Advancements in Aptamer Discovery Technologies

Abstract

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies. This is primarily because conventional aptamer-discovery techniques such as SELEX are time-consuming and labor-intensive and often fail to produce aptamers with comparable binding performance to antibodies. This Account describes a body of work from our laboratory in developing advanced methods for consistently producing high-performance aptamers with higher efficiency, fewer resources, and, most importantly, a greater probability of success. We describe our efforts in systematically transforming each major step of the aptamer discovery process: selection, analysis, and characterization. To improve selection, we have developed microfluidic devices (M-SELEX) that enable discovery of high-affinity aptamers after a minimal number of selection rounds by precisely controlling the target concentration and washing stringency. In terms of improving aptamer pool analysis, our group was the first to use high-throughput sequencing (HTS) for the discovery of new aptamers. We showed that tracking the enrichment trajectory of individual aptamer sequences enables the identification of high-performing aptamers without requiring full convergence of the selected aptamer pool. HTS is now widely used for aptamer discovery, and open-source software has become available to facilitate analysis. To improve binding characterization, we used HTS data to design custom aptamer arrays to measure the affinity and specificity of up to ∼10(4) DNA aptamers in parallel as a means to rapidly discover high-quality aptamers. Most recently, our efforts have culminated in the invention of the "particle display" (PD) screening system, which transforms solution-phase aptamers into "aptamer particles" that can be individually screened at high-throughput via fluorescence-activated cell sorting. Using PD, we have shown the feasibility of rapidly generating aptamers with exceptional affinities, even for proteins that have previously proven intractable to aptamer discovery. We are confident that these advanced aptamer-discovery methods will accelerate the discovery of aptamer reagents with excellent affinities and specificities, perhaps even exceeding those of the best monoclonal antibodies. Since aptamers are reproducible, renewable, stable, and can be distributed as sequence information, we anticipate that these affinity reagents will become even more valuable tools for both research and clinical applications.

Real-time measurement of small molecules directly in awake, ambulatory animals

“…Created using well-established in vitro selection methods (16,17), aptamers can be generated that bind a wide range of analytes (18) and can be rationally reengineered such that they undergo a large-scale conformational change upon binding these analytes (19) over arbitrarily broad (20,21) or narrow (20,22) concentration windows. E-AB sensors use this conformational change to generate an easily measureable electrochemical signal without the need for the target to undergo a chemical transformation (23).…”

Molecular Elucidation of Disease Biomarkers at the Interface of Chemistry and Biology

“…111 Many other advances utilizing novel technologies, such as microfluidic devices and DNA microarray technologies, for SELEX have already been reported by the Soh group and others. 24,112 All of these efforts redound to greater control of the chemistry/biology interface, such that the resultant molecular tools can be put into clinical use to expand the practice of precision medicine.…”

Localized Chemical Remodeling for Live Cell Imaging of Protein‐Specific Glycoform

“…Given the ever-expanding available repertoire of proteinbinding aptamers [15] and carbohydrate-processing enzymes, [10c, 16] the LCM strategy should be easily adaptable to the live cell imaging of aw ide range of protein-specific glycoforms. Compared with currently used MOE method, desired assay features associated with LCM approach include minimum target cell perturbation, short reaction timeframe,e ffectiveness as am olecular ruler,a nd quantitative analysis capability.…”