Cyan-Excitable Orange-Red Fluorescent Proteins for Reduced Phototoxicity
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Solution Overview
Problem
Current fluorescent and bioluminescent proteins for imaging in living cells and tissues face challenges such as low detection efficiency, toxicity from blue light excitation, and limited ability to simultaneously image two biological processes due to their excitation and emission properties, as well as inefficiencies in bioluminescence imaging through tissues.
Innovation Solution
Engineered orange-red fluorescent proteins excitable with cyan light, which have increased emission intensity and reduced toxicity, are developed for use in bioluminescent resonance energy transfer systems and fluorescence imaging, allowing for improved detection and reduced background interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If blue light (420-460 nm) is used to excite RFP for simultaneous dual-event imaging with CFP, then both CFP and RFP can be excited with the same light source, but phototoxicity and autofluorescence occur due to absorbance by flavin compounds
Solution Approach 1:
The patent engineers RFP variants (mPlum, mCherry, mNeptune, mCrimson) with modified amino acid sequences that shift their excitation spectra from blue (420-460 nm) to cyan-green (470-530 nm) wavelengths. This parameter change in excitation wavelength allows simultaneous dual-event imaging while avoiding the phototoxicity and autofluorescence caused by blue light absorption from flavin compounds.
2Ease of operation
If blue-excitable RFPs are used for simultaneous dual-event imaging with CFP, then single excitation source can be used, but quantum yields are at most 0.27 which is far lower than the >0.6 of commonly used GFPs and CFPs
Solution Approach 1:
The patent develops RFP variants (mPlum with QY=0.36, mCherry with QY=0.38, mNeptune with QY=0.41, mCrimson with QY=0.43) through amino acid sequence engineering that simultaneously improves quantum yield and shifts excitation wavelength to cyan-green range, resolving the contradiction between single excitation source capability and detection efficiency.
3Object-affected harmful factors
If FLuc2 is used for bioluminescence imaging in animals, then red photons (peak emission near 600 nm) are produced that can transmit through tissue, but catalysis rates are very low at 1.6 reactions per second
Solution Approach 1:
The patent merges the high catalysis rate of NanoLuc (100× faster than FLuc2) with the red-shifted emission properties by creating fusion proteins where NanoLuc is genetically fused to red fluorescent protein acceptors (mPlum, mCherry, mNeptune, mCrimson). This combines the high productivity of NanoLuc with the tissue-penetrating capability of red emission through BRET energy transfer.
Solution Approach 2:
The patent uses red fluorescent proteins as intermediary molecules in BRET systems, where they accept energy from NanoLuc and re-emit at red wavelengths. This mediator approach allows the high catalysis rate of NanoLuc to be converted into red photon emission that transmits through tissue effectively.
4Productivity
If NanoLuc is used for bioluminescence imaging, then catalysis rate is 100× faster than FLuc2, but emission peaks at 460 nm which is even bluer and has not been demonstrated to improve detectability in vivo
Solution Approach 1:
The patent employs red fluorescent proteins (mPlum, mCherry, mNeptune, mCrimson) as intermediary molecules in BRET fusion proteins. NanoLuc generates blue light at 460 nm with high catalysis rate, the red fluorescent protein acceptor absorbs this energy and re-emits at red wavelengths (560-620 nm) that transmit through tissue effectively, thus converting high-productivity blue emission into tissue-penetrating red emission.
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 proteins enhance imaging capabilities by increasing emission intensity in the orange-red spectrum, reducing phototoxicity, and enabling more efficient bioluminescence imaging through tissues, improving the sensitivity and accuracy of dual-event imaging.
Implementation Method 1
engineered orange-red fluorescent proteins excitable with cyan light having increased emission intensity
Implementation Method 2
BLI refers to imaging of light produced by luciferase enzymes by oxidation of chemical substrates
Implementation Method 3
bioluminescent resonance energy transfer systems
Data Source
AI summary
Engineered orange-red fluorescent proteins with enhanced fluorescent properties, obtained by mutagenesis of mNeptune2, are disclosed. In particular, the invention relates to engineered orange-red fluorescent proteins excitable with cyan light having increased emission intensity and their use in bioluminescent resonance energy transfer systems and fluorescence and bioluminescence imaging.