A Pair of DNA Aptamers That Can Selectively Bind to Bilirubin and Biliverdin.

Bilirubin and biliverdin are two important metabolites from the degradation of heme. Development of aptamers for them will not only help with the measurement of their concentrations for diagnosing diseases such as neonatal jaundice and liver dysfunction, but may also aid in developing molecular switches for the regulation of gene expression. In this work, we report the selection of DNA aptamers against bilirubin and biliverdin. For the biliverdin selection, the tightest affinity aptamer has a dissociation costant (K_d) value of 6 nM determined using isothermal titration calorimetry (ITC), and using a fluorescent strand-displacement assay, a limit of detection of 0.7 nM was achieved. This strand-displacement sensor also showed a response to bilirubin, although with a 10-fold lower affinity. For the bilirubin selection, many sequences obtained were also present in the biliverdin selection, and it was attributed to the oxidation of a fraction of bilirubin to biliverdin by air. This oxidation was confirmed by a visual color change of bilirubin and by UV-vis spectroscopy. The tightest binding bilirubin aptamer has a K_d value of 203 nM based on ITC, and a detection limit of 47 nM was achieved using the strand-displacement assay. This pair of aptamers offer insights into molecular recognition of heme breakdown products and may be useful for developing biosensors and intracellular molecular switches.