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

Mihaila, T. S., Bäte, C., Ostersehlt, L. M., Pape, J. K., Keller-Findeisen, J., Sahl, S. J., et al. (2022). Enhanced incorporation of subnanometer tags into cellular proteins for fluorescence nanoscopy via optimized genetic code expansion. Proceedings of the National Academy of Sciences of the USA, 119(29): e2201861119. doi:10.1073/pnas.2201861119.

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 Creators:
Mihaila, T. S.1, Author           
Bäte, C.1, Author           
Ostersehlt, L. M.1, Author                 
Pape, J. K.1, Author           
Keller-Findeisen, J.1, Author           
Sahl, S. J.1, Author           
Hell, S. W.1, Author                 
Affiliations:
1Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350048              

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Free keywords: nanoscopy; genetic code expansion; fluorescence; click chemistry; actin
 Abstract: 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. Welldefined
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
(chPylRS2020) 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 molecularscale
optical three-dimensional resolution.

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Language(s): eng - English
 Dates: 2022-07-13
 Publication Status: Published online
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1073/pnas.2201861119
 Degree: -

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Project name : T.S.M. was the recipient of a 2019–2020 Fulbright Scholarship.
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Title: Proceedings of the National Academy of Sciences of the USA
Source Genre: Journal
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Pages: 9 Volume / Issue: 119 (29) Sequence Number: e2201861119 Start / End Page: - Identifier: ISSN: 0027-8424
ISSN: 1091-6490