Development of Unnatural Nucleotides for the Improved Detection of N6-Methyladenosine in Messenger RNA

开发非天然核苷酸以改进信使 RNA 中 N6-甲基腺苷的检测

• 批准号: 8981069
• 负责人: Alyson Weidmann
• 金额: $ 5.05万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-03 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981069
• 关键词: 2-hydroxypyridine; Adenosine; Alzheimer' s Disease; Amines; Base Pairing; Base Sequence; Biochemical; Biological; Cell physiology; Cells; Chemical Dynamics; Chemicals; Complement; Complementary DNA; DNA; DNA Sequence; DNA biosynthesis; Detection; Development; Disease Progression; Early Diagnosis; Enzymes; Epigenetic Process; Family; Fatty acid glycerol esters; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Goals; High-Throughput Nucleotide Sequencing; Human; Hydrogen Bonding; Hydrophobicity; Hypermethylation; Kinetics; Libraries; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Methodology; Methods; Methylation; Modification; Mutation; Nucleic Acids; Nucleic acid sequencing; Nucleosides; Nucleotides; Obesity; Physiological Processes; Play; Positioning Attribute; Proteins; Public Health; RNA; RNA Sequences; RNA Stability; RNA methylation; RNA-Directed DNA Polymerase; Regulator Genes; Resolution; Ribose; Role; Sensitivity Training Groups; Sequence Alignment; Shapes; Site; Spliced Genes; Structure; Techniques; Technology; Testing; Thermodynamics; Thymidine; Transcript; Translating; Translations; Uridine; Variant; base; cancer type; design; epigenetic regulation; human disease; improved; insight; methyl group; methylome; nervous system disorder; nucleobase; public health relevance; research study; success; transcriptome sequencing

 DESCRIPTION (provided by applicant): Dynamic chemical modifications to DNA, RNA, and proteins serve as essential regulators of gene expression. Modifications to messenger RNA (mRNA) are purported to regulate RNA stability and structure, translation efficiency, and gene splicing; the most abundant of these modifications is the methylation of the N6 position of adenosine to form N6-methyladenosine (m6A). Significantly, m6A is a major substrate of the fat mass and obesity-associated protein (FTO), and RNA methylation is implicated in a variety of human diseases associated with FTO, including Alzheimer's disease and several types of cancer. However, the specific function of m6A is not currently well understood, as it is exceptionally challenging to identify the modification using traditional RNA sequencing techniques. To fully elucidate the biological role of m6A in mRNA, it is necessary to achieve global mapping of the RNA methylome at single nucleotide resolution. Previous studies have shown that m6A hybridization to canonical adenosine base pairs, thymidine (T) and uridine (U), is thermodynamically destabilized due to unfavorable interactions with the extraneous methyl group. We hypothesize that an unnatural nucleobase that is sterically complementary to m6A can be developed as a sequencing alternative to native base pairs, thereby providing a unique chemical marker for specific m6A sites in RNA. In this proposal, we will synthesize a library of nonstandard nucleobases and identify a complement that can selectively hybridize to m6A using duplex thermal denaturation methods. By employing the principle of steric complementarity in base pairing, we will synthesize a family of unnatural nucleosides derived from 2-pyridone, designed to relieve the steric clashing encountered by the N6-methyl group of m6A and the O4 keto group of T and U. Once a suitable complement to m6A has been identified by thermal denaturation studies, we will incorporate the synthetic base into RNA sequencing methods, first establishing that the complement can be reverse transcribed into cDNA opposite m6A in an RNA template, followed by detection of m6A in native RNA transcripts. This methodology can be applied broadly toward global mapping of the RNA methylome, which will enhance understanding of the role of m6A in cells and, consequently, the implications of aberrant RNA methylation in human disease.

 描述（由申请人提供）：DNA、RNA和蛋白质的动态化学修饰是基因表达的重要调节因子。对信使RNA（mRNA）的修饰旨在调节RNA的稳定性和结构、翻译效率和基因剪接;这些修饰中最丰富的是腺苷的N6位置的甲基化以形成N6-甲基腺苷（m6 A）。值得注意的是，m6 A是脂肪量和肥胖相关蛋白（FTO）的主要底物，并且RNA甲基化涉及与FTO相关的多种人类疾病，包括阿尔茨海默病和几种类型的癌症。然而，m6 A的具体功能目前还没有得到很好的理解，因为使用传统的RNA测序技术来鉴定修饰是非常具有挑战性的。为了充分阐明m6 A在mRNA中的生物学作用，有必要以单核苷酸分辨率实现RNA甲基化组的全局映射。先前的研究已经表明，m6 A与规范腺苷碱基对胸苷（T）和尿苷（U）的杂交由于与外来甲基的不利相互作用而被破坏。我们假设，一个非天然的核碱基，是空间互补的m6 A可以开发作为一种替代天然碱基对的测序，从而提供了一个独特的化学标记的特定m6 A网站在RNA中。在这个提议中，我们将合成一个非标准的核碱基库，并确定一个互补，可以选择性地杂交到m6 A使用双重热变性方法。利用碱基配对中的空间互补性原理，我们将合成一个由2-吡啶酮衍生的非天然核苷家族，旨在减轻m6 A的N6-甲基与T和U的O 4酮基所遇到的空间冲突。一旦通过热变性研究确定了m6 A的合适互补物，我们将把合成碱基纳入RNA测序方法中，首先确定互补物可以在RNA模板中逆转录成与m6 A相对的cDNA，然后检测天然RNA转录物中的m6 A。这种方法可以广泛地应用于RNA甲基化组的全球定位，这将增强对m6 A在细胞中的作用的理解，从而增强对异常RNA甲基化在人类疾病中的影响的理解。