Selection and Characterization of Vimentin-Binding Aptamer Motifs for Ovarian Cancer

Abstract

The application of aptamers in biomedicine is emerging as an essential technology in the field of cancer research. As small single-stranded DNA or RNA ligands with high specificity and low immunogenicity for their targets, aptamers provide many advantages in cancer therapeutics over protein-based molecules, such as antibodies. Vimentin is an intermediate filament protein that is overexpressed in endothelial cells of cancerous tissue. High expression levels of vimentin have been associated with increased capacity for migration and invasion of the tumor cells. We have selected and identified thioated aptamers with high specificity for vimentin using human ovarian cancer tissues. Tentative binding motifs were chosen for two vimentin aptamers based on predicted secondary structures. Each of these shorter, tentative binding motifs was synthesized, purified, and characterized via cell binding assays. Two vimentin binding motifs with high fidelity binding were selected and further characterized via cell and tissue binding assays, as well as flow cytometric analysis. The equilibrium binding constants of these small thioated aptamer constructs were also determined. Future applications for the vimentin binding aptamer motifs include conjugation of the aptamers to synthetic dyes for use in targeted imaging and therapy, and ultimately more detailed and precise monitoring of treatment response and tumor progression in ovarian pathology.

Keywords: aptamer; binding motifs; ovarian cancer.

Figure 1

Secondary structures of selected (V3) and (V5) thio-aptamers. Proposed binding motifs are shown in red.

Figure 2

Screening of synthesized aptamer motifs. Synthesized biotinylated motifs of V3 and V5 were incubated with IGROV cells. Fluorescein isothiocyanate (FITC) conjugated streptavidin is used to detect biotinylated motifs. The binding affinity of the aptamer motifs was assessed based on the fluorescence intensity using flow cytometry. Histogram graphs demonstrated the fluorescence intensity of V3 aptamer motifs (A), and V5 aptamer motifs (B) binding to IGROV cells. Vimentin expression was evaluated and confirmed using an anti-human vimentin antibody before screening the binding affinity of motifs (C). A scrambled aptamer was used as a control.

Figure 3

The binding affinity of V3, V5, selected motifs and their equilibrium dissociation constant. Filter-binding assays were performed with the biotinylated V3, V5, V3M2, V5M2 thioaptamers and purified vimentin protein. Chemiluminescent detection of spot intensities on the nitrocellulose membranes was used to quantitate the thio-aptamer binding affinity. (A) Saturation binding curves were generated and the equilibrium dissociation constants, Kd, were calculated from the equation Y = Bmax × X/(Kd + X), assuming a single binding site. Bmax represents the maximum binding capacity of aptamer bound to vimentin protein. X is the protein concentrations and Y is the calculated spot intensity. (B) Representative spot image of biotinylated V3M2 and V5M2 binding with vimentin protein retained on the nitrocellulose membrane. Non-binding motifs stained on a nylon membrane.

Figure 4

Dose-dependent binding of selected motifs. Biotin conjugated V3M2 and V5M2 were incubated with vimentin-expressing IGROV cells at various concentrations and followed by streptavidin-FITC staining. Their binding affinity was analyzed by flow cytometry. Histograms presenting the fluorescence intensity above the background were shown for V3M2 (A) and V5M2 (B). A scrambled control aptamer with non-specific and low binding affinity is also assessed (C).

Figure 5

Validating specific binding of selected motifs with the human cell line. Vimentin-expressing IGROV cells were incubated with biotinylated V3M2, V5M2, anti-human vimentin antibody (VIM Ab) or scrambled control (SA) aptamer. Fluorescein isothiocyanate (FITC) conjugated streptavidin was used to detect biotinylated aptamers. The binding proficiencies were determined by fluorescence intensity using fluorescence microscopy. Fluorescence intensity is quantified by normalizing the fluorescence intensity of pixel per area (intensity of motif/intensity of DAPI) and presented as a bar graph with mean ± SE of three replicates. Hoechst 33,342 was used to stain the cell nuclei (blue).

Figure 6

Detection of vimentin expression in human ovarian tumor tissue. Tissue sections of human ovarian tumor or normal ovarian tissue were incubated with biotinylated V3M2 or V5M2 at a concentration of 250 nM, followed by streptavidin-FITC to detect their binding affinity. Anti-human vimentin antibody was also used as a positive control for both ovarian tumor tissue and normal ovarian tissue. Images are representative of three samples of ovarian tumor or normal ovarian tissue. Fluorescence intensity is quantified by normalizing the fluorescence intensity of pixel per area (intensity of motif/intensity of DAPI) and presented as a bar graph with mean ± SE of three replicates. Hoechst 33,342 was used to stain the cell nuclei (blue).