• Korean Journal of Microbiology
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• v.47 no.3
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• pp.188-193
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• 2011

As a specific and effective therapeutic genetic material against hepatitis C virus (HCV) multiplication, HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was constructed. The allosteric ribozyme was composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nucleotide of HCV IRES. With real-time PCR analysis, the ribozyme was found to efficiently inhibit HCV replicon replication in cells. Of note, the allosteric ribozyme was shown to inhibit HCV replicon replication more efficiently than either HCV genome-targeting ribozyme or NS5B aptamer only. This allosteric ribozyme can be used as a lead genetic agent for the specific and effective suppression of HCV replication.

• BMB Reports
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• v.34 no.1
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• pp.51-58
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• 2001

Attempts using in vitro and in vivo selection procedures have been made to search for hammerhead ribozymes that have higher activities than the wild-type ribozyme and also to determine whether other sequences might be possible in the catalytic core of the hammerhead ribozyme. Active sequences selected in the past conformed broadly to the consensus core sequence except at A9, and no sequences were associated with higher activity than that of the hammerhead with the consensus core, an indication that the consensus sequence derived from viruses and virusoids is probably the optimal sequence [Vaish et al. (1997) Biochemistry 36, 6495-6501]. Recently, during construction of ribozyme expression vectors, we isolated a mutant hammerhead ribozyme, with an insertion of G between A9 and G10.1, that appeared to show significant activity [Kawasaki et al. (1996) Nucleic Acids Res. 24, 3010-3016; Kawasaki et al. (1998) Nature 393, 284-289]. We, therefore, characterized kinetic properties of the G-inserted mutant ribozymes in terms of the NUX rule. We demonstrate that the NUX rule is basically applicable to the G-inserted ribozymes and, more importantly, one type of G-inserted ribozyme was very active with $k_{cat}$, value of $6.4\;min^{-1}$ in 50 mM Tris-HCl (pH 8.0) and 10 mM $MgCl_2$ at $37^{\circ}C$.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.159-165
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• 2007

For the development of basic genetic materials for specific and effective therapeutic approach to suppress multiplication of hepatitis C virus (HCV), HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was developed. The ribozyme targeted most effectively to +382 nucleotide (nt) site of HCV IRES RNA. The allosteric ribozyme was designed to be composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nt of HCV IRES. Noticeably, we employed in vitro selection technology to identify the most appropriate communication module sequence which can induce ribozyme activity depending on the US5B protein. We demonstrated that the ribozyme was nonfunctional either in the absence of any proteins or in the presence of control bovine serum albumin. In sharp contrast, the allosteric ribozyme can induce activity of cleavage reaction with HCV IRES RNA in the presence of the HCV NS5B protein. This allosteric ribozyme can be used as lead compound for specific and effective anti-HCV agent, tool for highthroughput screening to isolate lead chemicals for HCV therapeutics, and ligand for biosensor system for HCV diagnosis.

• Applied Biological Chemistry
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• v.37 no.1
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• pp.56-63
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• 1994

Oligonucleotides for a conserved region of the coat protein gene of cucumber mosaic virus (CMV) and a hammerhead structure ribozyme against CMV RNA were synthesized using a DNA synthesizer. Both strands of oligonucleotides were annealed and restricted with BamHI/SacI, then cloned into a plasmid pBS SK (+). The cloned CMV substrate and ribozyme were sequenced to verify correct constructions. In vitro transcriptions were carried out by using T7 RNA polymerase with BssHII or SspI digests of $1\;{\mu}g$ of substrate and ribozyme clones. The size of substrate RNA was 176 nucleotides (nt) containing 50 nt of CMV RNA sequence, 6 nt of XbaI restriction site and 120 nt of vector-derived sequence in the case of BssHII digest. The size of ribozyme RNA was 164 nt containing 40 nt of ribozyme RNA sequence and same sequences of substrate. Substrate RNA was efficiently cleaved into two fragments (96 nt and 80 nt) by ribozyme RNA. This endonucleolytic cleavage occurred more efficiently at $55^{\circ}C$ than $37^{\circ}C$. SspI digest-derived substrate RNA (2234 nt) was also cleaved into two fragments by the same ribozyme. SspI digest-derived ribozyme RNA (2222 nt) cleaved the above substrate to two fragments. In vitro-tested ribozyme construct is being cloned into a plant transformation vector to develop virus-resistant plants.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.522-527
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• 1995

