Ribozyme-based "Ribopepzyme"

The discovery of RNA enzyme (or ribozymes) in the 1980s reignited interest in the chemical basis of the origin of life and resulted in formulation of RNA World hypothesis. If RNA, in principle, can be both a genome and a primordial biocatalyst, it seems possible that naturally occurring ribozymes transitioned to protein enzymes via transferring both genetic information and catalytic function. Previously, we employed a DNAzyme-RNAzyme combination strategy to construct a 10-23 deoxyribozyme-hammerhead ribozyme combination thatrecognizes and cleaves different sites of theβ-lactamase mRNA. The combinantgene was cloned into the phagemid vector pBlue-script II KS (+). The single-stranded recombination phagemid vector exhibited 10-23 deoxyribozyme activity in vitro, and the linear transcript also displayed hammerhead ribozyme activity. Subsequently, we chemically synthesized a 25-residue peptide encoded by the combinant gene and found that the peptide possesses single-strandedRNA cleavage activity. To further explore the catalytic mechanism of thispeptide, we analyzed the RNA cleavage activities of chemically synthesized 12-and 13-residue peptides that were encoded by the 10-23 deoxyribozyme and the hammerhead ribozyme, respectively. Results showed that the 13-residue peptide exhibits single-stranded RNA cleavage activity, while the 12-residue peptide does not. Because the 13-residue peptide arises from a ribozyme-based code and exhibits catalytic activity similar to that of ribonucleases, we have designated the catalytic peptide"ribopepzyme R"(RPZR).Given that there are no naturally occurring DNAzymes, and that no RNA cleavage activity was detected from the peptide encoded by the 10-23 deoxyribozyme, we focused our analysis on the activities of peptides encoded from naturally occurring ribozymes. The peptides encoded by the hammerhead ribozyme had single-stranded RNA cleavage activity. We wanted to know if this holds true for peptides encoded by other naturally occurring ribozymes. The hammerhead ribozyme, the hairpin ribozyme, and the hepatitis delta virus (HDV) ribozyme all show an extreme degree of convergence in sequence, structure, and reaction mechanism. We thus analyzed the peptides encoded by the genomic hepatitis delta virus (HDV(+)) ribozyme, the antigenomic HDV (HDV(-)) ribozyme, and the hairpin ribozyme. Applying the same approach described above, we produced a19-residue peptide and a 25-residue peptide from the HDV (+) and HDV (-) ribozymes (RPZHDV(+) and RPZHDV(-)), respectively. Both demonstrated single-stranded RNA cleavage activities. The smallest trans-acting sequence of genomic HDV ribozyme contains a conserved nucleotide sequenceand demonstrated the same cleavage activity as in wild-type HDV ribozyme. A 15-residue peptide encoded by this small sequence also exhibitedsingle-stranded RNA cleavage activity (RPZSHDV). However, perhaps owing to a loss of functional information, a 17-residue peptide encoded by the hairpin ribozyme exhibited no single-stranded RNA cleavage activity (PHP).The hammerhead, hairpin, and HDV ribozymes all have relatively simplestructures and their catalytic mechanisms are clear. We examined anothernaturally occurring ribozymes, RNase P, a ribonucleoprotein that catalyze the maturation of the 5'end of tRNAs. The ribosome and RNase P are the only two known universal ribozymes, as they have been found in all organisms in all three domains of life. The RNA component of RNase P from all organisms shows marked similarities at the primary and secondary structure level, strongly suggesting evolutionary conservation. E. coli RNase P consists of a small protein and a catalytic RNA containing an ORF (125–193). The 22-residue peptide encoded by the ORF demonstrated single-stranded RNA cleavage activity (RPZP).The Neurospora Varkud satellite ribozyme (VS ribozyme) is the largest of the naturally occurring nucleolytic ribozymes (or small ribozymes, including the hammerhead, hairpin, hepatitis delta virus, and VS ribozyme), and the last member of this class of ribozyme for which we explored. It occurs in the 881 nt VS RNA found in the mitochondria of Neurospora, which is transcribed from the Varkud satellite DNA. Like the other members of the class, it undergoes a site-specific self-cleavage reaction by means of a transesterification reaction arising from the attack of a 2'-hydroxyl to generate 2', 3'-cyclic phosphate and 5'-hydroxyl termini. The VS ribozyme can also catalyse the reverse ligation reaction. The complementary sequence of the VS ribozyme is an intact reading frame, there is no terminator in it. So we cloned DNA sequence of the VS ribozyme into the E. coli expression vector pET21a(+) reverse, expressed and purified the encoded 56-residue peptide, and analyzed it's activity. The peptide (RPZVSC) performs the same single-stranded RNA cleavage reaction more effectively than other aforementioned ribopepzymes, but no ligation activity was tested.Using this approach, we obtained a family of novel ribopepzymes: RPZR, RPZ_HDV(+), RPZ_{HDV (-)}, RPZ_SHDV, RPZ_P, and RPZ_VSC.Compared with other synthesized catalytic peptides, these ribozyme-encoded ribopepzymes are extraordinary. They are almost all arginine- and proline-rich peteides. Pro, Arg, Trp, and Gly were common to all the ribopepzymes. Of these, Pro, Trp, and Gly were favorable to the stretching of the peptide chain. In contrast to ribozymes, ribopepzymes do not require divalent metal ions for catalysis, as Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺, Ba²⁺, Cu²⁺ and Co²⁺ inhibited the catalytic activity of ribopepzymes to different degrees. According to the searching results from BLAST, members of the family of ribopepzymes we generated do not have homology with other known peptides or proteins. Structural prediction results from PEPstr and robetta indicate that ribopepzymes primarily consist of a random coil. Circular dichroism (CD) analysis demonstrated that ribopepzymes exist mainly in an unfolded state in aqueous solution.Because the ribopepzymes we generated all appear to have similar structuresand functions, we used RPZR as a representative to conduct a more detailedcharacterization of their catalytic mechanisms. To test whether only the active ribozymes could encode ribopepezymes, we investigated the hammerhead ribozyme mutant-based peptides. Given the 11 conserved bases of the hammerhead ribozyme, there were 11 possible mutants corresponding to the mono-base transition (A(?)G, U(?)C). Owing to degeneracy of the genetic code, these 11 changes resulted in 7 hammerhead ribozyme mutant-based peptides(RPZ_RM1–RPZR_M7). Results different from our imagine, some mutant-based peptides lost activities but others still remained. However, activity analysisresults confirmed that peptides exhibited no catalytic RNA cleavage activitywhen they contained fewer than three arginine residues.Since ribopepzymes are arginine-rich, and an arginine residue is present in many active sites of enzymes, we examined the possibility that arginine is required for catalytic activity. Likewise, cleavage activity was present only when peptides contained 3 or more arginine residues. To confirm the relationship between RNA cleavage activity and the number of arginine residues, we produced a mutant of RPZ_R in which three arginine residues were replaced by alanine residues (RPZ_RM8). As expected, this mutant exhibited no single-stranded RNA cleavage activity. Further experiments showed that any mutation of arginine residues resulted in a loss of ribopepzyme activity(RPZ_RM9–RPZ_RM11). However, substituting other, non-arginine residues with alanine still left the ribopepzyme's RNA cleavage activity, although activity was weakened, likely from disrupting the original ribopepzyme scaffold (RPZ_RM12). The above results indicate that three arginine residues are involved in RPZRcatalysis.In order to further confirm the catalytic mechanism, we next analyzed the cleavage sites of the RPZR. RPZR cleaved substrate RNA predominantly at5'-U↓G-3', 5'-C↓G-3', and 5'-G↓C-3. Similarly, RNase A mainly hydrolyzes RNA at C and U residues15 and RNase T1 mainly hydrolyzes RNA at G residues16. Interestingly, we also found that rRNasin (Promega) inhibited the activities of the RPZR (Fig. 4a). Taken together, these results indicate that RPZRemploys a ribonuclease-like catalytic mechanism in which three arginine residues could be involved in hydrolyzing phosphodiester bonds. However, the active sites of RNase A and RPZR are chemically different: RNase A consists of histidine and lysine residues, while RPZ_R has arginine, and no histidine residues. We replaced the arginine residues of RPZ_R with the active site amino acids from RNase A. The mutant (RPZ_RA) displayed single-stranded RNAcleavage activity. The pH profile curve for RPZ_R activity was bell-shaped, with an optimal cleavage rate at pH 7.0. Thus, RPZ_R activity could be characteristic of concerted general acid-base catalysis.Both the low activity and low substrate specificity of ribopepzymes are probably due to the peptide's flexibility. Because of their relative simplicity and small size, it is difficult to form efficiently catalytic and binding domains.Induced fit did not occur easily between the ribopepzyme and the substrate.During early life on earth, RNAs were capable of both encoding genetic information and performing catalytic functions. At some point, peptides became the repository for both. One possible explanation is that both genetic information and catalytic function were directly transferred to a primordial enzyme. We propose that the relationship between ribozymes and ribopepzymes might represent such a transfer. Thus, ribopepzymes may represent the transition state in a possible evolutionary process (ribozymes→ribopepzymes→protein enzymes), and as such could be a possible ancestor of the modern ribonucleases. Another possibility is that RNA-based catalytic function was lost or diverged along with the loss or divergence of genetic information as part of the process of evolution. Owing to the loss or divergence of genetic information, peptidesbased on the hairpin ribozyme possessed no single-stranded RNA cleavageactivity. Divergent evolution could cause ribopepzymes based on one ancestor to evolve diversified catalytic functions.Based on the theories of molecular evolution and our experimental observations, we proposed an approach for the design of RNA-based biocatalysts. We successfully obtained a family of novel ribopepzymes. These results suggest that: (1) at the chemical level, both genetic information and catalytic function could be transferred from some ribozymes to protein enzymes; (2) the ribopepzyme could be a link between RNA- and protein-based biocatalysts in the early origin and evolution of enzymes; (3) the emergence of the ribopepzymes could drive biocatalyst evolution, and provide a basis for designed, divergent evolution of enzymatic function. The developed process might even be extended to other ribozymes and create a larger variety of catalytic lineages.