Superluminescent Variants of Marine Luciferases for Bioassays

Sep 27, 2011 - Inset B indicates relative optical intensities of firefly luciferase (FLuc), GLuc, and I90L. Inset C provides the color photographs. In...
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Superluminescent Variants of Marine Luciferases for Bioassays Sung Bae Kim,*,† Hideyuki Suzuki,‡ Moritoshi Sato,‡ and Hiroaki Tao† †

Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan ‡ Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan

bS Supporting Information ABSTRACT: In this study, a rational synthesis of superluminescent variants from marine luciferases with prolonged bioluminescence has been demonstrated. A putative active site of a model marine luciferase, Gaussia princeps Luciferase (GLuc), was assigned and modified by a site-directed mutagenesis. The potent variants were found to generate up to 10 times stronger bioluminescence, emitting red shifts of up to 33 nm with natural coelenterazine than native GLuc, rendering an efficient optical signature in bioassays. The advantageous properties were demonstrated with mammalian two-hybrid assays, single-chain probes, and metastases of murine B16 melanoma in BALB/c nude mice. The unique ideas for engineering GLuc are proved to be valid even for other marine luciferases.

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ioluminescence generated by a luciferase-catalyzed oxidation of luciferins is broadly utilized as an optical signature for quantifying the molecular events in cell lines and living subjects.1,2 In contrast to the popular use of luciferases in general bioanalysis, their common limitations, such as (i) the poor intensity, (ii) deficiency in the red-shifted luciferases, and (iii) rapid decline in the luminescence intensity after substrate addition, still need to be addressed. An interesting mutagenesis study on marine luciferases was conducted with Renilla reniformis luciferase (RLuc).3 The remarkable red-shifted bioluminescence was achieved by the mutants and also combining the mutants with coelenterazine-v (CTZ-v). Another trial was conducted to synthesize a stable bioluminescent variant of Gaussia princeps luciferase (GLuc) through a cellfree protein synthesis.4 The potent variant, M43I, effectively prolonged the bioluminescence. However, the intensities were not significantly improved. An autoillumination from yellow fluorescent protein (YFP)linked RLuc was previously employed for cell imaging, which was named “BRET-based autoilluminated fluorescent protein on EYFP” (eBAF-Y).5 The optimized pair was successfully utilized for single cell imaging. A random mutagenetic approach of luciferase engineering is normally slow and tedious and intensively consumes time-andlabor. Especially, in the absence of the crystallographic data, the researcher’s measures for luciferase engineering are highly restricted. As a conventional strategy to access clues for protein engineering, homologous protein sequences are aligned for finding consensus amino acids and mutagenesis, named “a semi-rational, consensus sequence driven mutagenesis strategy”.6 r 2011 American Chemical Society

In the present study, we demonstrate a semirational synthesis of potent marine luciferases exerting outstanding optical properties, such as superluminescence and/or a prolonged emission half-life. The chemical structures sharing an imidazolone ring are very similar between the chromophore, 65SYG67, of Aequorea green fluorescent protein (GFP) and native coelenterazine (CTZ) as the common substrate of marine luciferases. The comparison shows that the modification schemes similar to those for GFPs7 may lead to efficient alteration of marine luciferases. In specific, native GFP was initially modified by mutagenesis to improve the optical properties:8 S202F, I167V, T203I, Y66W, and T66H. Generation of enhanced yellow fluorescent protein (eYFP) and Venus was contributed by S72A and T203Y (1st generation), V68L and Q69K (2nd generation), and F46L and V163A (3rd generation).9 These references teach that alteration of chromophore-neighboring amino acids to F, V, W, H, Y, and L may be even effective for modification of the optical properties of marine luciferases. As a model marine luciferase, we selected GLuc, which is the smallest luciferase discovered (∼20 kDa) and which emits bursting bioluminescence intensities.10 GLuc comprises two conserved catalytic domains in the sequence.11 GLuc has multiple disulfide bonds and can be retained in the endoplasmic reticulum (ER) with an ER localization signal, i.e., “Lys-Asp-Glu-Leu” (KDEL) peptide.10 The crystallographic information has not been reported to date. Received: August 23, 2011 Accepted: September 27, 2011 Published: September 27, 2011 8732

