A novel approach using aptamers for histone code analysis

本研究旨在建立一种基于适体的染色质亲和纯化-质谱分析组蛋白密码子的新方法。首先，选择三种不同的组蛋白修饰（H3 K4 Me 3、H3 K9 Me 3和H3 K27 Me 3）作为靶组蛋白密码子，因为它们大量存在并且被认为是参与基因调控的重要组蛋白PTM。因此，本研究的第一个目标是开发针对组蛋白修饰后修饰的DNA适体，为此，利用随机DNA库和组蛋白修饰肽靶向包被的磁珠（MB）引入了SELEX（指数富集配体系统进化）过程。经过几轮筛选后，将最终的DNA池移至下一代测序（NGS）步骤进行DNA鉴定，分析每个组蛋白PTM靶标的经序列鉴定的DNA适体候选物的结合亲和力和靶标特异性，以验证适体。作为表征的结果，已发现适体1336为具有13 nM Kd的H3 K4 Me 3特异性适体。最佳适配体最终应用于ISET平台进行组蛋白编码的亲和纯化和MALDI-MS分析。

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.