To develop an antiviral agent for the rice black-streaked dwarf virus (RBSDV), a hammerhead type ribozyme, which has a potential target site on the genome segment 3, was designed. Oligonucleotides for the ribozyme and its substrate were synthesized, annealed, and cloned into a plasmid pBluescript II KS(+). Ribozyme and substrate RNAs were then synthesized by in vitro transcription with $T_3$ RNA polymerase, obtaining RNAs in expected size, 193 and 182 nucleotides, respectively. The substrate RNA was efficiently cleaved into two fragments when incubated with the ribozyme at $55^{\circ}C$, while the cleavage was not detected at $37^{\circ}C$. In addition, the segment 3 RNA of RBSDV was also cleaved into two fragments by the same ribozyme at $55^{\circ}C$. Taken together, our results demonstrated that the hammerhead ribozyme has an in vitro endonucleolytic activity and may be used as an antiviral agent in transgenic plants.

• BMB Reports
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• v.31 no.2
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• pp.177-182
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• 1998

A pool of cis-acting hammerhead ribozymes randomized in their substrate recognition sequences was constructed. A variety of active cis-acting ribozymes which had various structures of stems I and III was selected from the pool by in vitro selection. The selected ribozymes were cloned and sequenced. The relationship between the cleavage efficiency and base-pairing in stems I and III of the selected ribozymes was investigated. The ribozymes with the smaller difference in folding energies between the active conformation and the stable but inactive conformation showed a tendency to have the better cleavage efficiency. The optimum length of stem I was 5 or 6 bases while the longer stem III, in general, appeared to be required for efficient cleavage. The specificity of the ribozyme reaction is discussed in terms of the length of stems I and III.

• Journal of fish pathology
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• v.37 no.1
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• pp.1-8
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• 2024

The advantages of replicon vectors of RNA viruses include a high ability to stimulate innate immunity and exponential amplification of target mRNA leading to high expression of foreign antigens. The present study aimed to construct a DNA-layered nervous necrosis virus (NNV) replicon vector system in which the capsid protein gene was replaced with a foreign antigen gene and to compare the efficiency of foreign antigen expression between the conventional DNA vaccine vector and the present replicon vector. We presented the first report of a nodavirus DNA replicon-based foreign antigen expression system. Instead of a two-vector system, we devised a one-vector system containing both an NNV RNA-dependent RNA polymerase cassette and a foreign antigen-expressing cassette. This single-vector approach circumvents the issue of low foreign protein expression associated with the low co-transfection efficiency of a two-vector system. Cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 (with the capsid gene ORF replaced with VHSV glycoprotein ORF) exhibited significantly higher transcription of the VHSV glycoprotein gene compared to cells transfected with either a vector without hammerhead ribozyme or a conventional DNA vaccine vector expressing the VHSV glycoprotein. Furthermore, the transcription level of the VHSV glycoprotein in cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 showed a significant increase over time. These results suggest that NNV genome-based DNA replicon vectors have the potential to induce stronger and longer expression of target antigens compared to conventional DNA vaccine vectors.

• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.114-114
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• 2005

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• BMB Reports
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• v.33 no.5
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• pp.359-365
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• 2000

Ribozymes are catalytic RNAs that can cleave RNAs at specific sites, thus they have been employed to degrade a target mRNA in vivo. Development of allosterically controllable ribozymes is of great current interest, but it remained difficult to furnish such functions to ribozymes in cultured cells or in animals. Recently, we designed allosterically controllable ribozymes termed maxizymes, which have sensor arms that recognize target mRNA sequences and, in the presence of such target sequences only, they form a cavity that can capture catalytically indispensable $Mg^{2+}$ ions, cleaving the target. The maxizyme was applied to therapy for chronic myelogenous leukemia (CML). It cleaved specifically the chimeric BCR-ABL mRNA, which caused CML, without damaging the normal ABL or BCR mRNA in mammalian cells and also in mice, providing the first successful example for allosteric control of the activity of artificial ribozymes in vivo.