Aptamers with Tunable Affinity Enable Single-Molecule Tracking and Localization of Membrane Receptors on Living Cancer Cells

Abstract

Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.

Keywords: PAINT; aptamers; cell-surface receptors; live-cell imaging; single-molecule tracking.

Conflict of interest statement

The authors declare no conflict of interest.

© 2020 The Authors. Published by Wiley-VCH GmbH.

Figures

Figure 1

a) The fluorescently conjugated aptamer probe. The aptamer tail (green) is annealed to its complementary anti‐tail (grey) bearing a fluorophore (green sphere). b) Schematic of live‐cell imaging using aptamers. Thanks to TIR illumination, only aptamers (green fluorophores) that bind cell‐surface receptors are selectively excited and detected. In contrast, freely diffusing aptamers (grey fluorophores) are not observed. Transient binding of aptamers to target receptors enables single‐molecule imaging on nearly unperturbed living cells. c) Single‐molecule tracking was performed using sub‐nanomolar concentrations of probes (0.05–0.20 nm) to study receptor diffusion and aptamer binding kinetics. Examples of single EGFR trajectories on the surface of an A431 cell are shown (top image). Scale bar: 5 μm. X‐Y coordinates of a representative trajectory are also displayed. Diffusive status of a receptor can be studied by analysis of thousands of trajectories. Additionally, aptamer binding kinetics can be assessed by analyzing the distribution of trajectory durations, which follows a single‐exponential decay (black line). d) Membrane receptor densities are obtained by live‐cell PAINT using low nm concentrations (1–20 nm). Time‐lapse sequences are recorded (on the left), then a PAINT image (on the right) is reconstructed. Scale bar: 5 μm.

Figure 2

a–c) SMT of EGFR on living A431 cells exposed to 0.05 nm of anti‐EGFR aptamer (MinE07) conjugated to Atto647N. a) Distribution of measured D values for MinE07 is shown in green. b) Examples of selected long trajectories for each of these four diffusive states are shown: Brownian (black), confined (green), directed (red), and immobilization (blue). Scale bar: 2 μm. c) Corresponding mean‐square displacement (MSD) plots for four different types of EGFR motion. d–f) Two‐color single‐molecule tracking on living A431 cells exposed simultaneously to 0.05 nm AF488‐conjugated MinE07 (EGFR, green) and 0.20 nm Atto647N‐Waz aptamer (TfR, magenta). d) Representative example of one acquisition frame is displayed. Scale bar: 5 μm. e) Examples of trajectories of single EGFR (green) or TfR (magenta) on the cell surface. Scale bar: 2 μm. f) Distributions of D values calculated for the two channels (EGFR in green and TfR in magenta).

Figure 3

a) Reconstructed PAINT images of living A431 cells incubated with 3 nm MinE07 (high‐affinity MinE07). Scale bars: 5 μm (left) and 2 μm (right). Yellow arrows indicate aberrant irregular distribution of PAINT localizations at the edges of the cell. The image on the right shows a zoomed‐in view of the area within the dashed square. b) Workflow for aptamer engineering . Variant sequences were designed to destabilize the minimum free energy (MFE) secondary structure of MinE07 (top panel) and/or to promote alternative conformations, as shown for the MFE structure exhibited by MinE07_G6U/A33C (top panel, see also Figure S3). For each MinE07 variant, flow cytometry was used to assess binding of three cell lines that display differential expression of EGFR. Results and experimental details are reported in Figure S4‐S6. To assess retention of selectivity, candidate sequences were further tested in competition binding assays (Figure S7). c) Apparent dissociation constants (KD app) on HeLa cells were determined by flow cytometry using increasing concentrations of Cy5‐labeled anti‐EGFR aptamers (MinE07 and MinE07_G6U/A33C). Plots of median fluorescence intensity (MFI) versus aptamer concentration and apparent KD values are shown for MinE07 (top) and MinE07_G6U/A33C (bottom). Representative flow cytometry curves for all aptamer samples are shown in Figure S8. d) Reconstructed PAINT images of living A431 cells incubated with 3 nm MinE07_G6U/A33C (low‐affinity MinE07). Scale bars: 5 μm (left) and 2 μm (right). The image on the right shows a zoomed‐in view of the area within the dashed square.

Figure 4

a–c) Representative reconstructed PAINT images obtained upon incubation of living cells with Atto647N‐conjugated low‐affinity MinE07 (7 nm). Scale bars 5 μm. d) Normalized localization densities on the membrane are reported for A431, MDA‐MB‐231 and MCF‐7 cells upon incubation with 7 nm low‐affinity MinE07 (green) or CD4BA (blue). Means and standard deviations are reported in histograms. Data were obtained from 10 different cells in each of two independent experiments. Statistical analysis was performed with non‐parametric Mann‐Whitney test: ***: p<0.001; **: p<0.05; *: p<0.1; ns: not statistically significant.

Figure 5

a,b) Reconstructed PAINT image (a), and density map of trajectories (>5 frames) detected by SMT (b) obtained upon incubation of living MDA‐MB‐231 cells with low‐affinity MinE07 aptamer (17 nm). Scale bars: 5 μm. c–e) Density maps of sub‐populations of trajectories were obtained after applying a filtering process (to the same field of view shown in a–c) to select molecules with (c) slow diffusion (2×10−3 μm2 s−1<D<2×10−2 μm2 s−1), (d) fast diffusion (10−1 μm2 s−1<D<1 μm2 s−1), or (e) long distance covered (1.3 μm<d<1.7 μm). Colors indicate the number of trajectories centered within each pixel of the map (size 1 μm). The color scale is reported on the bar on the right of each image, while the total number of trajectories in the map is at the bottom.