Sensors, methods and kits for detecting nicotinamide adenine dinucleotides
10221439 · 2019-03-05
Assignee
Inventors
Cpc classification
C12Q1/008
CHEMISTRY; METALLURGY
C12Y101/01153
CHEMISTRY; METALLURGY
G01N33/542
PHYSICS
International classification
C12Q1/00
CHEMISTRY; METALLURGY
G01N33/542
PHYSICS
Abstract
The invention relates to the in vitro and in cellulo detection of the cofactors nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Provided is a sensor molecule for fluorescence- or luminescence-based detection of a nicotinamide adenine dinucleotide analyte, in particular for detecting the concentrations of NAD.sup.+, NADP.sup.+ and/or the ratios of the concentrations of NAD.sup.+/NADH and NADP.sup.+/NADPH, the sensor comprising (i) a binding protein (BP) for the nicotinamide adenine dinucleotide analyte, the BP being derived from sepiapterin reductase (SPR; EC 1.1.1.153) (ii) an SPR ligand (SPR-L) capable of intramolecular binding to said BP in the presence of the oxidized form of said analyte; and (iii) at least one fluorophore.
Claims
1. A sensor molecule for fluorescence or luminescence-based detection of a nicotinamide adenine dinucleotide analyte, in particular for detecting the concentration of NADP.sup.+ or the ratio of the concentrations of NADP.sup.+ and NADPH, NADP.sup.+/NADPH, the sensor comprising (i) a binding protein (BP) for the nicotinamide adenine dinucleotide analyte, the BP being sepiapterin reductase (SPR), wherein the SPR is human sepiapterin reductase (hSPR) according to SEQ ID NO:4, rat sepiapterin reductase according to SEQ ID NO:3, mouse sepiapterin reductase according to SEQ ID NO:2, or a variant thereof having at least 95% sequence identity with SEQ ID NO:4, SEQ ID NO: 3 or SEQ ID NO:2, provided that the variant comprises the residues corresponding to Ser157, Tyr170, Lys174, and Asp257 in hSPR; (ii) an SPR ligand (SPR-L) capable of intramolecular binding to said BP in the presence of the oxidized form of said analyte, wherein said SPR-L is based on a benzenesulfonamide; and (iii) at least one fluorophore.
2. Sensor molecule according to claim 1, for resonance energy transfer (RET) based analyte detection, the sensor comprising a segment A connected via a linker to a segment B, wherein each of segment A and segment B comprises a member of a RET pair comprising a donor moiety and an acceptor moiety, further characterized in that (i) segment A comprises the BP (ii) segment B comprises the SPR-L such that the donor moiety and the acceptor moiety are in a suitable juxtaposition to yield a RET signal when SPR-L is bound to BP, and wherein an analyte-induced change in SPR-L binding to BP results in a change in RET efficiency.
3. Sensor molecule according to claim 1, wherein said BP is human sepiapterin reductase (hSPR) having the amino acid sequence of SEQ ID NO:4 or a variant thereof having at least 98% sequence identity with SEQ ID NO:4, provided that the variant comprises the residues corresponding to Ser157, Tyr170, Lys174, and Asp257 in hSPR.
4. Sensor molecule according to claim 1, wherein said BP comprises an amino acid sequence showing at least 99%, sequence identity with SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO:3.
5. Sensor molecule according to claim 1, wherein BP comprises an SPR wherein the residues corresponding to positions 17 and 42 of SEQ ID NO:4 are basic residues.
6. Sensor molecule according to claim 1, wherein said SPR-L is a benzenesulfonamide having a pKa below 6 or above 9.
7. Sensor molecule according to claim 2, wherein the RET pair is a FRET pair.
8. Sensor molecule according to claim 2, wherein the RET pair is a BRET pair, and wherein the BRET pair comprises a luciferase and a fluorescent acceptor.
9. Sensor molecule according to claim 8, wherein the luciferase is NLuc luciferase that is provided at its C-terminus with a circular-permutated dihydrofolate reductase (cpDHFR).
10. Sensor molecule according to claim 2, wherein the linker is a proteinaceous linker.
11. Sensor molecule according to claim 1, wherein the sensor molecule is immobilized or absorbed to a solid carrier.
12. A method for fluorescence or luminescence-based in vitro detection of the concentration of a nicotinamide adenine dinucleotide analyte in a sample, the method comprising (a) contacting the sample with a sensor molecule according to claim 1 under conditions allowing for an analyte-dependent binding of said SPR-L to SPR or SPR mutant modulates the spectroscopic properties of the fluorophore or emission spectra of the sensor molecule; and (b) analyzing a change in a signal generated by modulation of the spectroscopic properties of the fluorophore or emission spectra of the sensor molecule and relating the signal change to the concentration of a nicotinamide adenine dinucleotide analyte in the sample.
13. Method according to claim 12, wherein the concentration of NADP.sup.+ or the NADP.sup.+/NADPH ratio in the sample is detected.
