Vaish Narendra K, Jadhav Vasant R, Kossen Karl, Pasko Christopher, Andrews Lori E, McSwiggen James A, Polisky Barry, Seiwert Scott D
Sirna Therapeutics, Inc., Boulder, Colorado 80301, USA.
RNA. 2003 Sep;9(9):1058-72. doi: 10.1261/rna.5760703.
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-activated 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.
我们描述了一种使用“半”核酶进行核酸超灵敏检测的策略，这种“半”核酶除非由反式作用靶核酸完成，否则没有催化活性。半核酶的概念最初是使用源自多周转I类连接酶的构建体来证明的。进行了迭代RNA筛选，以使这种半核酶进化为由丙型肝炎病毒（HCV）基因组中存在的保守序列激活的核酶。在对底物RNA进行序列优化后，这种HCV激活的半核酶显示出最大周转速率为69 min⁻¹（pH 8.3），并且被HCV靶标诱导速率提高了约2.6×10⁹倍。它在zeptomole范围内（6700个分子）检测到HCV靶标寡核苷酸，检测灵敏度比以前寡核苷酸激活的核酶所证明的灵敏度大约高2.6×10⁶倍，这一灵敏度足以用于分子诊断应用。
