Tang J, Breaker R R
Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, USA.
Proc Natl Acad Sci U S A. 2000 May 23;97(11):5784-9. doi: 10.1073/pnas.97.11.5784.
In vitro selection was used to isolate Mg(2+)-dependent self-cleaving ribozymes from random sequence. Characterization of representative clones revealed the emergence of at least 12 classes of ribozymes that adopt distinct secondary structure motifs. Only one class corresponds to a previously known structural motif, that of the naturally occurring hammerhead ribozyme. Each ribozyme promotes self-cleavage via an internal phosphoester transfer reaction involving the adjacent 2'-hydroxyl group with a chemical rate enhancement of between 10(3)- and 10(6)-fold greater than the corresponding uncatalyzed rate. These findings indicate that RNA can form a multitude of secondary and tertiary structures that promote cleavage by internal phosphoester transfer. Upon further in vitro selection, a class I ribozyme that adopts an "X motif" structure dominates over all other ribozymes in the population. Thus, self-cleaving RNAs isolated by in vitro selection from random-sequence populations can rival the catalytic efficiency of natural ribozymes.
体外筛选用于从随机序列中分离出依赖镁离子的自我切割核酶。对代表性克隆的表征揭示了至少12类采用不同二级结构基序的核酶的出现。只有一类对应于先前已知的结构基序，即天然存在的锤头状核酶的结构基序。每个核酶通过涉及相邻2'-羟基的内部磷酸酯转移反应促进自我切割，化学速率增强比相应的未催化速率高10³至10⁶倍。这些发现表明RNA可以形成多种二级和三级结构，通过内部磷酸酯转移促进切割。经过进一步的体外筛选，一种采用“X基序”结构的I类核酶在群体中比所有其他核酶占优势。因此，通过体外筛选从随机序列群体中分离出的自我切割RNA可以与天然核酶的催化效率相媲美。
