A novel approach using aptamers for histone code analysis

本研究旨在建立一种新的组蛋白编码分析方法，即基于适体的染色质亲和纯化-质谱法。首先，我们选择了三种不同的组蛋白修饰（H3K4Me3、H3K9Me3和H3K27Me3）作为靶组蛋白编码，因为它们丰富存在，参与基因调控，被认为是重要的组蛋白ptm。因此，本研究的第一个目标是为这些靶组蛋白ptm开发DNA适体。为了解决这一问题，采用随机DNA文库和组蛋白修饰肽靶包覆磁珠（mb），引入了SELEX（配体的指数富集系统进化）过程。经过几轮筛选，最终的DNA池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适体候选体进行了结合亲和力和靶标特异性分析，以验证适体。通过表征，发现适配体1336是具有13 nM Kd的H3K4Me3特异性适配体。最终将最佳适配体应用于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.