NOTE High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device

device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96microcolumns.We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding com-pared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins ’ aptamers, as well as two suspected background binding sequences, to eight targets running serially. The tar-gets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device’s plate-based format with other complementary plate-based systems for all downstream biochemical processes and analy-sis, high-throughput selections, characterizations, and optimi-zation were performed to significantly reduce the time and cost for completing large-scale aptamer selections