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

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.