• Title/Summary/Keyword: RNA aptamer

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Development of Hepatitis C Virus (HCV) Genome-Targeting Hammerhead Ribozyme Which Activity Can Be Allosterically Regulated by HCV NS5B RNA Replicase (C형 간염바이러스(HCV)의 NS5B RNA Replicase에 의해 그 활성이 조절되는 HCV지놈 표적 Hammerhead 리보자임 개발)

  • Lee, Chang-Ho;Lee, Seong-Wook
    • 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.

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Analysis of In Vivo Interaction of HCV NS3 Protein and Specific RNA Aptamer with Yeast Three-Hybrid System

  • HWANG BYOUNGHOON;LEE SEONG-WOOK
    • Journal of Microbiology and Biotechnology
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    • v.15 no.3
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    • pp.660-664
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    • 2005

  • We have previously isolated specific RNA aptamers with high affinity against the helicase domain of hepatitis C virus (HCV) nonstructural protein 3 (NS3). The RNA aptamers competitively and efficiently inhibited the helicase activity, partially impeding HCV replicon replication in human hepatocarcinoma cells. In this study, the RNA aptamers were tested for binding to the HCV NS3 proteins in eukaryotic cells, using a yeast three-hybrid system. The aptamers were then recognized by the HCV NS3 proteins when expressed in the cells, while the antisense sequences of the aptamers were not. These results suggest that the in vitro selected RNA aptamers can also specifically bind to the target proteins in vivo. Consequently, they could be potentially utilized as anti-HCV lead compounds.

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Development of Trans-Splicing Aptazyme Which Can Specifically Modify Hepatitis C Virus Genome (C형 간염바이러스(HCV) 유전체를 특이적으로 변형할 수 있는 Trans-Splicing Aptazyme 발굴)

  • Kim, Ju-Hyun;Lee, Chang-Ho;Jang, Sun-Young;Lee, Seong-Wook
    • Korean Journal of Microbiology
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    • v.44 no.3
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    • pp.186-192
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    • 2008

  • For the development of specific and effective basic genetic materials to inhibit replication of hepatitis C virus (HCV), HCV genome-targeting trans-splicing aptazyme, which activity is allosterically regulated by a specific ligand, was developed. The aptazyme was designed to be comprised of sequence of RNA aptamer to the ligand, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding the ligand to the aptamer, and trans-splicing ribozyme targeting +199 nt of HCV IRES. Especially, when the aptamer and the communication module was inserted at both P6 and P8 catalytic domain of the specific ribozyme, allosteric activity of the aptazyme was the most induced. The aptazyme was shown to induce activity of trans-splicing reaction specifically and efficiently only in the presence of the specific ligand, but neither in the absence of any ligand nor in the presence of control ligand. This aptazyme can be used as a specific and effective genetic agent against HCV, and a tool for the isolation of anti-HCV lead compounds.

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Examination of specific binding activity of aptamer RNAs to the HIV-NC by using a cell-based in vivo assay for protein-RNA interaction

  • Jeong, Yu-Young;Kim, Seon-Hee;Jang, Soo-In;You, Ji-Chang
    • BMB Reports
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    • v.41 no.7
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    • pp.511-515
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    • 2008

  • The nucleocapsid (NC) protein of the Human Immunodeficiency Virus-1 plays a key role in viral genomic packaging by specifically recognizing the Psi($\Psi$) RNA sequence within the HIV-1 genome RNA. Recently, a novel cell-based assay was developed to probe the specific interactions in vivo between the NC and $\Psi$-RNA using E.coli cells (J. Virol. 81: 6151-55, 2007). In order to examine the extendibility of this cell-based assay to RNAs other than $\Psi$-RNA, this study tested the RNA aptamers isolated in vitro using the SELEX method, but whose specific binding ability to NC in a living cellular environment has not been established. The results demonstrate for the first time that each of those aptamer RNAs can bind specifically to NC in a NC zinc finger motif dependent manner within the cell. This confirms that the cell-based assay developed for NC-$\Psi$interaction can be further extended and applied to NC-binding RNAs other than $\Psi$-RNA.

  • https://doi.org/10.5483/BMBRep.2008.41.7.511 인용 PDF