14. Method according to claim 12, wherein the sample is a biological sample or a fraction thereof.
15. Method according to claim 12, wherein the sample comprises (i) an unknown concentration of NADP.sup.+ or NADPH (ii) an unknown ratio of the concentrations of NADP.sup.+ and NADPH, or (iii) an unknown concentration of an enzyme that generates or consumes NADP.sup.+.
16. Kit of parts comprising a sensor molecule according to claim 1 and a solid carrier.
17. Kit according to claim 16, comprising a BRET-based sensor, further comprising a luciferase substrate.
18. The sensor molecule according to claim 1, wherein said SPRL based on a benzenesulfonamide is selected from the group consisting of sulfasalazine, sulfathiazole, sulfamethoxazole, sulfamethizole, phthalylsulfathiazole, sulfapyridine, sulfadiazine, sulfamerazine, sulfachloropyridazine, sulfameter, chlorpropamide, glibenclamide and tolbutamide.
19. The sensor molecule according to claim 7, wherein the FRET pair is selected from (i) a fluorescent protein and a self-labelling tag tethered with a synthetic molecule containing a fluorophore; (ii) two orthogonal self-labelling tags, one for the attachment of the synthetic molecule containing a donor moiety, the other to attach an acceptor moiety fluorophore; and (iii) two fluorescent proteins.
20. The sensor molecule according to claim 11, wherein the sensor is immobilized or absorbed to glass, a transparent plastic, a gold surface, paper or a gel.
21. Method according to claim 12, wherein (i) ex vivo clinical or diagnostic testing is performed; (ii) an ex vivo enzymatic assay that involves the formation or consumption of NADP.sup.+ is performed; (iii) ex vivo high-throughput screening is performed for compounds that can modulate NADP(H) in cells or for the validation of the toxicity profile of therapeutic drugs; or (iv) live cell measurements are performed comprising the use of a widefield fluorescence microscope, confocal fluorescence microscope or a Fluorescence Lifetime Imaging Microscopy (FLIM) system with appropriate excitation and emission filters.
22. A sensor molecule for fluorescence or luminescence-based detection of a concentration of NAD.sup.+ or an NAD.sup.+/NADH ratio, the sensor comprising (i) a binding protein (BP) capable of binding NAD.sup.+, the BP being a variant of sepiapterin reductase (SPR) having at least 95% sequence identity with SEQ ID NO:4, SEQ ID NO: 3 or SEQ ID NO:2, provided that the variant comprises residues corresponding to Ser157, Tyr170, Lys174, and Asp257 of SEQ ID NO:4, and provided that a residue corresponding to position 41 of SEQ ID NO: 4 is Asp or a residue corresponding to position 42 of SEQ ID NO:4 is Val, Ile, or Trp; (ii) an SPR ligand (SPR-L) capable of intramolecular binding to said BP in the presence NAD.sup.+, wherein said SPR-L is based on a benzenesulfonamide; and (iii) at least one fluorophore.
Description
LEGEND TO THE FIGURES
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EXPERIMENTAL SECTION
(9) The examples below described the design and construction of different SPR-based sensors for NADP(H) and NAD(H).
(10) All chemical reagents and dry solvents for synthesis were purchased from commercial suppliers (Sigma-Aldrich, Fluka, Acros, Calbiochem) and were used without further purification or distillation. Peptide couplings were performed by activation of the respective carboxylic acid with N,N,N,N-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) or N,N,N,N-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) in the presence of N,N-diisopropylethylamine (DIPEA) as base in anhydrous dimethylsulfoxide (DMSO) at room temperature. Preparative HPLC was performed on a Waters 600 controller and with a Waters 2487 dual absorption detector using a SunFire Prep C18 OBD 5 m 19150 mm Column.
Example 1: SNAP-p30-EGFP-SPR
(11) This example describes the design and construction of the initial FRET-based sensor capable of sensing the concentration of NADP(H). The sensor comprises the human sepiapterin reductase (SPR) as NADP(H)-specific binding protein and sulfapyridine or sulfamethoxazole as intramolecular ligand. EGFP and TMR form respectively the FRET donor and acceptor (
(12) A synthetic molecule containing an O.sup.6-benzylguanine (BG) moiety for the specific labelling of SNAP-tag, the flurophore tetramethylrhodamine (TMR) and sulfapyridine (SPY) as intramolecular SPR ligand was synthesized according to Scheme 1.
(13) ##STR00011## ##STR00012##
6-amino-N-[4-(pyridin-2-ylsulfamoyl)phenyl]hexanamide (i-2)
(14) To a solution of Fmoc-6-aminohexanoic acid (100 mg, 0.28 mmol, 1 eq.) in dry DMSO (1.0 mL, 280 mM), DIPEA (52 L, 0.31 mmol, 1.1 eq.) and sulfapyridine (i-1) (200 mg, 0.8 mmol, 2.9 eq.) were added. HBTU (130 mg, 0.34 mmol, 1.2 eq.) was added to the reaction mixture and stirred for 2 h at RT. Then, 200 L H.sub.2O was added to the reaction mixture and stirred for 30 min. Finally, the reaction was quenched with 80 L AcOH, purified by HPLC and lyophilized to yield a white solid (64.6 mg, 39%).
