Network of functions and structures of ribozymes

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Onto an artificially designed and constructed RNA, a reaction site for RNA-RNA ligation and a catalytic module that conducts the ligation was installed. The catalytic module was prepared by conducting in vitro selection from a pool consisting of 30 nucleotides. The constructed RNA ligase ribozyme was further molecularly designed and engineered from cis to trans type ligase successfully. In addition, the ribozyme was transformed into a allosterically controllable ribozyme by installing receptor RNA unit(s) that alters their conformation due to the binding of a small molecule. The technique developed for constructing artificial RNA with defined 3D structure was further employed for design and construction of RNP (RNA-protein complex) by using RNA design and protein molecules with known 3D structures. An RNP that consists of a scaffold RNA and two protein molecules, CFP and YFP, attached to the RNA was constructed to test whether FRET can be observed between the two proteins. The FRET was observed as anticipated. It was also found that the FRET energy can be controlled by adjusting the distance between two proteins on the RNA by simply adjusting the size of the RNA. The molecular engineering that we have developed here should serve as a useful tool to develop a new field in Synthetic Biology.