Dual-Mode Aptamer AP1-F Achieves Molecular–Morphological Precision in Cancer Diagnostics via Membrane NCL Targeting

TL;DR

A new aptamer called AP1-F improves cancer diagnostics by targeting membrane-bound nucleolin with high specificity and accuracy.

Contribution

AP1-F is a G4-structured aptamer that targets membrane NCL, achieving superior cancer cell discrimination compared to existing probes.

Findings

AP1-F showed a >10-fold fluorescence signal ratio between malignant and normal cells in co-cultures.

AP1-F achieved over 98.78% specificity in heterogeneous cell populations using dual-channel flow cytometry.

AP1-F provided 40.5-fold fluorescence intensity ratio in tumor vs. normal tissue in pathological sections.

Abstract

Nucleic acid aptamers leverage defined tertiary structures for precise molecular recognition, positioning them as transformative biomedical tools. We engineered AP1-F, a G-quadruplex (G4)-structured aptamer that selectively binds membrane-anchored nucleolin (NCL) non-permeabilizing, overcoming a key limitation of conventional probes. Microscale thermophoresis confirmed nanomolar affinity to NCL. By means of rigorous optimization, AP1-F attained a greater than ten-fold fluorescence signal ratio between malignant and normal cells in co-cultures, exceeding the extensively researched AS1411. Dual-channel flow cytometry demonstrated over 98.78% specificity at single-cell resolution within heterogeneous cell populations, owing to AP1-F’s unique membrane localization—unlike AS1411’s intracellular uptake, which elicited erroneous signals from cytoplasmic NCL. Competitive binding experiments and Laser Confocal Imaging confirmed that AP1-F specifically identifies cancer cells by binding to the NCL recognition site on the membrane. In pathological sections, AP1-F exhibited a 40.5-fold fluorescence intensity ratio between tumor and normal tissue, facilitating accurate tissue-level differentiation. Significantly, it delineated molecular subtypes by associating membrane NCL patterns with morphometric analysis: luminal-like MCF-7 displayed consistent staining in cohesive clusters, whereas basal-like MDA-MB-468 revealed sporadic NCL with irregular outlines—characteristics imperceptible to intracellular-targeted antibodies, thus offering subtype-specific diagnostic insights. This combination biochemical–morphological approach accomplished subtype differentiation with a single-step, non-permeabilized process that maintained lower cytotoxicity and tissue integrity. AP1-F enhances diagnostic accuracy by utilizing spatial confinement to eradicate intracellular interference, connecting molecular specificity to intraoperative margin evaluation or biopsy categorization.

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