Fluorogenic Amino Acids for Low-Background Biomolecular Labeling
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Solution Overview
Problem
Current biomolecular labeling approaches face challenges due to high background signal from unbound probes, which limits the effectiveness of imaging and detection of biomolecules and cells, particularly due to the substantial size and slow kinetics of existing fluorogenic protein labels.
Innovation Solution
Development of new fluorogenic amino acids (FgAAs) based on the fluorescent molecular rotor concept, which are designed to activate and fluoresce only when spatially constrained within biomolecules or cells, minimizing background noise and allowing for real-time monitoring with minimal perturbation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If small fluorogenic probes are used to reduce background signal, then background noise is minimized, but the probes may have slow incorporation kinetics or require specific maturation conditions
Solution Approach 1:
The patent modifies the chemical structure of fluorogenic probes by incorporating amino acid derivatives with specific molecular rotor characteristics. These structural parameter changes enable the probes to maintain small size for low background signal while achieving rapid incorporation kinetics through optimized chemical reactivity and enzymatic recognition patterns.
Solution Approach 2:
The invention uses amino acid analogs that mimic natural substrate structures, allowing them to be recognized and incorporated by cellular enzymes without requiring complex maturation processes. This copying of natural biomolecular patterns enables rapid incorporation while maintaining the fluorogenic properties needed for low-background imaging.
2Illumination intensity
If fluorogenic protein labels are used for tracking proteins in vivo, then real-time imaging is achieved, but the substantial size and slow maturation kinetics limit the effectiveness
Solution Approach 1:
The patent extracts the essential fluorogenic function from large protein labels and implements it using small molecule amino acid derivatives. This extraction eliminates the substantial size and slow maturation requirements of protein-based fluorophores while retaining the ability to produce fluorescence signals for real-time imaging of biomolecules.
Solution Approach 2:
The invention employs small, rapidly metabolized amino acid derivatives that can be quickly incorporated and cleared, replacing persistent protein labels. These small molecular weight probes provide transient fluorescence signaling without the long-term presence and complexity of protein-based labels, enabling cleaner temporal resolution in imaging experiments.
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 FgAAs enable efficient and precise labeling and detection of biomolecules and cells by activating fluorescence upon incorporation, reducing background noise and providing clear imaging with minimal disruption to cellular processes.
Implementation Method 1
small probes that activate, or 'turn on,' only upon incorporation into biomolecules or macromolecules
Implementation Method 2
Sometimes known as fluorescent molecular rotors (FMRS), these fluorophores have an emission intensity that is sensitive to the ability of the environment the fluorophore is in to restrict bond rotation
Data Source
AI summary
Provided herein are compounds (i.e., fluorogenic amino acids (FgAAs), e.g., compounds of Formulae (I), (II), (III), and (IV)) that can be used in fluorescent labeling of biomolecules (e.g., proteins) and/or cells. Also described herein are methods of labeling and detecting biomolecules and/or cells by incorporating the FgAA compounds described herein into the biomolecules and/or cells (e.g., by enzymatic incorporation). Also provided herein are biomolecules, cells, compositions, and kits comprising the FgAA compounds described herein.