Figure 1: Structure and mechanism of fluorescent Zn²⁺ sensor platforms.

A) The FRET sensors ZapCY and ZnT72R and dual BRET/FRET sensors BLZinCh and BLCALWY are shown. The Zn²⁺-sensing mechanisms of BLZinCh and BLCALWY are identical to the FRET sensors eZinCh and eCALWY, respectively. The dual BRET/FRET sensors BLZinCh and BLCALWY contain NanoLuc (NL), whereas eZinCh and eCALWY do not. B) The single fluorescent protein (FP)-based Zn²⁺ sensors GZnP (top example), ZnGreen1 and ZIBG1 couple ZF1 and ZF2 derived from Zap1 with cpGFP, mTFP1 and GFP, respectively. In response to Zn²⁺, GZnP1 and ZIBG1 are fluorescence turn-on and ZnGreen1 is fluorescence turn-off. The single FP-based Zn²⁺ sensors ZnGreen2 and ZIBG2 (middle example) couple Zn²⁺ hook peptides derived from Rad50 with cpTFP1 and cpGFP, respectively. In response to Zn²⁺, ZIBG2 is fluorescence turn-on and ZnGreen2 is fluorescence turn-off. ZnRed (bottom example) couples Zap1’s ZF1 and ZF2 with mApple and is fluorescence turn-on. C) The ratiometric Zn²⁺ sensor mCherry-GZnP2 couples mCherry with GZnP2. GZnP2 fluorescence is directly modulated by Zn²⁺, and mCherry fluorescence is independent of Zn²⁺ changes. D) A chemigenetic Zn²⁺ sensor based on addition of a chloroalkane moiety (yellow box) to the small-molecule Zn²⁺ sensor ZP1 (ZP1-12Cl). ZP1-12Cl can interact with HaloTag (ZP1-HaloTag) or HaloTag-mCherry (ZP1-HaloTag-mCherry) to create targetable intensiometric or ratiometric versions, respectively. E) Multiplexing a Zn²⁺-dependent DNAzyme with genetically encoded fluorescent proteins was achieved by selectively-targeting mRNA encoding mClover2. In the presence of Zn²⁺, the Zn²⁺ DNAzyme binds and degrades mClover2 mRNA, but not mRNA encoding mRuby2, leading to an increased mRuby2/mClover2 ratio with increasing concentrations of Zn²⁺.