选择性标记长链RNA指定位点的PLOR-2.0方法

RNA是生物体重要的遗传信息载体，RNA结构决定其功能。RNA选择性标记是开展RNA结构生物学研究的基础，而长链RNA选择性位点标记技术是RNA研究和技术应用的瓶颈。申请者前期研发的PLOR-1.0方法成功地将同位素、荧光团、化学官能团等修饰基团标记到长链RNA指定位点上，但对核苷酸变化频率高的RNA序列标记效率低，并受限于某些RNA序列和标记位点。本项目基于前期研究工作，改进PLOR-1.0的不足，发展可靠、高效、灵活的PLOR-2.0方法，可将不同的修饰基团引入长链RNA的任意位点，并将标记范围扩大到无法转录的RNA；拟采用HPLC、MS、PAGE、NMR、X-射线衍射、稳态荧光、FRET等生物物理技术，分析PLOR-2.0对多种长链RNA的标记效率和准确性。本项目将为选择性标记长链RNA任意位点提供原创性新方法，推进RNA基础和应用研究，具有重要的科学意义和应用价值。

RNA is critical genetic messager in cells, whose functions are dependent on structures. Position-specific labelling is fundamentally important for obtaining structural insights, however, position-specific labelling of large RNA is still challenging, and hence restricts research and applications of RNA. PLOR-1.0 developed by us has been used to introduce isotopes, fluorophores, functional groups to specific positions at long RNA, but it is ineffective in labelling sequences with frequent nucleotide change, and it is also limited in choosing labeled RNA and positions. The project is to develop a improved method, PLOR-2.0 with reliability, efficiency and flexibility from previous study. PLOR-2.0 is capable of labelling untranscribed RNA and any position at RNA potentially. The project will apply HPLC, PAGE, MS, NMR, X-ray crystallography, steady fluorescence, FRET and other biophysical methods to detect efficiency and accuracy of PLOR-2.0 on various RNA. This project is to provide a novel method for labelling any position at long RNA, which is of great scientific significance and application value to promote the basic and applied research of RNA.