Endonucleases are enzymes that cleave internal phosphodiester bonds within double-stranded DNA or RNA and are essential for biological functions. Herein, we use genetic code expansion to create an unnatural endonuclease that cleaves non-coding RNAs including short interfering RNA (siRNA) and microRNAs (miRNAs), a function that does not exist in nature. We introduce a metal-chelating unnatural amino acid, (2,2′-bipyridin-5-yl)alanine (BpyAla) to impart endonuclease activity to the viral suppressor of RNA silencing protein p19. Upon binding of copper, the mutant p19-T111BpyAla displays catalytic site-specific cleavage of siRNA and human miRNAs. Catalysis is confirmed using fluorescence polarization and fluorescence turn-on. Global miRNA profiling reveals that the engineered enzyme cleaves miRNAs in a human cell line. The therapeutic potential is demonstrated by targeting miR-122, a critical host factor for the hepatitis C virus (HCV). Unnatural endonuclease function is shown to deplete miR-122 levels with similar effects to an antagomir that reduces HCV levels therapeutically.

核酸内切酶是切割双链 DNA 或 RNA 内内部磷酸二酯键的酶，对于生物功能至关重要。在这里，我们使用遗传密码扩展来创建一种非天然核酸内切酶，可以切割非编码RNA，包括短干扰RNA (siRNA) 和微小RNA (miRNA)，这是自然界中不存在的功能。我们引入了一种金属螯合非天然氨基酸（2,2'-联吡啶-5-基）丙氨酸（BpyAla），以赋予 RNA 沉默蛋白 p19 的病毒抑制子核酸内切酶活性。与铜结合后，突变体 p19-T111BpyAla 显示出 siRNA 和人类 miRNA 的催化位点特异性切割。使用荧光偏振和荧光开启来确认催化作用。全局 miRNA 分析表明，工程酶可切割人类细胞系中的 miRNA。通过靶向丙型肝炎病毒 (HCV) 的关键宿主因子 miR-122，证明了其治疗潜力。非自然核酸内切酶功能可降低 miR-122 水平，其效果与治疗性降低 HCV 水平的 antagomir 类似。