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首页> 外文期刊>Journal of Molecular Biology >In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues.
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In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues.

机译：使用具有两个核苷酸类似物的诱变PCR，将锤头状核酶体外进化为嘌呤特异性核酶。

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摘要

The conventional hammerhead ribozyme cleaves RNA 3' to nucleotide triplets with the general formula NUH, where N is any nucleotide, U is uridine and H is any nucleotide except guanosine. In order to isolate hammerhead ribozyme sequences capable of cleaving 3' to the GUG triplet, we performed a mutagenic selection protocol starting with the conventional sequence of an NUH-cleaving ribozyme. The 22 nucleotides in the core and the stem-loop II region were subjected to mutagenic PCR using the two nucleotide analogues 6-(2-deoxy-beta-d-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C)][1, 2] oxazin-7-one and of 8-oxo-2'-deoxyguanosine. After five repetitions of the selection cycle, several clones showed cleavage activity. One sequence, having one deletion, showed at least a 90 times higher in trans cleavage rate than the starting ribozyme. It cleaved 3' to GUG and GUA. The sequence of this ribozyme is essentially identical with that obtained previously by selection for AUG cleavage starting with a randomised core and stem-loop II region. This identical result of two independent selection procedures supports the notion that sequences for NUR cleavage, where R is a purine nucleotide, are not compatible with the classical hammerhead structure, and that the sequence space for this cleavage specificity is very limited. The cleavage of NUR triplets is not restricted to the sequence of the substrate that was used for selection but is sequence-independent for in trans cleavage, although the sequence context influences the value for the cleavage rate somewhat. Analysis of cleavage activities indicates the importance of A at position L2.5 in loop II. Copyright 2000 Academic Press.
机译：常规锤头状核酶将RNA 3'切割为具有通式NUH的核苷酸三联体，其中N为任何核苷酸，U为尿苷，H为除鸟苷外的任何核苷酸。为了分离能够裂解3'到GUG三联体的锤头状核酶序列，我们从常规的NUH裂解核酶序列开始进行了诱变选择方案。使用两个核苷酸类似物6-（2-deoxy-beta-d-rifurfuranosyl）-3,4-dihydro-8H-pyrimido- [4，核和茎环II区的22个核苷酸进行了诱变PCR 5-C）] [1，2]恶嗪7-1和8-氧-2'-脱氧鸟苷。选择循环重复五次后，几个克隆显示出切割活性。具有一个缺失的一个序列显示出比起始核酶至少高90倍的反式切割速率。它切割了3'到GUG和GUA。该核酶的序列与先前从随机核心和茎环II区开始选择AUG裂解获得的核酶序列基本相同。两个独立选择过程的相同结果支持以下观念：NUR裂解的序列（其中R为嘌呤核苷酸）与经典锤头结构不兼容，并且这种裂解特异性的序列空间非常有限。 NUR三联体的切割不限于用于选择的底物的序列，而是在反式切割中与序列无关，尽管序列上下文在一定程度上影响切割速率的值。裂解活性的分析表明在环II中L2.5位置的A的重要性。版权所有2000学术出版社。

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