(15) The fmoc-protected product was dissolved in 1 mL of a solution mixture of piperidine 20% in acetonitrile. The reaction mixture was stirred 1 h at RT, quenched with 0.2 mL AcOH, evaporated under reduced pressure, purified by preparative HPLC and lyophilized to yield a white solid (39.8 mg, quant.). TMR(6)-COOMe (i-3) was synthesized accordingly to the previously described procedure (Masharina et al., J Am Chem Soc. 2012 Nov. 21; 134(46):19026-34). BG-NH.sub.2 (i-4) was synthesized accordingly to the previously described procedure (Keppler et al Nat Biotechnol. 2003 Jan.; 21(1):86-9.).
(16) BG-TMR(6)-COOH (i-5)
(17) To a solution of TMR(6-isomer)-COOMe (i-3) (6.1 mg, 11.8 mol, 1 eq.) in dry DMSO (200 L), DIPEA (20 L, 115 mol, 10 eq.) and HBTU (5.2 mg, 13.6 mol, 1.15 eq.) were added. After 5 min, BG-NH.sub.2 (i-4) was added to the reaction mixture. The reaction was stirred 15 min at RT. Then, 200 L 1M NaOH was added and the reaction was stirred for another 15 min. 50 L AcOH were added and the reaction was purified by HPLC and lyophilized to yield a red solid (4 mg, 45%).
(18) BG-TMR-C6-SPY (i-6)
(19) To a solution of BG-TMR(6)-COOH 1 eq. in dry DMSO, 10 eq. DIPEA and 1.2 eq. TSTU were added successively. The activation was checked by TLC (80/20 v/v ACN/H.sub.2O). After 5 min activation, i-2 was added and the reaction was allowed to stir for 1 h at RT. The reaction mixture were treated with H.sub.2O (100 L) and stirred for another 20 min to hydrolyze the remaining NHS ester. Then, the reaction was quenched with AcOH, purified by HPLC and lyophilized to yield a red solid.
(20) A synthetic molecule containing an O.sup.6-benzylguanine (BG) moiety for the specific labelling of SNAP-tag, the flurophore tetramethylrhodamine (TMR) and sulfamethoxazole (SMX) as intramolecular SPR ligand was synthesized according to Scheme 2.
(21) ##STR00013## ##STR00014##
(22) The synthesis of i-8, i-4 and i-5 is already described in the aforementioned example.
6-amino-N-{4-[(5-methyl-1,2-oxazol-3-yl)sulfamoyl]phenyl}hexanamide (ii-2)
(23) To a solution of Fmoc-6-aminohexanoic acid (100 mg, 0.28 mmol, 1 eq.) in dry DMSO (1.0 mL, 280 mM), DIPEA (52 L, 0.31 mmol, 1.1 eq.) and sulfamethoxazole (215 mg, 0.85 mmol, 3 eq.) were added. HBTU (130 mg, 0.34 mmol, 1.2 eq.) was added and the reaction was stirred 2 h at RT. Then, 200 L H.sub.2O was added to the reaction mixture and stirred for 30 min. Finally, the reaction was quenched with 80 L AcOH, purified by HPLC, lyophilized to yield a white solid (54 mg, 33%).
(24) The fmoc-protected product was dissolved in 1 mL of a solution mixture of piperidine 20% in acetonitrile. The reaction mixture was stirred 1 h at RT, quenched with 0.2 mL AcOH, evaporated under reduced pressure, purified by preparative HPLC and lyophilized to yield a white solid (33.5 mg, quant.).
(25) BG-TMR-C6-SMX (ii-6)
(26) To a solution of BG-TMR(6)-COOH (220 L, 0.58 mol, 1 eq.) in dry DMSO (2.9 mM), DIPEA (4 L, 12 mol, 41 eq.) and TSTU (7 L, 0.7 mol, 1.2 eq.) were added successively. The activation was checked by TLC (80/20 v/v ACN/H.sub.2O). After 5 min activation, ii-2 (34 L, 2.32 mol, 4 eq.) was added and the reaction was allowed to stir for 1 h at RT. The reaction mixture were treated with H.sub.2O (100 L) and stirred for another 20 min to hydrolyze the NHS ester. Then, the reaction was quenched by the addition of 20 L of AcOH, purified by HPLC and lyophilized to yield a red solid (0.4 mol, 70%).
