Third‐Generation Covalent TMP‐Tag for Fast Labeling and Multiplexed Imaging of Cellular Proteins

Abstract

Self-labeling protein tags can introduce advanced molecular motifs to specific cellular proteins.Here we introduce the third-generation covalent TMPtag (TMP-tag3) and showcase its comparison with HaloTag and SNAP-tag. TMP-tag3 is based on a proximity-induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP-tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18-kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal NÀ C termini of eDHFR also enable facile insertion into various protein loops. TMP-tag3, HaloTag, and SNAP-tag are orthogonal to each other, collectively forming a toolbox for multiplexed live-cell imaging of cellular proteins under fluorescence nanoscopy.

Gentle rhodamines for live-cell fluorescence microscopy

“…To meet the growing demands of fluorescence nanoscopy, the photophysical properties of dyes such as brightness 29,30 , photostability [31][32][33][34] , and blinking 35,36 have been further engineered. Most importantly, rhodamine can be used in conjunction with a variety of labeling techniques for instance self-labeling protein tags (HaloTag 37 , SNAP-tag 38,39 , and TMP-tag 40 ), tetrazine for click chemistry 41 , biomolecular ligands 25 and specific ligands for organelles 25,26 . This synergistic development has also consolidated rhodamine as a mainstream tool for bioimaging.…”