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

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Single-cell RNA sequencing (scRNA-seq) faces limitations in multiplexing fidelity due to reversible noncovalent labels, leading to signal crosstalk and data loss.
• Current methods struggle with background noise and heterogeneity, hindering precise analysis of rare cell populations.

Purpose of the Study:

• To introduce Covalent Photo-Aptamer Tagging (CPAT) as a novel strategy to achieve high-fidelity sample multiplexing in scRNA-seq.
• To enable permanent molecular anchoring via covalent bond formation, thereby minimizing signal crosstalk and data attrition.

Main Methods:

• CPAT utilizes diazirine-mediated carbene insertion to convert transient molecular recognition into stable covalent anchors.
• Photoreactive moieties are site-specifically integrated into DNA aptamer scaffolds, creating a monomolecular architecture for high-affinity binding and genomic indexing.
• A 365 nm irradiation triggers "kinetic locking" for rapid stabilization and stringent purification, eliminating nonspecific binding.

Main Results:

• CPAT demonstrated a superior signal-to-noise ratio in chemical transcriptomic screens of pharmacological perturbations.
• The method successfully resolved rare, drug-resistant cell subpopulations with enhanced precision.
• CPAT offers a robust, chemically defined, trifunctional framework surpassing the limitations of stochastic antibody conjugates.

Conclusions:

• CPAT overcomes the thermodynamic limitations of noncovalent labeling in scRNA-seq by establishing permanent covalent anchors.
• This technique significantly improves signal fidelity and reduces background noise, enabling more accurate multimodal single-cell profiling.
• CPAT provides a versatile platform for advanced applications in chemical transcriptomics and precision medicine.