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Chem Sci. 2017 Apr 1;8(4):3080-3091. doi: 10.1039/c6sc05420j. Epub 2017 Jan 30.

DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging.

Agasti SS1,2,3, Wang Y1,2,4, Schueder F1,2,5,6, Sukumar A1, Jungmann R1,2,5,6, Yin P1,2.

Author information

1
Wyss Institute for Biologically Inspired Engineering , Harvard University , Boston , Massachusetts , USA . Email: py@hms.harvard.edu ; Email: jungmann@biochem.mpg.de.
2
Department of Systems Biology , Harvard Medical School , Boston , Massachusetts , USA.
3
New Chemistry Unit and Chemistry & Physics of Materials Unit , Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) , Bangalore , India.
4
Program of Biological and Biomedical Science , Harvard Medical School , Boston , Massachusetts , USA.
5
Department of Physics and Center for Nanoscience , Ludwig Maximilian University , 80539 Munich , Germany.
6
Max Planck Institute of Biochemistry , 82152 Martinsried near Munich , Germany.

Abstract

Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled 'imager' strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells.

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