Covalent Photo-Aptamer Tagging (CPAT) for Single-Cell Multiplexing and Chemical Transcriptomic Screens

High-fidelity sample multiplexing in single-cell RNA sequencing is fundamentally limited by the thermodynamic reversibility of noncovalent labels, which causes signal crosstalk and data attrition. Here, we report Covalent Photo-Aptamer Tagging (CPAT), a strategy that converts transient molecular recognition into permanent covalent anchors via diazirine-mediated carbene insertion. Leveraging the synthetic programmability of DNA, we site-specifically integrated photoreactive moieties into aptamer scaffolds, creating a monomolecular architecture that couples high-affinity binding with genomic indexing. This "kinetic locking" mechanism enables near-instantaneous stabilization upon 365 nm irradiation, facilitating ultrastringent purification to eliminate nonspecific background. Validated in a chemical transcriptomic screen of pharmacological perturbations, CPAT's superior signal-to-noise ratio resolved rare, drug-resistant subpopulations. By surpassing the heterogeneity of stochastic antibody conjugates, CPAT provides a robust, chemically defined, trifunctional framework for precision multimodal single-cell profiling.