Imaging in mammals using fluorescent proteins (FPs) is an important technique to quantitatively and non-invasively track tumor growth and metastasis, gene expression, angiogenesis, and bacterial infection2
. Deep tissues visualization of the conventional FPs derived from the Green Fluorescent Protein family (GFP-like FPs) is still hindered by the high absorbance of hemoglobin and skin melanin. An optimal FP for in vivo
imaging should have both excitation and emission maxima within a NIRW from approximately 650 nm to 900 nm, which has the lowest tissue absorbance1
. However, to this moment even the most far-red shifted GFP-like proteins still have excitation spectra outside of the NIRW.
To circumvent these problems, near infra-red (NIR) FPs can be engineered on the basis of phytochromes3
. Phytochromes are photosensory receptors absorbing light in the red and far-red part of spectrum4
. The family of phytochromes shares a conserved photosensory protein core consisting of a PAS domain, a GAF domain, and a PHY domain. A linear tetrapyrrole chromophore, such as biliverdin IXα (BV), phycocyanobilin or phytochromobilin, is covalently bound to one of the first two domains. Bacteriophytochromes are more advantageous for use as design templates for NIR FPs since BV, an obligatory co-factor of bacteriophytochromes, is a component of normal mammalian heme metabolism5
Fluorescent properties of phytochromes have been known for a long time3,6-8
but only recently a NIR fluorescent mutant of the Dr
BphP bacteriophytochrome from Deinococcus radiodurans
named IFP1.4 was reported to be useful for in vivo
. However the properties of IFP1.4 remain suboptimal and require development of new superior probes.
In order to engineer NIR FP we turned to another template – bacteriophytochrome Rp
from the photosynthetic bacterium Rhodopseudomonas palustris
. The full-length Rp
BphP2 protein is weakly fluorescent at 725 nm when excited at 710 nm10
First, we truncated Rp
BphP2 to retain the PAS and GAF domains (Rp
BphP2-PAS-GAF; 316 amino acids in length) and introduced D202H mutation, since substitutions of this aspartic acid had been shown to improve fluorescent properties of the phytochromes8,11
. Expression of this Rp
BphP2-PAS-GAF/D202H variant in bacteria co-transformed with a plasmid bearing a heme-oxygenase gene (to produce BV co-factor) proved its initial fluorescence.
BphP2-PAS-GAF/D202H variant was subjected to three rounds of random mutagenesis followed by a round of saturating mutagenesis of the identified key residues. The final mutant had the following 13 substitutions: S13L, A92T, V104I, V114I, E161K, Y193K, F198Y, D202T, I203V, Y258F, A283V, K288T, and N290Y (Supplementary Fig. 1
). This variant was named an iRFP (infra-Red Fluorescent Protein).
Compared to IFP1.4, iRFP exhibited a higher extinction coefficient ( and ) as determined by direct measurement of the protein concentrations, while extinction coefficients calculated based on the comparison9
of absorbance of the proteins and free BV at 391 nm were similar. iRFP fluorescence exhibited the excitation/emission maxima at 690/713 nm (), slightly red-shifted compared to IFP1.4. Quantum yields at pH 7.5 were measured to be 5.9% for iRFP and 7.7% for IFP1.4. Based on these measurements the relative molecular brightness of iRFP is 1.2 of that of IFP1.4 ()
In vitro properties of iRFP (solid lines and circles) and IFP1.4 (dashed lines and triangles)
In vitro properties of iRFP in comparison with IFP1.4.
iRFP had slightly slower maturation at 37°C than IFP1.4 with a maturation half-time of 2.8 hours versus 1.9 hours for IFP1.4 ( and ). The fluorescence of iRFP was pH stable with pKa value of 4.0, compared to pKa of 4.6 for IFP1.4 ( and ).
Size-exclusion chromatography demonstrated that iRFP was a dimer while IFP1.4 contained two fractions: the monomeric and oligomeric with an apparent MW of ~190 kDa (Supplementary Fig. 2
). Revealed IFP1.4 oligomers may also exist in mammalian cells potentially limiting IFP1.4 use as a fusion tag. In contrast, iRFP exhibited a clear dominant dimer peak and subsequent tandem engineering strategy12
may enable iRFP to be useful as a fusion tag.
Quasi-equilibrium curves of guanidinium chloride (GndCl) induced protein unfolding13
demonstrated that iRFP had higher, compared to IFP1.4, conformation stability ( and Supplementary Fig. 3a
). The calculated difference in free energies of unfolding14,15
between the iRFP and IFP1.4 proteins was 3.5 kcal/mole. Since iRFP and IFP1.4, as other phytochromes5
, bind BV covalently (Supplementary Fig. 4
), GndCl-induced fluorescence decrease indicates the loss of proteins’ tertiary structures rather than BV dissociation indicating that iRFP is substantially more thermodynamically stable.
The normalized photostability of iRFP, measured in aqueous drops in oil, was substantially higher than that for IFP1.4, with the difference being ~10-fold (Supplementary Fig. 3b
and ). To exclude the possibility that both proteins revealed phytochromes photoswitching properties instead of bleaching, the aqueous drops irradiated with photobleaching light were left in the dark for additional 30 minutes and were then imaged again (Supplementary Fig. 3b
). Neither protein showed any increase in fluorescence suggesting that both remained in the main, non-photoswitched state and that the observed loss of fluorescence was caused by photobleaching.
