Supporting Information

Luciferases with Tunable Emission Wavelengths

Julien Hiblot, Qiuliyang Yu, Marina D.B. Sabbadini, Luc Reymond, Lin Xue, Alberto Schena,
Olivier Sallin, Nicholas Hill, Rudolf Griss, and Kai Johnsson*

anie_201708277_sm_miscellaneous_information.pdf
 Supplementary Information for “Luciferases with tunable emission wavelengths”

Supplementary information table of content:

Methods: 2

Chemical synthesis 2

Biology 6

Supplementary Figures 9

Figure 1: Self-labelling crystal structures presenting NanoLuc insertion trials 9

Figure 2: Additional characterization of the LUCIDRS-MTX sensor 10

Supplementary Tables 11

Table 1: Screening results for the selection of semi-synthetic luciferases 11

Table 2: Comparative labeling kinetics by fluorescence polarization 11

Supplementary Note 1: Amino acid sequence of proteins 12

Supplementary Movie 1: H-Luc emission color change upon labeling Separated file

1

Chemical synthesis.

All chemical reagents and anhydrous solvents for synthesis were purchased from commercial suppliers (Sigma-Aldrich, Fluka, Acros)
1
and were used without further purification or distillation. The composition of solvent mixtures is given by the volume ratio (v/v). H and
13 1 13
C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX 400 (400 MHz for H, 100 MHz for C, respectively),
1 13 1
Bruker AVANCE III 400 Nanobay (400 MHz for H, 100 MHz for C, respectively) or Bruker DRX-600 (600 MHz for H, 151 MHz for
13 1
C, respectively), with chemical shifts (δ) reported in ppm relative to the solvent residual signals of CDCl3 (7.26 ppm for H, 77.16 ppm
13 1 13 1 13
for C), CD3OD (3.31 ppm for H, 49.00 ppm for C), DMSO-d6 (2.50 ppm for H, 39.52 ppm for C). Coupling constants are reported
in Hz. High resolution mass spectra (HRMS) were measured on a Micromass Q-TOF Ultima spectrometer with electrospray ionization
(ESI) or Bruker MicroTOF with ESI-TOF (time-of-flight). LC-MS was performed on a Shimadzu MS2020 connected to a Nexerra UHPLC
system equipped with a Waters ACQUITY UPLC BEH C18 1.7 µm 2.1x50 mm column. Buffer A: 0.05% HCOOH in H2O Buffer B:
0.05% HCOOH in acetonitrile. Analytical gradient was from 5% to 95% B within 5.5 min with 0.5 mL/min flow. Preparative RP-HPLC
was performed on a Dionex system equipped with an UVD 170U UV-Vis detector for product visualization on a Waters SunFire™ Prep
C18 OBD™ 5 µm 10×150 mm Column (Buffer A: 0.1% TFA in H2O Buffer B: acetonitrile. Typical gradient was from 0% to 100% B
within 30 min with 4 mL/min flow.) After lyophilization of HPLC purified compounds, the solid residue was generally dissolved in dry
DMSO and the concentration of the compounds was measured by UV-Vis spectroscopy in PBS containing 0.1% SDS, using a molar
-1 -1 -1 -1
extinction coefficient of 100'000 M cm at 652 nm for SiR; and 90'000 M cm at 608 nm for CPY; 73'000 at 495 nm for Alexa488;
-1 -1 -1 -1 -1 -1 -1 -1
112'000 M cm at 556 nm for Alexa546; 90'000 M cm at 549 nm for TMR; 120'000 M cm at 554 nm for Cy3; 92'000 M cm at 590
-1 -1 -1 -1 -1 -1
nm for Alexa594; 120'000 M cm at 563 nm for Atto565; 120'000 M cm at 593 nm for Atto590; 150'000 M cm at 646 nm for
-1 -1
Atto647N and 125'000 M cm at 663 nm for Atto655.

