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

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.