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Analytical Chemistry The initial clue for the core region of a luciferase was taken from a hydrophobicity search, e.g., the scale of Kyte and Doolittle.12 This scale generally reveals a remarkably hydrophilic region, which may favorably recruit the specific substrate. This characteristic region is interestingly highly conserved among marine luciferases. Furthermore, we separately assumed the putative core region through a consecutive fragmentation and reconstitution strategy of GLuc in a single-chain probe as shown in our previous study.13 Surprisingly, some GLuc fusions suddenly tolerate an insertion of an exogenous protein, indirectly reflecting the structural interface of GLuc. This structural interface is interestingly superimposed with the most hydrophilic region in the scale of Kyte and Doolittle,12 to which the substrate may favorably access. This region is highly conserved among marine luciferases (Suppl. Figure 2A in the Supporting Information). The putative core region found by the above strategy was site-directly mutated to F, Y, W, V, H, G, L, etc. under the protocol of “Quickchange”14 for enhancing the hydrophobicity and exerting a “conservative amino acid change.” This rational mutagenesis generated several potent variants, I90L, 8990 (meaning F89W/I90L), 90115 (I90L/I115L), and Monsta (F89W/I90L/H95E/Y97W). I90L and 8990 emit 6- and 10-times stronger and/or prolonged bioluminescence, respectively, than intact GLuc in mammalian cells derived from African green monkey kidney fibroblast (COS-7 cells). For reasoning the improved optical properties, the Michaelis Menten constants, turnover rates, and quantum yields of columnpurified GLuc variants were precisely evaluated. The general validity of the present methodology on marine luciferases was demonstrated with Metridia longa luciferase (MLuc) and Metridia pacifica luciferase 1 (MpLuc1).

’ EXPERIMENTAL SECTION Mutagenesis of Marine Luciferases. All the present variants were originally designed to be sequestered into the ER through tagging a KDEL peptide to them. The cDNA template encoding GLuc was custom-synthesized on the basis of public information (GenBank accession number, AAG54095.1), where a KDEL sequence was genetically fused at the C-terminus for retention in the ER. The cDNA templates encoding Metridia pacifica luciferase 1 (MpLuc1) and Metridia longa luciferase (MLuc) were synthesized from the database information (GenBank accession number, AB195233 and AAR17541, respectively) and were genetically linked with the cDNA encoding a KDEL sequence. The constructs were subcloned into pcDNA 3.1(+) (Invitrogen) using the specific restriction sites, HindIII and XhoI, for initial mutagenesis studies and expression confirmation in mammalian cells. Site-directed mutagenesis was performed using a “QuickChange” protocol with a LA Taq polymerase (Takara) and customsynthesized primers. The competent mutants comprised the following mutation points: F89W/I90L (named 8990 for simplicity), I90L/I115L (90115), C94W/Y97W (9497), H95Q/D100N (95100), R93G/G99S (9399), Y97T/98G (9798), and F89W/ I90L/H95E/Y97W (Monsta). The corresponding plasmids were named p8990, p90115, p9497, etc. The pcDNA 3.1(+) vector encoding GLuc and I90L was named pGLuc and pI90L, respectively. The names in the parentheses indicate the mutation sites. Similarly, RLuc variants were generated using the protocol of QuickChange. The mutation sites were decided on the basis of the alignment of Suppl. Figure 2 in the Supporting Information. They were named I140L, V141L, V146L, and V146L/E151A,