1 BG-CPY

[1] [2]
CPY-COOH (3.5 mg, 9.5 μmol) was treated with DIPEA (10 µL, 58 μmol) and TSTU (3.8 mg, 12.6 μmol). After 5 min, BG-NH2 (3.5
mg, 13.0 μmol) was added. The mixture was incubated for 2 H at room temperature (RT). The product was purified by RP-HPLC,
1
lyophilized and dissolved in dry DMSO. 250 μL of 22.6 mM solution of 1 were obtained (59% yield) as a violet solution. H NMR (600
MHz, DMSO-d6) all signals were broadened by a slow exchange between forms δ 9.35 (t, J=5.8 Hz, 1 H), 8.41 (br. s, 1 H), 8.21 (s, 2
H), 7.72 (br. s, 1 H), 7.49 (d, J=8.0 Hz, 2 H), 7.35 (d, J=7.9 Hz, 2 H), 7.16 (br. s, 2 H), 6.76 (br. s, 4 H), 5.51 (s, 2 H), 4.47 (d, J=5.4 Hz,
+ +
2 H), 3.18 (br. s, 12 H), 1.86 (s, 3 H), 1.72 (s, 3 H); HRMS (ESI) calcd for C41H41N8O4 [M] 709.3245; found 709.3244.

2

2 Halo-Alexa594

Alexa594 carboxylic acid (3x triethylammonium salt, 5 & 6 isomers mixture) (20 mg, 20 µmol) was dissolved in 0.25 mL DMSO. The
solution was treated with DIPEA (20 uL, 110 µmol) and TSTU (9 mg, 30 µmol) and stirred for 10 min at RT. Halolinker-amine (TFA
salt, 13 mg, 40 µmol) was added and the mixture was stirred for 1 H at RT. The product was purified by C18 RP SiO2 (LiChroprep®
RP-18, 20 cm x 1 cm column) using eluents A: 0.1% TFA in water and B: 0.1% TFA in MeCN. Gradient from 0% B to 30% B with 10%
1
steps gave Halo-Alexa594 2 (16 mg, 86% yield). H NMR (400 MHz, MeOD) signals were broadened by a slow exchange between
forms δ 8.73-8.73 (br. s, 1 H), 8.40-8.06 (m, 2 H), 7.74-7.55 (br. m, 2 H), 7.38-7.19 (br. m, 2 H), 6.82 (s, 2 H), 5.86 (s, 2 H), 3.81-3.35
+
(m, 16 H), 3.25-3.08 (s, 6 H), 1.82-1.23 (m, 20 H); HRMS (ESI) calcd for C45H55ClN3O12S2 [M] 928.2916; found 928.2916.

3 Halo-Atto655

Atto655-NHS ester (0.5 mg, 0.56 μmol) was dissolved in 100 µL DMSO and treated with Halolinker-amine (TFA salt, 0.3 mg, 1 μmol)
and DIPEA (0.5 µL, 3 μmol). The mixture was stirred at RT for 30 min. The product was purified by RP-HPLC, lyophilized and dissolved
1
in dry DMSO. 100 μL of 3.7 mM solution of Halo-Atto655 3 were obtained (66% yield) as a blue solution. H NMR (400 MHz, DMSO-
d6) δ 8.03 (t, J = 5.3 Hz, 1 H), 7.97 (s, 1 H), 7.59 (s, 1 H), 7.03 (s, 2 H), 3.61 (t, J = 6.7 Hz, 2 H), 3.53-3.45 (m, 8 H), 3.37-3.22 (m, 7
H), 2.90 (m, 2 H), 2.66 (dd, J = 12.9, 4.9 Hz, 1 H), 2.41 (dd, J = 3.6, 13.5 Hz, 2 H), 2.32 (m, 3 H), 1.98-1.85 (m, 4 H), 1.67 (m, 3 H),
+
1.49-1.43 (m, 5 H), 1.39-1.24 (m, 10 H); HRMS (ESI) calcd for C37H54ClN4O7S [M ] 733.3402; found 733.3394.

