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

 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基本调节因素。信使RNA(MRNA)的修饰被认为是调节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N6位的甲基化形成N6-甲基腺苷(M6A)。值得注意的是，m6A是脂肪团和肥胖相关蛋白(FTO)的主要底物，RNA甲基化与多种与FTO相关的人类疾病有关，包括阿尔茨海默病和几种类型的癌症。然而，m6A的具体功能目前还不清楚，因为使用传统的RNA测序技术识别修饰是非常具有挑战性的。为了充分阐明m6A在mRNA中的生物学作用，有必要在单核苷酸分辨率下实现RNA甲基组的全球定位。先前的研究表明，m6A与典型的腺苷碱基对胸苷(T)和尿苷(U)的杂交，由于与外来甲基的不利作用，在热力学上是不稳定的。我们假设，可以开发一个与m6A空间互补的非天然核苷酸碱基作为天然碱基对的替代测序，从而为RNA中特定的m6A位点提供一个独特的化学标记。在这个方案中，我们将合成一个非标准碱基库，并利用双重热变性方法鉴定一个可以选择性地与M6A杂交的补体。通过碱基配对中的空间互补原理，我们将合成一类源自2-吡啶酮的非天然核苷，旨在缓解m6A的N6-甲基和T和U的O4酮基所遇到的空间碰撞。一旦通过热变性研究确定了m6A的合适补体，我们将把合成碱基纳入RNA测序方法，首先建立补体可以在RNA模板中反转录为相对的cDNAm6A，然后在天然RNA转录本中检测m6A。这种方法可以广泛应用于RNA甲基组的全球定位，这将增强对m6A在细胞中的作用的理解，从而增强对RNA异常甲基化在人类疾病中的意义的理解。

 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.