Komatsu Y, Yamashita S, Kazama N, Nobuoka K, Ohtsuka E
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan.
J Mol Biol. 2000 Jun 23;299(5):1231-43. doi: 10.1006/jmbi.2000.3825.
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.
发夹环和与该环结合的寡核苷酸构成假结结构的一半。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来激活它。锤头状核酶的茎II被非自我互补的环序列（环II）取代以消除切割活性。新的核酶对靶RNA几乎没有切割活性。然而，在存在与核酶环II互补的辅因子寡核糖核苷酸的情况下，它表现出切割活性。该活性被认为源自辅因子寡核苷酸与环II之间假茎结构的形成。包含该环的结构可能类似于假半结结构。辅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，并且具有较长环II的核酶比具有短环II的核酶更具活性。具有3'端悬垂嘌呤碱基的寡核糖核苷酸作为核酶的有效辅因子，并且2'-O-甲基寡核苷酸最有效地增强了核酶的切割活性，与不存在该寡核苷酸时相比提高了约750倍。在3'端具有胞嘧啶碱基的辅因子寡核苷酸也激活了具有G10.1.G11.1突变的核酶，该突变消除了野生型中的切割活性。通过光交联实验确定了寡核苷酸的结合位点，发现其为环中的预测位点。这是首次报道通过采用结构基序来正向控制核酶活性的设计。该方法可应用于控制其他具有发夹环的功能性RNA的活性。