4 Halo-Cy3

Sulfo-Cy3-biscarboxylic acid (15 mg, 23 μmol) was dissolved in 250 µL DMSO, treated with DIPEA (20 μL, 116 μmol) and TSTU (7
mg, 23 μmol) and stirred for 10 min at RT. Halolinker-amine (TFA salt, 10 mg, 30 μmol) was then added and the reaction mixture was
stirred for 1 H at RT. The product was purified by C18 RP SiO2 (LiChroprep® RP-18, 20 cm x 1 cm column) using eluents A: 0.1%

3

TFA in water and B: 0.1% TFA in MeCN. Gradient from 0% B to 40% B with 10% steps, a subsequent purification by RP-HPLC gave
1
Halo-Cy3 4 (7 mg, 36% yield). H NMR (400 MHz, DMSO-d6) δ 8.36 (t, J = 13.4 Hz, 1 H), 8.18 (t, J = 5.5 Hz, 1 H), 7.80 (d, J = 1.3 Hz,
2 H), 7.66 (dt, J = 1.7, 8.2 Hz, 2 H), 7.42 (d, J = 8.4 Hz, 1 H), 7.34 (d, J = 8.4 Hz, 1 H), 6.52 (dd, J = 13.4, 5.7 Hz, 2 H), 4.35-4.32 (m,
4 H), 3.61 (t, J = 6.6 Hz, 2 H), 3.13 (m, 3 H), 2.71-2.59 (m, 4 H), 1.70 (m, 14 H), 1.45 (m, 2 H), 1.38-1.25 (m, 4 H), 1.01. HRMS (ESI)
+
calcd for C39H53ClN3O11S2 [M ] 838.2810; found 838.2810.

5 Halo-Atto647N

Atto647N-NHS ester (1.1 mg, 1.3 μmol) was dissolved in 50 µL DMSO and treated with Halolinker-amine (TFA salt, 0.6 mg, 1.8 μmol),
DIPEA (5 μL, 29 μmol). After 30 min at RT the product was purified by RP-HPLC, lyophilized and dissolved in dry DMSO. 380 μL of
1
3.0 mM solution of Halo-Atto647N 5 were obtained (87% yield) as a blue solution. H NMR (500 MHz, MeOD) δ 7.73-7.71 (m, 2 H),
7.63 (m, 1 H), 7.44 (m, 1 H), 6.95 (s, 1 H), 6.85 (s, 1 H), 6.81 (s, 1 H), 3.85 (m, 1 H), 3.64-3.47 (m, 15 H), 3.20 (m, 5 H), 2.94 (s, 2 H),
+
2.80 (m, 1 H), 2.63 (m, 2 H), 2.08 (m, 2 H), 1.92-1.32 (m, 30 H), 1.07 (d, J = 6.5 Hz, 3 H); HRMS (ESI) calcd for C52H72ClN4O4 [M]
851.5242; found 851.5217.

6 Alexa546-COOH

Synthetic route of 6: (a) 3-mercaptopropionic acid, DIEA, DMF, 50 °C.

[3]
A mixture of tetrachloroderivative of Alexa546 (mixture of stereoisomers, synthesized as previously described ) (320 mg, 0.4 mmol),
3-mercaptopropionic acid (60 μL, 0.6 mmol) and DIPEA in DMF (1.5 mL) was stirred for 4 h at 50 °C. The mixture was evaporated and
purified by SiO2 column chromatography using MeCN and H2O as eluents. From 100% MeCN to MeCN/H2O 7:3. The eluted product
still contained impurities and 1/5 of this material was further purified by RP-HPLC to obtain Alexa546-COOH 6 as a mixture of
-
stereoisomers (10 mg, 15% yield). HRMS (ESI) calcd for C35H34Cl3N2O11S3 [M-H]- 859.0396; found 859.0363.

4

7 BG-Alexa546

Alexa546-COOH 6 (1.7 mg, 2.0 μmol, mixture of stereoisomers) was treated with DIPEA (2 μL, 12 μmol) and TSTU (0.8 mg, 2.6 μmol).
[2]
After 5 min, BG-NH2 ( 0.65 mg, 2.4 μmol) was added. The mixture was incubated for 2 H at RT. The product was purified by RP-
HPLC, lyophilized and dissolved in dry DMSO. 100 μL of 9.0 mM solution of BG-Alexa546 7 was obtained (45% yield) as a pink
+
solution. HRMS (ESI) calcd for C48H48Cl3N8O11S3 [M+H]+ 1113.1665; found 1113.1667.

[1, 4]
The others fluorophore substrates were purchased from available suppliers or synthesized as previously reported .

