The 10–23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10–23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10–23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10–23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10–23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10–23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary.

中文翻译：

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通过与合成 DNA 寡核苷酸杂交，使 RNA 裂解 DNA 酶可接近大结构 RNA 中的靶位点


10-23 DNA 酶是基于 DNA 的最活跃的酶之一，理论上可以设计为靶向 RNA 序列内的任何嘌呤-嘧啶连接进行切割。然而，生物来源的大结构 RNA （lsRNA） 分子内的嘌呤-嘧啶连接并不总是被 10-23 接近，从而否定了它作为 RNA 切割分子剪刀的一般用途。在此，我们报道了一种可推广的策略，该策略允许 10-23 访问 lsRNA 中的任何嘌呤-嘧啶连接。使用长度为 566、584 和 831 个核苷酸的三个大 SARS-CoV-2 mRNA 序列作为模型系统，我们表明，使用靶向切割位点两侧上游和下游区域的反义 DNA 寡核苷酸 （ASO） 可以将先前活性较差的 10-23 DNA 酶系统的活性 （kobs） 恢复高达 2000 倍。我们使用在线探测从机械上证实了这些发现，以证明 ASO 减少了 lsRNA 底物内 10-23 个 DNAzyme 靶位点结构。这种方法代表了一种简单、高效、经济且可推广的方法，可以提高 lsRNA 分子中 10-23 个位点对选定靶位点的可及性，尤其是在需要直接进入基因组 RNA 靶标的情况下。