A novel approach using aptamers for histone code analysis

使用适体进行组蛋白代码分析的新方法

• 批准号: 18F18330
• 负责人: 川村 猛
• 金额: $ 1.34万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2018
• 资助国家: 日本
• 起止时间: 2018-10-12 至 2021-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18F18330/
• 关键词: DNA aptamers; histone modification; SELEX; MALDI-MS; ISET; DNAアプタマー; エピゲノム; ヒストン修飾; MIK-MS

In this study, the development of new histone code analysis method was aimed by using aptamer based chromatin-affinity purification-mass spectrometry. First of all, three different histone modification (H3K4Me3, H3K9Me3, and H3K27Me3) was chosen as a target histone code since they are abundantly existed and are known as important histone PTMs by involving in gene regulations. Therefore, the first goal of this study was DNA aptamers development for those target histone PTMs.To address it, the SELEX (Systematic evolution of ligands by exponential enrichment) process was introduced by using a random DNA library and the histone modification peptide target coated magnetic beads (MBs). After several selection rounds, the final DNA pool was moved to next generation sequencing (NGS) step for DNA identification.Sequence-identified DNA aptamer candidates for each histone PTM targets were analyzed their binding affinity and target-specificity for verification of the aptamers. As a result of the characterization, aptamer 1336 has been found as H3K4Me3 specific aptamer with 13 nM of Kd. The best aptamers are ultimately applied to ISET platform for the affinity-purification and MALDI-MS analysis of histone code.

本研究旨在利用基于核酸适体的染色质亲和纯化-质谱法开发新的组蛋白密码分析方法。首先，选择三种不同的组蛋白修饰（H3K4Me3、H3K9Me3 和 H3K27Me3）作为目标组蛋白编码，因为它们大量存在并且通过参与基因调控而被称为重要的组蛋白 PTM。因此，本研究的首要目标是开发针对这些目标组蛋白 PTM 的 DNA 适体。为了解决这个问题，通过使用随机 DNA 文库和组蛋白修饰肽目标包被磁性，引入了 SELEX（指数富集配体系统进化）过程。珠子 (MB)。经过几轮选择后，最终的 DNA 库被转移到下一代测序 (NGS) 步骤进行 DNA 鉴定。对每个组蛋白 PTM 靶标的序列鉴定的 DNA 适体候选物进行分析，分析其结合亲和力和靶标特异性，以验证适体。表征结果表明，适配体 1336 被发现为 H3K4Me3 特异性适配体，Kd 为 13 nM。最好的适配体最终应用于ISET平台，用于组蛋白代码的亲和纯化和MALDI-MS分析。