Expanding the toolbox of modified nucleobases and click chemistry for investigations on non-coding RNA

Abstract

Non-coding ribonucleic acid (RNA) consists of highly structured molecules whose arrangement and function can be investigated using different chemical labeling approaches. Contemporary labeling techniques show restrictions either in labeling specificity or oligonucleotide length. Literature describes highly efficient and fast labeling strategies as well as different approaches of unnatural base pairs (ubp) specifically recognized by enzymes, but previous work failed to address a combined technique for labeling. In this thesis, an enzymatic template-directed labeling approach is shown based on unnatural ribonucleoside triphosphates either suitable for structural investigations using electron paramagnetic resonance (EPR) spectroscopy or modified for subsequent inverse electron demand Diels-Alder (IEDDA) click chemistry on non-coding RNA. Chemical synthesis of ubp and IEDDA click building blocks, successful template-directed T7 in vitro transcriptions with subsequent fluorescent labeling or EPR-based distance measurement will be described. This novel approach reveals a site-specific labeling technique of long RNA strands to get a better insight in catalysis and regulation on RNA level.