. In this thesis a Click-SELEX approach was applied for THC, which had been immobilised on magnetic particles for the selection procedure. A benzyl-functionalised DNA-library was utilized for the selection of clickmers. The click- SELEX resulted in a G-rich sequence, capable to bind THC-modified beads with a dissociation constant of about 100 nM. Further characterisation of its binding properties resulted in a 41 nt long oligonucleotide only containing one relevant benzyl-modified position. Binding was clearly dependent on the introduced aromatic residues. However, the determined affinity of the identified sequence to THC in solution is not sufficient for the construction of a road side test-device. Nonetheless, it could be demonstrated, that click-SELEX is a suitable method for the selection of ligands for difficult to address target molecules like THC. Additional click-selections with adapted selection conditions potentially could lead to clickmers that fulfil the criteria for a roadside test. <br /> To study the location and dynamics of RNA sequences of interest fluorescent, light-up aptamer (FLAP) systems have been developed. FLAPs consist of RNA aptamers that specifically bind and enhance the fluorescence of inherently almost non-fluorescent organic dyes. To date, green, yellow, and red fluorescent light-up aptamer systems have been developed and the development of more FLAPs is in progress. One prominent FLAP is the Spinach-DFHBI complex. It has been shown, that slight modifications on the DFHBI structure in combination with already existing aptamers result in slightly red-shifted fluorescence emission of these complexes. <br /> In this thesis a derivative of DFHBI (called eDFHBI) with an enlarged aromatic system was synthesized with the aim to develop a FLAP with red-shifted emission properties. In complex with Baby-Spinach eDFHBI showed a distinct red-shifted fluorescence. However, the affinity of Baby-Spinach to eDFHBI was drastically reduced. Thus, to develop a new FLAP system an RNA SELEX for eDFHBI has been undertaken. Yet the selection did not lead to the identification of the desired aptamer, thus the development of a red FLAP system was not successful. <br /> Furthermore, a photoactivatable derivative of DFHBI, called PA-DFHBI was synthesized here. The combination of Spinach or Spinach-derived aptamers with PADFHBI would result in a photoactivatable FLAP. Such photoactivatable systems might be advantageous in super resolution microscopy techniques. Indeed, in vitro characterisation of PA-DFHBI with DFHBI binding aptamers revealed the functionality of such a photoactivatable system. However, in mammalian cells the PA-DFHBIaptamer complex immediately emitted green fluorescence and thus the photoactivatable character of this system was lost. The reason for this problem could not be determined.