(2016). Nature Methods, 14(1) , 53-56. doi: 10.1038/nmeth.4074. Article Pubmed
| 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 |
| 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 | - |
| 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 | - |
| mCherry | 80.0 (369 cells) | 2.0 ± 0.3 (38 cells) | U-2 OS | - |
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.
Negligible photochromic behavior was measured for the mScarlet variants, while TagRFP-T, mRuby2, mRuby3 and mApple showed 15%, 19%, 41%, and 51% photochromic behavior, respectively. Hence, extreme care must be taken when using the latter four RFP variants as acceptors in FRET studies, since a photochromic effect is easily confused with a changed FRET state, especially if one considers that the typical FRET contrast in many sensors is in the range of only 5–20%. The photochromic behavior can also interfere with characterization of FPs, like determination of photostability (Supplementary Fig. 8) or brightness (Supplementary Fig. 5l).
Negligible photochromic behavior was measured for the mScarlet variants, while TagRFP-T, mRuby2, mRuby3 and mApple showed 15%, 19%, 41%, and 51% photochromic behavior, respectively. Hence, extreme care must be taken when using the latter four RFP variants as acceptors in FRET studies, since a photochromic effect is easily confused with a changed FRET state, especially if one considers that the typical FRET contrast in many sensors is in the range of only 5–20%. The photochromic behavior can also interfere with characterization of FPs, like determination of photostability (Supplementary Fig. 8) or brightness (Supplementary Fig. 5l).
Negligible photochromic behavior was measured for the mScarlet variants, while TagRFP-T, mRuby2, mRuby3 and mApple showed 15%, 19%, 41%, and 51% photochromic behavior, respectively. Hence, extreme care must be taken when using the latter four RFP variants as acceptors in FRET studies, since a photochromic effect is easily confused with a changed FRET state, especially if one considers that the typical FRET contrast in many sensors is in the range of only 5–20%. The photochromic behavior can also interfere with characterization of FPs, like determination of photostability (Supplementary Fig. 8) or brightness (Supplementary Fig. 5l).
Negligible photochromic behavior was measured for the mScarlet variants, while TagRFP-T, mRuby2, mRuby3 and mApple showed 15%, 19%, 41%, and 51% photochromic behavior, respectively. Hence, extreme care must be taken when using the latter four RFP variants as acceptors in FRET studies, since a photochromic effect is easily confused with a changed FRET state, especially if one considers that the typical FRET contrast in many sensors is in the range of only 5–20%. The photochromic behavior can also interfere with characterization of FPs, like determination of photostability (Supplementary Fig. 8) or brightness (Supplementary Fig. 5l).