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，由于其长度的限制，不能完全通过固相合成方法制备，仍然是一个挑战。本研究的主要目的是开发新的酶方法用于RNA分子的位点特异性标记，并利用半合成方法研究这些RNA的结构和动力学。非自然碱基对将被用作实现功能基团的工具，能够进行环加成反应，酶促地进入RNA，以允许合成后的报告基团附着。利用这一原理，可以通过正交逆电按需Diels-Alder和铜催化叠氮-炔环加成反应在转录RNA的特定位置引入两个荧光团。因此，经过修饰的非天然三磷酸核苷应该允许在体外转录的标准条件下引入特定位点的反应基团。在本项目提案的框架内，人类CPEB3蛋白应该用两个荧光团特异性标记，用于FRET研究由各种因素引起的全局构象变化。提出了通过电子顺磁共振波谱法将自旋标签引入RNA以进行距离测量的概念的扩展。所计划的方法代表了一种有用的替代方法，用于制备长序列特异性修饰RNA分子的基于连接的策略。因此，它可以作为功能性非编码RNA分子结构研究的理想工具。

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.