Programmable Self-Priming Lanthanide-DNA Probes Enabling Multiplexed Single-Cell Quantification of Glioma Splice Variants

Alternative splicing generates multiple mRNA isoforms, driving protein diversity and tumor heterogeneity. However, achieving multiplexed quantification of splice variants at single-cell resolution remains a significant challenge. Herein, we present a programmable self-priming lanthanide-labeled DNA probe (PSPLn) strategy for sensitive, multiplexed single-cell quantification of MDM2 splice variants in glioma-related cell models. By precisely tuning the number of adenines in the probe sequence and conjugating distinct lanthanide–DOTA complexes through click chemistry, we achieve junction-specific recognition and tunable signal amplification for splice-variant-resolved analysis. Coupling these probes with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), PSPLn enables high-throughput, single-cell readout of multiple MDM2 splice variants within individual cells. Application to U87 and U251 glioblastoma cell lines and the oligodendrocytic MO3.13 cell line reveals variant-dependent expression profiles and intervariant correlation patterns reflecting tumor heterogeneity. This platform overcomes the limitations of bulk and sequencing methods, offering a versatile tool for splice biomarker discovery and precision diagnostics in oncology.