The Synthesis of Selenium-modified RNA

The growing knowledge about the high significance of small RNAs in many biological processes is accompanied by a growing demand for structure elucidation of the participating RNAs by X-ray crystallography. In this context, Se-modification of RNAs is more and more requested, in particular, Se-modification of 30 to 100 nt RNAs that do not possess, or possess only a limited number of sites that allow for selective binding of metal ions with anomalous scattering propensity. The proper accessibility of Se-modified RNA offers a great alternative to obtain derivatives for facile phasing of diffraction data via the selenium sites. Robust methods for the preparation of Se-RNA would therefore have a major impact on nucleic acid X-ray structure analysis and, in general, on the field of structural biology. In preliminary studies, we have demonstrated this for a novel RNA fold of 49 nucleotides. In cooperation with other research teams, we have solved the structure of the Diels-Alder ribozyme and obtained the first molecular insights into RNA`s role in catalyzing a reaction other than phosphodiester chemistry. Goal of this project is the development of robust methodologies for the synthesis of selenium-derivatized RNAs. In our laboratory, we will focus on an approach involving the chemical synthesis of 2`-Se-methyl nucleoside phosphoramidite building blocks and their incorporation into oligoribonucleotides by solid-phase synthesis. In addition, we will enzymatically ligate the selenium-derivatized RNAs to obtain specific, biologically important RNAs and RNA domains. These RNAs will then be target of X-ray crystallographic structure analysis in cooperation with the laboratory of Dinshaw Patel (Sloan Kettering Memorial Cancer Center, NY). Our main RNA targets are G-rich triplet repeats, U6/U2 snRNA motifs, and metabolite-binding riboswitch mRNA domains. The latter are highly attractive, and biologically most important candidates: in the past two years it has been found that specialized domains within certain mRNAs directly bind metabolites in a selective and concentration dependent manner, and thereupon, control gene expression without the need of protein factors. Our studies should reveal the structural principles of this novel mode of gene regulation.

The growing knowledge about the high significance of small RNAs in many biological processes is accompanied by a growing demand for structure elucidation of the participating RNAs by X-ray crystallography. In this context, Se-modification of RNAs is more and more requested, in particular, Se-modification of 30 to 100 nt RNAs that do not possess, or possess only a limited number of sites that allow for selective binding of metal ions with anomalous scattering propensity. The proper accessibility of Se-modified RNA offers a great alternative to obtain derivatives for facile phasing of diffraction data via the selenium sites. Robust methods for the preparation of Se-RNA would therefore have a major impact on nucleic acid X-ray structure analysis and, in general, on the field of structural biology. In preliminary studies, we have demonstrated this for a novel RNA fold of 49 nucleotides. In cooperation with other research teams, we have solved the structure of the Diels-Alder ribozyme and obtained the first molecular insights into RNA`s role in catalyzing a reaction other than phosphodiester chemistry. Goal of this project is the development of robust methodologies for the synthesis of selenium-derivatized RNAs. In our laboratory, we will focus on an approach involving the chemical synthesis of 2`-Se-methyl nucleoside phosphoramidite building blocks and their incorporation into oligoribonucleotides by solid-phase synthesis. In addition, we will enzymatically ligate the selenium-derivatized RNAs to obtain specific, biologically important RNAs and RNA domains. These RNAs will then be target of X-ray crystallographic structure analysis in cooperation with the laboratory of Dinshaw Patel (Sloan Kettering Memorial Cancer Center, NY). Our main RNA targets are G-rich triplet repeats, U6/U2 snRNA motifs, and metabolite-binding riboswitch mRNA domains. The latter are highly attractive, and biologically most important candidates: in the past two years it has been found that specialized domains within certain mRNAs directly bind metabolites in a selective and concentration dependent manner, and thereupon, control gene expression without the need of protein factors. Our studies should reveal the structural principles of this novel mode of gene regulation.