Miyamoto Yoshitaka, Teramoto Naozumi, Imanishi Yukio, Ito Yoshihiro
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Biotechnol Bioeng. 2005 Apr 5;90(1):36-45. doi: 10.1002/bit.20360.
A ligase ribozyme that accelerates the ligation reaction with an oligonucleotide under low pH conditions was identified by in vitro adaptation in a previous study. We examined the effects of further rounds of evolution to isolate a more active ribozyme. The ribozyme, which was obtained after four rounds of evolution, was randomly mutated, and the resultant RNA library was subjected to in vitro selection at low pH. One ribozyme isolated from the pool was found to react 8,000 times faster than the original b1 ribozyme at pH 4. The reaction rate of the isolated ribozyme was enhanced at various pH values, and its pH dependence was less than that of the original ribozyme or the ribozyme selected with four rounds of evolution. The reaction rate of the isolated ribozyme was reduced in the presence of 3' primer, the sequence of which is complementary to the 3' primer-binding site of the ligase ribozyme. This inhibition induced by the primer oligonucleotide binding to the ribozyme 3' region implies that the 3' region plays a role in the ligation reaction of the ribozyme.
在之前的一项研究中，通过体外适应性进化鉴定出了一种在低pH条件下能加速与寡核苷酸连接反应的连接酶核酶。我们研究了进一步的进化轮次对分离出更具活性核酶的影响。经过四轮进化后获得的核酶被随机突变，所得RNA文库在低pH条件下进行体外筛选。从文库中分离出的一种核酶在pH 4时的反应速度比原始的b1核酶快8000倍。分离出的核酶在不同pH值下反应速率均有所提高，其对pH的依赖性小于原始核酶或经过四轮进化筛选出的核酶。在存在3'引物（其序列与连接酶核酶的3'引物结合位点互补）的情况下，分离出的核酶的反应速率降低。引物寡核苷酸与核酶3'区域结合所诱导的这种抑制作用表明，3'区域在核酶的连接反应中起作用。
