Zeptomole detection of a viral nucleic acid using a target-activated ribozyme
Zeptomole detection of a viral nucleic acid using a target-activated ribozyme
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DOI：
10.1261/rna.5760703
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发表时间：
2003-09-01
期刊：
影响因子：
4.5
通讯作者：
Seiwert, SD
中科院分区：
文献类型：
--
作者：
Vaish, NK;Jadhav, VR;Seiwert, SD
我们描述了一种使用“半”核酶的核酸超灵敏检测策略，所述“半”核酶缺乏催化活性，除非由反式作用靶核酸完成：半核酶的概念最初证明了使用一个多营业额I类连接酶衍生的构建体。进行迭代RNA选择以将该半核酶进化成由丙型肝炎病毒（HCV）基因组中存在的保守序列激活的半核酶。在优化底物RNA序列后，这种HCV激活的双半核酶显示出69 min（-1）（pH 8.3）的最大转换速率，并且被HCV靶诱导的速率约为2.6 × 10（9）倍。它能检测到zeptomole范围内的HCV靶寡核苷酸（6700个分子），其检测灵敏度比以前用利多卡因激活的核酶所证明的高约2.6 × 10（6）倍，足以用于分子诊断应用。
We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid:. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activatedi half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.