RNA: Tools for Cell Biology

RNA：细胞生物学工具

• 批准号: 10253884
• 负责人: Adrian Ferre-D'Amare
• 金额: $ 94.64万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10253884
• 关键词: Behavior; Biochemical; Biophysics; Cells; Cellular biology; Crystallization; Custom; Development; Dimerization; Effectiveness; Engineering; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Green Fluorescent Proteins; Image; Mammalian Cell; Mango - dietary; Mutagenesis; Property; Proteins; Protomer; RNA; RNA analysis; Reporting; Research; Research Project Grants; Side; Spinach - dietary; Structure; System; Time; Variant; Visualization; alpha helix; analog; aptamer; base; cross reactivity; design; dimer; experimental study; fluorophore; improved; in vivo; insight; interest; next generation; novel; thiazole orange; tool

This year we reported the crystal structure of the fluorogenic aptamer Mango-IV, and the results of structure-guided engineering and re-selection efforts that have yielded an RNA-based FRET pair. Unlike other Mango-family fluorescence turn-on aptamers, our biophysical and structural studies revealed Mango-IV to be a domain-swapped dimer. In addition, mutagenesis and biochemical studies suggested that the basis for this dimerization lies with a tendency for the RNA to explore several nearly iso-energetic folding registers. While this results in complicated biophysical behavior, it appears that the ability to function in multiple folded forms underlies the effectiveness of Mango-IV as a fluorescent tag in live cell analysis of RNA. It has been shown previously that among the four well-characterized Mango aptamers derived from the same selection experiment, Mango-IV is best for imaging in mammalian cells. Taking advantage of the dimerization of Mango-IV through a duplex region that we discovered in our crystal structure, we designed an RNA-based FRET pair. The fluorescence spectra, and fluorophore cross-reactivity of Mango-IV and Spinach are such that by connecting these two RNAs through complementary segments that form a helix, an efficient FRET system can be generated. Since Spinach can be helix-internal (that is, its fluoromodule is flanked on both sides by helices) it could be placed near the end of a helix of an RNA of interest, and the Mango-IV protomer with the complementary single-stranded segment used to report on expression or localization.

今年，我们报道了荧光适体芒果IV的晶体结构，以及结构引导的工程和重新选择工作的结果，产生了基于RNA的FRET对。 与其他芒果家庭的荧光转机适体不同，我们的生物物理和结构研究表明，芒果IV是域折叠的二聚体。 此外，诱变和生化研究表明，这种二聚化的基础在于RNA倾向于探索几乎几乎具有等异源折叠寄存器。 尽管这会导致复杂的生物物理行为，但似乎多种折叠形式起作用的能力是芒果IV作为RNA的活细胞分析中荧光标签的有效性。 先前已经表明，在从相同选择实验中得出的四个特征良好的芒果适体中，芒果-IV最适合在哺乳动物细胞中成像。 利用我们在晶体结构中发现的双链区域的芒果-IV二聚化，我们设计了一个基于RNA的FRET对。 芒果IV和菠菜的荧光光谱以及荧光团交叉反应，可以通过通过形成螺旋的互补段连接这两个RNA，可以产生有效的FRET系统。 由于菠菜可以是螺旋内部的（即，其荧光模块在两侧的两侧都可以通过螺旋螺旋置），因此可以将其放置在感兴趣的RNA的螺旋端附近，而芒果IV原型器具有互补的单链段，用于报告表达或本地化。