Primary Proteins:
  1. mRed7
  2. mRed7Q1
  3. mRed7Q1S1
  4. mRed7Q1S1BM
  5. mScarlet
  6. mScarlet-H
  7. mScarlet-I
    Secondary Proteins:
  1. mApple,
  2. mKate2,
  3. mRuby2,
  4. TagRFP-T

Bleach Measurements

Protein (state) t1/2 (s) Power Light Mode In Cell Fusion ˚C
TagRFP-T 337.0
mApple 4.8
mKate2 84.0
mRuby2 123.0
mScarlet 277.0 6.9 W/cm2 Arc-lamp Widefield H2A 37.0
mScarlet 161.0 1.35 W/cm2 Laser Spinning Disc Confocal H2A 37.0
mScarlet-I 225.0 6.9 W/cm2 Arc-lamp Widefield H2A 37.0
mScarlet-I 190.0 1.35 W/cm2 Laser Spinning Disc Confocal H2A 37.0
mScarlet-H 574.0 6.9 W/cm2 Arc-lamp Widefield H2A 37.0
mScarlet-H 368.0 1.35 W/cm2 Laser Spinning Disc Confocal H2A 37.0
A caution on interpretation of photostability measurements
Add photostability measurements

OSER Measurements

Protein % Normal Cells OSER/NE ratio Cell Type Temp (˚C)
mScarlet 80.0 (215 cells) 1.7 ± 0.3 (41 cells) U-2 OS -
mScarlet-I 76.0 (199 cells) 1.4 ± 0.2 (23 cells) U-2 OS -
mScarlet-H 82.0 (284 cells) 2.2 ± 0.2 (33 cells) U-2 OS -
dTomato 31.0 (258 cells) 5.7 ± 0.7 (110 cells) U-2 OS -
SGFP2 77.0 (104 cells) 1.7 ± 0.2 (40 cells) U-2 OS -
mCherry 80.0 (369 cells) 2.0 ± 0.3 (38 cells) U-2 OS -
mApple 87.0 (103 cells) 2.2 ± 0.2 (14 cells) U-2 OS -
mKate2 59.0 (102 cells) 3.6 ± 0.8 (42 cells) U-2 OS -
mRuby2 14.0 (113 cells) - U-2 OS -
mRuby3 14.0 (70 cells) - U-2 OS -

Excerpts

To start the development of a new monomeric RFP, a new synthetic template was designed based on the good maturating monomeric RFP mCherry... While designed to adopt the general fold of DsRed and mCherry (specifically concerning the conserved internal alpha helix), mRed7 harbors 30 amino acid changes as compared to mCherry and 44 as compared to DsRed.

The fluorescence lifetime of mScarlet is 3.9 ns, the highest value recorded to date for mRFPs, and it shows monoexponential decay. The quantum yield of mScarlet is 0.70; much higher than the quantum yield of other monomeric RFPs. The extinction coefficient of mScarlet is 100,300 M−1 cm−1, resulting in the highest calculated brightness in the mRFP spectral class, with a >3.5-fold increase relative to mCherry. Brightness analysis in mammalian cells confirmed that mScarlet is an extremely bright mRFP , especially when compared with mRuby2, mRuby3 and TagRFP-T.

The single amino acid substitution T74I found in mScarlet-I results in a marked maturation acceleration in cells, but at the cost of a moderate decrease in fluorescence quantum yield (0.54) and fluorescence lifetime (3.1 ns), although both values are still higher than those of all previously engineered bright mRFPs.

Screening for a more photostable mScarlet variant yielded mScarlet-H, with a single amino acid substitution (M164H) causing a >2-fold improvement in photostability, albeit accompanied by a serious decrease in quantum yield and fluorescence lifetime.