Figure 6.

Characterization of Aptamer Specificity against TCR-CD3ε

(A) Flow-cytometric analyses of binding of the highest affinity aptamer, ZUCH-1, against Jurkat.E6 cells used in SELEX (left), against wild-type Jurkat cells used for generating CRISPR knockout cell lines (middle), against CRISPR double-knockout Jurkat cells (right), and the overall conclusion from six independent specificity analyses (far right). Aptamer ZUCH-1 does not bind to knockout cells, thereby demonstrating epitope specificity (ordinary one-way ANOVA, using Tukey’s multiple comparisons test performed on GraphPad Prism to obtain statistical significance: ****p ≤ 0.0001). (B) Flow-cytometric analysis of competitive binding of ZUCH-5 against Jurkat.E6 cells in the presence of anti-CD28 antibody (left), OKT3 (middle) and UCHT1 (right) and overall conclusion from three independent analyses (far right) (ordinary one-way ANOVA, using Tukey’s multiple comparisons test performed on GraphPad Prism to obtain statistical significance: **0.001 ≤ p ≤ 0.01; ***0.0001 ≤ p ≤ 0.001; ns, not significant). In the presence of OKT3 and UCHT1 antibodies, aptamer ZUCH-5 binding is diminished, indicating that both OKT3 and UCHT1 displaced the aptamer, but not anti-CD28. (C) Flow-cytometric analysis of binding of the APC-labeled anti-CD3 antibody against T cells (left) and flow-cytometric analysis of ZUCH-3 (middle) and ZUCH-1 (right) aptamers against human T cells isolated from donor PBMCs, indicating that both aptamers bind to human T cells.