Enzyme Encode Enzyme: DNAzymeâ†'RNAzymeâ†'Pepzyme

Ribozyme, discovered by Cech last 80's, are catalytic nucleic acids thatrecognize and catalyze a target RNA in a highly sequence-specific manner. Theconcept and species of enzyme were developed by the finding of ribozymes.There are two types of ribozymes catalyzing different reactions. Cleavingribozymes include hammerhead ribozyme, HDV ribozyme, hairpin ribozymeand RNase P. Splicing ribozymes include group I intron and group II intron.10-23 deoxyribozyme, the most efficient deoxyribozyme, was obtained throughin vitro selection in 1994. Beside 10-23 deoxyribozyme, there are 8-17deoxyribozyme, bipartite DNAzyme, class I and class II DNAzyme. Until now,no natural DNAzyme was discovered. The hammerhead ribozyme and 10-23deoxyribozyme are the best representation for their simple small structures,easily being designed, synthesized and transferred. Furthermore, they haveapplicable value in gene therapy.The RNA hammerhead ribozyme is unstable and easily degraded.Furthermore, hammerhead ribozyme lacks the diversity of functional groupscharacteristic of protein enzymes, which are important in the formation ofcomplicated spatial structures with moderate rigidity and flexibility. Toovercome these disadvantages, in previous works we used in vitro selection toobtain a circular RNA-DNA enzyme composed of the hammerhead ribozymecatalytic core as well as backbone DNA containing a regulating sequence.Compared to the linear hammerhead ribozyme, the stability of the circularRNA-DNA enzyme was greatly improved. We then substituted the hammerheadribozyme with the more efficient and more stable 10-23 deoxyribozyme andreplaced the backbone DNA with the single-strand replication-competent vectorM13mp18, successfully constructing a novel replicating circulardeoxyribozyme that displayed 10-23 deoxyribozyme activity both in vitro andin vivo.Here, we report that a 10-23 deoxyribozyme-hammerhead ribozymetargeted the β-lactamase mRNA in initiation and coding regions. The 10-23deoxyribozyme-hammerhead ribozyme gene was cloned into single-strandphagemid vector pBlue-script Ⅱ KS (+) (2.96kb). In vitro the recombinantsingle-strand phagemid vector showed 10-23 deoxyribozyme activity. Thecatalytic activity of CDR is less than the activity of linear 10-23 deoxyribozyme.Kinetics parameters measured in 10mmol/L MgCl2, at 30℃, with a RNAsubstrate, yielded a KM value of ~35.8nmol/L, kcat value of ~0.42min-1 andKobs value of 1.18×107 mol-1·L·min-1, respectively. The activity of CDRdepends on the present of divalent metal ions which was the same as otherdeoxyriboszymes. The lined recombinant phagemids were used as templates forin vitro transcription with MAXIscript T3 Kit. We measured the cleavageactivity of the 175 nt transcript (LDR) using the 32P-labeled RNA substrate. Thedenaturing 16% PAGE was used to isolate the substrates and the products. Theresult demostrates that LDR can cleave the RNA substrate. Kinetic parameterswere measured in 100mmol/L MgCl2, 50mmol/L (pH7.5) Tris-HCl, at 30℃.TheKM was 2801nmol/L, kcat was 0.12min-1 and Kobs was ~0.43×105 mol-1·L·min-1,respectively. The activity of CDR and LDR displayed metal ion dependence. Mg2+,Mn2+ greatly stimulated their activities, and Co2+, Ba2+ didn't cause any increasein activity. Since CDR and LDR required Mg2+ for optimal activities, we furthertested the dependence of relative cleavage rates for CDR and LDR on differentconcentrations of Mg2+. The experiments exhibited, in a certain range, theactivities of CDR and LDR increased with increasing concentrations of Mg2+.From the dependence of CDR and LDR on divalent metal ions in vitro, weconclude that CDR and LDR may employ similar mechanisms as the 10-23deoxyribozyme and the hammerhead ribozyme, respectively. Since the replicaand the transcript of the recombinant vector both had RNA-cleaving activities,we chemically synthesized a 25-residue peptide (pepzyme, PDR) encoded by theDR gene. The experiments demonstrated that PDR cleaved single-strand RNAbut did not cut single-strand DNA or double-strand DNA. Thus through rationaldesign, we successfully achieved 'enzyme encode enzyme': DNAzyme→RNAzyme→pepzyme. In vivo the β-lactamase expression and the growth of thedrug-resistance strains (TEM1 and TEM3) were both obviously inhibited by therecombinant single-strand phagemid vectors. The experiments demonstrate thatthe recombinant phagemid vector containing the 10-23deoxyribozyme-hammerhead ribozyme gene sequence are capable ofreplication, transcription and translation in E.coli cells and show RNA-cleavingactivities of the 10-23 deoxyribozyme, the hammerhead ribozyme and thepepzyme with different levels. CDR more efficiently inhibited the growth ofdrug-resistant strains than recombinant CD and CR.PDR are composed of two models, PD and PR. A 13-residue Arginine-richbasic peptide encoded by the hammerhead ribozyme (PR), showed single-strandRNA-cleaving activity. But PD had no activity. Here, the peptide encoded byribozyme was designated as ribopepzyme (RP). We investigated the peptidesencoded by the inactive mutants of the hammerhead ribozyme. There were 11inactive mutants corresponding to the mono-base transition (A?G, U?C) ofthe 11 conserved bases of hammerhead ribozyme. From degeneracy, 7 peptideswere encoded by these inactive mutants. The experiments revealed that thepeptides containing three Args or more had single-strand RNA-cleavingactivities. Thus, a ribopepzyme family, with Arginine-rich basic peptidesencoded by the hammerhead ribozyme and its inactive mutants was obtained.These results demonstrated that the informational and catalytic properties werenot always combined in the ribozymes. We investigated the peptides encodedby the hepatitis delta virus (HDV) ribozymes, the hairpin ribozyme and theRNase P ribozyme respectively. We found a 19-residue and a 25-residue peptideencoded by the genomic (+) and antigenomic (-) strands of HDV ribozymesboth had RNA-cleaving activities. For the RNase P ribozyme, a 22-residuepeptide encoded by an open reading frame (ORF, 125-193) of the ribozymedisplayed RNA-cleaving activity. While the same activity was not detected forthe 17-residue peptide (P) encoded by the hairpin ribozyme. Just like RPR,RPHDV(+), RPHDV(-) and RPP were all Arginine-rich basic peptides, but PHP wasnot. Ribopepzyme is a new type of pepzyme that has no homologous sequenceswith other catalytic peptides.Genetic information is highly conserved, and although the structure of theinformation carrier is different, the exhibited function is conserved. In this work,despite the different cleaving sites, the DNAzyme, the RNAzyme and thepepzyme (ribopepzyme) all cleaved single-strand RNA. Basing on theribozymes encode the ribopepzymes, we suggest 'enzyme encode enzyme',which means the conversion between the DNAzyme, the RNAzyme and thepepzyme (protein enzyme). The ribozymes encode ribopepzymes was only a'phenotype' of the 'enzyme encode enzyme'. Both active and inactiveribozymes encoding ribopepzymes may show an evolutionary transition from'enzyme encode enzyme' to 'gene encode enzyme' at the beginning ofevolution. The finding that ribozymes encode ribopepzymes supports the RNAworld hypothesis. On the other hand, 'enzyme encode enzyme' enrich the RNAworld hypothesis. The 'enzyme encode enzyme' develops the function and thespecies of enzyme. The 'enzyme encode enzyme' closely connected the DNA,RNA, peptide (protein) in the early evolution.