Enabling precision distance measurements in long RNAs

探索核糖核酸(RNA)等复杂生物分子的形状和运动的技术对于理解这种生物分子在人类生物学或疾病中的作用至关重要。开发新的分析方法是非常重要的，因为这增加了生物学家可用于精确研究生物分子结构和行为的工具的范围。该项目寻求开发一种表征RNA的新技术-利用生物合成将非自然功能基团引入RNA，这些基团可以与含有稳定自由基的探针反应，这些探针被称为“自旋标记”。这项工作将为“长”RNA(即比通过化学合成可以可靠地制备的RNA更长)的分析提供一种新的、完全未知的方法。关键的生物合成方法被称为“逐步转录”，它使用某些RNA聚合酶接受非天然核苷三磷酸盐(NTPs，大自然用来构建RNA的构件)的能力作为RNA链进行生物合成。这些非天然的核苷含有一种聚合酶可以容忍的化学功能，但它可以在生物合成后对RNA进行标记。在我们的自旋标记的情况下，这使得使用电子顺磁共振光谱(EPR)来分析生物分子成为可能。这项技术已经成功地应用于小RNA和DNA基序的分析，这些基序可以通过化学合成来制备，但由于目前制备它们的困难，很少用于旋转标记的长RNA(&gt；50核苷酸)。EPR技术本身信息量很大，不仅可以测量距离，还可以测量运动和方向。该项目本身将涉及NTP的合成，这些NTP包含安装自旋标签生物合成后的适当功能。虽然已经存在许多经过修改的NTP(我们将在本项目中使用它作为起点)，但为了在EPR实验中获得最佳信息，我们需要在NTP中设计新的、更僵化的标签手柄。这是一项令人兴奋的挑战，其后果超出了EPR(可以在这些相同的手柄上安装其他探头)。我们还将合成一系列自旋标记，以粘贴到生物合成的RNA上。为了测试该方法，我们已经确定了一个71个核苷酸的RNA基序，该基序在特定的条件下会发生结构变化。我们将标记这个基序，并通过EPR光谱探索其形状的变化，从而将这一新技术确立为一种可行且易于应用的RNA自旋标记方法。

Technologies that explore the shape and motion of complex biomolecules such as ribonucleic acids (RNA) are fundamentally important for understanding the role of such biomolecules in human biology, or disease. The development of new analytical methods is of high importance, as this increases the range of tools available to the biologist for the precise investigation of biomolecule structure and behaviour.This project seeks to develop a new technology for the characterisation of RNA - the use of biosynthesis to introduce unnatural functional groups into RNA that can be reacted with probes that contain 'stable free radicals', which are termed 'spin labels'. This work will offer a new and totally unexplored method for the analysis of 'long' RNA (that is, RNA which is longer than can reliably be prepared through chemical synthesis).The key biosynthesis methodology is called 'Stepwise transcription', which uses the ability of certain RNA polymerase enzymes to accept unnatural nucleoside triphosphates (NTPs, the building blocks used by Nature to construct RNA) as an RNA chain is biosynthesised. These unnatural nucleosides contain a chemical functionality which is tolerated by the polymerase, but which enables post-biosynthesis 'labelling' of the RNA. In the case of our spin labels, this enables the use of electron paramagnetic resonance spectroscopy (EPR) to analyse the biomolecule. This technique has been applied with success for the analysis of small RNA and DNA motifs which can be prepared through chemical synthesis, but only very rarely to spin labelled long RNAs (>50 nucleotides) due to the current difficulties with their preparation. The EPR technique itself is highly informative, not only measuring distances, but also motion and orientation. The project itself will involve the synthesis of NTPs that contain appropriate functionality to install the spin label post-biosynthesis. While many modified NTPs already exist (which we will use in this project as a starting point), to obtain the best information in the EPR experiments, we will need to design new, more rigidified labelling handles in the NTP. This is an exciting challenge, and one that has consequences beyond EPR (other probes could be installed on these same handles). We will also synthesize a range of spin labels to attach to the biosynthesized RNA. To test the methodology, we have identified a 71 nucleotide RNA motif that is known to undergo structural change under defined conditions. We will label this motif, and explore by EPR spectroscopy the change in its shape, thus establishing this new technology as a viable and easily applied method for RNA spin labelling.