(27) The fusion protein comprising of SNAP-tag, a 30-proline linker, EGFP and sepiaterin reductase (SPR), was constructed by first replacing the coding sequence of HCA in the previously described sensor SNAP-p30-CLIP-HCA (Brun et al. J Am Chem Soc. 2011; 133(40): 16235-42) by the coding sequence of SPR using standard cloning techniques yielding SNAP-p30-CLIP-SPR. In a second cloning step, the CLIP-tag was replaced by the coding sequence of EGFP using standard cloning techniques yielding SNAP-p30-EGFP-SPR. The fusion protein was expressed in the E. coli strain Rosetta-Gami 2 (DE3)pLysS (Novagen, Merck KGaA, Darmstadt, Germany) and purified using a C-terminal His-tag as well as an N-terminal Strep-tag II to obtain the full construct.
(28) The purified fusion protein SNAP-p30-EGFP-SPR (5 M) was labelled with 2 eq. of BG-TMR-C6-SPY or BG-TMR-C6-SMX (10 M) in a buffer (50 mM HEPES, 150 mM NaCl, pH 7.5) for 1 h at room temperature. After the incubation, the excess of ligand was removed by three washing steps (3400 L) with the aforementioned buffer using a centrifugal filter spin column with a 50 kDa cut-off membrane (Amicon Ultra-0.5 Centrifugal Filter, Merck KGaA, Darmstadt, Germany). The purified sensor is then diluted to a concentration of 5 M in HEPES buffer (in 50 mM HEPES, 150 mM NaCl, pH 7.5).
(29) To evaluate the performance of the sensor, titrations experiments were performed with different concentrations of NADP.sup.+. The labelled sensor was diluted to a concentration of 50 nM in 100 L of HEPES buffer (50 mM HEPES, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.5) containing defined concentrations of NADP.sup.+ and NADPH in black non-binding 96-well plates (Greiner Bio-One, Kremsmnster, Austria).
(30) The solutions were incubated at room temperature for at least 15 minutes to ensure that the sensor had reached equilibrium. Fluorescence measurements were done on an Infinite M1000 spectrofluorometer (TECAN). Excitation was carried out at 450 nm with a bandwidth of 10 nm and spectra were recorded from 480 to 610 nm using a step size of 1 nm and bandwidths of 10 nm.
(31) As shown in
(32) Another titration was performed using the aforementioned procedure, where the labelled sensor was diluted in buffer containing different ratio of NADPH/NADP.sup.+ with a fixed total concentration of cofactors (100 M) and a fixed concentration of N-acetylserotonin as competitive SPR ligand to tune the sensor response. We observed that the sensor can be efficiently use to measure ratio of NADPH/NADP.sup.+ but when labelled with sulfapyridine as intramolecular ligand, the sensor is sensitive to pH variations (
Example 2: SPR-EGFP-p80-SNAP
(33) This example describes an alternative optimized geometry of the previously described FRET sensor.
(34) As before, the sensor contains the same FRET pair (EGFP and TMR). SNAP-tag is labelled with the synthetic molecule BG-TMR-C6-SMX previously described. The fusion protein comprising of SPR, EGFP, a 30-proline linker and SNAP-tag was constructed by first replacing the coding sequence of RLuc8 in a previous plasmid SPR-RLuc8-SNAP by the coding sequence of EGFP using standard cloning techniques yielding SPR-EGFP-SNAP. In a second cloning step, an annealed 30-proline oligonucleotides was inserted between EGFP and SNAP using standard cloning techniques yielding SPR-EGFP-p30-SNAP. The fusion protein was expressed in the E. coli strain Rosetta-Gami 2 (DE3)pLysS (Novagen, Merck KGaA, Darmstadt, Germany) and purified using a C-terminal His-tag as well as an N-terminal Strep-tag II to obtain the full construct.
(35) The purified fusion protein SPR-EGFP-p30-SNAP (5 M) was labelled with 2 eq. of BG-TMR-C6-SMX or BG-TMR-C6-SPY (10 M) in a buffer (50 mM HEPES, 150 mM NaCl, pH 7.5) for 1 h at room temperature. After the incubation, the excess of ligand was removed by three washing steps (3400 L) with the aforementioned buffer using a centrifugal filter spin column with a 50 kDa cut-off membrane (Amicon Ultra-0.5 Centrifugal Filter, Merck KGaA, Darmstadt, Germany). The purified sensor is then diluted to a concentration of 5 M in HEPES buffer (in 50 mM HEPES, 150 mM NaCl, pH 7.5).
