Zhang Qingqing, Hao Jian, He Yuqiu, Weng Benrui, Yu Shanshan, Li Chunsen, Wang Fuan
College of Chemistry and Molecular Sciences, Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430072, P. R. China.
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, P. R. China.
J Am Chem Soc. 2025 Sep 24;147(38):34944-34958. doi: 10.1021/jacs.5c11409. Epub 2025 Sep 10.
The in-depth integration of gene regulation with protein modulation can enhance cellular information processing, yet it is significantly constrained by ineffective and complex protein-to-gene transduction strategies. Herein, we developed a simple protease-guided autocatalytic gene silencing platform named iPAD (intelligent peptide-programmed deoxyribonuclease) that converts the protease recognition events into versatile DNA readout signals by rationally designing a native protease-responsive cationic peptide (PP) to efficiently modulate the DNAzyme (Dz) activity. Without requiring additional chemical modifications, the multifunctional PP regulator consists simply of one cell-specific targeting peptide segment and two cationic peptide segments isolated by one protease-specific peptide substrate. The catalytic activity of Dz can be potently disrupted via the cooperatively stabilized electrostatic interactions between cationic PP and the anionic Dz. Subsequently, the Dz activity is efficiently restored via the protease-specific cleavage of the PP, which disrupts the cooperative stabilization between PP and Dz, thus achieving robust and accurate monitoring of protease activity. Molecular dynamic simulations theoretically validate that the on-demand regulation of Dz activity was indeed acquired from the programmed oligomerization of oligo-peptides. As a universal protease sensing platform, this method was successfully applied to probe Caspase-3 and thrombin, facilitating the early evaluation of tumor therapeutic efficacy. Moreover, the endogenous Caspase-3-activated Dz facilitates the construction of an autocatalytic gene regulation platform that self-adaptively upregulates the expression of apoptotic proteases, accelerating tumor cell apoptosis. This simple yet intelligent iPAD system provides a versatile toolbox for high-performance biosensing and bioengineering applications, paving the way for new strategies in smart theragnostic research.
基因调控与蛋白质调节的深度整合可增强细胞信息处理能力，但却受到低效且复杂的蛋白质到基因转导策略的显著限制。在此，我们开发了一种名为iPAD（智能肽编程脱氧核糖核酸酶）的简单蛋白酶引导的自催化基因沉默平台，通过合理设计一种天然蛋白酶响应阳离子肽（PP），将蛋白酶识别事件转化为通用的DNA读出信号，以有效调节脱氧核酶（Dz）的活性。无需额外的化学修饰，多功能PP调节剂仅由一个细胞特异性靶向肽段和两个由一个蛋白酶特异性肽底物隔开的阳离子肽段组成。Dz的催化活性可通过阳离子PP与阴离子Dz之间协同稳定的静电相互作用而被有效破坏。随后，通过PP的蛋白酶特异性切割有效地恢复Dz活性，这破坏了PP与Dz之间的协同稳定作用，从而实现对蛋白酶活性的稳健而准确的监测。分子动力学模拟从理论上验证了Dz活性的按需调节确实是从寡肽的程序化寡聚化获得的。作为一种通用的蛋白酶传感平台，该方法成功应用于探测Caspase-3和凝血酶，有助于肿瘤治疗疗效的早期评估。此外，内源性Caspase-3激活的Dz有助于构建一个自催化基因调控平台，该平台可自适应地上调凋亡蛋白酶的表达，加速肿瘤细胞凋亡。这个简单而智能的iPAD系统为高性能生物传感和生物工程应用提供了一个多功能工具箱，为智能诊疗研究的新策略铺平了道路。
