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, 6 (12), 191268
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tagPAINT: Covalent Labelling of Genetically Encoded Protein Tags for DNA-PAINT Imaging

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tagPAINT: Covalent Labelling of Genetically Encoded Protein Tags for DNA-PAINT Imaging

Daniel J Nieves et al. R Soc Open Sci.

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.

Conflict of interest statement

We declare we have no competing interests.

Figures

Figure 1.
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.
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 ad and large panel in e, and 500 nm for i–iii.

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