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

Agasti, Sarit S.; Wang, Yu; Schüder, Florian; Sukumar, Aishwarya; Jungmann, Ralf und Yin, Peng (2017): DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. In: Chemical Science, Bd. 8, Nr. 4: S. 3080-3091

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Physik
• Fakultätsübergreifende Einrichtungen: Center for NanoScience (CENS)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 530 Physik ; 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
• ISSN: 2041-6520
• Sprache: Englisch