tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging

Abstract

Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging; however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

Keywords: DNA-PAINT imaging; covalent; monovalent; protein tags; superresolution.

Figure 1.

tagPAINT imaging of CD3ζ and LAT proteins. LAT and CD3ζ knockout (KO) Jurkat cells were transfected; LAT-SNAP (a), CD3ζ-SNAP (b), LAT-Halo (c) and CD3ζ-Halo (d) constructs. (a) LAT-SNAP transfected cells were stained with anti-SNAP-ATTO488 antibody to determine expressing cells (top left). Gaussian convolved images of positive cells imaged using DNA-PAINT (top right; 2 nM, P01-ATTO655 imager). Zoomed regions of DNA-PAINT imaging are shown (yellow boxes, i and ii). (b) CD3ζ-SNAP transfected cells were stained with anti-SNAP-ATTO488 antibody to determine expressing cells (top left). Gaussian convolved images of positive cells imaged using DNA-PAINT (top right; 2 nM, P01-ATTO655 imager). Zoomed regions of DNA-PAINT imaging are shown (yellow boxes, i and ii). (c) LAT-Halo transfected cells were stained with anti-Halo-ATTO568 antibody to determine expressing cells (top left). Gaussian convolved images of positive cells imaged using DNA-PAINT (top right; 2 nM, P03-ATTO655 imager). Zoomed regions of DNA-PAINT imaging are shown (yellow boxes, i and ii). (d) CD3ζ-Halo transfected cells were stained with anti-Halo-ATTO568 antibody to determine expressing cells (top left). Gaussian convolved images of positive cells imaged using DNA-PAINT (top right; 2 nM, P03-ATTO655 imager). Zoomed regions (yellow boxes, i and ii). Scale bars for all larger images are 5 µm and for zoomed images 1 µm.

Figure 2.

Dual-tagPAINT imaging. CD3ζ knockout (KO) Jurkat cells were transfected with both CD3ζ-Halo and LAT-SNAP constructs. Cells were stained with both (a) anti-SNAP-ATTO488 and (b) anti-Halo-ATTO568 to identify cells expressing both constructs. Convolved images of Exchange-PAINT imaging of (c) LAT-SNAP (2 nM, P01-ATTO655 imager) and (d) CD3ζ-Halo (2 nM, P01-ATTO655 imager), which are overlaid in (e). Zoomed regions of the overlaid images are shown from regions i–iii (yellow boxes) in e (right). Scale bars are 5 µm for a–d and large panel in e, and 500 nm for i–iii.