BODIPY Fluorogenic Agents for Specific Protein Labelling
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Solution Overview
Problem
Current methods for protein labelling in living cells are limited by non-specificity, sterical perturbation, and the need for UV or blue light excitation, which can cause photodamage and are not suitable for visualizing proteins in green and red channels of fluorescence microscopes.
Innovation Solution
Development of novel fluorogenic labelling agents based on a BODIPY fluorophore with a dimaleimide core that emit at long wavelengths, allowing for specific and site-directed labelling of proteins using the FlARe strategy, enabling visualization in green and red channels without photodamage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If fluorescent dyes bearing reactive functional groups like maleimides are used for protein labelling, then site-specific labelling can be achieved, but non-specific labelling of surface-exposed functional groups of many different proteins occurs
Solution Approach 1:
The fluorogenic probe is divided into two functional parts: a fluorophore for detection and a maleimide group for selective thiol labelling. This segmentation allows the probe to specifically target cysteine residues while maintaining detectability, resolving the contradiction between specific labelling and non-specific background labelling.
Solution Approach 2:
The invention changes the fluorescence parameter from always-on to conditional (fluorogenic). The maleimide group quenches fluorescence until it reacts with a thiol group, at which point fluorescence is restored. This parameter change enables specific detection only at the labelling site, eliminating non-specific background signals.
2Measurement precision
If fusion proteins are used to label proteins of interest, then detection capability is improved, but the significant size of fusion proteins alters the biological function of the target protein
Solution Approach 1:
The invention extracts only the essential detection function from large fusion proteins by using small-molecule fluorogenic probes. These probes can be administered systemically and will specifically bind to the target protein through the FlARe tag, providing detection capability without the steric burden of large fusion proteins.
Solution Approach 2:
A small peptide tag (FlARe tag) serves as an intermediary between the target protein and the fluorogenic probe. This intermediary is small enough not to disrupt protein function but provides a specific binding site for the probe, enabling detection without direct fusion of large fluorescent proteins.
3Measurement precision
If UV or blue light excitation is used for fluorescence microscopy, then detection sensitivity is improved, but photodamage occurs and visualization in green and red channels is not possible
Solution Approach 1:
The invention changes the excitation wavelength parameter from UV/blue to green/red wavelengths. The BODIPY fluorophore is specifically designed to absorb in the green region and emit in the red region, allowing excitation with lower-energy light that causes less photodamage while maintaining detection sensitivity.
4Manufacturing precision
If maleimide groups are used for thiol-specific labelling, then site-specific reaction with cysteine residues is achieved, but maleimides quench fluorescence through photoinduced electron transfer
Solution Approach 1:
The invention converts the harmful fluorescence quenching effect of the maleimide group into a beneficial fluorogenic switch. The maleimide quenches the fluorophore until it reacts with a thiol group, at which point the quenching is relieved and fluorescence is restored. This transforms a defect into a functional feature that enables specific detection.
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
The BODIPY-based fluorogenic agents provide high selectivity and specificity for protein labelling, reducing background reactivity and photodamage, enabling robust and efficient labelling of proteins in living cells with minimal disruption, and are compatible with standard fluorescence microscopy.
Implementation Method 1
BODIPY-based fluorogenic agents... that emit at long wavelengths
Implementation Method 2
fluorogenic labelling agents based on a BODIPY fluorophore... enabling visualization in green and red channels
Implementation Method 3
Maleimide groups have long been used in applications that exploit their propensity to react selectively with thiol groups, undergoing Michael addition reactions through their C2=C3 double bond
Implementation Method 4
Maleimides are also known to quench fluorescence, probably due to their participation in a photoinduced electron transfer (PeT), allowing non-radiative relaxation of the fluorophore's excited state
Implementation Method 5
The thiol addition reaction breaks the conjugation of the maleimide group, altering the energy levels of its molecular orbitals and removing its capacity to quench fluorescence
Data Source
AI summary
There are provided fluorogenic labelling agents comprising a dimaleimide core connected to a BODIPY-based fluorophore, processes for preparation thereof, and uses thereof for labelling and/or detection of specific protein targets. Fluorogenic labelling agents having an excitation wavelength that can be visualized in the green or red channels of fluorescence microscopes are provided. In some embodiments, fluorogenic labelling agents comprising a compound having the structure of Formula I or II, and salts thereof, are described.