Targetable Red Ca2+ Indicators via HaloTag Mediator
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Solution Overview
Problem
Current fluorescent Ca2+ indicators face limitations in brightness, sensitivity, and spectral properties, particularly in the far-red region, and often compromise cellular performance when incorporating ligand moieties, which affects their ability to measure calcium dynamics effectively.
Innovation Solution
The design of small-molecule Ca2+ indicators with systematically explored fluorophore and chelator motifs, utilizing the HaloTag labeling system, results in bright and targetable red indicators that enhance fluorescence properties and compatibility with cellular imaging, especially in the primary cilium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If ligand moieties are incorporated into small-molecule indicators to enable targetability, then cell-type specificity is improved, but cellular performance is compromised
Solution Approach 1:
The patent uses HaloTag as an intermediary protein that binds specifically to the ligand-modified indicator molecule. The HaloTag-Ca2+ indicator complex then targets specific cell types through the HaloTag protein, while the indicator molecule itself maintains its optimized photophysical properties. This mediator approach allows targetability without directly modifying the indicator's core structure in a way that would compromise performance.
2Illumination intensity
If red-shifted fluorophores are used to extend imaging into far-red region, then spectral coverage is improved, but fluorescence quantum yield and brightness decrease
Solution Approach 1:
The patent systematically varies key parameters of the fluorophore structure, including the xanthene core substitution patterns, heteroatom positions, and conjugation extent. By changing these parameters, the patent achieves red-shifted emission wavelengths while maintaining higher fluorescence quantum yields compared to conventional far-red dyes. The optimized molecular structure reduces non-radiative decay pathways that typically limit quantum yield in the far-red region.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These indicators provide improved brightness, sensitivity, and spectral properties, enabling precise measurement of calcium dynamics, including in small subcellular locations like the primary cilium, with enhanced fluorescence quantum yield and selectivity over other divalent cations.
Implementation Method 1
fluorescent indicators... fluorescence emission maxima... fluorescence quantum yield
Implementation Method 2
Ca2+ chelation alters the electronic structure of the BAPTA, resulting in reduced PeT quenching and increased fluorescence
Implementation Method 3
In the Ca2+-free state, the fluorescence emission of 1 is quenched by photoinduced electron transfer (PeT) from the BAPTA moiety to the fluorophoric system
Data Source
AI summary
The presently-disclosed subject matter includes fluorescent indicators, including bright and targetable red Ca2+ indicators. The presently-disclosed subject matter also includes kits comprising the same as well as methods for using the same to detect a target substance. Fluorescent indicators of the presently-disclosed subject matter include a compound of the formula: