DNA Aptamers Precisely Identify Senescent Cells Without Prior Target Knowledge

Cellular senescence — irreversible cell-cycle arrest driven by stress or damage — contributes to aging and age-related disease through the pro-inflammatory senescence-associated secretory phenotype (SASP). A longstanding problem in the field is the absence of a single universal biomarker; researchers must combine multiple indicators (p16, p21, SA-β-gal, SASP factors, morphology) to confidently identify senescent cells. This gap hampers both basic research and the development of senolytics that must selectively eliminate senescent cells without harming healthy tissue.

To address this, the Mayo Clinic team applied Systematic Evolution of Ligands by EXponential Enrichment (SELEX) — an iterative in vitro selection process — to evolve single-stranded DNA aptamers directly against senescent mouse adult fibroblasts (MAFs). Senescence was induced with etoposide and rigorously validated by SA-β-gal staining, morphological changes, RT-qPCR of p16/p21/SASP markers, and loss of Ki67. Naïve libraries of trillions of random 80-mer DNAs were cycled nine rounds: aptamers were first depleted against healthy MAFs (counter-selection), then positively selected on senescent MAFs. Library enrichment was tracked by qPCR and next-generation sequencing.

By round 7, recovery increased markedly and was sustained through round 9. Deep sequencing confirmed library convergence beginning at round 5. Ten candidate aptamers were synthesized and screened. All ten bound senescent MAFs more than control cells by qPCR recovery assay; six showed statistically significant preferential staining using fluorescent streptavidin-biotin imaging on an IncuCyte platform. Crucially, binding was not an artifact of prolonged culture: serum-starved control cells did not acquire aptamer binding, but trypsin treatment of senescent cells sharply reduced binding, implicating a cell-surface or extracellular matrix protein as the target.

Two aptamers — 6757 and 6758 — were found to bind fibronectin, specifically a form present on senescent cells, with sub-nanomolar affinity even in complex protein mixtures. The aptamers showed broad selectivity across multiple senescent cell types (including radiation-induced and oncogene-induced senescence) and multiple mouse cell lineages, suggesting the fibronectin epitope they recognize is a general feature of the senescent phenotype rather than specific to one induction method. In aged mouse lung tissue, one aptamer showed significantly elevated staining compared to young controls, and this staining was markedly reduced in INK-ATTAC transgenic mice in which p16-expressing senescent cells had been pharmacogenetically eliminated, providing compelling in vivo validation.

This study establishes that unbiased cell-based SELEX can generate senescence-specific DNA reagents without requiring prior knowledge of the molecular target. The identified aptamers, particularly those targeting fibronectin, could serve as diagnostic imaging agents, tools for purifying or quantifying senescent cells, or as targeting moieties for aptamer-drug conjugate senolytics — analogous to antibody-drug conjugates but potentially cheaper and more chemically tractable.