Figure 1 Binding Assay Layout: Triplicate reactions were filtered using a nitrocellulose filter. To test for positive binding, R1, R3, and R5 protein and RNA was placed in three wells in the regions 1, 3, and 5 respectively. To determine how much of the binding was a result of RNA affinity for the nitrocellulose, R1, R3, and R5 RNA minus protein was placed in three wells in regions 2, 4, and 6 respectively.
Figure 2 Binding Assay: Nitrocellulose filter was used to separate the protein and bound RNA away from the unbound RNA which was collected on the nylon filter. The dots are a result from exposure of the 32P labeled RNA to phosphor plates.
Binding was minimal as shown by the faintness of the samples collected on the nitrocellulose filter in figure 2 and the comparison of the percent binding in figure 3. However, it must be noted that the results may be slightly skewed due to prolonged exposure between the 32P labeled RNA and phosphor plates which may account for the increased desensitization, and subsequently higher percent bound volumes, on the nitrocellulose filter. Nevertheless, the decreased average percentage of negatively binding RNA on the nitrocellulose filter (1.125% to 0.584%) does indicate the RNA with higher affinity for the protein, rather than the beads or filter, is gradually being selected. The average percent bound of high affinity RNA can be increased by progressively more stringent selection conditions to further reduce the amount of low affinity sequences and background binders.
Figure 3 Percent Binding: Percent binding is the amount of bound RNA and EphA2 on the nitrocellulose filter. It is calculated by dividing the total volume of protein and both bound and unbound RNA by the volume of bound RNA and EphA2.
Ansuini, H., et al (2009) “Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer.” Journal of Oncology 2009: 1-10.
Kinch, M. S. (2005). Targeted drug delivery using EphA2 or EphA4 binding moieties. Patent No. 20050153923. Laytonsville, MD, US.
Phillips, J. A., et al (2008) “Applications of aptamers in cancer cell biology.” Analytica Chimica Acta 621 Review: 101-108.
Walker-Daniels, J., et al (2003) “Differential Regulation of EphA2 in Normal and Malignant Cells.” American Journal of Pathology 162: 1037-1042.