Directed Evolution of Aptamer Discovery Technologies

Abstract

ConspectusAlthough antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of “non-nattural” aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

Future of Digital Assays to Resolve Clinical Heterogeneity of Single Extracellular Vesicles

“…Generating highly specific aptamers has proven challenging with conventional processes like SELEX (Systematic Evolution of Ligands by EXponential Enrichment). 112 Modern highthroughput screening platforms will be required to design and select aptamers for epitopes of multiplexed protein targets with optimal binding affinities. 113,114 Nanobodies.…”

Tailoring electrode surface charge to achieve discrimination and quantification of chemically similar small molecules with electrochemical aptamers

“…Such sensors can deliver low-cost, sensitive analyte detection while obviating the need for cumbersome sample preparation. There have been many innovations in selecting high-affinity aptamers using directed evolution, [11][12][13][14] and also in improving the signaling of these aptamers by rationally designing switches 15,16 and engineering electrode interfaces to tune transduction. [17][18][19][20][21] However, one major challenge is that these sensors often suffer from poor specificity, such that the presence of structurally-similar interferents and metabolites can potentially make accurate molecular quantification of the target difficult or impossible.…”