(36) To evaluate the performance of the sensor, titrations experiments were performed with different concentrations of NADP.sup.+ and NADPH using the enzyme glucose-6-phosphate dehydrogenase (Baker yeast, type IX, Sigma) and its substrate glucose-6-phosphate to convert the NADP.sup.+ measured in 96-well plate into NADPH. This enzyme cycling method was performed to assure that there is almost no remaining NADP.sup.+ present in the solution but only NADPH. Indeed, it was found that the commercially available NADPH contains traces of NADP.sup.+ up 2%. The labelled sensor was diluted to a concentration of 50 nM in 100 L of HEPES buffer (50 mM HEPES, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.5 and 1 mM glucose-6-phosphate) containing defined concentrations of NADP.sup.+ in black non-binding 96-well plates (Greiner Bio-One, Kremsmnster, Austria). The solutions were incubated at room temperature for at least 15 minutes to ensure that the sensor had reached equilibrium. Fluorescence measurements were done on an Infinite M1000 spectrofluorometer (TECAN). Excitation was carried out at 450 nm with a bandwidth of 10 nm and spectra were recorded from 480 to 610 nm using a step size of 1 nm and bandwidths of 10 nm. After this first measurement, 1 L of glucose-6-phosphate dehydrogenase from baker's yeast (Type IX, Sigma) reaching a final concentration of 26 nM. The solutions were incubated for 1 h at room temperature to ensure the maximal conversion of NADP.sup.+ into NADPH. The result shows clearly that the intramolecular ligand can only bind to SPR in presence of NADP.sup.+ and not in presence of NADPH (
(37) The slight decrease of the FRET ratio at the highest concentration of cofactor is due to the inhibition of the glucose-6-phosphate dehydrogenase by the high concentration of NADPH, which cannot reach complete conversion (
(38) In another titration, the sensor's specificity was tested by diluting the labelled sensor to a concentration of 50 nM in 100 L of HEPES buffer (50 mM HEPES, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.5) containing defined concentration of different nucleotides structurally close to NADP.sup.+: NAD.sup.+, NADH, ATP, ADP, GTP (
Example 3: SPR-Halo-p30-SNAP
(39) This example describes the design and construction of an optimized FRET-based sensor capable of sensing the concentration of NADP(H). The sensor comprises the human sepiapterin reductase (SPR) as NADP(H)-specific binding protein and sulfamethoxazole as intramolecular ligand. Halo-tag, another self-labelling tag, is used for the specific labelling of the fluorophore SiR (
(40) For the labelling of Halo-tag, a molecule containing a chloroalcane group linked to the fluorophore silicon-rhodamine was synthesized accordingly to the described procedure (Lukinavicius et al, Nat Chem. 2013 Feb.; 5(2):132-9) (
(41) To evaluate the performance of the sensor, titrations experiments were performed with different concentrations of NADP.sup.+. The labelled sensor was diluted to a concentration of 50 nM in 100 L of HEPES buffer (50 mM HEPE, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.5) containing defined concentrations of NADP.sup.+ in black non-binding 96-well plates (Greiner Bio-One, Kremsmnster, Austria). The solutions were incubated at room temperature for at least 15 minutes to ensure that the sensor had reached equilibrium. Fluorescence measurements were done on an Infinite M1000 spectrofluorometer (TECAN). Excitation was carried out at 520 nm with a bandwidth of 10 nm and spectra were recorded from 540 to 740 nm using a step size of 1 nm and bandwidths of 10 nm. As seen in
Example 4: SPR(D41W42)-Halo-p30-SNAP
(42) This example describes the design and construction of an optimized FRET-based sensor capable of sensing the concentration of NAD(H). The sensor comprises a mutant of SPR as NAD(H)-specific binding protein and sulfamethoxazole as SPR-L. Halo-tag, another self-labelling tag, is used for the specific labelling of the fluorophore SiR. TMR and SiR form respectively the FRET donor and acceptor pair. SNAP-tag is labelled with the synthetic molecule BG-TMR-C6-SMX previously described and SiR-Halo is used for the specific labelling of Halo-tag. The fusion protein comprising of a mutant of SPR(D41W42), Halo-tag, a 30-proline linker and SNAP-tag was constructed by using PCR site-directed mutagenesis using designed primers to performed several point mutations in order to change two bases; namely the following mutations A41D and R42W to yield SPR(D41W42)-Halo-p30-SNAP. The fusion protein was expressed in the E. coli strain Rosetta-Gami 2 (DE3)pLysS (Novagen, Merck KGaA, Darmstadt, Germany) and purified using a C-terminal His-tag as well as an N-terminal Strep-tag II to obtain the full construct.
(43) The purified fusion protein SPR(D41W42)-Halo-p30-SNAP (5 M) was labelled with 2 eq. of BG-TMR-C6-SMX (10 M) in a buffer (50 mM HEPES, 150 mM NaCl, pH 7.5) for 1 h at room temperature. After the incubation, the excess of ligand was removed by three washing steps (3400 L) with the aforementioned buffer using centrifugal filter spin column with a 50 kDa cut-off membrane (Amicon Ultra-0.5 Centrifugal Filter, Merck KGaA, Darmstadt, Germany). The purified sensor is then diluted to a concentration of 5 M in HEPES buffer (in 50 mM HEPES, 150 mM NaCl, pH 7.5).
