A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interactions.

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs.

Concerted effects of two activator modules on the group I ribozyme reaction.

Group I intron ribozymes have a modular architecture and structural elements essential for catalysis. The elements are located in the conserved modular domain P3-P7 that is stabilized by another conserved module, P4-P6. It has been reported that artificial modules can complement the function of the native P4-P6. To exploit the modular architecture of group I ribozyme, we have constructed a hybrid ribozyme by attaching an artificial activator module to the wild-type T4 td ribozyme. Kinetic analysis of the hybrid ribozyme revealed that the artificial module and P4-P6 have unusual positive and negative concerted effects in activating the ribozyme.