Conformational dynamics of RNAs as well as their interactions with proteins in RNP complexes will be investigated by a variety of different:
Time-resolved NMR-techniques are applied to obtain a three-dimensional description at atomic resolution of the conformational states of regulatory RNA molecules under various conditions. In addition, NMR relaxation measurements, paramagnetic relaxation enhancement (PRE) and residual dipolar couplings (RDC) will be used to explore local dynamics of RNA molecules and to characterize the three-dimensional structure of the molecules as well as the structural changes within ligand binding.
Pulsed Electron Paramagnetic Resonance (EPR) techniques such as PELDOR (Pulsed Electron Electron Double Resonance) will be used and improved to characterize global conformational changes in larger RNAs or RNPs by obtaining long-range distances (1-6 nm) and for the investigation of the conformational flexibility of RNA and RNA/protein complexes.
Sophisticated photophysical properties of structurally sensitive molecular probes such as fluorescence quantum yields or quenching properties are utilized in order to understand RNA dynamics. IR-spectroscopy is combined with selective RNA-labelling techniques borrowed from NMR-spectroscopy to characterize ligand binding events and conformational changes in RNA. It will be explored whether fluorescent pyrenes can be utilized to monitor RNA conformational transitions in a biological context. Pyrene fluorophores have great potential because they form distance- and orientation-dependent excimer complexes upon di- or oligomerization, and their fluorescence can be quenched in complexes with perylenes in a well understood manner.
Methods were developed that allow one to quantify protein and RNA copy numbers directly in cells by Super-Resolution Fluorescence Microscopy and neuron-wide landscape analysis with single-molecule resolution is performed. In particular, the copy numbers in individual synapses is determined, the copy numbers to activity and plasticity are correlated, and individual synapses within the same and among different cells are compared.
The cross-talk between experimental and simulation approaches will achieved by the re-use the structural and dynamical data accumulated in the previous/current funding period from the work of Schwalbe, Wachtveitl and Prisner at atomistic simulations. The data will be used to validate and enhance the quality of force fields available for RNA and RNA-ligand complexes and then use these force fields to investigate structural transitions in riboswitches and RNA-thermometers by Atomistic and Coarse-grained Molecular Dynamics Simulations.
As a very novel approach for the characterization of RNA-ligand interactions, Site-specific Dynamic Nuclear Polarization (DNP) – a sensitivity enhancing technique – in conjunction with solid-state NMR will be used. The structure and dynamics of ligand binding sites in RNA-regulatory elements such as riboswitches and ribozymes will be investigated. The approach will use either ligands or bound metal ions as “Trojan horses” to introduce paramagnetic centers into the RNA of interest at a strategically chosen site to selectively enhance the NMR-signals of atoms in the binding site.
Small molecules that do not occur naturally in cells but are non-toxic and easily taken up can regulate gene expression through interactions with non-natural RNA elements that bind to them. The pioneer approach led to the development of Artificial Riboswitches triggered by neomycin and tetracycline which work in translational regulation in lower eukaryotes. These riboswitches were successfully adapted to work in other classes of organisms and most importantly shown to be able to regulate constitutive and alternative splicing in mammalian cells.
Light is apparently a bio-orthogonal trigger for RNA conformational changes since in contrast to the protein world so far no natural light-responsive RNAs are known. Light-triggered RNA-based regulatory elements based on the selective interactions between an RNA and a small molecule ligand was develdoped that switches between two different conformations in response to light – so called Photoswitches.