Group Ⅱ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg²⁺ concentration. Finally, we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group Ⅱ introns in biotechnology.
DNA ; Eukaryota ; Gene Targeting ; Introns/genetics* ; RNA, Catalytic/genetics*

Hepatitis C ; genetics ; Humans ; RNA, Catalytic ; genetics ; RNA, Small Nuclear ; genetics

Overexpression of procollagen gene can cause the extraordinary increase of collagen's synthesis and therefore lead to the keloid and hypertrophic scar. To utilize ribozyme to suppress the expression of procollagen genes, a eukaryotic expression recombinant plasmid containing a dual-ribozyme gene against alpha 1 (I) and alpha 1 (III) procollagen genes was constructed. The ribozyme from in vitro transcription was incubated with target transcripts from recombinant plasmids which separately contained the fragments of the second exons of pro alpha 1 (I) and pro alpha 1 (III) collagen genes under various experimental conditions. The results showed that the dual-ribozyme could efficiently catalyze the specific cleavage of the target RNAs at 37 degrees C, 42 degrees C, 50 degrees C and Mg2+ concentration from 10 mmol/L to 20 mmol/L. This work provided a basis for further study on the ribozyme to suppress the expression of procollagen genes and control the cicatrization.
Base Sequence ; Exons ; Molecular Sequence Data ; Procollagen ; genetics ; RNA ; metabolism ; RNA, Catalytic ; genetics ; metabolism ; Temperature

With the rapid development of genetic engineering and metabolic regulation, antisense technology displays its fascination to the world as a mild regulation genetic tool. Compared with other loss-of-function research methods (e.g. gene knockout), antisense technologies have advantages such as low cost, short period, and easy operation. It has been increasingly used in bacterial metabolic regulation as a powerful genetic tool. This review briefly summarized the latest progress and problems in antisense technologies that are recently used in metabolic engineering of bacteria, and compares the advantages and disadvantages of these technologies.
Bacteria ; genetics ; metabolism ; Genes, Bacterial ; Genetic Engineering ; Metabolic Networks and Pathways ; genetics ; Oligonucleotides, Antisense ; genetics ; RNA, Antisense ; genetics ; RNA, Catalytic ; genetics

A self-cleaving hammerhead ribozyme gene containing a 14nt target sequence of ASSVd at the 3' end of hammerhead ribozyme was synthesized, amplified and cloned at the Xho I-Hind III site of pGEM7Zf(+). The ends produced by Xho I or Sal I can link together, thus the recognition sites of both enzymes vanish and can't be cut by either one. We used this property to get the recombinant plasmid bearing 2, 4, 6, 8, 10 and 12 copies of self-cleavable ribozyme respectively after successively sub-cloning five times. Linearized recombinat plasmid model catalyzed by T7 RNA polymerase was transcribed in vitro. The multimeric ribozyme molecules efficiently self-cleaved via cis-acting to release many ribozyme molecules It indicates that the concentration of ribozyme transcripts has been enhanced during transcription. Trans-cleavage reaction was carried out by incubating monomeric and multimeric ribozymes with same mol concentration and 32P labeled target ASSVd. Both ribozymes and target transcripts were mixed in 1:1 ratio. Autoradiograms showed the transcripts of multimeric ribozyme were substantially more effective against the ASSVd target RNA than the monomeric ribozymes. We confer that the multimeric self-clevable ribozyme is likely to provide more valuable application in vivo.
Malus ; virology ; RNA, Catalytic ; chemistry ; genetics ; metabolism ; RNA, Viral ; metabolism ; Viroids ; metabolism

OBJECTIVETo explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.

METHODSAccording to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.

RESULTSBoth the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.

CONCLUSIONSThe cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.

DNA, Antisense ; genetics ; Genome, Viral ; Hepatitis B virus ; genetics ; Hepatitis Delta Virus ; enzymology ; genetics ; Humans ; RNA, Catalytic ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Viral ; genetics ; Transcription, Genetic

OBJECTIVETo study the cleavage activity of specific deoxyribozyme to hepatitis C virus in vitro.

