Projekt Nr. 05

Chemical tools for RNP isolation and for gene regulation via riboswitches

 

Institute of Organic Chemistry & Center for Molecular Biosciences Innsbruck
State of the Art

The project is focused on tool development for the isolation of RNPs and for gene regulation via small artificial riboswitches

 

Objective A - Methods for RNP isolation based on the Staudinger ligation


The isolation and characterization of the protein components that interact with ncRNAs is fundamental in order to understand their functions. We will elaborate novel chemical methods for the isolation of ribonucleoprotein complexes (RNPs): Referring to ncRNAs of known base sequence, we will synthesize derivatives of these RNAs (50 to 100 nts) and site-specifically introduce chemical anchor groups to equip them for covalent attachment to solid beads. The choice of these functional groups is most stringent and will be based on the so-called Staudinger ligation.

 
The Staudinger ligation is one of the rare examples of bio-orthogonal reactions, in which the functional groups introduced (azides, phosphines) neither interfere with endogenous cellular functions nor be inappropriately recognized by the host. The modified RNAs can then be used directly for transfection of target cells or applied to cell lysates. Harvesting of the RNPs occurs via solid-phase beads possessing properly functionalized surfaces suitable for the covalent attachment. The major advantage of the Staudinger ligation is the high chemical selectivity. Therefore, compared to other affinity purification approaches, significantly less unspecific interference with the host cell machinery can be expected. To exploit the benefits of this reaction for RNA methodologies has not yet been achieved. Once we have verified our approach on a model RNP system (e.g. siRNA/p19) the method should serve as tool for the ncRNAs discovered in the course of this project cluster (Hüttenhofer/Polacek, Barta, Schroeder, Kovar, Martinez) in order to identify or validate the interactions with protein components.

Objective B - Artificial riboswitches for gene regulation


Natural metabolite-responsive riboswitches have been recognized in recent years to play a major role in gene regulation in prokaryal genomes.4 We aim at the development of small artificial riboswitches that are controllable by simple chemical reactions. Preliminary investigations have demonstrated that functionalized nucleobases, such as trichloroethyl guanines, possess the potential to trigger RNA secondary structure rearrangements.5 This concept will be expanded to nucleotides with functional groups that are sensitive for stimulators such as light, pH, or metal ions. Consequently, illumination or a change in pH or in metal ion concentration should induce a different shape of the RNA. Riboswitch sequences of this kind will be designed (together with Hofacker) and experimentally verified. The perspective is to exploit these modular artificial riboswitch tools for gene regulation at the ncRNA/mRNA level. This concept stands align with recent attempts for the regulation of gene expression by tailor-made ribozymes, with the aptamer-antidote system for controlling blood coagulation in animals, or with ligand-controlled riboregulators for eukaryal gene expression. In addition, the artificial riboswitch modules will serve as ‘lead sequences’ in genomic screens for the identification of tiny, naturally occurring riboswitches.

Business Area

Teaching and research at the Leopold-Franzens University of Innsbruck

 

For further information please contact Marius Koppler