Correlative Single-Molecule FRET and DNA-PAINT Imaging

• Published in: Nano letters Vol. 18; no. 7; pp. 4626 - 4630
• Main Authors: Deußner-Helfmann, Nina S, Auer, Alexander, Strauss, Maximilian T, Malkusch, Sebastian, Dietz, Marina S, Barth, Hans-Dieter, Jungmann, Ralf, Heilemann, Mike
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.07.2018
• Subjects: Binding Sites; DNA - chemistry; DNA - ultrastructure; Fluorescence Resonance Energy Transfer; Nanotechnology - methods; Oligonucleotides - chemistry; Single Molecule Imaging; DNA origami; multiplexing; Single-molecule FRET; DNA-PAINT; super-resolution microscopy
• ISSN: 1530-6984; 1530-6992
• DOI: 10.1021/acs.nanolett.8b02185

DNA-PAINT is an optical super-resolution microscopy method that can visualize nanoscale protein arrangements and provide spectrally unlimited multiplexing capabilities. However, current multiplexing implementations based on, for example, DNA exchange (such as Exchange-PAINT) achieves multitarget detection by sequential imaging, limiting throughput. Here, we combine DNA-PAINT with single-molecule FRET and use the FRET efficiency as parameter for multiplexed imaging with high specificity. We demonstrate correlated single-molecule FRET and super-resolution on DNA origami structures, which are equipped with binding sequences that are targeted by pairs of dye-labeled oligonucleotides generating the FRET signal. We futher extract FRET values from single binding sites that are spaced just ∼55 nm apart, demonstrating super-resolution FRET imaging. This combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging with low background and opens the door for accurate distance readout in the 1–10 nm range.