Datensatz

Zusammenfassung

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.