Isomeric Tuning Yields Bright and Targetable Red Ca²⁺ Indicators

Abstract

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca 2+ indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a farred targetable indicator to visualize Ca 2+ fluxes in the primary cilium.

Enhancing biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

“…Isomeric mixtures of 5/6-carboxyrhodamines and fluoresceins are usually obtained by the classical condensation process of phthalic anhydrides and 3-dimethylaminophenol or resorcinol respectively 17, 18 . But condensation between resorcinol and 1,2,3-benzenetricarboxylic acid or its homologs yields solely 7’-carboxyfluorescein and not 4’-carboxyfluorescein 19 . Another widely applied and regioselective synthesis strategy, which can be applied in the synthesis of 4’-carboxyrhodamines, involves addition of aryl-lithium species to the 3,6-substituted-xanthone 20 .We used formation of di- tert -butyl ester 1 for the protection of 3-bromophtalic acid carboxyl groups for the halogen-lithium exchange reaction (Fig.…”

Advanced Near‐Infrared Light for Monitoring and Modulating the Spatiotemporal Dynamics of Cell Functions in Living Systems

“…Thus, the hybrid voltage indicators have attracted great attentions among researchers due to their unique advantages to combine the merits of genetically encoded proteins and small molecule dyes. [ 113–115 ] For example, Abdelfattah and colleagues recently developed a hybrid voltage indicator by combining the rhodopsin and small molecule JF525 (Voltron525). [ 113 ] In this study, the HaloTag was fused to the rhodopsin.…”

Tetrazine as a general phototrigger to turn on fluorophores

“…Photoactivatable uorophores, also called turn-on uorophores, [1][2][3][4][5][6][7] have numerous advanced biological applications, including detection and release of ions [8][9][10][11] and metabolites, [12][13][14][15] monitoring of enzyme activity, [16][17][18][19] and multiple types of specialized microscopy. 7,[20][21][22][23][24][25] Most of these photoactivatable uorophores are masked by a reactive "cage" group intimately attached to the uorophore to alter its photophysical properties or to serve as an energy-transfer agent for use in a uorescence resonance energy transfer (FRET) pair.…”