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首页> 外文期刊>Journal of Molecular Biology >Construction of new ribozymes requiring short regulator oligonucleotides as a cofactor.
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Construction of new ribozymes requiring short regulator oligonucleotides as a cofactor.

机译：新的核酶的构建需要短的调节寡核苷酸作为辅因子。

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摘要

A hairpin loop and an oligonucleotide bound to the loop form one-half of the pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short complementary oligonucleotide as a cofactor. Stem II of the hammerhead ribozyme was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage activity of the target RNA. However, it exhibited the cleavage activity in the presence of a cofactor oligoribonucleotide, which is complementary to loop II of the ribozyme. The activity is assumed to be derived from the formation of a pseudo-stem structure between the cofactor oligonucleotide and loop II. The structure including the loop may be similar to the pseudo-half-knot structure. The activation efficiencies of the cofactor oligonucleotides were decreased as the lengths of the oligonucleotides increased, and the ribozyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3'-dangling purine bases served as efficient cofactors of the ribozyme, and a 2'-O-methyloligonucleotide enhanced the cleavage activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligonucleotides with a cytidine base at the 3'-end also activated a ribozyme with the G10.1.G11.1 mutation, which eliminates the cleavage activity in the wild-type. The binding sites of the oligonucleotide were identified by photo-crosslinking experiments and were found to be the predicted sites in the loop. This is the first report of a design aimed at positively controlling the activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin loops. Copyright 2000 Academic Press.
机译：发夹环和与该环结合的寡核苷酸形成假结结构的一半。我们设计了一种变构锤头状核酶，通过使用短互补寡核苷酸作为辅因子引入该基序，可以激活该酶。锤头状核酶的茎II被非自互补的环序列（环II）取代，以消除切割活性。新的核酶几乎没有靶RNA的切割活性。然而，它在辅酶寡核糖核苷酸的存在下表现出切割活性，该辅酶寡核糖核苷酸与核酶的环II互补。假定该活性源自辅因子寡核苷酸与环II之间假茎结构的形成。包括环的结构可以类似于伪半结结构。辅助因子寡核苷酸的激活效率随着寡核苷酸长度的增加而降低，具有较长环II的核酶比具有短环II的核酶更具活性。具有3'悬空的嘌呤碱基的寡核糖核苷酸是核酶的有效辅因子，与不存在寡核苷酸的情况相比，2'-O-甲基寡核苷酸最有效地增强了核酶的裂解活性，约为750倍。寡核苷酸。在3'末端带有胞苷碱基的辅因子寡核苷酸还激活了具有G10.1.G11.1突变的核酶，从而消除了野生型的切割活性。通过光交联实验鉴定寡核苷酸的结合位点，发现其是环中的预测位点。这是旨在通过利用结构基序积极控制核酶活性的设计的首次报道。此方法可用于控制具有发夹环的其他功能性RNA的活性。版权所有2000学术出版社。

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