Engineering a Bright Near-Infrared Fluorescent Protein by Screening a Comprehensive Phenotypic Landscape
Devon L Kulhanek1, Qiyao Wei2, Jared Head3
1Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX.
Biorxiv : the Preprint Server for Biology
|December 15, 2025
Summary
Researchers enhanced near-infrared fluorescent proteins (niRFPs) using deep mutational scanning. Novel mutations in small ultra red fluorescent protein (smURFP) significantly boosted in vivo fluorescence, yielding the brightest niRFP yet reported.
Area of Science:
- Biophysics
- Molecular Biology
- Biotechnology
Background:
- Near-infrared fluorescent proteins (niRFPs) are crucial for deep-tissue imaging due to low background and invasiveness.
- Small ultra red fluorescent protein (smURFP) is a leading niRFP, but its characteristics can be further improved.
Purpose of the Study:
- To systematically explore the mutational landscape of smURFP.
- To identify novel smURFP mutations that enhance in vivo fluorescence.
- To characterize the biophysical properties of improved smURFP variants.
Main Methods:
- Deep mutational scanning (DMS) library construction for smURFP.
- Analysis of codon substitutions, insertions, and deletions via relative abundance changes.
- Biophysical and photophysical characterization of mutant niRFP sequences.
Main Results:
- Identified seven single-codon substitutions that increase in vivo fluorescence threefold in E. coli when combined.
- Two novel smURFP variants exhibit higher molecular brightness than wild-type.
- Developed the brightest near-infrared fluorescent protein reported to date.
Conclusions:
- Deep mutational scanning is effective for optimizing niRFP brightness.
- The identified mutations significantly enhance smURFP performance for in vivo imaging.
- These improved niRFPs represent valuable tools for advanced biological research.
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