De novo Design of Near Infrared Fluorescent Proteins

Fluorescent proteins have revolutionized the field of molecular imaging, enabling the visualization of diverse biological processes. Biological imaging in the near-infrared (NIR, 800-1000 nm) and shortwave infrared (SWIR, 1000-2000 nm) ranges confers a number of photophysical advantages, but remains a challenge in practice due to the dearth of suitable fluorescent proteins for these optical windows. To address this limitation, we sought to develop a general approach integrating computational protein design with organic synthesis for creating long-wavelength fluorescent proteins from scratch. We used this approach to design a new class of proteins that specifically bind to synthetic merocyanine dyes, forming Schiff base covalent linkages, which when protonated activate fluorescence with large redshifts in both excitation and emission wavelengths. Our de novo designed far-red fluorescent protein MC7BP34 exhibited a brightness greater than that of existing protein probes in a similar wavelength range. The de novo designed NIR fluorescent protein MC9BP81 with excitation at 892 nm and emission extending into the SWIR range demonstrated higher contrast and imaging sensitivity in vivo than the previously developed iRFP720 (excitation 672 nm) owing to the reduced tissue autofluorescence at longer wavelengths. Our results are a substantial step towards genetically encodable probes in the SWIR region, and our approach lays the groundwork for the development of NIR biosensors for specific biological applications.