Directed Evolution of Aptamer Discovery Technologies

CONSPECTUS: Although 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 laboratory 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 multiparametric 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 matrixes such as serum. The third method is an alkyne-azide chemistry ("click chemistry")-based particle display (click-PD) that enables the generation and screening of "non-natural" 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. Finally, we describe the non-natural aptamer array (N2A2) platform in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in high throughput, 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 for affinity and specificity 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.