Figure 1.
Schematic of Adherent Cell-SELEX (AC-SELEX) Procedure.
A starting library of >1015 aptamers was utilized and eighteen rounds of AC-SELEX were carried out to purify and amplify the aptamers which recognized epitopes present on HF cells and not present on HeLa cells. The starting library was first incubated with the HeLa cell line and the unbound aptamers were recovered and utilized for the AC-SELEX procedure.
Figure 2.
Predicted aptamer secondary structures.
The secondary structures for each of the aptamers investigated. The structure(s) with the lowest free energy (dG) are presented. Secondary structure predictions were determined using UNAfold software and are sir_graph ® output [13].
Table 1.
Aptamer sequences.
Figure 3.
The target (HF) and non-target (HeLa) cell lines after incubation with FAM labeled aptamers.
Images of HF and HeLa cells incubated with fluorescently tagged aptamers. The aptamers are specific for the non-tumorigenic HF cell line. They recognize epitopes on or in the HF cells and do not recognize their binding sites on the tumorigenic HeLa cells. All images are 40× and are of cells within 24 hours after incubation with 2000 nM aptamers and subsequent fixation and coverslipping with Aquamount.
Figure 4.
Confocal images of aptamer-HF cell binding sites.
Images of HF cells with FAM labeled aptamers. Aptamers 13, 20 and 28 appear to be binding solely to the cell surfaces, while aptamer 14 is also being internalized into the cell cytoplasm. All images are 40× and are of cells within 24 hours after incubation with 2000 nM aptamers and subsequent fixation and coverslipping with Aquamount.
Figure 5.
Binding equilibrium for aptamers and their HF cell epitopes.
Fluorescence quantification of FAM-labeled aptamers bound to HF cells. Aptamer binding curves (line and data points) were generated by graphing the average fluorescence with the error bars representing the standard error of the mean for each data point (n = 4 per data point). These data were fitted to the model Y = Bmax*X(Kd+X) (solid line) which was generated using nonlinear regression analysis for one site specific binding: aptamer 13 (Kd = 2.5±0.5 nM); aptamer 14 (Kd = 7.1±0.4 nM); aptamer 20 (Kd = 1.6±0.4 nM); and aptamer 28 (Kd = 6.9±0.2 nM).
Figure 6.
The nontumorigenic, HA and tumorigenic SiHa cervical cancer cells after aptamer incubation.
Images depicting the non-transformed revertant, HA cell line and the HPV-transformed cervical cancer cell line, SiHa, after incubation with FAM labeled aptamers. The aptamers generated to target the non-transformed revertant, HF cell line also recognize the non-transformed revertant, HA cell line. Also notice that these aptamers do not recognize epitopes on the SiHa cervical cancer cell line, similar to the results we observed with the HeLa cervical cancer cell line. These results are consistent with the hypothesis that these aptamers are recognizing epitopes that are lost as a result of transformation of cervical epithelial cells by HPV. All images are 40× and are of cells within 24 hours after incubation with 2000 nM aptamers and subsequent fixation and coverslipping with Aquamount.