Novel DNA Aptamers to Dickkopf-1 Protein and Their Application in Colorimetric Sandwich Assays for Target Detection...

Chronic immunoinflammatory rheumatic diseases, such as axial spondyloarthritis (AxSpA), are accompanied by a dysregulation of bone remodeling. Among potential biomarkers of bone metabolism, the Wnt pathway antagonist, Dickkopf-1 (DKK-1), is of particular interest because of its potential to reflect a shift towards joint ossification or osteoporosis, but its diagnostic value needs validation. There is still a lack of stable and efficient methods of measuring serum DKK-1 levels suitable for longitude studies. The use of aptamer-based diagnostic assays could be very promising for this purpose. We generated novel anti-DKK-1 DNA aptamers from a combinatorial library with a pre-defined sequence pattern in the randomized region. This approach showed high efficacy, as only four SELEX rounds of selection produced high-affinity aptamers with dissociation constants ranging from 1.3 to 3.7 nM. A family of their truncated versions was also developed by rational design. Novel DNA aptamers functioned as capture components in a microplate ELISA-like assay with HRP-conjugated anti-DKK-1 antibody as a reporter component. We succeeded in revealing the aptamer/aptamer sandwich pairs that provided an aptamer-only sandwich colorimetric assay. The aptamer/antibody colorimetric test systems were also examined in the analyses of blood serum from AxSpA patients and shown sufficient workability. However, in a number of cases we registered significant differences between assays based on TD10 and DK4 aptamers and made some suggestions about the origin of this effect.