title: Isolation of single-stranded DNA aptamers that distinguish influenza virus hemagglutinin subtype H1 from H5.
abstract: Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.
authors.affiliation: Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Hye-Min
authors.initials: HM
authors.lastname: Woo
authors.affiliation: Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Jin-Moo
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Sanggyu
authors.initials: S
authors.lastname: Yim
authors.affiliation: Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Yong-Joo
authors.initials: YJ
authors.lastname: Jeong
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000235:genetics*; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006388:Hemagglutinins / Q000235:genetics*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics; D007251:Influenza, Human / Q000235:genetics; D052156:SELEX Aptamer Technique / Q000379:methods
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006388:Hemagglutinins