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Synthesis of Selenium-modified RNA by engineered RNA Polymerases

通过工程 RNA 聚合酶合成硒修饰的 RNA

基本信息

批准号：
132770984
负责人：
Professor Dr. Andreas Marx
金额：
--
依托单位：
Institut für Organische Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2009
资助国家：
德国
起止时间：
2008-12-31 至 2014-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/132770984?language=en
关键词：
Synthesis Selenium modified RNA engineered

项目摘要

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.

RNA 传统上与翻译装置相关，分为三大类：mRNA、tRNA 和 rRNA。然而，近年来，非蛋白质编码RNA（ncRNA）的重要作用已得到认识。大量的 ncRNA 及其功能日益浓厚，伴随着对结构表征的需求不断增加。迄今为止，测定核酸结构最重要的方法是X射线晶体学。一旦新型 RNA 晶体生长并记录衍射图样，数据定相可能会成为一个严重的障碍。为了有效地实现这一目标，现代技术需要在各自的 RNA 晶体内设置异常散射中心。有多种选择；其中之一是 Se 修饰的 RNA，其化学合成方法是由我们之一开发的。然而，通过化学固相合成制备Se-RNA非常耗时，并且RNA大小的限制是目前的另一个严重缺点。为了克服这些障碍，我们的目标是使用工程 RNA 聚合酶开发 Se-RNA 的酶促合成策略。通过结合两个团队在有机化学、生物技术和生物化学方面在核糖核苷酸合成和核酸修饰酶定向进化方面的优势，我们将进化出能够有效合成硒修饰RNA的新型RNA聚合酶。只有通过两个小组的合作，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’-甲基硒基修饰的三磷酸的化学合成，而另一小组将开发新的方法来生成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’-甲基硒代修饰核苷酸的长 RNA 用于 X 射线分析，从而刺激这些方面的进展。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The synthesis of 2′-methylseleno adenosine and guanosine 5′-triphosphates

2β-甲基硒腺苷和鸟苷5β-三磷酸的合成

DOI：
10.1016/j.bmc.2012.01.044
发表时间：
2012
期刊：
Bioorganic & Medicinal Chemistry
影响因子：
3.5
作者：
T. Santner;V. Siegmund;A. Marx;R. Micura
通讯作者：
R. Micura

Enzymatic synthesis of 8-vinyl- and 8-styryl-2'-deoxyguanosine modified DNA--novel fluorescent molecular probes.

酶法合成8-乙烯基-和8-苯乙烯基-2-脱氧鸟苷修饰的DNA--新型荧光分子探针

DOI：
10.1016/j.bmcl.2012.03.056
发表时间：
2012
期刊：
Bioorganic & medicinal chemistry letters
影响因子：
2.7
作者：
B. Holzberger;J. Strohmeier;V. Siegmund;U. Diederichsen;A. Marx
通讯作者：
A. Marx

Solution structure and conformational dynamics of deoxyxylonucleic acids (dXNA): an orthogonal nucleic acid candidate.

脱氧木糖核酸（dXNA）的溶液结构和构象动力学：正交候选核酸

DOI：
10.1002/chem.201102509
发表时间：
2012
期刊：
Chemistry
影响因子：
0
作者：
M. Maiti;V. Siegmund;M. Abramov;E. Lescrinier;H. Rosemeyer;M. Froeyen;A. Ramaswamy;A. Ceulemans;A. Marx;P. Herdewijn
通讯作者：
P. Herdewijn

Screening mutant libraries of T7 RNA polymerase for candidates with increased acceptance of 2'-modified nucleotides.