Protein-specific localization of a rhodamine-based calcium-sensor in living cells

Abstract

A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.

Fluorogenic Protein Probes with Red and Near‐Infrared Emission for Genetically Targeted Imaging**

“…It has been used to imagep roteins with av ariety of fluorescent reporters (including some commercial ones) or to target biosensors such as Ca 2 + or Zn 2 + probes. [8][9][10][11] Even with an efficient targeting strategy,o ne still has to remove unbounddyes that may interfere with the experiments by emitting an off-target signal, which can be ac riticalf law in experiments in which the excess dye cannote asily be washed away.T he use of fluorogenicp robes that specifically light-up upon binding to their targetb ut are otherwise dark, circumvents this issue since unbound dyes will remain undetected, thus ensuringh igh-contrast images. [12][13][14] The fluorogenic targetingo fp rotein self-labelingtagsi st hus av ery useful strategy to image proteinsw ith high contrast and in no-wash conditions.…”

“…HaloTag has emerged as one of the most useful technology: as a modified haloalkane dehalogenase enzyme, it reacts selectively with a chemically simple halogenoalkane ligand to form a covalent complex with very fast reaction kinetics. It has been used to image proteins with a variety of fluorescent reporters (including some commercial ones) or to target biosensors such as Ca 2+ or Zn 2+ probes [8–11] …”

The Collective Power of Genetically Encoded Protein/Peptide Tags and Bioorthogonal Chemistry in Biological Fluorescence Imaging

“…HaloTag on the other hand could accept a much larger inventory of cargos for protein‐specific labelling than a single color, photophysically limited emitter as the sole produce of an FP. In addition to fluorescent labels (e. g., 14 , TMR Halo ligand in Figure 5b), synthetic indicators targeting Zn(II), [60] Ca(II), [61] Mg(II) (e. g., 15 , MGH in Figure 5b), [62] microviscosity, [63] or other substances or properties, bright and stable dyes that enable super‐resolution imaging, [64] and other functional molecules [65] have been delivered to a POI via the HaloTag. The second order rate constant of labeling is up to 3×10 6 M −1 s −1 , which is 100 times larger than the SNAP‐tag [57] .…”

“…Similar to other tags, HaloTag is useful in attaching novelty synthetic dyes, such as indicators for ions (a Mg(II) probe MGH is shown in Figure 5b) [60–62,68] or microviscosity, [63] photochemical agents such as sensitizers, and those useful in super‐resolution fluorescence microscopies, [64] to POIs that are found in tight subcellular locations. As a highlight of the applications of HaloTag, voltage‐sensitive fluorescent indicators have been delivered to the membranes of neurons via this technology.…”

Fluorescent and bioluminescent calcium indicators with tuneable colors and affinities

“…This approach has been used to create a number of localizable synthetic calcium indicators, e.g. BG3-Indo-1, 12 BOCA-1-BG 13 or RhoCa-Halo 14 and the far-red indicator JF646-BAPTA. 5,15 However, these probes have limited cell permeability and solubility, and furthermore require washing steps to remove unreacted probes, greatly limiting their applicability.…”