In vitro selection of DNA aptamers binding to the recombinant human interleukin-6

Abstract

ILr6 is known to inhibit growth and induce differentiation of several myeloid leukemia cell lines. With the recombinant human interleukin-6 (BL-6) as a target protein, DNA aptamers specifically binding to IL-6 were isolated from a random otigodeoxyribonucleotide pool. A large pool of 96-mer oligodeoxyribonucleotides, made up of 60 bases of random sequences flanked by the defined regions of primer binding sites at the ,5' and 3' ends, was synthesized. This randomly generated oligodeoxyribonucleotide pool was denatured to generate single-stranded DNA and subjected to in vitro selection with affinity chromatography and in vitro amplification with polymerase chain reaction (PCR) for enrichment in single-stranded DNA aptamers specifically binding to Hr6. Multiple rounds of successive affinity chromatography and PCR resulted in the continued purification of binding species. The gel retardation experiment showed that aptamers had affinity to IL-6 with dissociation constants of the order of magnitude of 10-6 M. The sequences of the aptamers were identified. A homology search revealed that they were categorized into at least four groups according to the consensus sequences among them.