5

Biology

Cloning, protein expression and purification. NanoLuc variants, chimeric luciferase candidates and sensors were cloned in a
pET51b(+) vector (Novagen) for production in Escherichia coli and in a pcDNA3.1(+) vector (Thermo Fisher Scientific) for expression
in mammalian cells; two cytochrome c oxidase subunit 8A (Cox8A) sequences and three nuclear localization signal (NLS) sequences
[5]
were fused to the genes for mitochondrial and nuclear localization, respectively. Cloning was performed by Gibson assembly . Proteins
were expressed in E. coli strain Rosetta-gami (DE3) (Novagen). Luria-Bertani broth (LB) cultures were grown at 37 °C to optical density
at 600 nm (OD600nm) of 0.8, induced by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside and grown at 17 °C overnight in
the presence of 1 mM MgCl2. The cells were harvested by centrifugation and lysed by sonication. The cell lysate was cleared by
centrifugation before tandem affinity-tag purification using Ni-NTA (Qiagen) and Strep-Tactin (IBA) resins following the suppliers’
instructions. Proteins were finally concentrated using an Ultra-0.5 mL centrifugal filter device (Amicon) with a molecular weight cut-off
(MWCO) according to the protein size and then stored in a glycerol 45% solution at -20 °C. NanoLuc variants, SNAP-tag and HaloTag7
proteins were purified using Ni-NTA only. NanoLuc variants were fused to maltose-binding protein (MBP), which was removed by
[6]
overnight cleavage with Tobacco Etch Virus (TEV) protease at 30 °C using previously described conditions . After buffer exchange
using an Ultra-4 mL centrifugal filter device with a MWCO of 10 kDa, NanoLuc variants were submitted to a second Ni-NTA purification
where the flowthrough containing the cleaved protein was kept. Proteins were then concentrated as previously described and
conserved in a glycerol 45% solution at -20 °C.

Protein labeling. For screening, reactions were performed in Activity Buffer (HEPES 50 mM pH 7.3, NaCl 50 mM) in presence of 0.5
µM of protein and 4-fold molar excess of substrate. The DMSO concentration was maintained below 2% and reactions were considered
finished after 3 H of incubation at room temperature (RT). H-Luc, S-Luc and sensors were labeled for 1 H at 1 µM protein concentration
and 4-fold molar excess of substrates at RT maintaining the DMSO concentration below 5%. Labeled sensors were purified using an
Ultra-0.5 mL centrifugal filter device with a 50 kDa MWCO performing 4 exchanges of 450 µL Activity Buffer prior to further experiments,
to eliminate the excess of remaining intramolecular ligand. Proteins were then stored in a glycerol 45% solution at -20 °C.

Kinetics of labeling. Labeling kinetics of SNAP-tag and HaloTag7 derivatives were performed by following the polarization of
fluorescence of TMR substrates on an EnVision Multilabel Reader (PerkinElmer) using the filters BODIPY TMR FP 531 ± 30 nm for
excitation, BODIPY TMR FP P/S-pol 595 ± 30 nm for emission and a general dichroic mirror (G-factor of 1 was kept by default). Kinetics
were followed at RT in 100 µL Activity Buffer supplemented with 5 mg/mL Bovine Serum Albumin (i.e. Activity Buffer BSA) with
fluorophore concentration of 50 nM and protein concentration of 200 nM. Experiment results were fitted to a one phase association
curve (equation (1)) using GraphPad Prism 6 where FP0 is the initial value of fluorescence polarization and FPmax the value at the end
of the reaction. k is the first order kinetic constant of reaction; the second order kinetic constants k2 were calculated from the equation
(2) where [S] corresponds to the initial substrate concentration. Value are reported as mean ± s.d. from triplicate experiments.

𝐹𝑃 = 𝐹𝑃$ + 𝐹𝑃&'( − 𝐹𝑃$ ∗ (1 − 𝑒𝑥𝑝 −𝑘 ∗ 𝑡 ) (1)

