Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10–23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10–23, overcomes the problem of product inhibition, which limited previous 10–23 designs, using molecular chemotypes with DNA, 2′-fluoroarabino nucleic acid and α-l-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10–23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.

由于其在临床试验中的低功效，在治疗应用中使用 RNA 切割 DNA 酶 (DNAzymes) 作为基因沉默剂的努力已经停滞不前。在这里，我们报告了经典 DNAzyme 10-23 的异种核酸修饰版本，它在细胞条件下实现多周转活性并抵抗核酸酶消化。新试剂 X10-23 克服了产物抑制问题，该问题限制了以前的 10-23 设计，使用具有 DNA、2'-氟阿拉伯核酸和 α -l的分子化学型-苏呋喃糖核酸骨架结构，可平衡增强的生物稳定性与 RNA 杂交和二价金属离子配位的影响。在培养的哺乳动物细胞中，X10-23 通过有效降解外源性和内源性信使 RNA 转录物来促进持久的基因沉默。总之，这些结果表明新的分子化学型可以提高脱氧核糖核酸酶的活性和稳定性，并可能为核酸酶进入临床提供新的途径。