In vitro selection of DNA aptamers binding to interleukin-6

Abstract

Aptamers are DNA or RNA molecules that bind specific molecular targets. With the recombinant human interleukin-6 (IL-6) as a target protein, which has been known to inhibit growth and induce differentiation of several myeloid leukemia cell lines, a set of DNA aptamers binding to IL-6 from a random oligodeoxyribonucleotide pool was isolated. A large pool of 96-mer oligodeoxyribonucleotides was chemically synthesized that was made up of 60 bases of random sequences flanked by the defined regions of primer binding sites at the $5^\prime$ and $3^\prime$ ends. This randomly generated oligodeoxyribonucleotide pool renaturated at $25\,^\circ\!C$ for proper folding of single-stranded DNAs after denatured at $95\,^\circ\!C$ was subjected to in vitro selection with affinity chromatography and in vitro amplification with polymerase chain reaction (PCR) for enrichment in DNA aptamers specifically binding to IL-6. Multiple rounds of affinity chromatography and PCR resulted in the continued purification of binding species. The sequences of the aptamers and their binding properties for IL-6 will be determined. Since a binding strand can not be chosen from the sequencing data, binding affinity of labeled two single-stranded DNAs was tested by gel retardation assay, respectively. The gel retardation experiment showed that aptamers had affinity to IL-6 and their dissociation constants were about $\sim10^{-6}$ M. A homology search revealed that they were catalogued into at least three groups according to the consensus sequences among them. The consensus sequences are likely to participate in forming IL-6 binding motifs.