DNA point accumulation in nanoscale topography (DNA-PAINT) increases the resolution and multiplexing capabilities of super-resolution imaging, but cellular DNA interferes with DNA–DNA hybridization between target and probe in the nucleus. Here, we introduce left-handed DNA (L-DNA) oligomers that do not hybridize to natural right-handed DNA (R-DNA) and demonstrate that L-DNA-PAINT has the same specificity and multiplexing capability as R-DNA-PAINT, but improves the imaging of nuclear targets by substantially reducing background signal.
The microscopy data that support the findings of this study will be available from the corresponding author upon reasonable request, due to storage limitations. Source data are provided with this paper.
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We thank all members of the Ewers laboratory for helpful discussions. This study was supported by the ERC (starting grant no. Stg 677673 awarded to B.B.K.) and Deutsche Forschungsgemeinschaft (project no. 278001972 – TRR 186 and SFB 958 to H.E.).
The authors declare no competing interests.
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a, Titration experiment of L-DNA imagers (LP3) on L-DNA binders (LB3) and R-DNA imagers (P3) on R-DNA binders (LB3). Single molecule images are 5 µm x 5 µm. b, Plot of total number of localizations per frame in the titration experiment was quantified for L- and R-DNA and found to be very similar. No single molecules were detected for 100 nM of imagers and as such excluded from these graphs. Boxplots (centre is the median; box are the IQR; whiskers are 1.5 × IQR; mean values are represented by open squares; X represent the 1% and 99% percentiles). c, Fluorescence on-times as a result of imager-binder hybridization were extracted. The histogram represents the distribution of on-times for R-DNA and L-DNA and was fitted with a single exponential, yielding a half time of 230 ± 20 ms and 220 ± 10 ms, respectively. Data were gathered from 3 independent measurements. d, The time between subsequent imager-binder hybridization event is defined as the off-time. For 1 nM of imager, the off-times were found to be 61 ± 4 s and 59 ± 6 s for R- and L-DNA, respectively. Data were gathered from 3 independent measurements.
P1 and LP1 as well as P12 and LP12, that are coupled to an Atto655 fluorophore, show a significant difference in the ratio of localization density in the nucleus over the cytoplasm. No significant difference was detected between Atto655 or Cy3B as fluorescent dyes. P-values are obtained from two-sided paired t-Tests 16 cells stained with Cy3B-P1 and 33 cells for P1 and LP1 in 3 independent experiments and 20 cells in 2 independent measurements for P13 and LP13. Boxplots (center is the median; box are the IQR; whiskers are 1.5 × IQR; mean values are represented by open squares).
Extended Data Fig. 3 Plot of relative localization density of R-DNA over L-DNA for multiplexing experiments initiated with either R-DNA or L-DNA.
Cells were imaged with sequence-identical R-DNA and L-DNA imagers, sequentially. No significant difference in the relative localization densities of R-DNA over L-DNA was found when the image sequence was initiated with R-DNA (pink) or L-DNA (blue). P-value was obtained by two-tailed t-test of 61 and 52 cells for R-DNA and L-DNA initiation, respectively, in 17 independent experiments. Boxplots (centre is the median; box are the IQR; whiskers are 1.5 × IQR; mean values are represented by open squares).
FISH was performed on HEK293T cells harboring two copies of an integrated HHV-6A genome as described previously18. The two viral integration sites were detected in the cell nuclei using HHV-6A specific probes by FISH, matching the detection using the DNA-PAINT approach, and could be successfully reproduced in 30 independent FISH experiments. Scale bar is 2 µm.
R-DNA imagers strongly localize to the nucleus of FISH samples, both without DNA-FISH probes and with DNA-FISH probes coupled to complementary R-DNA binders. L-DNA imagers are not detected in the FISH samples omitting the FISH probes, but show specific loci where the samples are labeled with DNA-FISH probes. Scale bars are 5 µm. All images are reconstructed from 5000 frames and 2 nM imager concentrations.
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Geertsema, H.J., Aimola, G., Fabricius, V. et al. Left-handed DNA-PAINT for improved super-resolution imaging in the nucleus.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-020-00753-y