Synthesis of Selenium-modified RNA by engineered RNA Polymerases

传统上，RNA与翻译装置相关，并分为三大类，即mRNA、tRNA和rRNA。然而，近年来，非蛋白质编码RNA（ncRNA）的重要作用已被认识到。大量的ncRNA和对它们功能的日益关注伴随着对结构表征的日益增长的需求。到目前为止，确定核酸结构最重要的方法是X射线晶体学。一旦一种新的RNA晶体生长出来，并记录了衍射图案，数据的定相可能是一个严重的障碍。为了有效地实现这一点，现代技术是可用的，需要在相应的RNA晶体内的异常散射中心。有几种选择;其中一种是指硒修饰的RNA，其化学合成已由我们中的一个开发。然而，通过化学固相合成制备Se-RNA是非常耗时的，并且RNA大小的限制是目前另一个严重的缺点。为了克服这些障碍，我们的目标是开发一种酶促合成策略Se-RNA使用工程RNA聚合酶。通过结合有机化学，生物技术和生物化学的优势，在核糖核苷酸合成和核酸修饰酶的定向进化，我们将开发新的RNA聚合酶，合成硒修饰的RNA有效。只有在这两个小组的合作下，才能获得该项目成功所需的广泛方法和知识。一个小组将提出2 '-甲基硒修饰的三磷酸的化学合成，另一个小组将开发新的方法来产生接受化学修饰的核苷酸作为底物的RNA聚合酶变体。预期的系统将在前所未有的短时间内提供大量含有2 '-甲基硒修饰的核苷酸的长RNA用于X射线分析，从而推动沿着这些路线的沿着进展。

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.