Binding assay to determine binding affinity between N34 RNA and EphA2

Lack of binding between the ephrin A1 ligand and the EphA2 receptor, 130 kDa tyrosine kinase receptor found in adult human epithelial cells, causes unstoppable cell growth, and subsequently, development of tumors associated with epithelial cancers (Kinch, 2005; Ansuini et al, 2009). Inhibiting the kinase activity and phosphorylation of EphA2 protein receptor has been associated with a decrease in the growth of malignant cells (Ansuini et al, 2009). Ligand based approaches have been utilized by attaching a ligand to phosphorylating EphA2 protein which inhibits phosphorylation and subsequently decreases tumor growth (Walker-Daniels et al, 2002). A synthetic, highly modifiable ligand that can be used to inhibit EphA2 phosphorylation is an aptamer. Aptamers are nucleic acid fragments that have specific quaternary structures allowing them to bind with high specificity and affinity to certain protein targets, much like innate ligands, such as ephrin A1 (Phillips et al, 2008). Specific aptamers are filtered from a large nucleic acid pool through repeated, increasingly stringent tests, such as SELEX, which select for the highest affinity binders and amplification of those binders, and a binding assay can be performed to determine the progression of binding affinity through the rounds of selection.

A binding assay was performed to determine the affinity of filtered N34 RNA from R1, R3, and R5 to EphA2. 20 pmol of RNA, 40 pmol of EphA2 protein, and 200 pmol tRNA was used for the triplicate reactions which tested for both positive and negative binding. In order to detect the RNA, the double stranded DNA was labeled with radioactive beta 32P nucleotides during transcription. The radioactively labeled RNA was pipetted onto a nitrocellulose filter using the layout in figure 1. A nitrocellulose filter was used to filter the bound RNA and protein while a nylon filter collected the unbound RNA, as can be seen in figure 2.

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.