Kuwabara Tomoko, Warashina Masaki, Taira Kazunari
Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan.
J Biochem. 2002 Jul;132(1):149-55. doi: 10.1093/oxfordjournals.jbchem.a003193.
To overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.
为克服靶位点选择的障碍，我们最近构建了一种新型杂交核酶，它可以通过募集细胞内RNA解旋酶来作用于任何选定的位点[Warashina等人（2001年）《美国国家科学院院刊》98，5572 - 5577；Kawasaki等人（2002年）《自然生物技术》20，376 - 380]。我们之前还证明，具有两个底物结合臂且分别靶向靶mRNA内两个不同位点的RNA聚合酶III驱动的最大核酶在体内形成了非常活跃的异二聚体[Kuwabara等人（2000年）《生物技术趋势》18，462 - 468；Tanabe等人（2001年）《自然》406，473 - 474]。尽管二聚化过程复杂，但我们在培养细胞中测试的所有最大核酶都比亲本核酶具有更高的催化活性。为了研究最大核酶在细胞中的作用，我们设计了一种具有两个底物结合臂的特异性最大核酶，其靶向内源性表达的LTR - 荧光素酶嵌合mRNA，其中LTR指HIV - 1的长末端重复序列。最大核酶的一个底物结合臂被设计用于结合HIV - 1 TAR RNA内的一个位点，已知该位点形成稳定的茎结构，通常会阻止核酶结合。另一个底物结合臂靶向荧光素酶基因内一个相对容易接近的位点。正如预期的那样，传统核酶由于TAR区域稳定的二级结构而无法在体内切割该区域。然而，令我们惊讶的是，最大核酶在体内能高效切割茎内的TAR区域。最大核酶在体内增强的切割作用可能是由于在靶mRNA茎结构的呼吸过程中发生的一种熵有利的分子内二次结合过程。重要的是，我们的数据表明，这种最大核酶技术可能用作一种替代方法，用于在先前发现难以接近的切割位点募集RNA解旋酶。