(44) In order to evaluate, that these mutations were sufficient to fully switch the specificity of the cofactor binding site of SPR from NADP(H) to NAD(H), we prepared titration experiments according to the similar procedure explained herein (example 3). The labelled sensor were diluted in buffer solution containing defined concentrations of NAD.sup.+ and NADP.sup.+ and measure with the fluorimeter (TECAN) (
(45) The titration curves revealed that SPR mutant has a complete reverse cofactor specificity as seen for the wild-type (see
Example 5: Lysate Measurement
(46) For the measurement in lysate, SPR-Halo-p30-SNAP was labelled with BG-TMR-C6-SMX and SiR-Halo as described in the example No. 3.
(47) HEK293 cells in suspension were grown (37 C., 5% CO.sub.2, 100 rpm) in 15 mL Ex-cell 293 medium supplemented by GlutaMax for 4 days reaching typically 210.sup.6 cells/mL, 310.sup.7 total cells with viability >97% (trypan blue test). The cells were pellet by centrifugation (5 min, 750 rpm, 4 C.), The supernatant was removed and the cells were resuspended in 1 mL cold PBS. The cells were lyzed by freeze-thaw cycle (2 min in N.sub.2 liq., 2 min in a water bath at 37 C.) and shortly vortex. The cells debris were centrifuge at 13000 rpm, 5 min, 4 C. and the supernatant was filtered through centrifugal filter spin column with a 10 kDa cut-off membrane (Amicon Ultra-0.5 Centrifugal Filter, Merck KGaA, Darmstadt, Germany). The labelled sensor was then diluted to 50 nM in 100 L lysate and the ration was measured with a fluorimeter (TECAN) using the same parameter than described in the example 3. In parallel, a titration of the labelled sensor in solution containing defined ratio of NADPH/NADP.sup.+ was performed as calibration (total cofactor concentration: 100 M). Additionally, the lysate was also measured in a UV-Vis spectrophotometer to get a rough estimation of the concentration of [NADPH]+[NADH] giving an absorbance 1.1-1.7 at 340 nm (1=1 cm, =6220 M.sup.1 cm.sup.1) correlated to a concentration of 180-270 M. The lysate is therefore sufficiently concentrated for the sensor measurement to be reliable.
(48) Comparing the results that we obtained with the lysate measurement (ratio TMR/SIR=1.06) with the calibration curve, we obtained a ratio of NADPH/NADP.sup.+=40 (
Example 6: Live Cells Measurement
(49) SPR-Halo-p30-SNAP was expressed in U2OS cells and labelled with CP-TMR-C6-SMX and SiR-Halo. CP-TMR-C6-SMX (
(50) Semi-stable U2OS (SPR-Halo-p30-SNAP cloned in a pEBTet vector; see Bach et al. FEBS J. 2007 Feb.; 274(3):783-90 for the description of pEBTet) freshly trypsinated are diluted 5-fold with DMEM GlutaMax+10% FBS and 1 g/mL puromycin. They are plated (2 mL) in previously sterilized and poly-L-ornithine coated (1 h incubation) glass coverslips (2020 mm, VWR) in 12-well plate (TPP). On day 2, the expression is induced by 100 ng/mL doxycycline for 24 h. On day 3, the cells are labelled with 1 M CP-TMR-C6-SMX and 1 M SiR-Halo with 10 M (+/)-verapamil overnight. On day 4, the cells are washed 3 times with DMEM GlutaMax+10% FBS without phenol red (10 min incubation, 37 C., 5% CO.sub.2 between each step). After 30 min of incubation, the cells are ready for imaging.
(51) Glass coverslips with labelled U2OS cells were transferred to a Cytoo chamber (443410 mm). Gravity fed perfusion of the chamber was performed at a flow rate of 1 mL/min. Time-course experiments of sensor imaging were performed using a Leica LAS AF 7000 wide-field microscope equipped with a 63 plan Apochromat 1.40 NA oil immersion objective lens. A xenon arc lamp was used for imaging of the U2OS cells. For each frame, the two channels (donor and FRET) were measured consecutively with an interval of 10 s between the two emission channels. The filter set Cy3/Cy5 was used for FRET ratio imaging with excitation at 530/35 and emission at 580/40 (Cy3) as well as excitation at 635/30 and emission at 700/72 (Cy5). If not indicated otherwise, the image size was 194 M194 M. The reagent perfused during the experiment (H.sub.2O.sub.2, SPY) were dissolved in HBSS (Lonza). HBSS solution was continuously perfused during the other time point of the experiment.
(52) As a control for the sensor functionality, 2 mM of sulfapyridine (SPY) is perfused on the cells. The sulfapyridine, once entered in the cytosol, competes with the intramolecular ligand sulfamethoxazole and open the sensor. The concentration of sulfapyridine is sufficient to fully open the sensor and thus have a calibration point to quantify changes in NADPH/NADP.sup.+. Then, the sulfapyridine is wash out by HBSS and we can observe that the intracellular sensor goes back to its initial ratio (TMR/SiR). Then, a solution of hydrogen peroxide was perfused and as result a sharp decrease of the ratio can be monitored. The addition of H.sub.2O.sub.2 caused an oxidative stress activating the cellular oxidative system. This system comprises the glutathione peroxidase, which oxidizes the glutathione to decompose the hydrogen peroxide. The oxidized pool of glutathione will then be reduced by the activity of the glutathione reductase depleting the NADPH, thus decreasing the ratio NADPH/NADP.sup.+ and closing the sensor. Interestingly, we can observe that the cells are able to quickly return to their basal level of NADPH/NADP.sup.+ once the addition of H.sub.2O.sub.2 is stopped and washed out.