4
𝑘3 = (2)
5

Luciferase activity, BRET characterization and screening. Measurements of luciferase activity were performed in Activity Buffer
BSA. Proteins were diluted to 10 nM and reactions were performed with a final 800-fold dilution of NanoGlo furimazine substrate
(Promega). Classical read-outs were performed using a white flat bottom non-binding 96 well plate (Corning) in 100 µL volume at RT.
For screening, intensity based measurements were obtained using an EnVision Multilabel Reader (PerkinElmer) with 1 s integration
time and using the filters Umbelliferone (460 ± 12.5 nm) and Cy3 (595 ± 30 nm) for NanoLuc and TMR, respectively. The emission
spectra of the different labeled chimers were obtained using the luminescence scan mode of an Infinite M1000 spectrophotometer
(Tecan). S-Luc was labeled with fluorophore substrates (emission maximum, 𝜆7&
&'( ) Alexa488 (525 nm), Alexa546 (575 nm), Cy3 (570

nm), TMR (575 nm), Atto565 (590 nm), Atto590 (620 nm), CPY (635 nm), SiR (670 nm) and Cy5 (675 nm) and H-Luc with Alexa488,
Cy3, TMR, Alexa594 (600 nm), CPY, SiR, Atto647 (670 nm) and Atto655 (680 nm). H-Luc highlighted in Figure 1 corresponds to the
insertion of NanoLuc circular permuted at position 67-68 between the residues 154 and 156 of HaloTag7. Luminescence scans were
performed from 390 nm to 710 nm with a 1 nm step, 10 nm bandwidth and 10 ms integration time in Activity Buffer BSA at RT. Blood

6

experiments were performed in a mix of 50% blood (Chicken whole blood NaEDTA; Innovative Research Inc., USA) and 50% of Activity
Buffer BSA. In this case, luminescence scans were performed with a bandwidth of 20 nm, an integration time of 100 ms was used and
spectra were smoothened with Excel using a sliding window of 9 nm. BRET ratios were evaluated by calculating the ratio of fluorophore
intensity at 𝜆7& 7&
&'( over NanoLuc intensity (𝜆&'( = 460 nm). The intensities were extracted from the chimers emission spectra averaging

the values over 5 nm around 𝜆7&

&'( .

Ratio quantification of labeled proteins. In vitro evaluation of ratios [H-Luc-TMR]/[H-Luc-CPY] were performed recording emission
spectra analogously as above explained except that Perkin Elmer 96 well plates were used, and protein concentration was maintained
at 50 pM. Emission spectra were obtained using the Lumi-module of a Spark M20 microplate reader (Tecan) with integration time of 1
s. Specific TMR and CPY channels were defined as 585 ± 20 nm and 645 ± 20 nm, respectively. Emission spectra and specific channel
intensities of different ratios [H-Luc-TMR]/[H-Luc-CPY] were recorded (from 100% to 0%). The ratios of [H-Luc-TMR]/[H-Luc-CPY]
noted as RC versus the intensity ratios ChTMR/ChCPY noted as RI were plotted and fitted to equation (3). RMax and RMin correspond to the
RI values of pure H-Luc-TMR and H-Luc-CPY entities, respectively. F corresponds to the RC value at half value of RI. Values are
reported as mean ± s.d. from triplicate experiments.

;<=> ;<DE
𝑅: = + (3)
?@A/;C ?@;C /A

BRET sensor characterization and screening. The H-Luc used in LUCIDRS-MTX consists of the insertion of NanoLuc circular
permuted at position 65-66 in between the residues 154 and 156 of HaloTag7. The sensor LUCIDRS-MTX was characterized recording
the emission spectra of the sensor at different methotrexate concentration (0 and 400 µM) with a final concentration of 2% DMSO.
Spectra were obtained with an Infinte M1000 spectrophotometer as explained in section Luciferase activity, BRET characterization
and screening. The CPY and Cy5 channels were defined as 631 nm and 669 nm, respectively. The sensors were diluted to 20 nM in
Activity Buffer BSA. Sensors were also evaluated in the presence of bilirubin (10 µM) and in blood as above mentioned. The LUCID-
[7]
MTX sensor (5 nM), consisting of SNAP-Pro30-NanoLuc-cpDHFR, was characterized in in the presence and absence of bilirubin (10
µM) using the EnVision Multilabel Reader using channel intensities as described in section Luciferase activity, BRET
characterization and screening. The multiplex sensor measurements were performed using LUCID-MTX and LUCIDRS-MTX both at
10 nM concentration in Activity Buffer BSA by recording emission spectra as above mentioned. The additional channel for NanoLuc
and Cy3 of LUCID-MTX were defined as 460 nm and 570 nm, respectively. Measurements were performed 10 min after spiking the
substrate. The ratio R of donor and acceptor emission was plotted versus methotrexate concentration and then fitted to a single binding
[7]
isotherm (equation (4)) to obtain the C50 and ratio change (RC) values, as previously described ; using GraphPad Prism 6. The C50
and ratio changes are reported as mean ± s.d. from triplicate experiments.

