Green R, Szostak J W
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Science. 1992 Dec 18;258(5090):1910-5. doi: 10.1126/science.1470913.
The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.
SunY核酶源自自我剪接的I组内含子RNA。由于其尺寸小，该核酶被选作设计自我复制RNA的起点。作为促进自我复制过程的一种手段，通过删除非保守结构域减小了该核酶的尺寸；然而，进行此类删除时，酶活性严重丧失。体外基因筛选用于鉴定可重新激活几乎无活性的SunY缺失突变体的突变。在筛选条件下，一个在一级序列中分散有五个取代突变的选定突变体显示出比原始核酶更高的催化活性。SunY核酶及其选定的小变体均能催化模板导向的寡核苷酸组装。选定变体的小尺寸和减少的二级结构导致其自我复制速率相对于原始核酶有所提高。这种工程化核酶能够在自我复制反应中有效地作为催化剂和模板发挥作用。
