Mass Spectrometry of Modified RNAs

 描述(申请人提供)：对转录后修饰的核苷在核糖核酸(RNA)中的功能作用的科学理解在很大程度上受到缺乏方法的限制，这些方法可以常规地识别修饰并将修饰映射到初级RNA序列上。这项研究的长期目标仍然是开发适当的质谱学方法，使对修饰RNA的功能意义的生物学研究成为可能。这次更新集中在利用质谱学改进RNA修饰图谱的具体目标上。该学科中使用的现有方案是在30多年前开发的，对于寻求在复杂的细胞RNA混合物中发现新的修饰的研究人员来说，它有很大的局限性。这一更新分为三个目标，这三个目标将导致创建一种修饰映射协议，该协议可以使现代生物学研究RNA修饰模式，包括RNA表观遗传学的种植领域。第一个目标将通过提供三种新的碱基特定内切酶来实现基于发现的RNA修饰图谱。这一目标确保了在修改映射实验中适当的序列覆盖。接下来的两个目标集中在改进LC-MS/MS(LC-MS/MS)对修饰的RNA消化产物的检测和测序。第二个目的是修订LC-MS/MS鉴定较大RNA中修饰核苷的策略。通过消除对任何RNA的未修饰区域进行浪费的LC-MS/MS测序，所得到的MS/MS数据将丰富关于任何特定修饰的质量和序列位置的信息。最终目标是建立一种基于数据独立获取(DIA)的LC-MS/MS策略，用于RNA修饰图谱。这项研究计划将对多个研究领域产生重大影响。基于发现的修饰图谱可以用来扩展我们对真核RNA修饰模式的知识，包括对生物功能重要的修饰。新的协议将使对RNA的异生修饰的研究成为可能，包括RNA氧化损伤等过程，这可能在心血管疾病、癌症和神经损伤等与衰老相关的疾病中意义重大。此外，这项研究产生的生物分析发展可以应用于科学调查，试图了解细胞如何调节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.