Joseph S, Burke J M
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
J Biol Chem. 1993 Nov 25;268(33):24515-8.
We have applied in vitro selection methods to achieve a large increase in the catalytic activity of a hairpin ribozyme targeted against a highly conserved 14-nucleotide sequence within HIV-1 pol RNA. The substrate specificity was changed by mutating 8 bases within the substrate-binding domain of the parental (-)sTRSV ribozyme. The resulting enzyme cleaved the HIV substrate specifically but with a 20-fold reduction in catalytic efficiency (kcat/KM). Following random mutagenesis, ribozymes with increased activity against the target sequence were selected through 10 rounds of in vitro selection. Selective pressure was increased by decreasing MgCl2 and spermidine concentrations, and reducing reaction time. Variant ribozymes with base substitutions A11-->G and U39-->C were selected in the population. These mutations were introduced singly and in combination into the trans-acting anti-HIV ribozyme. Each of the single-base substitutions significantly increased ribozyme activity, while the activity of double mutant was increased to nearly the level of the parental ribozyme. These findings demonstrate that in vitro selection is a powerful and efficient method to optimize ribozymes for the catalytic inactivation of targeted RNA molecules.
我们应用体外筛选方法，使针对HIV-1 pol RNA中一段高度保守的14个核苷酸序列的发夹状核酶的催化活性大幅提高。通过对亲本（-）sTRSV核酶底物结合域内的8个碱基进行突变，改变了底物特异性。所得酶能特异性切割HIV底物，但催化效率（kcat/KM）降低了20倍。经过随机诱变后，通过10轮体外筛选，选出了对靶序列活性增强的核酶。通过降低MgCl2和亚精胺浓度以及缩短反应时间来增加选择压力。在群体中选出了碱基替换为A11→G和U39→C的变异核酶。将这些突变单独或组合引入反式作用抗HIV核酶中。每个单碱基替换都显著提高了核酶活性，而双突变体的活性则提高到接近亲本核酶的水平。这些发现表明，体外筛选是一种强大而有效的方法，可用于优化核酶以催化失活靶向RNA分子。
