Insight into the epigenetic modulation-correlated molecule interactions has significant implications for the in-depth understanding of intracellular intricate biological networks. However, there is currently a lack of reliable biological tools for elucidating the potential correlation between epigenetic regulators and relevant genes, e.g., microRNAs (miRNAs). Herein, an alkB homologue 5 (ALKBH5, a key epigenetic regulator)-modulated catalytic DNA circuit (ACD) was constructed by grafting a N6-methyladenosine (m⁶A)-caged I-R3 DNAzyme into the circuitry components for achieving the on-site miRNA imaging in living cells. Specifically, the catalytic activity of I-R3 DNAzyme could be effectively suppressed by the m⁶A modification situated at its highly sequence-conserved core region and then be selectively restored through the ALKBH5-mediated demethylation pathway. And the ALKBH5-activated I-R3 DNAzyme allowed the highly efficient DNA cleaving reaction in the presence of DNAzyme cofactors, resulting in the liberation of catalytic hairpin assembly (CHA) reactants. Subsequently, target miRNA triggered the CHA circuit to produce a duplex DNA product while releasing the miRNA analyte. The liberated miRNA could autonomously trigger the next round of the CHA assembly cycle for generating the amplified fluorescence readout. By virtue of the stimuli-responsive activation and the CHA amplification circuit, the ACD system achieved highly specific and sensitive imaging of miRNA in tumor cells. Moreover, this efficiently and reliably ALKBH5-activated DNA circuit is demonstrated to reveal the underlying relationship between activator ALKBH5 and miRNA. Overall, the developed ACD system provides a promising tool for the robust on-site profiling of epigenetic-involved signal pathways, thus displaying great potential in bioanalytical applications.

中文翻译：

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利用去甲基化酶调节的催化 DNA 电路对与 MicroRNA 的调节连接进行原位研究


深入了解表观遗传调控相关分子相互作用对于深入了解细胞内错综复杂的生物网络具有重要意义。然而，目前缺乏可靠的生物工具来阐明表观遗传调节因子与相关基因（例如 microRNA （miRNA））之间的潜在相关性。在此，通过将 N6-甲基腺苷 （m⁶A） 笼状 I-R3 DNA酶移植到电路组分中，构建了 alkB 同源物 5 （ALKBH5，一种关键的表观遗传调节因子）调节的催化 DNA 回路 （ACD），以实现活细胞中的 miRNA 现场成像。具体来说，位于其高度序列保守的核心区域的 m⁶A 修饰可以有效抑制 I-R3 DNAzyme 的催化活性，然后通过 ALKBH5 介导的去甲基化途径选择性恢复。ALKBH5 激活的 I-R3 DNAzyme 允许在 DNAzyme 辅因子存在下进行高效的 DNA 切割反应，从而释放催化发夹组装 （CHA） 反应物。随后，靶标 miRNA 触发 CHA 电路产生双链 DNA 产物，同时释放 miRNA 分析物。释放的 miRNA 可以自主触发下一轮 CHA 组装周期，以生成扩增的荧光读数。凭借刺激响应激活和 CHA 扩增电路，ACD 系统实现了肿瘤细胞中 miRNA 的高度特异性和灵敏成像。此外，这种高效可靠的 ALKBH5 激活的 DNA 回路被证明可以揭示激活剂 ALKBH5 和 miRNA 之间的潜在关系。 总体而言，开发的 ACD 系统为表观遗传学相关信号通路的稳健现场分析提供了一种有前途的工具，因此在生物分析应用中显示出巨大的潜力。