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Conference Paper: In vivo selection and characterization of DNA aptamers against H5N1 virus nucleoprotein
Title | In vivo selection and characterization of DNA aptamers against H5N1 virus nucleoprotein |
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Authors | |
Issue Date | 2010 |
Publisher | Blackwell Publishing. |
Citation | The 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 2010), Vienna, Austria, 10-13 April 2010. How to Cite? |
Abstract | OBJECTIVE: To develop DNA aptamers against H5N1 virus nucleoprotein and study the ability to inhibit H5N1 viruses production. METHODS: Nucleoprotein of influenza A virus (A/vietnam1194/2004 (H5N1)) was cloned and expressed in E. coli. The single strand DNA library is composed of 30 random nucleotides flanked by constant sequences for PCR amplification. Magnetic beads-based approach was used to screen DNA aptamers binding purified His-tagged nucleoprotein. After 20 rounds of selection, the enriched pool was cloned and 40 colonies were sequenced. The structure of the G-quadruplex aptamers was characterized by circular dichroism spectroscopy. MDCK cells were transfected with representative aptamers by Lipofectamin 2000. Four hours after transfection, cells were challenged with 100TCID50 influenza A virus (A/Vietnam/1194/2004(H5N1)). The viral titer was determined by HA assay at 48 hours post-infection. RESULTS: The sequences from in vitro selection revealed fairly conserved and could be classified into two groups, G-quadruplex and T-rich DNA aptamers. G-quadruplex aptamers were found dominant in the population and comfirmed by circular dichroism spectroscopy. Circular dichroism spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. In the early stage of post-transfection, DNA aptamers were mainly localized in the nuclei where the replication and transcription of influenza virus genome take place exclusively, which may facilitate successful antiviral efficacy. The T-rich aptamer had only mild effect on the inhibition of H5N1 virus, whereas G-quadruplex forming aptamers exhibited potent antiviral effect. CONCLUSIONS: This study has demonstrated that nucleoprotein could be a potential antiviral target for influenza H5N1 virus. Further investigation will aim to elucidate the interaction between the G-quadruplex aptamer and nucleoprotein, as well as the antiviral mechanism. |
Description | Abstract no. P1092 |
Persistent Identifier | http://hdl.handle.net/10722/126472 |
DC Field | Value | Language |
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dc.contributor.author | Lin, Y | en_HK |
dc.contributor.author | Tanner, JA | en_HK |
dc.contributor.author | Zheng, B | en_HK |
dc.date.accessioned | 2010-10-31T12:30:36Z | - |
dc.date.available | 2010-10-31T12:30:36Z | - |
dc.date.issued | 2010 | en_HK |
dc.identifier.citation | The 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 2010), Vienna, Austria, 10-13 April 2010. | en_HK |
dc.identifier.uri | http://hdl.handle.net/10722/126472 | - |
dc.description | Abstract no. P1092 | - |
dc.description.abstract | OBJECTIVE: To develop DNA aptamers against H5N1 virus nucleoprotein and study the ability to inhibit H5N1 viruses production. METHODS: Nucleoprotein of influenza A virus (A/vietnam1194/2004 (H5N1)) was cloned and expressed in E. coli. The single strand DNA library is composed of 30 random nucleotides flanked by constant sequences for PCR amplification. Magnetic beads-based approach was used to screen DNA aptamers binding purified His-tagged nucleoprotein. After 20 rounds of selection, the enriched pool was cloned and 40 colonies were sequenced. The structure of the G-quadruplex aptamers was characterized by circular dichroism spectroscopy. MDCK cells were transfected with representative aptamers by Lipofectamin 2000. Four hours after transfection, cells were challenged with 100TCID50 influenza A virus (A/Vietnam/1194/2004(H5N1)). The viral titer was determined by HA assay at 48 hours post-infection. RESULTS: The sequences from in vitro selection revealed fairly conserved and could be classified into two groups, G-quadruplex and T-rich DNA aptamers. G-quadruplex aptamers were found dominant in the population and comfirmed by circular dichroism spectroscopy. Circular dichroism spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. In the early stage of post-transfection, DNA aptamers were mainly localized in the nuclei where the replication and transcription of influenza virus genome take place exclusively, which may facilitate successful antiviral efficacy. The T-rich aptamer had only mild effect on the inhibition of H5N1 virus, whereas G-quadruplex forming aptamers exhibited potent antiviral effect. CONCLUSIONS: This study has demonstrated that nucleoprotein could be a potential antiviral target for influenza H5N1 virus. Further investigation will aim to elucidate the interaction between the G-quadruplex aptamer and nucleoprotein, as well as the antiviral mechanism. | - |
dc.language | eng | en_HK |
dc.publisher | Blackwell Publishing. | - |
dc.relation.ispartof | European Congress of Clinical Microbiology & Infectious Diseases, ECCMID 2010 | - |
dc.title | In vivo selection and characterization of DNA aptamers against H5N1 virus nucleoprotein | en_HK |
dc.type | Conference_Paper | en_HK |
dc.identifier.email | Tanner, JA: jatanner@hku.hk | en_HK |
dc.identifier.email | Zheng, B: bzheng@hkucc.hku.hk | en_HK |
dc.identifier.authority | Tanner, JA=rp00495 | en_HK |
dc.identifier.authority | Zheng, B=rp00353 | en_HK |
dc.description.nature | link_to_OA_fulltext | - |
dc.identifier.hkuros | 175110 | en_HK |
dc.description.other | The 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 20), Vienna, Austria, 10-13 April 2010. | - |