Oleynikov Maxim, Jaffrey Samie R
Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, USA.
bioRxiv. 2024 Aug 19:2024.04.11.589009. doi: 10.1101/2024.04.11.589009.
The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.
RNA的功能效应可能源于被称为三级结构的复杂三维折叠。然而，预测RNA的三级结构以及RNA是否采用不同的三级构象仍然具有挑战性。为了解决这个问题，我们开发了BASH MaP（一种单分子硫酸二甲酯（DMS）足迹法）和DAGGER（一种计算流程），以识别不同RNA分子采用的替代三级结构。BASH MaP利用硼氢化钾来揭示鸟苷N7位置的化学可及性，鸟苷是三级结构的关键介质。我们使用BASH MaP在体外和细胞中识别RNA G-四链体（一种重要的RNA三级基序）的多种构象状态和动力学。对荧光适体Spinach进行的BASH MaP和DAGGER分析表明，它采用了决定其荧光状态的替代三级构象。因此，BASH MaP通过揭示以前无法检测到的三级结构，为RNA的结构分析提供了一种方法。
