Functionalized tetrahedral DNA frameworks for the capture of circulating tumor cells

Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands’ position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed ‘n-simplexes’, can be used as probes to specifically target receptor−ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.