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LOCALIZATION MICROSCOPY; RESOLUTION; BINDINGSingle-molecule FRET; DNA-PAINT; super-resolution microscopy; DNA
origami; multiplexing;
Abstract:
Correlating DNA-PAINT (point accumulation for imaging in nanoscale topography) and single-molecule FRET (Forster resonance energy transfer) enables the multiplexed detection with sub-diffraction optical resolution. We designed pairs of short oligonucleotides, labeled with donor and acceptor fluorophores with various distances generating different FRET efficiencies. The strands can transiently bind to a target docking strand, simultaneous binding of both strands results in FRET signals which yield a super-resolved image via DNA-PAINT imaging. We demonstrate FRET-PAINT by designing and imaging DNA origami, which is a useful tool to establish super-resolution methods. The DNA origami structures were equipped with three target binding sites spaced by 55 nm, a sub-diffraction limited distance, however ensuring that no FRET between the target sites occurs. We resolved the individual binding sites in the donor and acceptor channels, and in addition extracted the FRET efficiency for each site in single and mixed populations. The combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging in conjunction with distance-sensitive readout in the 1 to 10 nm range.
