Artificial ligase ribozymes isolated by a “design and selection” strategy

We developed a new in vitro selection strategy “design and selection” to isolate effectively artificial ribozymes (catalytic RNAs). An overall RNA structure (scaffold) is initially designed, and then a relatively short randomized sequence is installed at the reaction point of the scaffold, followed by the in vitro selection. This method can reduce the length of randomized sequence, providing large coverage of the sequence space in contrast with the conventional way, which makes the selection experiment effectively. Additionally, further analysis of ribozymes obtained by this approach is practically easy since the overall molecular structure is predesigned and well known. Here we show the procedure to isolate artificial RNA ligase ribozymes by this strategy. We have succeeded in isolation of the designed and selected ligase (DSL) ribozymes.