• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1408-1413
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• 2005

Elevated expression of carcinoembryonic antigen (CEA) has been implicated in various biological aspects of neoplasia such as tumor cell adhesion, metastasis, blocking of cellular immune mechanisms, and antiapoptosis function. Thus, the CEA could be an important target for anticancer therapy. In this study, we developed Tetrahymena group 1 intron-based trans-splicing ribozymes that can specifically target and replace CEA RNA. To this end, we first determined which regions of the CEA RNA were accessible to ribozymes by employing an RNA mapping strategy that was based on a trans-splicing ribozyme library. Next, we assessed the ribozyme activities by comparing the trans-splicing activities of several ribozymes that targeted different regions of the CEA RNA, and then the ribozyme that could target the most accessible site was observed to be the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme was found to react with and altered the target CEA transcripts in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace CEA RNAs in tumors with a new RNA-harboring anticancer activity, thereby hopefully reverting the malignant phenotype.

• BMB Reports
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• 제36권6호
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• pp.538-544
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• 2003

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.

• 미생물학회지
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• 제35권3호
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• pp.211-217
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• 1999

표적 RNA 의 구조가 Tetrahymena thermophila 의 group I intron 에 의한 trans-splicing 반응에 미치는 영향을 분석하기 위해 강력한 stem-loop 형태의 안정된 구조를 갖고 있는 표적 RNA mapping 분석 방법을 이용한 결과 in vitro 뿐만 아니라 in vivo 에서도 stem 부위의 염기들에 반해 loop 부위의 염기들이 ribozyme 에 의해 잘 인지되었으며 이러한 결과는 그러한 부위들을 인지할 수 있는 ribozyme 들에 의한 trans-cleavage 그리고 trans-splicing 반응을 수행함으로써 검증하였다. 또한 이러한 trans-splicing 반응은 정확하게 일어남을 반응 산물의 염기서열 결정을 통해 확인하였다. 따라서 표적 RNA 의 구조가 in vitro 및 in vivo 에서의 ribozyme 활성에 매우 중요한 요인임을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.1070-1076
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• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.

• Journal of Microbiology
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• 제41권4호
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• pp.340-344
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• 2003

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

• 대한화학회지
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• 제61권3호
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• pp.122-124
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• 2017

• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.1038-1040
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• 2010

• 대한화학회지
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• 제59권4호
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• pp.341-343
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• 2015

• Bulletin of the Korean Chemical Society
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• 제29권10호
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• pp.1937-1940
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• 2008

The P1 duplex of Tetrahymena group I ribozyme is the important system for studying the conformational changes in folding of ribozyme. The formation of the P1 duplex between IGS and substrate RNA and the catalytic activity of ribozyme require a variety of metal ions such as $Mg^{2+}$ and $Mn^{2+}$. In order to investigate the effect of the $Mg^{2+}$ concentration on the conformation of the P1 duplex, the NMR study was performed as a function of $Mg^{2+}$ concentration. This study revealed that the less stable AU-rich region formed duplex at $50{^{\circ}C}$ under high $Mg^{2+}$ concentration condition but melts out under low $Mg^{2+}$ concentration condition. It was also found that in the active conformation under 10 mM $MgCl_2$ condition, the unstable central G${\cdot}$U wobble pair maintains the significant base pairing up to $50{^{\circ}C}$. This study provides the information of the unique feature of the P1 duplex structure and the roll of $Mg^{2+}$ ion on the formation of the active conformation.

• Genomics & Informatics
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• 제5권1호
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• pp.32-35
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• 2007

Telomerase reverse transcriptase (TERT) is an enzymatic ribonucleoprotein that prolongs the replicative life span of cells by maintaining protective structures at the ends of eukaryotic chromosomes. Telomerase activity is highly up-regulated in 85-90% of human cancers, and is predominately regulated by hTERT expression. In contrast, most normal somatic tissues in humans express low or undetectable levels of telomerase activity. This expression profile identifies TERT as a potential anticancer target. By using an RNA mapping strategy based on a trans-splicing ribozyme library, we identified the regions of mouse TERT (mTERT) RNA that were accessible to ribozymes. We found that particularly accessible sites were present downstream of the AUG start codon. This mTERTspecific ribozyme will be useful for validation of the RNA replacement as cancer gene therapy approach in mouse model with syngeneic tumors.