Fujita Yuki, Furuta Hiroyuki, Ikawa Yoshiya
Graduate School of Engineering, Kyusyu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.
Nucleic Acids Symp Ser (Oxf). 2006(50):231-2. doi: 10.1093/nass/nrl115.
A new strategy termed "Design & Selection" has been developed for construction of artificial ribozymes. In this strategy, two distinct approaches (de novo rational design and in vitro selection) were successfully combined. De novo rational design was employed for construction of a structural scaffold of a new ribozyme whereas in vitro selection was adopted for isolation of a catalytic unit, sequence and structure of which were unpredictable. Using this strategy, a ligase ribozyme (DSL ribozyme) has been isolated. To validate versatility of Design & Selection strategy, we attempted to isolate a new catalytic unit utilizing a nicotinamide mononucleotide as a leaving group in RNA-RNA ligation reaction. Into the de novo designed scaffold RNA, 45 random nucleotides were inserted. The resulting RNA library consisting of the scaffold and randomized regions was subjected to in vitro selection. Using the library with 10(14) sequence diversity, a new class of ribozyme bearing a novel catalytic unit sequence was successfully isolated.
一种名为“设计与筛选”的新策略已被开发用于构建人工核酶。在该策略中，两种不同的方法（从头理性设计和体外筛选）成功地结合在一起。从头理性设计用于构建新核酶的结构支架，而体外筛选则用于分离催化单元，其序列和结构是不可预测的。利用这一策略，已分离出一种连接酶核酶（DSL核酶）。为了验证“设计与筛选”策略的通用性，我们尝试在RNA-RNA连接反应中利用烟酰胺单核苷酸作为离去基团分离一个新的催化单元。在从头设计的支架RNA中插入了45个随机核苷酸。由支架和随机区域组成的所得RNA文库进行体外筛选。使用具有10¹⁴序列多样性的文库，成功分离出一类带有新型催化单元序列的新核酶。
