Mass Spectrometry of Modified RNAs

 描述（由申请人提供）：对核糖核酸（RNA）中转录后修饰核苷的功能作用的科学理解在很大程度上受到缺乏可常规识别和映射一级RNA序列修饰的方法的限制。这项研究的长期目标仍然是开发适当的质谱方法，使生物学研究成为修饰RNA的功能意义。这次更新的重点是通过质谱法改进RNA修饰图谱的具体目标。该学科中使用的现有协议是在30多年前开发的，对于寻求在细胞RNA的复杂混合物中发现新修饰的研究人员来说具有重大限制。这种更新分为三个目标，这将导致创建一个修饰映射协议，可以使现代生物学研究RNA修饰模式，包括RNA表观遗传学的增长领域。第一个目标是通过提供三种新的碱基特异性核酸内切酶来实现基于发现的RNA修饰图谱。这一目的确保了修饰作图实验中适当的序列覆盖率。接下来的两个目标集中在通过液相色谱-串联质谱法（LC-MS/MS）改进修饰的RNA消化产物的检测和测序。第二个目标是修改用于LC-MS/MS鉴定较大RNA内修饰核苷的策略。通过消除任何RNA的未修饰区域的浪费的LC-MS/MS测序，所得MS/MS数据将富含关于任何特定修饰的质量和序列位置的信息。最终的目标是建立一个基于数据独立采集（DIA）的LC-MS/MS策略，用于RNA修饰图谱。该研究计划将对多个研究领域产生重大影响。基于发现的修饰映射可以用于扩展我们对真核RNA修饰模式的知识，包括对生物功能重要的修饰。新协议将使研究对RNA的外源性修饰，包括RNA氧化损伤等过程，这可能在心血管疾病，癌症和神经功能障碍等衰老相关疾病中具有重要意义。此外，从这项研究中产生的生物分析发展可以应用于科学研究，以了解细胞如何调节RNA修饰模式以及可变的RNA修饰模式如何影响其他细胞调控过程。

 DESCRIPTION (provided by applicant): Scientific understanding of the functional role of post-transcriptionally modified nucleosides in ribonucleic acids (RNAs) is limited in large part by the lack of methods that can routinely identify and map modifications onto a primary RNA sequence. The long-term goal of this research continues to be to develop appropriate mass spectrometric approaches that enable biological studies into the functional significance of modified RNAs. This renewal is focused on the specific goal of improving RNA modification mapping by mass spectrometry. The existing protocol used in the discipline was developed over 30 years ago and has significant limitations for researchers seeking to discover new modifications within complex mixtures of cellular RNAs. This renewal is separated into three aims that will result in the creation of a modification mapping protocol that can enable modern biological studies into RNA modification patterns, including the growing area of RNA epigenetics. The first aim will enable discovery-based RNA modification mapping by making available three new base-specific endonucleases. This aim ensures appropriate sequence coverage in modification mapping experiments. The next two aims focus on improving detection and sequencing of modified RNA digestion products by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The second aim revises the strategy used for LC-MS/MS identification of modified nucleosides within larger RNAs. By eliminating wasteful LC-MS/MS sequencing of unmodified regions of any RNA, the resulting MS/MS data will be enriched in information about the mass and sequence location of any particular modification. The final aim will create a data independent acquisition (DIA)-based LC-MS/MS strategy for RNA modification mapping. This research plan will have significant impacts on multiple research fields. Discovery-based modification mapping can be used to expand our knowledge of eukaryotic RNA modification patterns, including modifications that are important for biological function. The new protocol will enable research into xenobiotic modifications to RNA including processes such as RNA oxidative damage, which may be significant in aging-related diseases such as cardiovascular disease, cancer and neurological impairment. Moreover, the bioanalytical developments to result from this research can be applied to scientific investigations that seek to understand how the cell regulates RNA modification patterns as well as how variable RNA modification patterns affect other cellular regulatory processes.