Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging

Abstract

Although current implementations of super-resolution microscopy are technically approaching true molecular-scale resolution, this has not translated to imaging of biological specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topography (DNA-PAINT). To demonstrate the achievable resolution, specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.

Figure 1. SOMAmers as labeling probes for quantitative, high resolution DNA-PAINT imaging of membrane receptors.

(a) Labeling scheme using SOMAmers for diffraction-limited imaging with a fixed dye. (b) Labeling scheme using SOMAmers for DNA-PAINT super-resolution imaging. Transient binding of a dye-labeled strand to a complementary docking strand is enabled by single-stranded extension of the SOMAmer sequence. (c) SOMAmer against EGFR labeled with a fixed Cy3 dye (SL1069, see scheme in a) allows specific detection of EGFR in A549 cells using confocal microscopy. (d) Diffraction-limited DNA-PAINT image using a complementary dye-labeled imager strand against a docking-site-modified SOMAmer (see scheme in b) against EGFR (standard deviation image) in A431 cells. (e) Zoom-in of highlighted area in the diffraction-limited image in d. (f) Corresponding DNA-PAINT super-resolution image of the highlighted area in d. (g, top) Zoom-ins of highlighted areas (i, ii, and iii) in f. (g, bottom) Cross-sectional histogram analysis in i, and iii, respectively, demonstrates high-resolution DNA-PAINT imaging of single EGFR proteins labeled using SOMAmers. (h) Fitting a Gaussian distribution to the center-of-mass-aligned single-molecule localizations of ~34000 SOMAmer-labeled EGFR proteins yields a localization precision of 3.2 nm. (i) qPAINT analysis of single EGFR proteins yields a unimodal distribution of binding events, confirming quantitative 1:1 labeling of EGFR proteins using SOMAmers. Scale bars: 10 µm (c), 2 µm (d), 200 nm (e, f), 20 nm (g). Experiments were repeated at least three times with similar results; representative data are shown.

Figure 2. Intracellular labeling of GFP-tagged Nup107 for DNA-PAINT imaging using GFP-SOMAmers.

(a) Confocal images of GFP-tagged Nup107 nucleoporins labeled with Cy3-modified GFP-SOMAmers. GFP (SL1070, left, green), SOMAmer-Cy3 (center, red), and merged signal (right) at the bottom of the nucleus (top) and center (bottom) showing specific binding of the SOMAmer to the GFP-tagged Nup107. (b) Diffraction-limited and corresponding DNA-PAINT super-resolution image of GFP-tagged Nup107 using a docking-strand-extended SOMAmer against GFP. (c) Representative zoom-ins of single nuclear pore complexes (NPCs). (d) xy-projection (left) and xz-projection (right) of a single NPC shows well-resolvable clusters of Nup107 in the xy-projection (left) and the nuclear and cytoplasmic rings in the xz-projection (right). Color indicates height. (e) Cross-sectional histogram analysis of highlighted areas in d reveals a distance of ~30 nm between Nup107 in xy (left) as well as a ~51 nm distance in z of the nuclear and cytoplasmic rings (right). Scale bars, 10 µm (a), 2 µm (b), 50 nm (c), 25 nm (d). Height scale, -100 nm to 400 nm (blue to red). Experiments were repeated at least three times with similar results; representative data are shown.

Figure 3. Intracellular labeling and DNA-PAINT imaging of catalase proteins in peroxisomes using SOMAmers.

(a) Confocal micrograph of PMP70 proteins in the peroxisomal membrane of A431 cells labeled using primary and Alexa647-conjugated secondary antibodies. (b) Confocal micrograph of catalase proteins labeled using Fluorescein-conjugated SOMAmers. (c) Overlay of PMP70 antibody and catalase SOMAmer (SL1071) signal demonstrates co-localization of both proteins to peroxisomes. (d) DNA-PAINT super-resolution image of catalase molecules in peroxisomes using docking-strand-extended SOMAmers. (e) Comparison of diffraction-limited (left) and super-resolved (right) zoom-in of the yellow-highlighted area in d reveals single catalase molecules in peroxisomes in the SR image. (f) Zoom-ins of areas highlighted in white in d. 3D localization information is color-coded, revealing distinct z localizations of peroxisomes. (g) xz-projection of catalase molecules in a single peroxisome from area i in d and f. (h) xz-projection of catalase molecules in a single peroxisome from area iii in d and f. Scale bars, 5 µm (a, b, c), 500 nm (d), 200 nm (e, f), 100 nm (g, h). Height scale, -100 nm to 400 nm (blue to red). Experiments were repeated at least three times with similar results; representative data are shown.