Results and discussion To develop a specific Akt inhibitor aptamer for MMP2 protein, we performed a modified DNA LY333531 ic50 SELEX technique as described in the ‘Methods’ section. To select a high-affinity aptamer, we used nucleotides chemically

modified by benzylaminocarbonyl-dU (Benzyl-dU) at the 5′ positions, which mimic amino acid side chains. After eight rounds of SELEX, the enriched DNA pool was cloned and sequenced according to standard procedures. After each round of SELEX, binding assays were performed to measure the dissociation constant (K d) value of the aptamer pool using [α-32P] ATP. The sequence and secondary structure of the best aptamer selected in this study were presented in Figure 1. The mean B max and K d values of the aptamer were 35% ± 0.8% and 5.59 ± 0.52 nM, respectively (Figure 2). Figure 1 Sequence and RXDX-101 mouse secondary structure of the MMP2 aptamer. (a) Sequence of the 40-nucleotide random region (N40, shaded) and of the two constant regions flanking the random region. (b) The hairpin-like secondary structure of the aptamer is presented in the lower panel. Figure

2 Affinity of the MMP2 aptamer. (a) 32P-labeled aptamers and different MMP2 protein concentrations were used to examine the binding affinity of the MMP2 aptamer. (b) Images of radiolabeled aptamer that interacted with proteins in the binding assay. To determine whether the MMP2 aptamer could be used to precipitate the target protein, we performed precipitation and then western blotting using anti-MMP2 antibody. To do this, we biotinylated the aptamer and used streptavidin beads for the precipitation. MMP2 in buffer containing 10% serum was incubated with the biotinylated MMP2 aptamer, and the protein-aptamer complex was then precipitated and detected by immunoblotting. The aptamer successfully precipitated MMP2 protein (Figure 3), whereas the biotinylated control Farnesyltransferase aptamer did not (data not shown). Figure 3 Precipitation of MMP2 protein by MMP2 aptamer. MMP2 protein in buffer containing 10% serum was incubated with the aptamer (0.2 μg/ml) overnight

at 4°C. The protein was detected by immunoblotting with anti-MMP2 antibody. Next, we examined whether the MMP2 aptamer could be applied for immunohistochemical purposes in pathological tissues, that is, atherosclerotic plaques and gastric cancer tissues. In both tissue types, the MMP2 aptamer successfully detected MMP2 (Figure 4), whereas the control aptamer did not (data not shown). To further confirm the specificity of the aptamer for immunohistochemistry, we performed peptide blocking. Immunohistochemistry was performed after incubating the aptamer for 2 h with the bare protein, and the intensities of positive signals were significantly reduced (Figure 5). Figure 4 Comparison of the tissue staining abilities of anti-MMP2 antibody and MMP2 aptamer. Normal aorta, atherosclerotic plaques, and gastric cancer tissues were stained with anti-MMP2 antibody and MMP2 aptamer. Similar staining patterns were observed.