Multiplexed Microcolumn-Based Process for Efficient Selection of RNA Aptamers

Abstract

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin—a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 108 with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection

“…Each round involves aptamer binding to either soluble or immobilized targets and a method that separates target-bound from -unbound aptamers. Immobilized targets have included proteins (9,10), peptides (11,12) and metabolites (13–15), and immobilization plays an essential role in new microfluidic SELEX technologies (10,16). Optimization of SELEX in these situations requires that we understand whether cooperative binding might change effective affinities, and if so, how.…”

Functionalized paper SERS (P-SERS) substrates for selective targeting of analytes in complex samples

“…25,26 More recently, nucleic acid aptamers have been introduced as novel recognition elements. [27][28][29][30][31] Aptamers are short artificial DNA and RNA sequences selectively chosen by their extremely strong binding affinity to their target proteins through a process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In the SELEX process, an extremely large pool of oligonucleotides of different sequences is incubated with the protein target of interest.…”

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…”

“…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.…”