Sequencing the Mono-Methylated Derivatives of Cytidine

项目摘要 这个项目的目标是开发一个原型测序平台和相关的方法学 直接对胞嘧啶的单甲基衍生物进行测序。近年来，LC-MS/MS方法用于评价 胞嘧啶的单甲基化异构体的存在，即5-甲基胞嘧啶(M5C)、4-甲基胞苷(M4C)、3-甲基胞苷(3- 甲基胞苷(M3C)、2‘-O-甲基胞苷(Cm)以及双甲基衍生物5-甲基-2’-O-甲基胞苷 (M5Cm)，他们发现当细胞暴露在氧化环境中时，这些修饰会发生变化 炎症应激、微量营养素应激、热休克应激。此外，该项目的位置和范围 甲基化的胞嘧啶RNA核苷酸现在被认为在神经发育中很重要，似乎影响了 智力，以及癌症、湿疹和代谢紊乱的变化。然而，目前还没有技术 有能力对这些化学标记进行排序和量化，严重限制了该领域的 理解和潜在利用这些修饰作为生物标记物(用于诊断、预后和 例如治疗目的)。为了实现拟议的壮举，在这个项目期间，我们将具体 正在开发一种新型的基于纳米孔的阅读器，具有直接区分单甲基化的灵敏度 胞嘧啶异构体及其相关测序平台的半自动、稳定、高精度 测序测量。然后，我们将演示和优化开发的测序方法 通过各种测序演示，包括对单甲基衍生物进行测序和作图 来自两个不同细胞系的胞嘧啶跨越RNA。这个项目建成后，我们将开发出， 经过优化，并充分展示了世界上第一台能够直接 对胞嘧啶的单甲基衍生物进行测序。

Project Summary The goal of this project is to develop a prototype sequencing platform and the associated methodology capable of directly sequencing the mono-methylated derivatives of cytosine. Recently, LC-MS/MS methods to assess the presence of the mono-methylated isomers of cytosine, i.e., 5-methylcytosine (m5C), 4-methylcytidine (m4C), 3- methylcytidine (m3C), 2`-O-methylcytidine (Cm), as well as the bismethyl derivative 5-methyl-2`-O-methylcytidine (m5Cm), have made promising discoveries that these modifications change when cells are exposed to oxidative and inflammatory stress, micronutrient stress, and heat shock stress. Moreover, the placement and extent of methylated cytosine RNA nucleotides is now believed to be important in neurodevelopment, appears to impact intelligence, and changes with cancer, eczema, and metabolic disorders. However, no technology currently exists that is capable of sequencing and quantifying these chemical marks, severely limiting the field’s understanding and potential utilization of these modifications as biomarkers (for diagnostics, prognostics, and therapeutic purposes, as examples). In order to enable the proposed feat, during this project, we will specifically be developing a novel nanopore-based reader with the sensitivity to directly differentiate the mono-methylated isomers of cytosine as well as the associated sequencing platform for semi-automated, stable, high-accuracy sequencing measurements. We will then demonstrate and optimize the developed sequencing methodology through various sequencing demonstrations, including sequencing and mapping the mono-methylated derivates of cytosine across RNA from two different cell lines. Upon the completion of this project, we will have developed, optimized, and fully demonstrated the world’s first functioning prototype sequencer capable of directly sequencing the mono-methylated derivatives of cytosine.