(53) It has to be noted that with the microscope, the purified sensor has a slightly reduced FRET ratio change between the open and closed conformation (Rmax=6). Due to the relatively high free ratio of NADPH/NADP.sup.+ in living cells, the sensor cannot be seen in a fully closed conformation, explaining the smaller FRET ratio changes observed in live cells compared to the in vitro experiments.
(54) To summarize, the sensor is very useful for live cell imaging. Especially, it can be used to quantity the different ratio NADPH/NADP.sup.+ in the different subcellular compartments (cytosol, mitochondria, nucleus, reticulum endoplasmic).
Example 7: SPR-TagGFP2-p30-SNAP-TagRFP
(55) This example describes the design and construction of a FRET-based sensor using two fluorescent proteins capable of sensing the concentration of NADP.sup.+ and ratio of NADPH/NADP.sup.+. The sensor comprises the human SPR as NADP(H)-specific binding protein and sulfamethoxazole as intramolecular ligand. TagGFP2 and TagRFP form respectively the FRET donor and acceptor (
(56) ##STR00015##
6-amino-N-{4-[(5-methyl-1,2-oxazol-3-yl)sulfamoyl]phenyl}hexanamide (ii-2)
(57) To a solution of Boc-6-aminohexanoic acid (150 mg, 0.65 mmol, 1 eq.) in DMF (2.0 mL), EDC HCl (140 mg, 0.72 mmol, 1.1 eq.) and HOBt (98 mg, 0.72 mmol 1.1 eq.) were added under stirring. After full dissolution of the reagents, sulfamethoxazole (494 mg, 1.9 mmol, 3 eq.) was added followed by DIPEA (0.11 mL, 0.65 mmol, 1 eq.). The reaction was stirred overnight at RT. Finally, the reaction was acidified with acetic acid (0.2 mL), purified by HPLC and lyophilized to yield a white solid (182.1 mg, 0.39 mmol, 59%).
(58) The Boc-protected product (60 mg, 0.128 mmol, 1 eq.) was dissolved in TFA (0.5 mL) and stirred for 30 min. TFA was then evaporated under reduced pressure. The product was dissolved in 0.5 mL DMSO, purified by HPLC and lyophilized to yield a white solid (30.1 mg, 81.9 mol, 64%).
(59) BG-NH.sub.2 (i-4) was synthesized accordingly to the previously described procedure (Keppler et al Nat Biotechnol. 2003 Jan.; 21(1):86-9.).
4-[(4-{[(2-amino-9H-purin-6-yl)oxy]methyl}benzyl)amino]-4-oxobutanoic acid (iii-1)
(60) To a suspension of BG-NH2 (300 mg, 1.1 mmol, 1 eq.) in dry DMSO (6 mL) were added DIPEA (0.4 mL, 2.4 mmol, 2.2 eq.) and succinic anhydride (133 mg, 1.3, 1.2 eq.) under stirring. The reaction mixture clarified during the process of the reaction. After 1 h, the reaction was shown to be complete. 1 mL H.sub.2O was added to the reaction mixture to hydrolyze the succinic anhydride. Finally, the reaction was quenched with 0.5 mL acetic acid, purified by HPLC and lyophilized to yield a white solid (388 mg, 1.05 mmol, 95%).
(61) BG-Suc-C6-SMX (iii-2)
(62) To a solution of compound iii-1 (19.5 mg, 52.7 mol, 1 eq.) in dry DMSO (0.6 mL), DIPEA (17.5 L, 105.4 mol, 2 eq.) and TSTU (23.8 mg, 79 mol, 1.5 eq.) in 200 L DMSO were added. After 5 min, the formation of the NHS-ester was checked by TLC (9/1 DCM/MeOH) and LC-MS. Then, the amine ii-2 (300 l, 63.2 mol, 1.2 eq.) with DIPEA (17.5 L, 105.4 mol, 2 eq.) was added and stirred for 30 min. Finally, the reaction was quenched first with 200 L H.sub.2O (15 min) and then with 50 L AcOH. The product was purified by HPLC and lyophilized to yield a white solid (7.1 mg, 9.9 mol, 19%).
(63) The fusion protein comprising of the human SPR, TagGFP2, a 30-proline linker, SNAP-tag and TagRFP was constructed by replacing the coding sequence of Halo-tag in a previous construct SPR-Halo-p30-SNAP by the coding sequence of TagGFP2 (Evrogen, Moscow, Russia) and inserting TagRFP (Evrogen, Moscow, Russia) at the end of the construct using Gibson Assembly (NEB, Ipswich, England) yielding SPR-TagGFP2-p30-SNAP-TagRFP. The fusion protein was expressed in the E. coli strain Rosetta-Gami 2 (DE3)pLysS (Novagen, Merck KGaA, Darmstadt, Germany) and purified using a C-terminal His-tag as well as an N-terminal Strep-tag II to obtain the full construct.
