Synthesis of Selenium-modified RNA by engineered polymerases

RNA is traditionally associated with the translational apparatus and is classified into three main categories that are mRNA, tRNA, and rRNA. However, in recent years, the important role of non-protein encoding RNAs (ncRNAs) has been recognized. The great number of ncRNAs and the growing interest into their functions are accompanied by the increasing demand for structural characterization. By far the most important method for nucleic acid structure determination is X-ray crystallography. Once crystals of a novel RNA have been grown and diffraction patterns recorded, phasing of the data can be a serious obstacle. To efficiently achieve this, modern techniques are available that require anomalous scattering centres within the respective RNA crystal. There are several options; one of them refers to Se-modified RNA whose chemical synthesis has been developed by one of us. However, preparation of Se-RNA by chemical solid-phase synthesis is highly time-consuming and the limitation with respect to the size of RNA represents another serious drawback at present. To overcome these hurdles we aim to develop an enzymatic synthesis strategy for Se-RNA using engineered RNA polymerases. By combining the strengths in organic chemistry, biotechnology and biochemistry of both groups in ribonucleotide synthesis and directed evolution of nucleic acid modifying enzymes, we will evolve new RNA polymerases that synthesize Se-modified RNA efficiently. Only in a collaboration of both groups the required broad methodologies and knowledge that are needed for the success of this project are available. While one group will put forward the chemical synthesis of 2`- methylseleno-modified triphosphates the other group will develop new means for the generation of RNA polymerase variants that accept the chemical modified nucleotides as substrate. The anticipated systems will provide large numbers of long RNAs that contain 2`-methylseleno-modified nucleotides for Xray analysis in an unprecedented short time span and thus spur progress along these lines.

In the course of the ERA Chemistry project Enzymatic Synthesis of Selenium-modified RNA by engineered RNA Polymerases, the Austrian partners (Micura research group, University of Innsbruck) funded by the Austrian Science Fund FWF successfully developed the chemical synthesis of Selenium modified ribonucleoside triphosphates. These derivatives represented the substrates for the successfully evolved new RNA polymerases with enhanced performance for 2-modified nucleoside triphosphates by the German research team (Marx research group, University of Konstanz) funded by the Deutsche Forschungsgemeinschaft DFG.RNA is traditionally associated with the translational apparatus and is classified into three main categories that are mRNA, tRNA, and rRNA. However, in recent years, the important role of non-protein encoding RNAs (ncRNAs) has been recognized. The great number of ncRNAs and the growing interest into their functions are accompanied by the increasing demand for structural characterization. By far the most important method for nucleic acid structure determination is X-ray crystallography. Once crystals of a novel RNA have been grown and diffraction patterns recorded, phasing of the data can be a serious obstacle. To efficiently achieve this, modern techniques are available that require anomalous scattering centers within the respective RNA crystal. There are several options; one of them refers to Se-modified RNA whose chemical synthesis was developed previously by one of us. However, preparation of Se-RNA by chemical solid-phase synthesis is highly time-consuming and the limitation with respect to the size of RNA represents another serious drawback at present. To overcome these hurdles we developed an enzymatic synthesis strategy for Se-RNA using engineered RNA polymerases. By combining the strengths in organic chemistry, biotechnology and biochemistry of both groups in ribonucleotide synthesis and directed evolution of nucleic acid modifying enzymes we evolved new RNA polymerases that synthesize Se-modified RNA efficiently. Because of the intensive collaboration of both groups, the required broad methodologies and knowledge was available and led to the success of this project. While one group put forward the chemical synthesis of 2-methylseleno-modified triphosphates the other group developed new means for the generation of RNA polymerase variants that accepted the chemical modified nucleotides as substrates. The system developed here guarantees efficient access to large numbers of long RNAs that contain 2-methylseleno-modified nucleotides for X-ray analysis in an unprecedented short time span.