## 4 Discussion  
In this study, we performed molecular modeling of kUrd to design a closed trimer, the formation of which was confirmed by EMSA and AFM analyses. On the other hand, significant formation of higher oligomers indicated that the current structural design was insufficient partly due to the 3D structure of the full-length _Tetrahymena_ ribozyme used for the molecular design in this study. In molecular modeling of kUrds (a dimeric form of an engineered _Tetrahymena_ ribozyme connected by the kink-turn motif), we used a model 3D structure of the full-length ribozyme and the X-ray crystallographic structure of a shortened form of the ribozyme lacking some peripheral elements [29]. There may have been some deviations from the full-length _Tetrahymena_ ribozyme 3D structure, which has yet to be solved by X-ray crystallography. Improvement of 3D structural design, however, may be possible because a new 3D model structure of the full-length ribozyme was constructed recently by combining cryo-EM data, chemical probing data and molecular modeling [54]. Although the resolution of this new model is modest (6.8 A), it will be useful to refine the design of ribozyme-based nanostructures. The formation of the closed dimer is also an issue to be resolved for precise design of kUrd-based RNA nanostructures. Formation of the closed kUrd dimer, which was unexpected from molecular modeling, suggested the structural flexibility of kUrd RNAs and entropic effects in RNA assembly. The flexible nature of the kink-turn RNA motifs has been analyzed and reported previously [55; 56; 57]. Therefore, we hypothesized that the formation of the closed dimer may also be supported by the structural flexibility of the KT-15 kink-turn motif. Distinct from our previous Urd with an extended linker, each kUrd can accept L7 Ae protein at its linker element bearing the KT-15 kink-turn motif. EMSA of the heterotrimer in the presence of L7 Ae suggested the formation of KT-15-L7 Ae complex at each corner of the triangle-shaped closed kUrd trimer. We also performed AFM analysis of the heterotrimer in the presence of L7 Ae but no significant differences were observed between images of kUrds with and without L7 Ae. This was conceivable because L7 Ae is much smaller than kUrd RNA. We also found that L7 Ae protein strongly inhibited the catalytic ability of kUrds. Collaboration between L7 Ae protein and the _Tetrahymena_ ribozyme unit in catalysis is still an open issue. In this study, we mainly analyzed the closed trimer and closed dimer of kUrds. The closed trimer contained six ribozyme units and three kink-turn motifs that interacted with L7 Ae proteins. In reactions promoted by the ribozyme unit in kUrd oligomers, _trans_-splicing reaction proceeded more efficiently in the presence of three kUrds forming a heterotrimer than two kUrds providing the core AP5 ribozyme and P5 abc activator. Although the extent of improvement was moderate, this was a promising result as an initial step toward RNA processing in vivo controlled by ribozyme-based nanostructures. The kUrd trimer containing six group I ribozyme units consists of nearly 2400 nucleotides, which is comparable to bacterial 16S and 23S ribosomal RNAs. The size of the triangle shape (with an edge of approximately 34 nm) was also comparable to the bacterial ribosome (approximately 20 nm in diameter). Although the structure and function of kUrd-based nanostructures are still primitive, this study represents an initial step toward artificial construction of complex RNA nanostructures capable of playing crucial roles in naturally occurring biological systems. _Trans_-splicing ribozymes have been investigated as a promising tool to repair mutated mRNAs causing genetic disorders [49; 50]. Ribozyme dimers employed in this study may be used to repair two distinct mutated mRNA simultaneously and cooperatively. Two ribozyme units in a dimer also target the same mRNA sequence, to which two _trans_-splicing ribozyme units perform distinct splicing reactions to yield different products.