New methods for sequence-specific modification of RNA to study structural and functional aspects of large ribozymes

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The site-specific labeling of RNA is of main interest for structural and functional studies on a plethora of long non-coding RNA molecules. The synthesis of sequence-specific modified long RNA molecules, which cannot entirely be prepared via solid phase synthesis methods due to its limitations in length, has remained a challenge. Main objective of the proposed research project is to develop new enzymatic methods for the site-specific labeling of RNA molecules to study structure and dynamics of these RNAs using a semi-synthetic approach. Unnatural base pairs will be employed as a tool to implement functional groups, capable of undergoing cycloadditions reactions, enzymatically into RNA to allow postsynthetic attachment of reporter groups. This principle should be used to introduce two fluorophores at specific positions in transcribed RNA via orthogonal inverse electron-demand Diels-Alder and copper-catalyzed azide-alkyne cycloaddition reactions. Accordingly modified unnatural nucleoside triphosphates should allow the site-specific introduction of reactive groups using standard conditions for in vitro transcription. In the framework of this project proposal, the human CPEB3 protein should site-specifically be labeled with two fluorophores for FRET studies of global conformational changes caused by various factors. The extension of the concept towards site-specific introduction of spin-labels into RNA for distance measurements via electron paramagnetic resonance spectroscopy is proposed. The planned method represents a useful alternative for ligation based strategies for the preparation of long, sequence-specific modified RNA molecules. Thus it could be applied as an ideal tool for structural investigation of functional, non-coding RNA molecules.