期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 142, 期 28, 页码 12069-12078出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.9b03418
关键词
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资金
- Human Frontier Science Program [0027/2013]
- Engineering and Physical Sciences Research Council of the UK [EP/N008235/1]
- Biotechnology and Biological Sciences Research Council [BB/P026508/1, BB/T007176/1]
- Royal Society University Research Fellowship [UF160152]
- European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (ERC-STG) [851667]
- EPSRC DTP studentship
- BBSRC [BB/T007176/1, BB/P026508/1] Funding Source: UKRI
- EPSRC [EP/N008235/1] Funding Source: UKRI
- European Research Council (ERC) [851667] Funding Source: European Research Council (ERC)
Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.
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