;JKLE M;NOJPL
𝑅 = 𝑅FGHI7 + CQR (4)
?@
<ST

Cell culture and imaging. For cell tracking experiments, HeLa cells were grown in glass-bottom poly-d-lysine coated 35 mm dishes
(coverslip thickness No. 1.5, MatTek Corporation) in DMEM Glutamax medium (Life Technologies/GIBCO) supplemented with 10%
fetal bovine serum (FBS, Life Technologies/GIBCO) at 37 °C and 5% CO2. Transient transfections with 1 µg of the appropriate plasmids
were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol in Opti-MEM (Life
Technologies/GIBCO) for 6 H. Cell recovery was performed in DMEM-FBS overnight. Then cells were mixed (S-Luc and H-Luc
populations) in a 1:1 ratio and re-seeded onto 35 mm dishes. After overnight recovery cells were labeled with chloropyrimidine-TMR
(CP-TMR) and chloroalkane-SiR (Halo-SiR) both at 1 µM for 1 H in Opti-MEM. The cells were then washed three times with Opti-MEM
at intervals of 10 min and washed twice with PBS (Lonza) prior to incubation in Opti-MEM for imaging. Images for luciferase activity
were obtained using NanoGlo substrate diluted 50 times and using a Leica LAS AF 7000 wide-field microscope equipped with a 63x
plan Apochromat 1.4 numerical aperture oil immersion objective. The images correspond to a field of view of 185.4 µm x 185.4 µm.
The whole emitted light was acquired over 10 s without excitation and emission filters, specific TMR and SiR signals were both acquired
over 20 s using emission filters for Cy3 (580 ± 20 nm) and Cy5 (700 ± 36 nm), respectively. During the bioluminescence imaging, the
excitation light guide of the microscope was unplugged. A xenon arc lamp was used for fluorescence imaging using the same emission
filters and excitation filters for Cy3 (530 ± 17.5 nm) and Cy5 (635 ± 15 nm). The sample was kept at 37 °C during imaging.

7

For sub-cellular imaging, an analogous protocol was used. However, the cells were grown in glass-bottom poly-d-lysine coated 12-
well plates (coverslip thickness No. 1.5, MatTek Corporation) and were not re-seeded. Labeling was performed directly after overnight
recovery of transfection using benzylguanine-TMR (BG-TMR) and Halo-SiR at 1 µM and MitoTracker Green (Invitrogen) at 100 nM.
Images were obtained with the same microscope, but using a 100x plan Apochromat 1.47 numerical aperture oil immersion objective,
resulting in image dimension of 116.8 µm x 116.8 µm. The whole emitted light was acquired over 20 s, specific TMR and SiR signals
were both acquired over 40 s using the same set of filters already described. For the MitoTracker Green the filter set for Alexa488 was
used (excitation 470 ± 20 nm and emission 520 ± 20 nm).

Picture and Supplementary movie. Photographs were taken with a Canon 600D camera from a mix of S-Luc or H-Luc pre-labeled
and diluted to 10 nM in Activity Buffer BSA in presence of 800 times dilution of NanoGlo substrate. The movie was shot taking
consecutive pictures of 0.6 s exposure using a Canon PowerShot G1X camera. Pictures were assembled as a movie using Movavi
video editor. The protein concentration in the final assay was of 16 nM with addition of 80 nM of labeling fluorophore substrates
(respectively left to right: no label, TMR, CPY and SiR); in presence of a 500 times dilution of NanoGlo substrate.

[8]
Structural analysis. Structure representation and protein design were performed using PyMol .

8

Figure S1: Self-labelling crystal structures presenting NanoLuc insertion trials. Structures of SNAP-tag (PDB ID: 3kzz) (a) and
HaloTag7 (PDB ID: 4kaj) (b) with residues target for insertion of NanoLuc highlighted in purple; stick residues representing the best
insertion positions. HaloTag7 inhibitor N-(2-ethoxy-3,5-dimethylbenzyl)-1H-tetrazol-5-amine and SNAP-tag substrate benzylguanine
are included as orange sticks to indicate location of labeling site. The zinc atom in the SNAP-tag structure is indicated as a blue sphere.

