Aptamer-mediated targeting of CXCR3-B in acute lymphoblastic leukemia nalm-6 cells: an in silico and in vitro study

The CXCR3-B receptor plays a crucial role in inducing apoptosis upon binding with its ligands. Here, we aimed to rationally design an ultra-short (5-base) DNA aptamer targeting the N-terminal domain of CXCR3-B and evaluate its therapeutic potential in Acute Lymphoblastic Leukemia (ALL).

An in-silico library of 32 pentanucleotide aptamers (comprising adenine and guanine) was constructed. The three-dimensional structure of the CXCR3-B N-terminus was modeled and docked with the aptamers, followed by targeted molecular docking and binding stability scoring to evaluate complex interactions. In vitro, Nalm-6 cells were treated with the lead aptamer (GAGGA), a scrambled control, and proteinase K. Cell viability, metabolic activity, and apoptosis were assessed via trypan blue, MTT, and Annexin V/PI flow cytometry. The expression of BAX, P53, and CDKN1A (p21) was quantified using qPCR.

The GAGGA aptamer exhibited the highest binding affinity and complex stability in silico. In vitro, GAGGA at 600 µM significantly reduced metabolic activity and viability after 24 h compared with untreated and scrambled aptamer controls. The apoptotic cell ratio increased significantly (13.65% vs. 2.98% in control), accompanied by the significant upregulation of BAX, P53, and CDKN1A. Pre-treatment with proteinase K abolished these effects, confirming receptor-specific binding.

Our findings demonstrate that the ultra-short GAGGA aptamer specifically targets CXCR3-B and triggers apoptotic pathways in ALL cells. While the effective concentration is high, likely due to the lack of nuclease resistance in unmodified ultra-short oligonucleotides, this study provides a novel molecular scaffold for future aptamer-based ALL therapies following appropriate chemical modifications.