Low activity has been the primary obstacle impeding the use of DNA enzymes (DNAzymes) as gene silencing agents in clinical applications. Here we describe the chemical evolution of a DNAzyme with strong catalytic activity under near physiological conditions. The enzyme achieves ~65 turnovers in 30 minutes, a feat only previously witnessed by the unmodified parent sequence under forcing conditions of elevated Mg²⁺ and pH. Structural constraints imposed by the chemical modifications drive catalysis toward a highly preferred UGUD motif (cut site underlined) that was validated by positive and negative predictions. Biochemical assays support an autonomous RNA cleavage mechanism independent of RNase H1 engagement. Consistent with its strong catalytic activity, the enzyme exhibits persistent allele-specific knock-down of an endogenous mRNA encoding an undruggable oncogenic KRAS target. Together, these results demonstrate that chemical evolution offers a powerful approach for discovering new chemotype combinations that can imbue DNAzymes with the physicochemical properties necessary to support therapeutic applications.

低活性一直是阻碍 DNA 酶 (DNAzymes) 在临床应用中用作基因沉默剂的主要障碍。在这里，我们描述了在接近生理条件下具有强催化活性的 DNAzyme 的化学演变。该酶在 30 分钟内实现了约 65 次转换，这一壮举之前只有在 Mg ²⁺和 pH 升高的强制条件下未修饰的亲本序列才能见证。化学修饰所施加的结构限制推动了对高度优选的 U GU的催化D 基序（切割位点加下划线），由正负预测验证。生化分析支持独立于 RNase H1 参与的自主 RNA 切割机制。与其强大的催化活性一致，该酶表现出持续的等位基因特异性敲低编码不可药物致癌 KRAS 靶标的内源性 mRNA。总之，这些结果表明，化学进化提供了一种强大的方法来发现新的化学型组合，这些组合可以使 DNAzyme 具有支持治疗应用所需的物理化学特性。