Sequencing the Mono-Methylated Derivatives of Cytidine

胞苷单甲基化衍生物的测序

基本信息

批准号：
10581093
负责人：
Eric Ervin
金额：
$ 40万
依托单位：
ELECTRONIC BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-13 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10581093
关键词：
Benchmarking Biological Biological Markers Cell Line Cells Charge Chemicals Clinical Communities Complex Custom Cyclodextrins Cytidine Cytosine Defect Derivation procedure Detection Development Devices Diagnostic Diameter Dimensions Disease Eczema Electrodes Electrostatics Ensure Evaluation Exonuclease Exoribonucleases Exposure to Goals Heart failure Heat-Shock Response Hemolysin Human Inflammatory Intelligence Isomerism Length Lipid Bilayers Malignant Neoplasms Malignant neoplasm of cervix uteri Malignant neoplasm of liver Maps Measurement Metabolic Diseases Methodology Methods Methylation Methyltransferase Micronutrients Modification Molecular National Human Genome Research Institute Nucleotides Phase RNA Reader Research Ribonucleotides Ribosomal RNA Sampling Small Business Innovation Research Grant Stress Surface Targeted Research Technology Therapeutic Therapeutic Intervention Time Translational Research Virus Diseases access restrictions base cancer cell directed differentiation epitranscriptomics high reward high risk innovation mutant nano nanopore neurodevelopment novel novel diagnostics personalized medicine precision medicine prevent prognostic prototype sequencing platform transcriptome sequencing voltage

项目摘要

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.

项目概要该项目的目标是开发原型测序平台和相关方法最近，LC-MS/MS 方法对胞嘧啶的单甲基化衍生物进行了直接测序。胞嘧啶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 (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）取得了有希望的发现，当细胞暴露于氧化环境时，这些修饰会发生变化以及炎症应激、微量营养素应激和热休克应激。甲基化胞嘧啶 RNA 核苷酸现在被认为在神经发育中很重要，似乎会影响智力，以及癌症、湿疹和代谢紊乱的变化，但目前还没有技术。存在能够对这些化学标记进行测序和量化的技术，严重限制了该领域的发展了解和潜在利用这些修饰作为生物标志物（用于诊断、预后和治疗目的，例如）为了实现拟议的壮举，在这个项目期间，我们将专门进行。正在开发一种新型的基于纳米孔的读取器，其灵敏度可以直接区分单甲基化胞嘧啶异构体以及相关的半自动化、稳定、高精度测序平台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，包括单甲基化衍生物的测序和作图该项目完成后，我们将开发出来自两种不同细胞系的胞嘧啶。优化并充分展示了世界上第一个功能原型测序仪，能够直接对胞嘧啶的单甲基化衍生物进行测序。