In vitro selection of ssDNA aptamers and specific binding activity of the ssDNA pool.
(A) Schematic representation of the counter-SELEX procedure. After removing ssDNA species binding nonspecifically to glutathione agarose beads, the ssDNA pool was incubated with H5-HA1 (negative control) and centrifuged to remove H5-HA1-binding ssDNAs, after which the unbound ssDNAs were incubated with H1-HA1 (target protein), and the H1-HA1-bound DNAs were extracted using phenol-chloroform and amplified by PCR. After 14 rounds of selection, the enriched DNA was PCR-amplified, cloned, and sequenced. (B) Specific binding activity as measured by ELISA after 8, 10, 12, and 14 rounds of selection. GST-tagged H1-HA1 (100 nM) incubated on selected DNA-coated plates and analyzed using anti-GST antibody-HRP with TMB color detection.