9

Figure S2: Additional characterization of the LUCIDRS-MTX sensor. (a) Titration curve of the sensor LUCID-MTX in presence and
absence of bilirubin (10 µM). (b) Emission spectra of the LUCIDRS-MTX sensor in presence of different methotrexate concentrations.
Emission curve (c) and titration curve (d) of LUCIDRS-MTX sensor obtained in blood. A zoom on the relevant part of the emission
spectra is presented in the inlay. (e) Emission spectra of a mix of sensor LUCID-MTX (10 nM) - LUCIDRS-MTX (10 nM) in presence of
different methotrexate concentrations. The blue background part of the graph highlights the emission profile of the LUCID-MTX sensor
with the two maximum peaks corresponding to NanoLuc and Cy3. The orange background part of the graph highlights the emission
profile of the LUCIDRS-MTX sensor with the two maximum peaks corresponding to CPY and Cy5. (f) Titration curve of the LUCID-MTX
sensor (red) and LUCIDRS-MTX (purple) sensor. For the complete figure, RC corresponds to the maximum ratio change of the sensors
and C50 the analyte concentration that results in 50% of the maximum ratio change.

10

Table S1: Screening results for the selection of semi-synthetic luciferases

Construct % of NanoLuc TMR/NanoLuc

emission BRET ratio
NanoLuc-SNAP 100 ± 51 0.57 ± 0.03
SNAP-NanoLuc 87 ± 43 0.69 ± 0.02
SNAP[30NanoLuc31] 3±2 0.10 ± 0.01
SNAP[31NanoLuc32] 11 ± 6 0.31 ± 0.01
SNAP[32NanoLuc33] 199 ± 102 2.19 ± 0.10
SNAP[33NanoLuc34] 195 ± 99 1.43 ± 0.09
SNAP[34NanoLuc35] 204 ± 102 1.69 ± 0.07
SNAP[35NanoLuc36] 50 ± 32 0.58 ± 0.11
SNAP[128NanoLuc129] 100 ± 55 1.29 ± 0.18
SNAP[159NanoLuc160] 12 ± 6 0.08 ± 0.00
SNAP[160NanoLuc161] 10 ± 5 0.06 ± 0.00
SNAP[163NanoLuc164] 12 ± 7 0.07 ± 0.01
SNAP[32(NanoLucCP65/68)34] 2±1 0.85 ± 0.04
SNAP[35(NanoLucCP65/68)36] 8±4 1.95 ± 0.04
SNAP[91(NanoLucCP65/66)93] 0.4 ± 0.2 0.49 ± 0.07
SNAP[91(NanoLucCP67/68)93] 22 ± 12 1.79 ± 0.18
SNAP[122(NanoLucCP65/68)126] 18 ± 9 1.40 ± 0.06
SNAP[151(NanoLucCP65/66)154] 42 ± 20 3.99 ± 0.03
SNAP[151(NanoLucCP67/68)154] 8±4 1.16 ± 0.00
NanoLuc-HaloTag7 107 ± 56 1.13 ± 0.10
HaloTag7-NanoLuc 45 ± 22 0.37 ± 0.02
HaloTag7[139NanoLuc140] 102 ± 54 1.15 ± 0.09
HaloTag7[164NanoLuc165] 106 ± 58 1.03 ± 0.10
HaloTag7[178NanoLuc179] 7±3 0.08 ± 0.00
HaloTag7[242NanoLuc243] 44 ± 30 1.08 ± 0.40
HaloTag7[248NanoLuc249] 34 ± 17 0.32 ± 0.01
(NanoLucCP65/68)-I4-HaloTag7 23 ± 11 3.28 ± 0.06
HaloTag7[142(NanoLucCP65/68)147] 15 ± 7 12.5 ± 0.7
HaloTag7[141-G-(NanoLucCP65/68)-G-148] 22 ± 11 8.22 ± 0.84
HaloTag7[141-G-(NanoLucCP65/68)148] 31 ± 16 6.41 ± 0.33
HaloTag7[142-G-(NanoLucCP65/68)-G-147] 61 ± 31 4.97 ± 0.66
HaloTag7[142-(NanoLucCP65/66)-145] 48 ± 24 2.61 ± 0.15
HaloTag7[142-(NanoLucCP67/68)-145] 46 ± 23 6.67 ± 0.15
HaloTag7[154-(NanoLucCP65/66)-156] 87 ± 42 10.8 ± 0.2
HaloTag7[154-(NanoLucCP67/68)-156] 149 ± 77 11.0 ± 0.8
HaloCP142/145-NanoLucCP65/68 28 ± 14 1.58 ± 0.04
NanoLucCP65/68-HaloCP142/145 8±4 1.10 ± 0.10
HaloCP142/145-NanoLucCP65/66 33 ± 17 1.42 ± 0.06
NanoLucCP65/66-HaloCP142/145 8±4 0.92 ± 0.03
Data represent the mean ± s.d. over triplicate experiments.