Two-photon (2P) excitation spectrum of purified iRFP measured in 1100-1340 nm spectral region revealed excitation peak at 1260 nm corresponding to the main one-photon absorbance maximum (Supplementary Fig. 5
). Thus iRFP is also suitable for the multiphoton imaging though its 2p properties remain to be studied further.
To characterize iRFP in mammalian cells, we FACS analyzed HeLa cells transiently transfected with iRFP and IFP1.4 encoding plasmids (no exogenous heme-oxygenase gene was used here). Cells were also co-transfected with EGFP plasmid for subsequent NIR signal normalization. Since amount of endogenous BV might not be enough to bind to all produced NIR proteins, where indicated the saturating concentration9
of 25 μM of BV was added to culture medium 2 hours before analysis.
Despite slight differences in molecular brightness of the phytochromes, the fluorescence signals of the iRFP and IFP1.4 expressing cells differed drastically (). While the iRFP cells showed bright fluorescence even without addition of exogenous BV, the IFP1.4 fluorescence was observed only in the cells expressing EGFP at high levels and in the presence of exogenous BV. Quantification of this difference, performed by normalizing phytochrome signal to EGFP signal, showed that iRFP cells were 13-fold brighter than the IFP1.4 cells without exogenous BV and 7-fold brighter after addition of BV ( and Supplemetary Table 1
). Therefore the effective brightness of iRFP in living cells, which is a combination of molecular brightness, intracellular stability, affinity for BV, and protein expression level, is substantially higher than that of IFP1.4. To compare relative concentrations of the produced fluorescent molecules of iRFP and IFP1.4, the normalized cellular fluorescence intensities were divided by the respective molecular brightness. The cellular amount of the iRFP fluorescent molecules was 5.9-fold and 11.0-fold greater than that of IFP1.4 with and without exogenous BV, respectively (Supplemetary Table 1
Epifluorescent microscopy of the transiently transfected HeLa cells showed evenly dispersed fluorescent signals without any intracellular aggregates for both proteins (). Addition of exogenous BV and 4-fold longer exposure times were typically required to obtain images of the IFP1.4 cells of the same brightness as images of the iRFP cells without exogenous BV. The normalized intracellular photostability of iRFP was even higher than in aqueous drops while the photostability of IFP1.4 was similar, with an overall difference between two proteins ~30-fold ( and Supplemetary Table 1
In order to assess the degradation kinetics of iRFP and IFP1.4 cells expressing one or another protein were treated with 1 mM of a puromycin to inhibit protein translation16
. The fluorescence of both proteins was stable in cells and exhibited similar degradation time-courses over a period of 20 hours ().
Since the brightness of the IFP1.4 cells increased more upon BV addition than that of the iRFP cells ()
, we studied whether the proteins had different BV binding efficiencies. Different BV concentrations were added to the HeLa cells expressing either IFP1.4 or iRFP and the BV-binding curves () were fitted and processed using a Scatchard equation17
. The BV dissociation constants for iRFP and IFP1.4 were 0.35 μM and 4.2 μM, respectively (Supplementary table 1
). The data suggest that the 12-fold higher iRFP binding affinity allows efficient formation of iRFP-BV fluorescent complexes utilizing relatively low concentrations of endogenous BV produced in cells.
To assess intracellular stability of the iRFP and IFP1.4 apoproteins, we expressed iRFP and IFP1.4 in HeLa cells without or with exogenous BV added for a short (2 hours) or long (42 hours) periods of time before the essay. Expression of IFP1.4 in presence of BV during 42 hours resulted in cells that were twice as fluorescent compared to cells maintained with BV just for 2 hours. In contrast, prolonged BV exposure had no effect on the amount of iRFP fluorescence ( and Supplementary table 1
). Similar results were obtained by expressing the proteins in bacteria bearing the heme-oxygenase without and with added heme precursors (Supplementary Fig. 6
). Overall, these data suggested that BV binding to IFP1.4 apoprotein was required to stabilize it, possibly by preventing from intracellular degradation. At the same time the majority of the iRFP apoprotein molecules remained intact during 2 days as suggested by the same brightness of the iRFP cells exposed to exogenous BV for short and long time periods ().
To study toxicity of both proteins in mammalian cells an approach used for GFP-like proteins was applied18
. iRFP, IFP1.4 and control GFP/S65T variant were transiently expressed for 1, 3, and 5 days, and the mean fluorescence intensity of the viable cells at each day was determined using FACS (Supplementary Fig. 7
). If the FPs were cytotoxic then the fluorescent intensity of the expressing cells would rapidly decrease18
. In agreement with the previous data19,20
the GFP-producing cells demonstrated a “bell-shaped” profile of the expression. In contrast, the apparent expression of iRFP and IFP1.4 steadily increased during 5 days. We attributed this increase to a combination of two processes: the high-level production of exogenous apoproteins and a ‘catching up’ synthesis of endogenous BV to bind it, thus, forming the fluorescent holoproteins.
