Kore A R, Carola C, Eckstein F
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany.
Bioorg Med Chem. 2000 Jul;8(7):1767-71. doi: 10.1016/s0968-0896(00)00103-6.
An in vitro selection was carried out to identify hammerhead ribozymes cleaving 3' to GAC triplets more efficiently than the wild type ribozyme. A double-stranded DNA containing the sequence for the hammerhead ribozyme with 10 randomizations in the catalytic core, designed for in cis cleavage, was transcribed and the cleavage product amplified by reverse transcription and PCR. After seven selection cycles, the DNA was cloned and 50 colonies sequenced. One sequence, appearing six times, was active for in cis cleavage of GAC. It was identical to the consensus sequence except for a mutation at position 7. Another cleaved GUC and two more, cleaved GUA. The cleavage rates of these ribozymes for in trans cleavage were slower than the rate of the consensus ribozyme. Interestingly, the consensus sequence was not found in the selection. This strongly suggests that the consensus hammerhead ribozyme has evolved to an optimal sequence.
进行了一项体外筛选，以鉴定比野生型核酶更有效地切割GAC三联体3'端的锤头状核酶。转录了一个双链DNA，其包含催化核心中有10个随机位点的锤头状核酶序列，用于顺式切割，切割产物通过逆转录和PCR进行扩增。经过七个筛选循环后，将DNA克隆并对50个菌落进行测序。有一个序列出现了六次，对GAC的顺式切割具有活性。除了第7位的一个突变外，它与共有序列相同。另一个切割GUC，还有两个切割GUA。这些核酶的反式切割速率比共有核酶的速率慢。有趣的是，在筛选中未发现共有序列。这强烈表明共有锤头状核酶已进化为最佳序列。
