Y-shaped trivalent aptamer for targeted visualization and tracking of reprogrammed astrocytes

Despite extensive research into the diverse transformations and functions of astrocytes, conventional fluorescence immunohistochemistry for their distinct identification remains challenging and time-consuming, primarily due to the lack of cell surface binders specific to these glial cells. To address this limitation, we developed a specific and straightforward imaging strategy for primary astrocytes using cell surface-targeting aptamers. We identified a novel anti-astrocyte DNA aptamer (designated Ast17-30) through a 17-round cell-SELEX process incorporating magnetic-activated cell sorting, designed to bypass the technical hurdles of SELEX when applied to short-lived cells. To enhance binding affinity, we further engineered this aptamer into a Y-shaped trimer (Tri-ΔAst17-30), enabling clear discrimination between astrocytes and neurons. Consequently, the engineered trivalent aptamer facilitated rapid astrocyte-specific endocytosis within minutes due to its increased binding avidity. This capability enabled longitudinal monitoring of astrocyte conversion into induced neural precursor cells, observing moderate pro-inflammatory gene expression in their transcriptomic profiles. Furthermore, Tri-ΔAst17-30 bound strongly to astrocytic glioblastoma cells (U87MG), with minimal binding to glial glioblastoma cells (C6), confirming specificity for astrocytic tumor cells. Given the current absence of reliable live-astrocyte-specific imaging techniques, we propose that this trivalent anti-astrocyte DNA aptamer has potential for investigating developmental pathways and targeted therapy of these crucial glial cells.