Smart aptamers facilitate multi-probe affinity analysis of proteins with ultra-wide dynamic range of measured concentrations

Protein concentration can vary over several orders of magnitude in many physiological, pathological, and biotechnological processes. Studies of these processes require affinity analysis of proteins with a wide dynamic range of accurately measured concentrations. The wide dynamic range can be achieved with multiple affinity probes that bind the target with significantly different equilibrium constants (K-d). Every probe in such a multi-probe affinity analysis is responsible for detection of the target in a range of concentrations around its K-d value. A multi-probe affinity analysis of proteins has not become practical so far due to the lack of generic affinity probes with a wide range of K-d and high selectivity. Kinetic capillary electrophoresis (KCE) has been recently proven to generate smart DNA aptamers with a wide range of predefined values of K-d and high selectivity. Here, we demonstrate, for the first time, that such aptamers can facilitate multi-probe affinity analysis of a protein with an ultra-wide dynamic range of measured concentrations. Our results showed that a three-aptamer analysis had a concentration dynamic range of more than 4 orders of magnitude. To the best of our knowledge, this is the widest dynamic range ever reported for affinity analyses of proteins. Advantageously, protein concentration in a multi-aptamers analysis can be determined using a simple calibration-free approach. This work proves that the wide range of predefined binding parameters of smart aptamers can bring new capabilities to quantitative affinity analyses. The same feature of smart aptamers makes them potentially indispensable molecular tools in studies of intracellular processes.