Rational Design of an Orthogonal Pair of Bimolecular RNase P Ribozymes through Heterologous Assembly of Their Modular Domains

Abstract

The modular structural domains of multidomain RNA enzymes can often be dissected into separate domain RNAs and their noncovalent assembly can often reconstitute active enzymes. These properties are important to understand their basic characteristics and are useful for their application to RNA-based nanostructures. Bimolecular forms of bacterial RNase P ribozymes consisting of S-domain and C-domain RNAs are attractive as platforms for catalytic RNA nanostructures, but their S-domain/C-domain assembly was not optimized for this purpose. Through analysis and engineering of bimolecular forms of the two bacterial RNase P ribozymes, we constructed a chimeric ribozyme with improved catalytic ability and S-domain/C-domain assembly and developed a pair of bimolecular RNase P ribozymes the assembly of which was considerably orthogonal to each other.

Catalytic RNA Oligomers Formed by Co-Oligomerization of a Pair of Bimolecular RNase P Ribozymes

“…Activity assays confirmed this prediction because the four complexes showed no detectable pre-tRNA cleavage activity ( Figure S4C ). This was consistent with the previous study performed with 50 mM Mg 2+ , under which conditions the mixture of Sβ RNA and αC RNA exhibited no catalytic activity and another mixture (SαRNA and Cβ RNA) had only residual activity [ 32 ].…”

“…Plasmids encoding sequences of S-domains and C-domains of bimolecular ribozymes (Rz-α, Rz-β, and Ch:hS) were described previously [ 32 ]. Introduction of KL elements into the above plasmids and extension of the pre-tRNA anticodon stem were achieved by site-directed mutagenesis using inverse-PCR.…”

“…To use the bimolecular form of bacterial RNase P ribozymes as a structural platform to develop ribozyme-based RNA nanostructures, we have developed a chimeric bimolecular ribozyme consisting of the S-domain RNA from the Thermus thermophilus (Tth) ribozyme and the C-domain RNA from the Escherichia coli (Ec) ribozyme [ 32 ]. The resulting chimeric ribozyme exhibited greater stability of the S-domain/C-domain complex than either the bimolecular Tth ribozyme or Ec ribozyme.…”

“…The resulting chimeric ribozyme exhibited greater stability of the S-domain/C-domain complex than either the bimolecular Tth ribozyme or Ec ribozyme. Based on the chimeric ribozyme, we further prepared a pair of bimolecular ribozymes (Rz-α and Rz-β) in which the RNA–RNA interfaces between the S-domain/C-domain were engineered to achieve orthogonal molecular recognition of the two interfaces ( Figure 1 B) [ 32 ]. Open-form co-oligomers of RNase P ribozyme, in which active Rz-α and Rz-β ribozymes were formed alternately, could be designed by developing unit RNAs ( Figure 1 C) [ 30 ].…”

Partial RNA Design

“…RNA design describes the problem of generating RNA sequences with certain properties to fulfill desired functions [Hammer et al, 2019]. The design endeavors often start from carefully selected RNA fragments with known functions to combine these so-called motifs and achieve new functionality [Wachsmuth et al, 2012, Li et al, 2018, Nozawa et al, 2019]. The designed construct is then evaluated in a wet laboratory experiment proofing the desired functionality.…”