Aptamer ligands capable of photocrosslinking

In the proposed project I am going to develop a new class of chemical compounds which can interact with so-called aptamers. Aptamers are nucleic acids (RNA or DNA) that can adopt complex structures thereby forming a pocket that specifically recognizes the compound of interest (ligand). Usually, the ligand binds to the aptamer in reversible manner via non-covalent interactions, i.e. hydrogen bonds, ion pairs, or by hydrophobic interactions e.g. stacking. Some naturally occurring RNA aptamers change their structure upon ligand binding. This way, nature utilizes aptamers for the purpose of gene regulation, switching genes on or off in response to varying ligand concentration ("riboswitches"). In other words, if the particular compound is present in the cellular environment, the switch interacts with it, changes its shape, and therefore affects the cellular machinery usually transcription (RNA synthesis) or translation (protein synthesis). These mechanisms are very common in bacteria but also in some higher organisms and they allow the cell to respond to changing environmental conditions. Among aptamers there are also artificial ones, which were created by scientists through in vitro selection approaches. An important class of such aptamers exhibit fluorescence after binding the dedicated (non-fluorescent) ligand. These so called light-up aptamers are particularly useful for biotechnology studies e.g. for detecting nucleic acids both, in vitro and in cells. In my project I intend to develop aptamers with advanced performance. Based on the known compounds which are substrates for the aptamers I will synthesize similar compounds, but these will be capable of photocrosslinking. This process starts upon UV irradiation which results in the fomation of a stable (covalent) bond between the ligand and the aptamer. This will keep the ligand in the aptamers pocket. Consequently, the aptamer becomes frozen and ideally a certain function is performed constantly. The novel photocrosslinking ligands developed in this project are promising tools for biosciences. In case of light-up aptamers I will develop ligand-RNA systems which will be useful for RNA imaging and localization in vitro and in vivo, for RNA tracing in cellular processes, for identification of novel cellular RNAs, and for RNA purification. Additionally, I intend to thoroughly investigate how covalent linkage between aptamer and chromophores will alter the absorbance/fluorescence behavior. Concerning the naturally occurring aptamers I will develop ligands useful for the isolation and identification of RNA riboswitches and their role in the metabolism and gene regulation.