Enhanced incorporation of subnanometer tags into cellular proteins for fluorescence nanoscopy via optimized genetic code expansion.

Autor: Mihaila TS; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany., Bäte C; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany., Ostersehlt LM; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany., Pape JK; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany., Keller-Findeisen J; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany., Sahl SJ; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany., Hell SW; Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, 37077, Germany.; Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, 69120, Germany.
Jazyk: angličtina
Zdroj: Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2022 Jul 19; Vol. 119 (29), pp. e2201861119. Date of Electronic Publication: 2022 Jul 13.
DOI: 10.1073/pnas.2201861119
Abstrakt: With few-nanometer resolution recently achieved by a new generation of fluorescence nanoscopes (MINFLUX and MINSTED), the size of the tags used to label proteins will increasingly limit the ability to dissect nanoscopic biological structures. Bioorthogonal (click) chemical groups are powerful tools for the specific detection of biomolecules. Through the introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair (tRNA: transfer ribonucleic acid), genetic code expansion allows for the site-specific introduction of amino acids with "clickable" side chains into proteins of interest. Well-defined label positions and the subnanometer scale of the protein modification provide unique advantages over other labeling approaches for imaging at molecular-scale resolution. We report that, by pairing a new N-terminally optimized pyrrolysyl-tRNA synthetase (chPylRS 2020 ) with a previously engineered orthogonal tRNA, clickable amino acids are incorporated with improved efficiency into bacteria and into mammalian cells. The resulting enhanced genetic code expansion machinery was used to label β-actin in U2OS cell filopodia for MINFLUX imaging with minimal separation of fluorophores from the protein backbone. Selected data were found to be consistent with previously reported high-resolution information from cryoelectron tomography about the cross-sectional filament bundling architecture. Our study underscores the need for further improvements to the degree of labeling with minimal-offset methods in order to fully exploit molecular-scale optical three-dimensional resolution.
Databáze: MEDLINE
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