Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT

Abstract

DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.

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Fig. 1. Faster DNA-PAINT through overlapping sequence motifs.

(a) A single speed-optimized DNA-PAINT sequence exhibits a certain number of binding events (e.g. two per unit time). (b) Concatenation leads to a linear increase in binding frequency and thus imaging speed (top). However, sequence length increases linearly with the number of binding sites (e.g. from 7 nt for one to 35 nt for five binding sites). Using periodic sequence motifs enables overlapping binding sites, thus allowing shorter docking sequences (e.g. from 7 nt to only 19 nt, while maintaining a 5x speed increase). (c) Proof-of-concept using two 20-nm-grid DNA origami carrying 1xR1 and 5xR1 sequence extensions, respectively, shows an increase in the number of binding events (insets: zoom-ins from highlights in the overview, n = 2226). (d) Analysis of binding events for whole DNA origami structures from c shows an increase in events for the 5xR1 sequence motif (n = 2226). (e) Comparing the number of binding events for single docking strands featuring 1x, 3x, 5x, and 10x binding motifs shows a linear increase in the number of binding events (n = 3805). Centers depict the mean value of the Gaussian fit and error bars the standard deviation of the fit. Scale bars, 500 nm (c, overview), 20 nm (c, zoom-ins). Each experiment was repeated three times independently with similar results.

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

(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.