Clickable HaloTag ligands for live cell labeling and imaging.

Self-labeling protein (SLP) tags, such as HaloTag, have gained considerable interest as advanced tools for live cell labeling. However, the chloroalkane-based substrates that can be directly used for protein labeling are limited. Here, we report two bioorthogonal small molecule linkers, chloroalkane-tetrazine (CA-Tz) and chloroalkane-azide (CA-N3), which can penetrate cell membranes and facilitate click chemistry-based labeling in live cells. We compare their labeling capability using two clickable silicon rhodamine dyes (SiR-PEG3-TCO and SiR-PEG4-DBCO). Confocal imaging results demonstrate that using CA-Tz and SiR-PEG3-TCO dye exhibits superior intracellular labeling with low nonspecific signals. We subsequently compared the photostability of SiR dyes with that of green fluorescent proteins (mEmerald). Total internal reflection fluorescence (TIRF) imaging indicates that SiR dyes exhibit superior photostability under identical excitation conditions, making them suitable for long-term cell imaging. Furthermore, SiR dyes labeling also shows high structure retention for the fourth-order super-resolution optical fluctuation imaging (SOFI) compared to fluorescent proteins. This study presents clickable HaloTag linkers as effective tools for live cell labeling and imaging, highlighting the high-quality labeling of chloroalkane linkers and clickable dyes for live cell imaging. [ABSTRACT FROM AUTHOR]