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respectively. The mutation was confirmed with a BigDye Terminators v1.1 cycle sequencing kit and a genetic sequencer (ABI PRISM 310 Genetic Analyzer, Applied Biosystems). Characterization of GLuc Variants. To determine the optical intensities of GLuc variants, the corresponding plasmids were transiently transfected into African green monkey kidney fibroblast-derived COS-7 cells raised on a 96-well black frame plate (Nunc) using a transfection reagent, TransIT-LT1 (Mirus). The cells on the plate were lysed with a lysis buffer (E291A; Promega) and placed in a bioluminescence microplate reader equipped with automatic substrate injectors (Mithras LB 940, Berthold). The substrate, CTZ, was dissolved in an assay buffer (E290B) carrying porcine gelatin and thiourea15 in a Renilla luciferase assay kit (E2820, Promega). The developed bioluminescence intensities after automatic injection of the substrate were integrated for 2 s. The consequent intensities are presented in Figure 1. All the experiments were conducted in the Renella luciferase assay buffer (E290B inside E2820, Promega), unless otherwise designated. The bioluminescence spectra were similarly monitored to determine the red-shifted variants in COS-7 cells cultured on a 12-well plate. The lysates on the plate were transferred into a quartz cell, with the addition of 400 μL of 0.5 μg/μL of CTZ, manually mixed and read in a spectrophotometer (F-7000, Hitachi) with a 2400 nm/min scan speed. The maximal light intensity (λmax) was detected using the equipped software (FL Solutions ver. 2.1) (Figure 1, inset A). The relative optical intensities of FLuc, GLuc, and I90L were manually visualized in test tubes (Figure 1, inset B). For the experiment, COS-7 cells cultured on a six-well plate were transfected with a pcDNA 3.1(+) vector encoding FLuc, GLuc, or I90L. After lysis, the cell lysates were transferred to 1.6 mL tubes to determine the integrated intensities for 5 s using an image analyzer (LAS-3000, Fuji Film). The color photographs shown in Figure 1, inset C, were taken by a digital camera (IXYdigital1000, Canon). The red-shifted property of GLuc variants was also examined with COS-7 cells raised in a 96-well black frame plate (Suppl. Figure 3B in the Supporting Information). The cells expressing a GLuc variant were lysed and placed in a microplate reader equipped with the automatic substrate injector and a 610 nm long-pass filter (Mithras LB940, Berthold). The bioluminescence intensities were integrated for 2 s. In the present study, the luminescence intensities were normalized by three methods: (i) one was by the amount of proteins in cell lysates. The unit of absolute luminescence is subsequently RLU/μg of protein (e.g., Figure 4B); (ii) the other was expressed by a relative luminescence unit (RLU) ratio ((), where RLU (+) and RLU ( ) represent the luminescence intensity from 1 μg of protein of cell lysate after COS-7 cells were stimulated with and without a ligand, respectively (e.g., Figure 3A); (iii) the third is that the optical intensities were expressed in RLU per cell on the basis of the briefly counted cell numbers before experiments (e.g., Figure 1, inset D). The present “transient transfection” protocol may cause a deviation in the transfection efficiency. We used native GLuc as the standard enzyme to normalize the transfection efficiency between batches (Y-axis of Figure 1). The variance in the protein amounts in Western blotting (Figure 1, inset E) was basically normalized with a Bradford reagent in practical experiments. Optical Stability of GLuc Variants. An enhanced light stability of luciferases is an attractive property for the application as an optical signature in bioanalysis. The light stability of GLuc 8733

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Figure 1. Optical properties of GLuc variants. Bioluminescence intensities of GLuc variants relative to intact GLuc. COS-7 cell lysates made by a Promega lysis buffer (E291A inside E2820) were assayed (n = 3). Intact GLuc was utilized as the internal reference. The asterisks highlight important mutants, λmax of which were specified on the bars. Inset A shows red-shifted spectra of several GLuc variants. Monsta carries four mutations, F89W/ I90L/H95E/Y97W. The numbers in parentheses show the percents of emitted photons greater than 600 nm. Inset B indicates relative optical intensities of firefly luciferase (FLuc), GLuc, and I90L. Inset C provides the color photographs. Inset D shows the uperluminescent properties of several GLuc variants. The specific substrate, native coelenterazine (CTZ), dissolved in a Renilla luciferase assay buffer (E290B, Promega) was consecutively injected using an automatically programmed injector. Inset E shows a Western blot analysis to determine the protein amounts of GLuc variants. The expressed protein amounts of the variants were compared using anti-GLuc antibody and anti-α-tubulin antibody.

variants was examined in two buffer conditions: one is a Renilla luciferase assay buffer (E290B, Promega; Figure 1, inset D) and the other is a phosphate-buffered saline (PBS, pH 7.2, 0.02 M) (Suppl. Figure 3A in the Supporting Information). All the ingredients in the assay buffer are unknown. According to Promega’s patent, gelatin and thiourea in the buffer contribute to the stability of the luminescence.15 To exclude such a reagent contribution, the simple PBS buffer was used. This approach is useful to highlight the sole effect of the point mutation. COS-7 cells expressing GLuc or one of its variants, I90L, 8990, and 90115, were raised in a 96-well black frame plate to 95% confluence and briefly counted before experiments. The cells were lysed and placed in a bioluminescence microplate reader equipped with automatic injectors (Mithras LB940; Berthold). The injectors were loaded with the substrate dissolved in the Renilla luciferase assay buffer comprising porcine gelatin and thiourea. A volume of 20 μL of the substrate solution (0.5 mM CTZ) was consecutively injected into each well for monitoring the stepwise elevation of bioluminescence (Figure 1, inset D). A more long-term experiment was conducted in a PBS buffer (pH = 7.2, 0.05 M; Suppl. Figure 3A in the Supporting Information). Enzymatic Properties of GLuc Variants. Enzymatic properties of several GLuc variants were examined for reasoning the superluminescence intensities (Supplementary Table 1 in the Supporting Information). Before the following experiments, the automatic microplate reader (Mithras LB940; Berthold) was calibrated using three different light standards (Glowell; LUX biotech). cDNAs of I90L, 8990, and 819096, besides GLuc, were first subcloned into a Cold-Shock expression vector (pCold I, Takara) exerting maximal folding quality and solubility through a low-temperature expression and slow folding and expressed in

