Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

Abstract

Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time.

Engineering BRET-Sensor Proteins

“…The feasibility of the BRET/FRET approach was explored by fusing NanoLuc to the donor Cerulean domain of two previously developed FRET-sensors for intracellular Zn 2+ imaging, the eCALWY and eZinCh-2 sensors (Aper, Dierickx, & Merkx, 2016). In these sensors, bioluminescent excitation of Cerulean takes place via BRET, and this energy is subsequently transferred to the yellow acceptor Citrine via FRET (Figs.…”

“…(B and C) Bioluminescence (B: normalized to emission at 455 nm) and fluorescence (C: normalized to emission at 513 nm; excitation at 430 nm) emission spectra of BLCALWY-1 in Zn 2+ -depleted (blue, straight) and Zn 2+ -saturated states (red, dashed). Adapted with permission from Aper, S. J., Dierickx, P., & Merkx, M. (2016). Dual readout BRET/FRET sensors for measuring intracellular zinc.…”

Chelating Rotaxane Ligands as Fluorescent Sensors for Metal Ions

“…In particular, the development of small‐molecule sensors2, 3 for the detection and quantification of Zn 2+ in vivo has attracted considerable recent attention due to the spectroscopically silent nature of the d 10 Zn 2+ ion, combined with the recognition that changes in zinc homeostasis are associated with high‐morbidity diseases such as Alzheimer's disease,4 Type II diabetes,5 and age‐related macular degeneration 6…”

Chelating Rotaxane Ligands as Fluorescent Sensors for Metal Ions

“…[1] In particular, the development of small-molecule sensors [2,3] fort he detection and quantification of Zn 2+ in vivo has attracted considerable recent attention due to the spectroscopically silent nature of the d 10 Zn 2+ ion, combined with the recognition that changes in zinc homeostasis are associated with high-morbidity diseases such as Alzheimersd isease, [4] Ty pe II diabetes, [5] and age-related macular degeneration.…”

“…We synthesized rotaxane 4 [18] in excellent yield (88 %) using the active-template [19] Cu-mediated alkyne-azide cycloaddition (AT-CuAAC) reaction [20,21] between azido fluorophore 3 and acetylene 2 in the presence of readily available bipyridine macrocycle 1 [22] and [Cu(MeCN) 4 ]PF 6 .Addition of one equivalent of Zn(ClO 4 ) 2 ·6 H 2 Ot oasolution of 4 in CD 3 CN resulted in large changes in the 1 HNMR spectrum (see Figure S39 in the Supporting Information), which is consistent with the binding of the metal ion into the cavity of the macrocycle,c onfirming that 4 is capable of acting as al igand. Theb ehavior of rotaxane 4 as am etal-responsive sensor for Zn 2+ was investigated by fluorescence titration with Zn(ClO 4 ) 2 ·6 H 2 O. Portion-wise addition of Zn 2+ to rotaxane 4 in MeCN led to monotonic quenching of the emission at 560 nm [23] that plateaued once af ull equivalent had been added ( Figure 1a).…”