An RNA Triangle with Six Ribozyme Units Can Promote a Trans-Splicing Reaction through Trimerization of Unit Ribozyme Dimers

Abstract

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product.

A Hexameric Ribozyme Nanostructure Formed by Double‐Decker Assembly of a Pair of Triangular Ribozyme Trimers

“…Selective formation of the double‐decker hexamer will allow us to exploit its potential applications by engineering the ribozyme units capable of trans ‐splicing reaction, which has been developed using the parent Tetrahymena ribozyme and its oligomeric derivatives. [40,41] . The double‐decker hexamer may be expected to have higher resistance against degradation by ribonuclease than its monomer components [42] .…”

“…[39] Such new information is useful and should be taken into consideration in optimization of T RNAbased nanostructures and also in the design of novel RNA nanostructures based on the Tetrahymena ribozyme. Selective formation of the double-decker hexamer will allow us to exploit its potential applications by engineering the ribozyme units capable of trans-splicing reaction, which has been developed using the parent Tetrahymena ribozyme and its oligomeric derivatives.. [40,41] The double-decker hexamer may be expected to have higher resistance against degradation by ribonuclease than its monomer components. [42] It should be also noted that closed forms of homodimer and homotrimers of the Tetrahymena ribozyme have constructed recently as a strategy for cryo-EM analyses of structured RNAs.…”

Nucleic Acids Chemistry and Engineering: Special Issue on Nucleic Acid Conjugates for Biotechnological Applications

“…They constructed an RNA triangle with six catalytic ribozyme units, in which the triangle complex was further supported by protein assembly on the corners. It is an attractive RNA-based nanoarchitecture and would be a unit for a system that exhibits more complex biological functions as the RNA triangle has succeeded to catalyze the trans-splicing of RNA with an arbitrary sequence [9]. S. Nishizawa et al have synthesized a chemical probe for fluorometric detection of an orphan cytosine, which has no base paring partner due to an abasic nucleotide on the opposite strand.…”