Escherichia coli DH5 cells (Invitrogen). The GLuc variants were purified with a His-tag affinity-column, dialyzed to a Tris-HCl buffered saline (pH 7.5, 0.05 M Tris-HCl, 0.15 M NaCl, 1 mM EDTA), and quantified using the Bradford assay with bovine serum albumin (BSA) as the standard. The solutions were finally loaded on each well of a 96-well plate in the automatic microplate reader for determining Michaelis Menten constants (KM), turnover rates, maximal rates (vmax), and quantum yields (QYs). For the measurement of the QYs, an excessive enzymatic condition was created upon the reaction with the substrate, coelenterazine. A volume of 50 μL of the purified enzymes (25 pmol) were placed on a 96-well black frame plate in the microplate reader equipped with an automatic injector, by which 10 μL of the specific substrate (1 pmol) was injected to the mix, and the total output of photons was integrated for 10 min. The kinetic properties, KM, turnover rates, and vmax were determined by injecting 20 μL of the GLuc variants (10 pmol) onto 40 μL of the substrate (ranging from 0.13 to 4 nmol) loaded on a 96-well black frame plate. The curve fitting based on the Michaelis Menten equation was processed using the kinetic curve fitting program, SigmaPlot 9.0 (Systat Software). Western Blot Analysis. The expression amounts of GLuc variants were evaluated by Western blot analysis (Figure 1, inset E). COS-7 cells expressing GLuc, I90L, 8990, or 90115 on a 12-well plate were washed once with a PBS buffer and lysed with 100 μL of lysis buffer (1% SDS, 10% glycerol, 10% 2-mercaptoethanol, 0.001% bromophenol blue, 0.05 M Tris-HCl, pH 6.8). Each 5 μL of the samples was electrophoresed in a 17% polyacrylamide gel, transferred to nitrocellulose membranes, and blotted with mouse anti-GLuc antibody (Nanolight) or rabbit anti-α-tubulin antibody (Sigma). The blots were incubated with horseradish 8734

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Figure 2. In vivo analysis: (A) A representative mouse image emitting bioluminescence (n = 2). The bioluminescence image was acquired using mice subcutaneously (sc) implanted with the same amounts of COS-7 cells (1  105) expressing GLuc or 8990. The image was taken after intravascular (iv) injection of CTZ to the mice via tail vein. (B) Bioluminescent imaging of metastasis of murine B16 melanoma cells expressing GLuc and I90L in living mice (n = 3). (Section A) Detailed histological analysis of metastasis of murine B16 melanoma cells. After intravenous (iv) injection of B16 melanoma to a mouse via tail vein, the organs were isolated from the mouse and imaged in the presence of 300 μL of CTZ.

Figure 3. Bioanalytical applications of potent GLuc variants. (A) A mammalian two-hybrid assay for investigating estrogenicity of ligands. Estrogenactivated ER LBD binds the Src SH2 domain, resulting in expression of GLuc, I90L, 8990, or RLuc8.6-535 (n = 3). Section A shows the relative intensities. (B) A single-chain probe for evaluating stress-hormonal activities of cortisol (n = 3). As illustrated, cortisol triggers the GR LBD LXXLL motif binding, reconstituting the adjacent fragments of GLuc variants. The probe carrying split-8990, named 8990N, sensitively enhanced the bioluminescence intensities in response to cortisol. The optical intensities were expressed in the RLU per 103 cells. SIMGR3 is a conventional probe carrying split-GLuc.26 Section A shows the relative intensities.

