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. Author manuscript; available in PMC: 2021 Mar 23.
Published in final edited form as: Nat Methods. 2020 Jun 29;17(8):789–791. doi: 10.1038/s41592-020-0869-x

Fig. 2. Multiplexing with concatenated speed-optimized motifs.

Fig. 2

(a) Designing six orthogonal binding motifs enables speed-optimized multiplexing in Exchange-PAINT experiments. (b) Proof-of-concept experiments using 20-nm-grid DNA origami with six orthogonal sequence motifs resolved in 5 minutes per round. (c) Exemplary structures from experiment depicted in b. (d) Imaging of 5-nm-features on ‘MPI’ origami structures carrying 5xR1 binding sites. 5-nm features are well-resolved, confirming that extending the length of docking sites to 19 nt does not impair spatial resolution. (e) EGFP-Nup96 proteins labeled with nanobodies that are site-specifically coupled to 5xR1 docking sites. DNA-PAINT imaging shows specific and efficient labeling of nuclear pore complexes with high quality and spatial resolution. (f) Cellular proof-of-concept study using four orthogonal overlapping sequence motifs targeting cell surface receptors (EGFR, Her2, ErbB3, and c-Met) using a combination of DNA-conjugated primary nanobodies (against EGFR-tagRFP and Her2-GFP) and secondary nanobodies against primary antibodies (ErbB3 and c-Met). (g) Four-plex Exchange-PAINT with improved docking site sequences enables single-protein resolution, revealing presumably homo- and heterodimers of Receptor Tyrosine Kinases (RTKs), highlighted by c-Met-EGFR- (i), Her2-ErbB3- (ii), EGFR-Her2-heterodimes, and EGFR homodimers (iv) with distances measures between 16 and 26 nm using a cross-sectional histogram analysis. Scale bars, 200 nm (b), 40 nm (c), 20 nm (d, e, g), and 200 nm (f). Each experiment was repeated three times independently with similar results.