MScarlet, a novel high quantum yield (71%) monomeric red fluorescent protein with enhanced properties for FRET ‐ and super resolution microscopy

Abstract

mScarlet, a novel red fluorescent protein was generated from a synthetic template based on a consensus amino acid sequence derived from naturally occurring red fluorescent proteins and purple chromoproteins and on consensus monomerization mutations. The encoded synthetic red fluorescent protein was optimized by molecular evolution through site directed and random mutagenesis. Improved variants were selected by quantitative multimode screening for increased fluorescence lifetime, increased photo stability, increased quantum yield and for increased chromophore maturation. Very bright variants were obtained with high fluorescence lifetimes up to 3.8 ns, quantum yields >70 % and complete maturation. The monomeric status of the variants was confirmed by OSER analysis and with a‐tubulin fusions. The brightness of mScarlet is >2‐ fold increased as compared to bright red fluorescent proteins such as mCherry, mRuby2 and tagRFP‐T as was analyzed with quantitative (single plasmid with viral 2A sequence) coexpression with mTurquoise2 in mammalian cells. mScarlet can be used as a bright red fluorescent fusion tag for staining various subcellular structures in live cells. Because of their efficient maturation and high quantum yield, mScarlet vastly outperforms existing monomeric red fluorescent proteins in ratiometric FRET‐microscopy applications due to seriously enhanced sensitized emission and lack of photochromism (figure 1). During evolution mScarlet variants with substantially altered spectroscopic properties were generated including fluorescence lifetime variants, photo labile variants and strongly spectrally shifted variants. Besides looking at generally optimized properties such as increased lifetime, maturation and brightness, some of these variants were rescreened for blinking properties. An mScarlet variant (7Q2BMs‐K) was identified that produced high spontaneous blinking upon illumination at 561 nm. This variant was fused to life‐act and co‐expressed with mVenus‐Lifeact. With 488 and 561 nm excitation, dual life cell single molecule localization microscopy produced perfectly colocalized yellow and red actin structures in living cells (figure 2), demonstrating the usefulness of the novel red fluorescent proteins for life cell super‐resolution microscopy.