Construction of an artificial ribozyme which ligates an RNA fragment activated by nicotinamide mononucleotide

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.