Hisamatsu S, Sonoki S, Kikuchi Y
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Biosci Biotechnol Biochem. 1995 Feb;59(2):294-7. doi: 10.1271/bbb.59.294.
We designed three hairpin ribozymes to cleave Escherichia coli beta-glucuronidase (GUS) mRNA and tested those activities in vitro. One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites. All ribozymes cleaved the model substrate (100 bases long) at the predicted target site. Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site. Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant cells. Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed.
我们设计了三种发夹状核酶来切割大肠杆菌β-葡萄糖醛酸酶（GUS）的mRNA，并在体外测试了它们的活性。其中一种核酶被设计为与目标GUS mRNA总共形成9个碱基对，另外两种核酶具有更长的底物结合位点。所有核酶都在预测的目标位点切割了模型底物（100个碱基长）。两种含有较长底物结合位点的核酶比另一种含有较短底物结合位点的核酶更有效地切割底物。此外，具有长底物结合位点的核酶对全长GUS mRNA（1.9千碱基）具有高活性，甚至在26℃的低温下也能保持活性，这是植物细胞的一般生长条件。本文讨论了核酶底物结合位点长度对切割活性的影响。
