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

Abstract

We describe a versatile 96-well microplate-based 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 96 microcolumns. 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 compared 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 targets 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 analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections.FigureSchematic breakdown of a microplate-based enrichment device for the selection of aptamers (MEDUSA), which can be customized and assembled in both parallel and serial configurations. Up to 96 selections can be performed simultaneously.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7661-7) contains supplementary material, which is available to authorized users.

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…The use of microcolumns was optimized for maximum enrichments of aptamers, revealing critical target concentrations that could be explained by geometric constraints for steric hindrance. 60,61 This concentration as well as the small column volume reduce the amount of target needed by several orders of magnitude. Furthermore, tests that varied the flow rates resulted in enrichment trends that scaled oppositely from the time-dependent kinetic binding model where the concentrations of unbound and bound molecules are also dependent upon their position x along the column…”

“…Recently, this technology was scaled up to a Microplate-based Enrichment Device Used for the Selection of Aptamers (MEDUSA) and has a 96-well microplate format to allow high-throughput and potentially automated platebased selections and processing to be performed. 61 In a similar modification of affinity chromatography, MonoLEX utilizes a narrow affinity column in the form of a capillary. 64 As is typical of chromatographic separations, the nucleic acid library can resolve itself along the capillary into different populations of binders (Figure 3(b)).…”

“…83 In addition, the 96-well microplatebased affinity microcolumns discussed earlier were also used to perform simultaneous processing and analysis. 61 These highly parallelized technologies are ideal compliments to automate microplate-based protocols and combining these two approaches may allow researchers to achieve automated and massively parallelized selections using similar robotic systems.…”

“…Recent studies have shown that in certain technologies, many of the core and intuitive assumptions of the SELEX model and basic binding kinetics are not well supported by the binding results. 36,37,[60][61][62]66,67,75 This is likely due to the added complexity of the more sophisticated technologies and immobilization schemes used. Selection techniques are currently evaluated primarily on the number of cycles needed and/or on the binding affinity of the resulting aptamer.…”

Detection and treatment of critical illnesses using oligonucleotides

“…In a particularly notable application, in vivo SELEX has been used to identify aptamers targeting entire tissues (147,148). The use of SELEX against such diverse targets has been described elsewhere (149)(150)(151)(152). In this chapter, we provide detailed methods for the rapid development of chemicallyoptimized RNA aptamers against recombinant, purified protein targets.…”