Kristallisation

The diverse functions of ribonucleic acids as information carrier (mRNA), adapter (tRNA), scaffold and catalyst (rRNA) reflect the key position within the living cell and their essential significance for life itself. New applications in biotechnology and molecular medicine arise from the catalytic features of ribozymes, the highly specific binding capability of aptamers and from small ribonucleic acids termed RNAi and regulators. The inherent structural and functional properties of these molecules are employed in the rapidly evolving area of RNA technologies underscoring a great need for folding motifs to design functional RNAs. So far, less than 2% of known structures belong to ribonucleic acids (NDB, PDB).

Starting with the 5S rRNA in the early 70-ies, the interest in this molecule initially came from the observation of a reduced peptidyl transferase activity if ribosomes lack 5S rRNA. However, it took three decades to understand that 5S rRNA is probably assisting in the assembly of the large ribosomal subunit. The milestone of ribosome structure by Steitz & Moore led to detailed insights into the RNA-protein interaction and the conformational change revealed by the first structure of free 5S rRNA (PDF file) proved the scaffold postulate.

We are currently contributing to the RNA technology by crystal analysis of high-affinity & catalytic (ribo)nucleic acids.

Spiegelmers were originally developed and covered by patents in our laboratory representing a promising approach in DNA or RNA drug stabilization. By exploiting chirality, these high-affinity L-enantiomeric oligonucleotide ligands exhibit a high resistence to enzymatic degradation compared with D-oligonucleotides (aptamers). Several Spiegelmers (binding targets like the gonadotropin-releasing hormone or the staphylococcal enterotoxin B) were identified by the pharmaceutical industry. The characterization of folding motifs and the definition of structural elements involved in target interactions include the knowledge for the optimization of these mirror-image molecules.

We are going to approach the phenomenon of RNA interference (RNAi) by crystallizing parts of the RNA-induced silencing complex (RISC). RNAi is a response to dsRNA which triggers the sequence specific gene silencing. It has been cultivated as means to manipulate gene expression experimentally and to probe gene function on a whole-genome scale. The structural studies will primarily include siRNA/dFXR complexes since the human protein homolog FMRP not only contains a RNA binding site but it is involved in the fragile X syndrome as well. Furthermore, the use of RNAi to control the expression of PHD provides an effective tool for the characterisation of angiogenic pathways and the understanding of the regulation mechanisms of HIF (hypoxia-inducible factor) hydroxylases.

Other catalytic activities to be structurally studied are the 10-23 DNA enzyme complexed with an RNA substrate and artificial ribozymes (Diels-Alderase ribozyme).

To enhance the probability of RNA crystallization and to improve the X-ray diffraction behavior, the following approaches are applied:

                                    - Nucleic acid engineering
                                    - Carrier-drived RNA crystallization
                                    - High-throughput screening
                                    - Crystal engineering
                                    - Microgravity experiments

The lab know-how includes state-of-the-art techniques from molecular biology, biochemistry and crystallography. Due to its unique position within the RNA Network, this structural unit additionally serves as interface for the management of customized projects from synthesis to structure.

Project Description