File Download

Conference Paper: In vivo selection and characterization of DNA aptamers against H5N1 virus nucleoprotein

TitleIn vivo selection and characterization of DNA aptamers against H5N1 virus nucleoprotein
Authors
Issue Date2010
PublisherBlackwell Publishing.
Citation
The 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 2010), Vienna, Austria, 10-13 April 2010. How to Cite?
AbstractOBJECTIVE: 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.
DescriptionAbstract no. P1092
Persistent Identifierhttp://hdl.handle.net/10722/126472

 

DC FieldValueLanguage
dc.contributor.authorLin, Yen_HK
dc.contributor.authorTanner, JAen_HK
dc.contributor.authorZheng, Ben_HK
dc.date.accessioned2010-10-31T12:30:36Z-
dc.date.available2010-10-31T12:30:36Z-
dc.date.issued2010en_HK
dc.identifier.citationThe 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 2010), Vienna, Austria, 10-13 April 2010.en_HK
dc.identifier.urihttp://hdl.handle.net/10722/126472-
dc.descriptionAbstract no. P1092-
dc.description.abstractOBJECTIVE: 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.languageengen_HK
dc.publisherBlackwell Publishing.-
dc.relation.ispartofEuropean Congress of Clinical Microbiology and Infectious Diseases, ECCMID 2010-
dc.titleIn vivo selection and characterization of DNA aptamers against H5N1 virus nucleoproteinen_HK
dc.typeConference_Paperen_HK
dc.identifier.emailTanner, JA: jatanner@hku.hken_HK
dc.identifier.emailZheng, B: bzheng@hkucc.hku.hken_HK
dc.identifier.authorityTanner, JA=rp00495en_HK
dc.identifier.authorityZheng, B=rp00353en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.hkuros175110en_HK
dc.description.otherThe 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID 20), Vienna, Austria, 10-13 April 2010.-

Export via OAI-PMH Interface in XML Formats


OR


Export to Other Non-XML Formats