Enabling precision distance measurements in long RNAs

探索复杂生物分子（如核糖核酸（RNA））的形状和运动的技术对于理解这些生物分子在人类生物学或疾病中的作用至关重要。开发新的分析方法非常重要，因为这增加了生物学家精确研究生物分子结构和行为的工具范围。该项目旨在开发一种表征RNA的新技术-利用生物合成将非天然官能团引入RNA，这些官能团可以与含有“稳定自由基”的探针反应，这被称为“自旋标记”。这项工作将提供一个新的和完全未开发的方法分析'长' RNA（即比通过化学合成可靠制备的RNA更长的RNA）。关键的生物合成方法被称为“分步转录”，它利用某些RNA聚合酶接受非天然核苷三磷酸的能力（NTPs，自然界用于构建RNA的构建块）作为RNA链被生物合成。这些非天然核苷含有聚合酶所耐受的化学官能团，但其能够实现RNA的生物合成后“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。该技术已成功应用于分析可通过化学合成制备的小RNA和DNA基序，但由于其制备的当前困难，仅很少用于自旋标记的长RNA（>50个核苷酸）。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。该项目本身将涉及含有适当功能的NTP的合成，以在生物合成后安装自旋标签。虽然已经存在许多经过修饰的NTP（我们将在本项目中将其作为起点），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，更刚性的标签手柄。这是一个令人兴奋的挑战，其后果超出了EPR（其他探头可以安装在这些相同的手柄上）。我们还将合成一系列自旋标签，以附着在生物合成的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，已知该基序在规定的条件下发生结构变化。我们将标记这个基序，并通过EPR光谱探索其形状的变化，从而建立这种新技术作为一种可行的和易于应用的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.