High-throughput identification of aptamer–target pairs with SPARK-seq

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Cell-surface proteins serve as the primary gateway for intercellular communication and account for over 60% of current therapeutic targets. Despite this clinical importance, the development of high-affinity molecular probes, such as aptamers, that target cell-surface proteins is a substantial bottleneck in precision medicine. Although Cell-SELEX (systematic evolution of ligands by exponential enrichment) enables the selection of aptamers within native cellular environments, identifying their precise molecular targets has remained a low-throughput and resource-intensive ‘black box’. This issue is now being addressed by the emerging field of ‘aptomics’, defined here as the high-throughput, systematic study of the entire aptamer interaction landscape, integrating large-scale sequence diversity with comprehensive target identification and kinetics characterization.

SPARK-seq has notable advantages over traditional mass spectrometry-based identification of aptamer–ligand pairs. It achieves nanomolar level sensitivity and unparalleled specificity because cells with distinct genetic perturbations serve as mutual internal controls to filter out nonspecific background noise. Indeed, we demonstrated that SPARK-seq was able to precisely discriminate targets with high structural homology, such as NRP1 and NRP2, while maintaining robust performance across protein expression levels spanning several orders of magnitude. Furthermore, SPARK-seq prioritizes stably bound aptamers (that is, those with slow dissociation rates), which is an essential feature for therapeutic and diagnostic applications.

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