Enabling precision distance measurements in long RNAs

实现长 RNA 的精确距离测量

• 批准号: BB/R021848/1
• 负责人: Edward Anderson
• 金额: $ 19.24万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR021848%2F1
• 关键词: Enabling; precision; distance; measurements; long

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.

探索复杂生物分子（例如核糖核酸（RNA））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。新分析方法的开发非常重要，因为这增加了生物学家可用的工具范围，以精确研究生物分子结构和行为。该项目旨在为RNA的表征开发新技术 - 生物合成的使用将不自然的官能团引入RNA中，这些官能团可以与包含“稳定自由基”的探针反应，这些探针称为“自旋标记”。这项工作将为“长” RNA分析（即，RNA比通过化学合成可以可靠制备的RNA）提供一种新的且尚未开发的方法。关键的生物合成方法称为“逐步转录”，使用该方法，它使用了该方法。某些RNA聚合酶接受不自然的三磷酸核苷（NTPS，自然用来构建RNA的构建块）作为RNA链的能力是生物合成的。这些不自然的核苷包含一个化学功能，该化学功能由聚合酶耐受性耐受性，但可以使生物合成后RNA的“标记”。在我们的自旋标签的情况下，这可以使用电子顺磁共振光谱（EPR）来分析生物分子。该技术已被成功地用于分析小的RNA和DNA基序，这些基序可以通过化学合成来制备，但由于目前的制备困难，很少将很少用于旋转标记的长RNA（> 50个核苷酸）。 EPR技术本身具有很高的信息丰富，不仅可以测量距离，而且还可以进行运动和方向。该项目本身将涉及NTP的合成，该NTP包含适当的功能以安装自旋标签生物合成。尽管许多修改的NTP已经存在（我们将在该项目中用作起点），但要获取EPR实验中的最佳信息，我们将需要在NTP中设计新的，更僵化的标签手柄。这是一个令人兴奋的挑战，它具有超出EPR的后果（其他探针可以安装在同一手柄上）。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RNA基序，该基序已知在定义条件下会发生结构变化。我们将标记此基序，并通过EPR光谱探索其形状的变化，从而确立这项新技术作为一种可行且易于应用的RNA自旋标记方法。