Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter

Abstract

Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ∼2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

“…Four positions near the phenol end of the chromophore-C159, M161, V196 and H198-were simultaneously mutated in FR to generate a ~7,000-member library (See Methods and Materials for details of library generation). We employed lifetime-based microfluidic flow cytometry 35 to select variants with increased brightness and values of  different from the parent (Figure 1). Sequencing of selected clones revealed that H198 and V196 were conserved but positions 159 and 161 showed sequence diversity (Supplementary Information S2: Table S2).…”

“…Library Targets: We targeted the positions 159, 161, 196 and 198 in FR (using FR numbering; Supplementary Information S1), expressed the libraries in yeast (Sacchromyces cerevisiae) and screened this library on a lifetime flow cytometer. 35 Screening revealed the presence of brighter clones with longer and shorter lifetime than parent FR (lifetime ~ 2.05 ns). We performed another two rounds of FACS enrichment on a BD FACSAria Fusion Cell Sorter to remove the dim/non-fluorescent clones.…”

“…Post-Microfluidic Sorting: After selection, the collected yeast cells were grown in liquid culture and then plated, and 25 clones with unique lifetimes were picked from these sorter-enriched libraries using a lifetime assisted plate based screen discussed in a previous work. 35 The FR site-directed (FSD) clones with unique DNA sequences were further characterized (Supplementary Information S2: Table S2).…”

“…34 FPs like FusionRed-M (FR-M) and mScarlet-I were developed to enhance cellular brightness over their molecularly brighter parents FR-1 and mScarlet, respectively. 20,35 Although there have been many efforts to improve the cellular and molecular brightness of FPs, performance in cell biology applications may still suffer from cellular toxicity, poor or over expression in certain cellular contexts and in cellulo oligomerization. 16,34 FusionRed (FR) was developed as a non-cytotoxic RFP that shows efficient and appropriate localization in multiple fusion constructs in different organisms.…”

“…We recently developed the FR-M variant (FR-L177M, numbered with respect to mCherry and FR-L175M numbered with respect to FR; See Supplementary Information S1: Table S1), which is 1.9-fold brighter than FR in HeLa cells. 35 In this study we use structure-guided engineering strategies to identify substitutions M42Q and C159V (sequence numbering with respect to FR) facing into the beta barrel that individually increase the  of FR. These mutations lead to the development of the bright triple mutant FR-MQV.…”