New methods for sequence-specific modification of RNA to study structural and functional aspects of large ribozymes

RNA的位点特异性标记是对大量长链非编码RNA分子进行结构和功能研究的主要兴趣。序列特异性修饰的长RNA分子的合成仍然是一个挑战，由于其长度的限制，其不能完全通过固相合成方法制备。 拟议研究项目的主要目标是开发新的酶方法用于RNA分子的位点特异性标记，以使用半合成方法研究这些RNA的结构和动力学。非天然碱基对将被用作实现官能团的工具，能够进行环加成反应，酶促进入RNA，以允许报告基团的合成后连接。这一原理可用于通过正交逆电子需求Diels-Alder和铜催化的叠氮化物-炔环加成反应在转录RNA的特定位置引入两个荧光团。因此，修饰的非天然核苷三磷酸应该允许使用体外转录的标准条件位点特异性引入反应基团。在本项目的框架下，人CPEB 3蛋白应位点特异性地标记两个荧光团，用于FRET研究各种因素引起的全局构象变化。延伸的概念，对特定位点的自旋标签引入RNA的距离测量，通过电子顺磁共振光谱提出。计划的方法代表了一个有用的替代连接为基础的策略，用于制备长，序列特异性修饰的RNA分子。因此，它可以作为一个理想的工具，功能性的，非编码RNA分子的结构研究。

The site-specific labeling of RNA is of main interest for structural and functional studies on a plethora of long non-coding RNA molecules. The synthesis of sequence-specific modified long RNA molecules, which cannot entirely be prepared via solid phase synthesis methods due to its limitations in length, has remained a challenge. Main objective of the proposed research project is to develop new enzymatic methods for the site-specific labeling of RNA molecules to study structure and dynamics of these RNAs using a semi-synthetic approach. Unnatural base pairs will be employed as a tool to implement functional groups, capable of undergoing cycloadditions reactions, enzymatically into RNA to allow postsynthetic attachment of reporter groups. This principle should be used to introduce two fluorophores at specific positions in transcribed RNA via orthogonal inverse electron-demand Diels-Alder and copper-catalyzed azide-alkyne cycloaddition reactions. Accordingly modified unnatural nucleoside triphosphates should allow the site-specific introduction of reactive groups using standard conditions for in vitro transcription. In the framework of this project proposal, the human CPEB3 protein should site-specifically be labeled with two fluorophores for FRET studies of global conformational changes caused by various factors. The extension of the concept towards site-specific introduction of spin-labels into RNA for distance measurements via electron paramagnetic resonance spectroscopy is proposed. The planned method represents a useful alternative for ligation based strategies for the preparation of long, sequence-specific modified RNA molecules. Thus it could be applied as an ideal tool for structural investigation of functional, non-coding RNA molecules.