peroxidase-conjugated secondary antibodies (GE Healthcare) and visualized by the ECL Western blotting detection system (GE Healthcare) and a luminescence image analyzer (LAS-3000, FujiFilm). Mouse Experiments with Lumazone FA. The optical intensities of GLuc and 8990 were compared in murine B16 melanoma cells implanted in a living mouse (Figure 2A) (n = 2). First, B16 melanoma cells grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) were transiently transfected with pGLuc and p8990. At 16 h after transfection, the cells were harvested, washed once, and resuspended in PBS. The cells were then counted before experiments using a cell counting chamber (Burker-Turk Deep, Erma Tokyo). An equivalent amounts of the cells (1  105) were subcutaneously (sc) implanted on the back of living mice (BALB/c nude mouse, female, 6 week old). The light emission, 2 min after intravenous (iv) injection of native CTZ to the mouse via tail vein (50 μg per mouse), was finally integrated for 2 min using Lumazone FA with an electron multiplying charge-coupled device (EM-CCD) camera (Nippon Roper). Mouse Experiments with IVIS System. The metastasis of tumors in living mice was illuminated with IVIS Lumina XR

(Xenogen) (Figure 2B) (n = 3). Murine B16 melanoma cells (Riken, Japan) were transiently transfected with pGLuc or pI90L using a lipid reagent (Lipofectamine 2000, Invitrogen). At 16 h after the transfection, the cells were washed and resuspended in PBS. After the cell counting, an equivalent number of the cells (50 μL, 1  105) was iv implanted into mice via the tail vein. After injection of native CTZ (50 μg per mouse) via the tail vein, the light emission was integrated for 2 min (mice) or 5 s (organs) using IVIS Lumina XR (Xenogen). The optical intensities at the sites of living mice and organs are summarized in the unit of photons/s/cm2/sr in Supplementary Table 3 in the Supporting Information. The results showed that almost equivalent amounts of photons were observed from the sites carrying I90L or Monsta as expected from the percentages of photons greater than 600 nm (>%600) (i.e., 8.8 ( 2.5  105 and 7.9 ( 0.5  105 photons/s/cm2/sr, respectively). Construction of Bioluminescent Probes. Some template plasmids carrying the cDNA constructs in Figure 3, and Suppl. Figures 2D and 4 in the Supporting Information were taken from our previous studies.13,16 For the brief explanation on the constructs, see the captions of Suppl. Figure 4A,B in the Supporting Information. 8735

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Figure 4. (A) Bioanalysis of the localization of GLuc variants. The locations of GFP-tagged GLuc variants and endoplasmic reticulum (ER) were imaged in green (first column) and red (second column). ER regions in the living cells were illuminated with ER-Tracker (Ex/Em, 587/615 nm; Molecular Probes). The images by GFP and ER tracker were superimposed in the third column. (B) The relative optical intensities of marine luciferases in the presence of native CTZ (n = 3). COS-7 cell lysates were assayed using a microplate reader right after automatic injection of native CTZ. The inset shows the localization of I90L in COS-7 cells. Section A shows the relative optical image on a microplate. (C) Pseudo-image of the relative optical intensities of marine luciferases. The bioluminescence was developed in a 96-well plate after autoinjection of an aliquot of the substrate.

Two-Hybrid Assays. The advantages of the GLuc variants as a reporter protein were examined with a mammalian two-hybrid assay expressing GLuc, I90L, 8990, or RLuc8.6-535 (Figure 3A). The present plasmids for the assays were modified from the commercial plasmids (Checkmate; Promega). COS-7 cells raised on a 96-well black frame plate were transiently cotransfected with one of the following mixtures: (i) pACT-lbd, pBIND-sh2, and pG5-gluc; (ii) pACT-lbd, pBIND-sh2, and pG5-i90l; (iii) pACTlbd, pBIND-sh2, and pG5 8990; or (iv) pACT-lbd, pBINDsh2, and pG5-rluc8.6-535. Following stimulation of the cells with 10 5 M 17β-estradiol (E2) for 12 h, the bioluminescence intensities could be efficiently elevated, demonstrating an estrogeninduced ER LBD SH2 domain interaction, where the developed optical intensities were determined using a microplate reader (Mithras LB 940, Berthold). Furthermore, the time course of the bioluminescence intensities was estimated after relatively short stimulation times of 0, 3, and 6 h (Suppl. Figure 4C in the Supporting Information). Figure 3A and Suppl. Figure 4D in the Supporting Information were independently determined but correspond to each other. Single-Chain Probe. We previously reported unique singlechain probes for illuminating the activities of hormones and chemicals.17,18 To demonstrate the advantage of the present GLuc variants in single-chain probes, we compared the previously reported probe, Simgr3, with a newly constructed single-chain probe carrying split-8990, named 8990n (Figure 3B and Suppl. Figure 4A in the Supporting Information). COS-7 cells raised in a 96-well black frame plate were transiently transfected with pSimgr3 or p8990n and were incubated for 16 h. The cells on the plate were stimulated with 10 6 M cortisol for 20 min, lysed, and placed in the microplate reader equipped with automatic substrate injectors. Before and after CTZ injection, the bioluminescence variances were determined. Localization of GLuc Variants. The folding efficiency of GLuc comprising many cysteines is poor in the cytosol because of the reducing condition. The endoplasmic reticulum (ER) is preferred for the maximal folding efficiency. To examine the retention site