(64) The purified fusion protein SPR-TagGFP2-p30-SNAP-TagRFP (5 M) was labelled with 2 eq. of BG-Suc-C6-SMX (10 M) in a buffer (50 mM HEPES, 150 mM NaCl, pH 7.5) for 1 h at room temperature. After the incubation, the excess of ligand was removed by three washing steps (3400 L) with the aforementioned buffer using centrifugal filter spin column with a 50 kDa cut-off membrane (Amicon Ultra-0.5 Centrifugal Filter, Merck KGaA, Darmstadt, Germany). The purified sensor is then diluted to a concentration of 5 M in HEPES buffer (in 50 mM HEPES, 150 mM NaCl, pH 7.5).
(65) To evaluate the performance of the sensor, titrations experiments were performed with different concentrations of NADP.sup.+. The labelled sensor was diluted to a concentration of 50 nM in 100 L of HEPES buffer (50 mM HEPES, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.5) containing defined concentrations of NADP.sup.+ in black non-binding 96-well plates (Greiner Bio-One, Kremsmnster, Austria). The solutions were incubated at room temperature for at least 15 minutes to ensure that the sensor had reached equilibrium. Fluorescence measurements were done on an Infinite M1000 spectrofluorometer (TECAN). Excitation was carried out at 450 nm with a bandwidth of 10 nm and spectra were recorded from 480 to 630 nm using a step size of 1 nm and bandwidths of 10 nm.
(66) As shown in
(67) One advantage of the sensors for NADP and NAD based on two fluorescent proteins is the use of a synthetic labelling molecule (containing the SPR ligand) with improved cell permeability since it does not contain a synthetic fluorophore. Furthermore, the synthetic scheme is significantly shorten and simplified. The same molecule functionalized with O.sup.4-benzyl-2-chloro-6-aminopyrimidine (CP) can also be used for the labelling of SNAP-tag and might further improve the cell permeability.
Example 8: SNAP_P30_hSPR(D41W42)_NLuc_cpDHFR
(68) This example describes the design and construction of an optimized BRET-based sensor capable of sensing the concentration of NAD.sup.+. The sensor comprises a mutant of SPR as NAD(H)-specific binding protein and BG-Peg11-Cy3-sulfamethoxazole as SPR-L. NanoLuc is used as the luciferase which forms a BRET pair with Cy3. SNAP-tag is labelled with the synthetic molecule BG-Peg11-Cy3-sulfamethoxazole (
(69) The fusion protein SNAP_P30_hSPR(D41W42)_NLuc_cpDHFR comprising of a mutant of SPR(D41W42), NanoLuc (NLuc), a 30-proline linker, SNAP-tag and circular-permutated dihydrofolate reductase (cpDHFR) was constructed using standard procedures as described above. cpDHFR is derived from wild-type E. coli DHFR by fusing the original N and C termini with a (glycine).sub.5 linker and splitting between Asn23 and Leu24. It was found that the attachment of an extra protein domain to the C terminus of NanoLuc decreased unspecific interactions of NanoLuc with other molecules. The fusion protein was expressed in the E. coli strain Rosetta-Gami 2 (DE3)pLysS (Novagen, Merck KGaA, Darmstadt, Germany) and purified using a C-terminal His-tag as well as an N-terminal Strep-tag II to obtain the full construct.
(70) The purified fusion protein SPR(D41W42)-Halo-p30-SNAP (5 M) was labelled with 2 eq. of BG-Peg11-Cy3-sulfamethoxazole (10 M) in a buffer (50 mM HEPES, 150 mM NaCl, pH 7.5) for 1 h at room temperature. After the incubation, the excess of ligand was removed by three washing steps (3400 L) with the aforementioned buffer using centrifugal filter spin column with a 50 kDa cut-off membrane (Amicon Ultra-0.5 Centrifugal Filter, Merck KGaA, Darmstadt, Germany). The following conditions were used for titration of the sensor with NAD.sup.+: 2.5 nM sensor was added to a buffer (500 mM HEPES, 500 mM NaCl, 10 mg/mL BSA, pH 7.5) containing NAD+ at various concentrations and 1000-fold diluted NanoLuc substrate. The assay was analyzed by recording the light emitted at 486 nm (from NanoLuc) and at 595 nm (from Cy3) and calculating the ratio of the two intensities. Measurements were done on an EnVision Multilabel Reader (Perkin Elmer). The apparent Kd of the sensor for NAD+ is 19 M. The observed ratio change when going from the open to the closed form of the sensor is fivefold (
(71) The sensor can also be used to measure the activity of lactate dehydrogenase (LDH) (