mRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis

Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.