Engineered HaloTag variants for fluorescence lifetime multiplexing

Abstract

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

A Locally Activatable Sensor for Robust Quantification of Organellar Glutathione

“…MD simulations using the spirocyclic form as starting point suggest that the side chain of residue F144 must rotate to allow for the ligand to occupy the alternative pocket and thus switch to its zwitterionic form (Figure S4, Movie S1). Although mutation of this residue does not seem to increase the brightness of SiR-carboxylate dyes 17 , this residue could be important to create HT mutants that induce larger fluorogenicity in other SiR-based spirocyclic dyes. Using MD simulations starting from the zwitterionic structure, we found that the structure rapidly relaxes to a minimum in which the cyanamide forms a persistent hydrogen bond with the backbone carbonyl of residue P243 (Extended Data Figure 2).…”

Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes

“… 13 − 15 However, they have only found limited use in fluorescence lifetime multiplexing, and their fluorescence lifetimes are often not characterized. 9 , 10 , 16 , 17 Proof-of-concept studies were restricted to fixed cell applications 9 , 10 and/or used probes with both differences in fluorescence lifetime and emission spectrum. 9 , 10 , 16 We recently demonstrated the combined use of synthetic probes and self-labeling protein tags for fluorescence lifetime multiplexing.…”

“… 9 , 10 , 16 We recently demonstrated the combined use of synthetic probes and self-labeling protein tags for fluorescence lifetime multiplexing. 17 However, our study was limited to only four probes and thus only partially exploited the potential of synthetic probes for fluorescence lifetime multiplexing.…”

“…We previously demonstrated the use of SLP-tags in fluorescence lifetime multiplexing and characterized the average fluorescence lifetime of different HaloTag7 fusion proteins conjugated to MaP555-CA (2.4 ns) or MaP618-CA (3.1 ns). 17 We hence performed a similar characterization for SNAP-tag localized to different subcellular localizations and labeled with MaP555-BG in living U-2 OS cells (e.g., histone 2B, Lamin B1, Tomm20 etc. ; Supporting Table S4 ).…”

“…In addition, we were able to perform a time course experiment by repeated FLIM measurements allowing us to follow the movement of all six species over 6 min ( Supporting Figure S12 ). Furthermore, by combining the SNAP-tag with two HaloTag variants (HaloTag7 and HaloTag11 (decreased fluorescence lifetime)), 17 eight species could be imaged. We labeled cells expressing HaloTag7 in the ER, HaloTag11 in the Golgi, and SNAP-tag at the plasma membrane with LysoTracker-Green, MitoTracker-Green, MaP555-BG, SPY555-Actin, MaP618-CA, SPY700-DNA, and SiR700-Tubulin and acquired all four spectral channels ( Figure 3 B, Supporting Figure S13 ).…”