Kuwabara T, Amontov S V, Warashina M, Ohkawa J, Taira K
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Tsukuba Science City, Japan.
Nucleic Acids Res. 1996 Jun 15;24(12):2302-10. doi: 10.1093/nar/24.12.2302.
A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem-loop II. In a previous study we demonstrated that a minizyme with high activity forms a dimeric structure with a common stem II. Because of their dimeric structure, minizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In order to examine the properties of different kinds of minizyme, we constructed a number of minizymes with short oligonucleotide linkers (2-5 bases) instead of stem-loop II and examined their cleavage activities against HIV-1 tat mRNA. Analyses of melting curves, as well as Arrhenius plots, revealed that, in general, the longer the oligonucleotide linkers, the more stable and more active were the dimer minizymes. All minizymes examined cleaved the target substrate at two sites simultaneously. The activity of the dimer minizyme with a 5 nt linker was higher than that of the parental hammerhead ribozyme because the latter full-sized ribozyme was able to cleave at one site only.
微型酶是一种锤头状核酶，其具有短寡核苷酸接头而非茎环II。在先前的一项研究中，我们证明具有高活性的微型酶会形成带有共同茎II的二聚体结构。由于其具有二聚体结构，微型酶有可能能够同时在两个不同位点切割底物。为了研究不同种类微型酶的特性，我们构建了一些带有短寡核苷酸接头（2 - 5个碱基）而非茎环II的微型酶，并检测了它们对HIV - 1 tat mRNA的切割活性。熔解曲线分析以及阿累尼乌斯曲线分析表明，一般来说，寡核苷酸接头越长，二聚体微型酶就越稳定且活性越高。所有检测的微型酶都能同时在两个位点切割目标底物。带有5个核苷酸接头的二聚体微型酶的活性高于亲本锤头状核酶，因为后者全长核酶仅能在一个位点切割。