Table S2: Comparative labeling kinetics by fluorescence polarization

-1 -1
Protein k2 (M s )
5
SNAP-tag 2.54 ± 0.65 x 10
5
S-Luc 3.72 ± 0.29 x 10
7
HaloTag7 2.02 ± 0.40 x 10
6
H-Luc [CP65-66] 9.19 ± 0.43 x 10
6
H-Luc [CP67-68] 7.58 ± 0.34 x 10
Kinetics were evaluated in presence of protein excess (200 nM) and limiting concentration of substrate (50 nM).
Data represent the mean ± s.d. of triplicate experiments.

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Note S1. Amino acid sequences of proteins

>MBP-NanoLuc

MHHHHHHHHHHMVKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG
LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG
GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQ
PSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSENLYFQGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGEN
GLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI
NPDGSLLFRVTINGVTGWRLCERILA

>MBP-NanoLuc circular permuted at position 65-66

MHHHHHHHHHHMVKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG
LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG
GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQ
PSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSENLYFQGGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGI
AVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVL
EQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

> MBP-NanoLuc circular permuted at position 65-68

MHHHHHHHHHHMVKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG
LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG
GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQ
PSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSENLYFQGSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV
FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQ
GGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

> MBP-NanoLuc circular permuted at position 67-68

MHHHHHHHHHHMVKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG
LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG
GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQ
PSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSENLYFQGSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV
FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQ
GGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL

> S-Luc(-CLIP)

MASWSHPQFEKGADDDDKVPHMDKDCEMKRTTLDSPLGKLELSGCEQGLHEIIMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSL
FQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG
KKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAFLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAYFHQPE
AIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
VKEWLLAHEGHRLGKPGLGGRLEVLFQGPKAFLEMDKDCEMKRTTLDSPLGKLELSGCEQGLHEIIFLGKGTSAADAVEVPAPAAVL
GGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILI
PCHRVVQGDSDVGPYLGGLAVKEWLLAHEGHRLGKPGLGGAPGFSSISAHHHHHHHHHH

> H-Luc with NanoLuc circular permuted at positions 65-66

MASWSHPQFEKGADDDDKVPHGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDL
IGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRT
GLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI
NGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII
PYEDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFW
GTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGAPGFSSISAHHHHHHHHHH

> H-Luc with NanoLuc circular permuted at positions 67-68

MASWSHPQFEKGADDDDKVPHGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDL
IGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRT
SGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN
GVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIP

12

YEGLDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLF
WGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGAPGFSSISAHHHHHHHHHH

>LUCIDRS-MTX protein sequence

MASWSHPQFEKGADDDDKVPMDKDCEMKRTTLDSPLGKLELSGCEQGLHEIIFLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWL
NAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDV
GGYEGGLAVKEWLLAHEGHRLGKPGLGPPPPPPPPPPPPPPPGGSGGSPPPPPPPPPPPPPPPPADLAWFKRNTLNKPVIMGRHT
WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWE
SVFSEFHDADAQNSHSYCFEILERRGGGGGMISLIAALAVDRVIGMENAMPWNGIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVL
FLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVK
GIAFMEFIRPIPTWDEWPEFARETFQAFRTGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK
KITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVS
SLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIA
GEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGAPG
FSSISAHHHHHHHHHH

Color code: His-tag - MBP - protease cleavage site - NanoLuc – linkers - STREP-tag II - SNAP-tag – CLIP-tag – HaloTag7 - eDHFR

References:

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