A novel approach using aptamers for histone code analysis

本研究旨在利用基于适体的染色质亲和纯化-质谱法建立新的组蛋白密码子分析方法。首先，我们选择了三种不同的组蛋白修饰物(H3K4me3、H3K9me3和H3K27me3)作为组蛋白编码的靶点，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，本研究的第一个目标是开发靶向组蛋白PTM的DNA适配子，为此，采用随机DNA文库和组蛋白修饰多肽靶向包被磁珠(MBS)，引入SELEX(系统进化指数富集法)过程。经过几轮筛选后，将最终的DNA池转移到下一代测序(NGS)步骤进行DNA鉴定，并分析每个组蛋白PTM靶标的DNA适体候选序列的结合亲和力和靶标特异性，以验证适体的有效性。鉴定结果表明，适配子1336为H3K4me3特异性适配子，Kd为13 nM。最优适配子最终应用于ISET平台，用于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.