Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

Abstract

Fö rster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra-and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.

Topography and motion of the acid-sensing ion channel intracellular domains

“…Fortunately, a novel GFP was described with a distinct chromophore and pKa of 3.4 52 . When combined with a relatively low pKa red fluorescent protein (TagRFP, pKa 3.8 53 ), this pairing should enable a FRET measurement that is relatively insensitive to the pH range examined while gaining the advantages of green/red FRET 39 . We therefore cloned the low pKa GFP into the N terminal position and the RFP into the C4 position (GFP-cA1 and cA1-RFP, Supplemental Figure 2) and tested the pH sensitivity of their fluorescence emission.…”

“…Fluorescent lifetime imaging (FLIM) was done as described previously 39 . Cells were transfected in either 35mm or 60 mm culture dishes and imaged using a water immersion 25x objective (XL Plan N, 1.05 NA) mounted on an Olympus IX61WI upright microscope.…”

Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness

“…The judicious choice of two monomeric FP scaffolds to act as downstream substitutes for APEX2 provides a straightforward and generic avenue for others in the field to fuse their recombinant antibodies of interest to mNeonGreen and mScarlet-I and interrogate other biological processes. It is noteworthy that mNeonGreen and mScarlet-I also make an ideal couple for Förster energy resonance transfer (FRET), 61 which should enable suitably paired recombinant antibody protein fusions to be developed into in vivo FRET probes and in vitro wash-less optical biosensors for rapid antigen detection.…”

Genetically Encoded Self-Assembling Iron Oxide Nanoparticles as a Possible Platform for Cancer-Cell Tracking

“…The latter, in particular, led to iron biomineralization and derived MR effects in the mammalian tumor cell line HepG2. As a fluorescent cargo protein, we chose mScarlet-I [ 39 , 40 ], targeted to the QtEnc FLAG nanoshell via QtSig encapsulation signal and modified by the DD domain (DD-mScarlet-I-QtSig). The latter ensures mScarlet-I fluorescence only within the encapsulin nanocompartment, while the fluorescent signal of transfected cells remains relatively high.…”