Target site selection for an RNA-cleaving catalytic DNA
Target site selection for an RNA-cleaving catalytic DNA
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DOI：
10.1038/8658
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发表时间：
1999-05-01
影响因子：
46.9
通讯作者：
Sun, LQ
中科院分区：
文献类型：
--
作者：
Cairns, MJ;Hopkins, TM;Sun, LQ
一种小的催化DNA，被称为10-23DNA酶或脱氧核酶，已被证明在体外有效地在嘌呤-嘧啶(R-Y)连接处水解RNA。虽然这些潜在的可切割连接是普遍存在的，但它们经常被RNA二级结构保护而不被脱氧核酶活性所影响。我们开发了一种多重切割试验，用于筛选目标RNA分子的全长脱氧核酶切割位点，在动力学和可获得性方面都是有效的。这一策略使我们能够同时比较模型靶基因(HPV16E6)的80个脱氧核酶和另外60个脱氧核酶对大鼠c-myc靶基因的RNA切割活性。人乳头瘤病毒(HPV)靶标主要用于表征多重系统并确定其有效性。C-myc靶点结合平滑肌细胞增殖实验，使我们能够评估体外切割效率和细胞培养中c-myc基因抑制之间的关系。多重反应方法简化了在功能分析中揭示有效脱氧核酶的过程，并提供了可获得的数据，该数据也可能适用于其他基于杂交的试剂的位点选择。
A small catalytic DNA, known as the 10-23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.