METHODSThree deoxyribozymes were designed to cleave at sites 157, 168, 173 in HCV 5'-noncoding region with the active region of 5'-GGCTAGCTACAACGA-3' respectively. Plasmid pCMV/T7-NCRC -Delta Luc was completely linearized with restriction endonuclease Xba I. HCV RNA5'-NCRC was transcribed in vitro from the linearized products and radiolabelled with [alpha-32P] UTP. Under the conditions of 37 degrees C, pH7.5, Mg2+ 10 mmol/L, the three deoxyribozymes were mixed with substrate RNA individually for 120 minutes and then the reactions were terminated. The cleavaged products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. DRz3 was mixed with the substrate RNA at different Mg2+ concentrations. The cleavage efficiency was analyzed with a gel document action analyzing systems.

RESULTSUnder the adopted conditions the three deoxyribozymes efficiently cleaved to the target RNA in vitro and the cleavage activity of DRz3 was increased with the increase of Mg2+ concentration.

CONCLUSIONThe designed deoxyribozymes can cleave 5'-NCR mRNA of HCV efficiently in vitro and it is dose-respondent to Mg2+ concentration.

DNA, Catalytic ; genetics ; DNA, Single-Stranded ; genetics ; Genetic Therapy ; Hepacivirus ; genetics ; Hepatitis C ; therapy ; Humans ; RNA, Messenger ; genetics

OBJECTIVETo study the cleavage activity on the HCV RNA of a chimeric recombinant of HCV specific ribozyme and U1 small nuclear RNA, which compartmentalizes within the nucleolus.

METHODSThe third stem-loop sequence of human U1 snRNA (position 95-116) within pBSIISK+ U1 was substituted by hammerhead ribozyme against HCV RNA by PCR and cloning methods, and the constructed plasmid was named pBSIISK+ (U1-Rz). Then the whole gene fragment of the chimeric ribozyme was cloned into a pGEM-T vector under the control of T7 promoter, and the constructed plasmid was named pGEM- (U1-Rz). The pGEM- (U1-Rz) and pGEM-Rz (containing the same ribozyme sequence as that in U1-Rz) transcripts as enzyme were transcribed in vitro. Also the (32)P-labeled pCMV/T7-NCRC luc (containing the gene sequence of the whole 5'-NCR and part core of HCV RNA) transcripts as target-RNAs were transcribed in vitro. The enzymes were incubated with the target RNAs under different conditions and autoradiographed after denaturing gel-electrophoresis.

RESULTSThe sequencing result showed that the construction of U1 snRNA chimeric ribozyme was correct. Compared with the ribozyme alone, both of them were active at 37 degree C and with Mg2+ (10 mmol/L) and TrisCl (10 mmol/L, pH7.9), and there was no remarkable difference between them. The cleavage activity of the chimeric ribozyme increased with the prolongation of reaction time and increment of enzyme concentration.

CONCLUSIONBoth ribozyme and U1 snRNA chimeric ribozyme exhibited specifically catalytic activity against HCV RNA in vitro. There was no remarkable difference between their cleavage efficiencies.

Chimera ; genetics ; Genetic Therapy ; Hepacivirus ; genetics ; Hepatitis C ; therapy ; RNA, Catalytic ; genetics ; metabolism ; RNA, Small Nuclear ; genetics ; pharmacology ; RNA, Viral ; genetics ; Recombinant Fusion Proteins ; pharmacology

Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this M1IGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.
Antiviral Agents ; pharmacology ; Escherichia coli ; genetics ; Genetic Engineering ; Hepacivirus ; genetics ; physiology ; RNA, Catalytic ; genetics ; pharmacology ; RNA, Guide ; genetics ; Ribonuclease P ; genetics ; Viral Core Proteins ; genetics

OBJECTIVETo analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.

METHODSTwo specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.

RESULTSAfter reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.

CONCLUSIONSCleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.

5' Untranslated Regions ; metabolism ; Base Sequence ; DNA, Catalytic ; genetics ; metabolism ; Hepacivirus ; enzymology ; genetics ; Humans ; Molecular Sequence Data ; RNA Processing, Post-Transcriptional ; RNA, Catalytic ; metabolism ; RNA, Viral ; metabolism ; mRNA Cleavage and Polyadenylation Factors ; genetics ; metabolism