Abstract
We designed and constructed a dimer of the Tetrahymena group I ribozyme the activity of which is regulated by self-dimerization. This dimer was rationally designed by utilizing the P5abc and ΔP5abc domains as large RNA motifs. This strategy enabled us to install large ribozyme functions into an RNA structure. This is a step toward expanding the field of RNA nanotechnology beyond the limitation of using only relatively small functional motifs. Self-dimerization can also be rationally programmed by modular engineering of RNA interaction motifs. In this chapter, we present the procedure for the rational design and construction of large ribozyme domains based on RNA tertiary structures. We also describe the electrophoresis mobility shift assay (EMS) and several ribozyme activity assays to confirm the ribozyme function and its regulation. We have succeeded in construction of tecto-GIRz.
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