Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

Abstract

19 Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to 20 profile intra-and inter-molecular interactions. Through fusion of genetically encoded 21 fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, 22 receptor activation, and protein translocation among other biophysical phenomena.23 Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have 24 been developed. These proteins offer much improved physical properties compared to 25 previous generations of monomeric red FPs that should help facilitate more general 26 adoption of Green/Red FRET. Here we assess the ability of these two proteins, along 27 with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP 28 mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging 29 Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a 30 stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We 31 then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP 32 tandem construct. With these constructs we found that mScarlet-I and mCherry are able 33 to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, 34 mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these 35 same assays. We explore possible explanations for this poor performance but are 36 unable to definitively determine the cause, all though protein maturation seems to play a 37 role. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry 38 and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with mScarlet-I out 39 performing mCherry due it's higher brightness. 40 41 Introduction 42 Genetically encoded Fluorescent Proteins (FPs) have advanced basic and translational 43 biology immensely. Starting with the cloning of the Aequorea victoria green FP[1], a 44 massive and continual effort to expand the number of available FPs with a different 45 physical and spectral properties began. Currently, there is an enormous variety of FPs 46 at all parts of the visible spectrum, and even some parts of the ultraviolet and infrared 47 spectrums. These new proteins were either cloned from other organisms[2-4], or48 developed through evolution of already identified FPs [4][5][6][7][8][9][10][11]. This ever expanding 49 catalog of FPs has been reviewed by others [12][13][14], and new efforts and archives have 50 been created to organize this information, such as the FPbase database [15]. 51As the catalogue of FPs has expanded, so have the potential uses. Of particular 52 note is the use of FPs as biosensors which can measure signaling events, cell 53 metabolites, pH, voltage and more [16]. Many of these biosensors employ Förster 54 Resonance Energy Transfer (FRET) as part of their reporting mechanism, producing a 55 change in acceptor emission upon donor excitation when the quantity of interest 56 changes. FRET is also orientation and distance dep...

An improved molecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under pathological conditions

“…569 nm/em. 593 nm) 23 as an acceptor protein, and named the sensor CRONOS (crowding sensor with mNeonGreen and mScarlet-I) (Figure . 1B) 17 . Both mNG and mScaI work as a monomer with minimal self-association 2223 .…”

Development of a dual fluorescent reporter system in Clostridioides difficile reveals a division of labor between virulence and transmission gene expression

“…This autofluorescence has traditionally limited the utility of green-emitting reporters like GFP and phiLOV, an FMN-based reporter to genes that are highly expressed (39, 44). However, mNeonGreen ( mNG ) is a rapidly maturing, yellow-green monomeric fluorescent protein made by Branchiostoma lanceolatum that is ∼4-fold brighter than GFP (45, 46), so we hypothesized that it might be bright enough to use as a fluorescent transcriptional reporter. Another advantage of mNeonGreen is that its spectral properties are closer to YFP, which reduces some of the autofluorescent signal from C. difficile .…”

“…Another advantage of mNeonGreen is that its spectral properties are closer to YFP, which reduces some of the autofluorescent signal from C. difficile . Finally, mNeonGreen is spectrally compatible with mScarlet ( mSc ), a derivative of mCherry that is ∼3-fold brighter than mCherry (47), such that mNeonGreen and mScarlet can be used in dual transcriptional reporter and protein localization systems(46, 48).…”

“…Finally, mNeonGreen is spectrally compatible with mScarlet (mSc), a derivative of mCherry that is ~3-fold brighter than mCherry (47), such that mNeonGreen and mScarlet can be used in dual transcriptional reporter and protein localization systems (46,48).…”

Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

“…4A and S3B ). mSc is a bright monomeric red fluorescent protein (Bindels et al, 2017; McCullock et al, 2020). AcGFP1 is a monomeric green fluorescent protein, obtained from Aequorea coerulescens , with similar spectral properties to EGFP.…”