These results prompted us to look for longer expression conditions where the holoprotein level could remain constant. For this purpose preclonal mixtures of HeLa cells expressing iRFP, E2-Crimson (non-cytotoxic standard)21
or mKate2 (cytotoxic standard)21
were made. Prolonged expression of IFP1.4 at detectable levels required constant BV addition that might affect the results; therefore, it was not assessed in this assay. Cells with iRFP, E2-Crimson, or mKate2 were maintained for 21 days after the transfection with a selection drug, then sorted, and finally analyzed after 20 more days being under the selection. E2-Crimson and iRFP sorted cell populations remained mostly within the original sorting gates while the majority of the sorted mKate2 cells lost their fluorescence (Supplementary Fig. 8
). Since iRFP expressing cells behaved similarly to the cells expressing non-cytotoxic control E2-Crimson we concluded that iRFP was not cytotoxic.
Next iRFP applicability for imaging in mammals was tested. Mice were infected with adenoviral particles containing either iRFP or IFP1.4 genes and then imaged using IVIS Spectrum imager. Fluorescence of the liver in the iRFP infected mice was detected starting the second day post-infection, with the peak intensity at day 5 (). The IFP1.4 expressing mice showed weak liver fluorescence during all days of imaging. At day 5 post-infection both mice were administrated 250 nmol of BV. After the injection, the IFP1.4 infected liver become ~4-fold brighter; however, it still was dimmer than the iRFP expressing liver. Calculation of total radiant efficiencies of the liver regions demonstrated the iRFP effective brightness in vivo being 22-fold higher without exogenous BV and 7-fold higher after the BV injection ().
Expression of iRFP in living mouse
Following BV administration, the IFP1.4 liver fluorescence lost half of its brightness after ~30 hours and returned to the initial brightness ~2 days after the injection (). The decrease in brightness of the iRFP liver during the 2 day period after the BV injection was ~10% only. These data suggest that in contrast to iRFP, prolonged mouse experiments involving IFP1.4 will require frequent BV injections.
Correct localization of both proteins was revealed by ex vivo imaging of the isolated livers (). Importantly, the iRFP fluorescence was easily detected in the liver 10 days post-infection without administrating BV during this period (), suggesting that iRFP is both stable and non-cytotoxic in vivo.
iRFP expression in other than liver tissues with no need for exogenous BV was demonstrated by ex vivo
imaging of the spleen excised from the infected mouse (Supplementary Fig. 9
In order to additionally support general applicability of iRFP for different body tissues expression, the liver cells isolated from iRFP-infected and control mice were subjected to FACS analysis. Then iRFP fluorescence level of these primary hepatocytes was compared to that of the stably iRFP-expressing in vitro
cultured cells originated from human cervix, rat brain, and rat mammary gland. The cultured cells stably expressed iRFP with the similar, compared to liver, or even higher fluorescence brightness without adding exogenous BV (Supplementary Fig. 10
) suggesting that iRFP is suitable for imaging of various organs and tissues, owing to its high affinity to endogenous BV.
Molecular evolution approach enabled us to develop an advanced NIR FP with excitation and emission maxima inside of the NIR window. This genetically-encoded iRFP probe should dramatically improve in vivo studies of small mammals.
iRFP has superior properties compared to the IFP1.4 protein. Firstly, iRFP has higher molecular brightness and greater photostability. Secondly, iRFP exhibits greater thermodynamic stability, lower pKa value, and higher binding affinity to BV. Thirdly, iRFP has significantly higher effective brightness in cells and in mice since IFP1.4 apoprotein has lower affinity to BV and is not stable without it. Lastly, iRFP does not require addition of an external BV when imaged in mammalian cells, and in this respect, it behaves similarly to GFP-like proteins.
Several GFP-like far-red FPs have been shown to be useful for the whole-body imaging22,23
however their spectral properties are suboptimal for this purpose. To directly compare iRFP deep-tissue imaging performance with that of far-red shifted FPs, such as mKate224
, and eqFP67023
, we imaged the same amount of purified proteins at 7.0 and 18.1 mm depth inside of a mouse phantom, which has the autofluorescence and light-scattering properties matching those of a mouse muscle tissue26,27
(). To compare brightness in different spectral channels, a signal-to-background ratio for the FPs for each channel was calculated. The highest ratio values among the different channels are shown (). iRFP has 2.4-fold and 3.2-fold larger ratio values at 7.0 mm and 18.1 mm depth, respectively, than the second in a row mNeptune. The data confirmed that the far-red shifted spectra allow iRFP to perform substantially better despite being less bright on the molecular level.
Comparison of iRFP with far-red GFP-like proteins in mouse phantom
In conclusion, currently iRFP is, both in terms of molecular and effective brightness, the brightest phytochrome-based and the most infra-red shifted FP. iRFP is stable, non-cytotoxic and utilizes the low concentrations of endogenous BV to be visualized in cells, tissues, and mammals. These features make its application as easy as the conventional GFP-like FPs, and hence should significantly broaden the possibilities of non-invasive in vivo imaging.