The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

中文翻译：

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含有5-溴尿苷的核酶的体外进化。

通过用5-溴尿苷替换所有99个组分尿苷残基，对四膜虫第I组核酶进行了修饰。与未修饰的核酶的行为相比，这导致RNA催化的磷酸酯转移反应的催化效率降低了13倍。构造了一个10（13）变异核酶群体，每个变异核酶都包含5-溴尿苷代替尿苷。进行了五个连续的“世代”体外进化，选择了改善的磷酸酯转移酶活性。与野生型含溴尿苷的核酶相比，进化出的分子表现出27倍的催化效率提高，甚至超过了非溴化形式的野生型核酶。发现了三种特定的突变与这种改变的行为有关。这些突变在5-溴尿苷的情况下增强了活性，但是在未修饰的尿苷的情况下是有害的。进化的RNA不仅可以耐受，而且还可以利用核苷酸类似物来执行其催化功能。