High-Throughput Multiplexed Quantification of Molecules by Aptamer Sequencing (Apt-seq) in Single Cells

Abstract

The advent of high-throughput sequencing technologies is transforming life sciences into a quantitative paradigm. However, existing sequencing technologies for nondirectly sequencable molecules, such as antibody-sequencing and glycan-sequencing, face certain challenges, including the complexity of antibody-oligonucleotide conjugation procedures, potential steric hindrance, and the labor-intensive chemoenzymatic labeling. Aptamers, as directly sequencable nucleic acids, offer exceptional specificity, broad target range, and small size, making them ideal tools for multimodal molecular profiling. In this work, we present Apt-seq, an aptamer-based, high-throughput platform for multimodal omics quantification at single-cell resolution. This integrative strategy─termed Aptomics─enables parallel profiling of cell surface proteins, glycans, and mRNA. The feasibility of the platform was validated using commercial cell lines, demonstrating strong concordance between sequencing data and flow cytometry results at both bulk and single-cell levels. When applied to complex clinical samples, Apt-seq exhibited exceptional sensitivity and enabled the precise profiling of tumor heterogeneity. Notably, the platform identified a subpopulation of tumor cells with elevated PTK7 surface expression, a marker of stemness. These findings underscore the role of PTK7 in tumor stemness and its potential as a stem-like biomarker. Moreover, using an aptamer targeting sialic acid, we tracked the dynamic changes in sialylation during T cell differentiation, observing an increase in sialic acid levels as resting T cells transitioned into functional T cells, followed by a subsequent decline upon maturity. Collectively, we have developed a new form of multiomics, termed Aptomics, which employs aptamers to enable the sequencing of molecules that are not directly sequenceable, such as proteins and glycans, etc. Apt-seq enables the simultaneous quantitation of both directly and nondirectly sequencable molecules, offering a versatile and scalable platform for the comprehensive quantification of molecules of life in complex biological systems and advancing quantitative science.