of KDEL-fused GLuc variants in COS-7 cells, the N-terminal of GLuc, I90L, and Monsta was genetically fused with green fluorescent protein (GFP) and subcloned into pcDNA 3.1(+) (Invitrogen) (Figure 4A). COS-7 cells expressing GFP-linked native GLuc or GFP-linked GLuc variants were stained with ERTracker Red (Invitrogen). The cells were washed and immersed in a HBSS buffer. Fluorescence images were acquired with a 60 oil immersion objective using a LSM 710 (Carl Zeiss MicroImaging). This study confirmed that the GLuc variants are dominantly sequestered into the ER (Figure 4A). An additional study using the supernatants (cell media) revealed that the mutagenesis to GLuc did not cause a biased secretion of the variants (data not shown). Relative Optical Intensities of Marine Luciferases. Relative optical intensities of marine luciferases were first verified with GLuc variants, M43I, RLuc8.6-535, and I90L (Figure 4B). COS-7 cells raised in a 96-well black frame plate (Nunc) were transfected with a plasmid encoding the GLuc variants as indicated. At 16 h after transfection, the cells were briefly counted, washed once, and lysed before CTZ injection. The optical intensities were recorded using the microplate reader (Mithras LB940, Berthold). The corresponding optical image to the relative bioluminescence intensities was taken independently using LAS-4000 (FujiFilm) (Figure 4C). COS-7 cells raised in a 96-well black frame plate (Nunc) were transfected with a plasmid encoding the same GLuc variants as indicated. At 16 h after transfection, the cells were washed and lysed. Furthermore, 40 μL of native CTZ was simultaneously injected to the wells using an eight-channel pipet (Gilson). The relative optical image was then taken using LAS-4000 (FujiFilm) with a 0.5 s exposure time. In these evaluations, all the reagents, including CTZ and the lysis buffer, were from the Renilla luciferase assay kit optimized for RLuc (E2820, Promega). The absolute transfection efficiency was examined with a microscope equipped with EM-CCD (Carl Zeiss) (Figure 4B, inset). COS-7 cells expressing I90L were illuminated on a glass-bottom plate in the presence of the substrate, CTZ. Optical Properties of MpLuc1, MLuc, and RLuc Variants. The bioluminescence intensities and stability of MpLuc1 and 8736

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Figure 5. (A) Optical properties of the variants of MpLuc1 and MLuc (n = 3). On the basis of the knowledge on GLuc variants, the corresponding amino acids were mutated as highlighted in the inset. (B) The sections A and B exhibit the prolonged bioluminescence intensities of MpLuc1 and MLuc, respectively.

MLuc variants were estimated with COS-7 cells expressing the variants (Figure 5). COS-7 cells raised in a 96-well black frame plate were transiently transfected with the plasmids encoding MpLuc1 or MLuc variants. The cells were briefly counted before the experiment, lysed, and placed in a microplate reader (Mithras LB940; Berthold) equipped with an automatic substrate injector. The bioluminescence intensities were integrated for 2 s after CTZ injection. The prolonged bioluminescence intensity was monitored for 5 s after CTZ injection (Figure 5B). Furthermore, relative optical intensities of RLuc variants were similarly verified with the plasmids encoding native RLuc, I140L, V141L, V146L, and V146L/E151A (Suppl. Figure 3D in the Supporting Information).

’ RESULTS Semirational Determination of Suitable Mutation Sites in GLuc. We initially tried a random mutagenesis for modifying

the properties of GLuc but obtained scanty outcomes. Thus, we conducted a site-directed mutagenesis on the basis of the following semirational approach: We considered the chemical structural similarity between the chromophore of GFPs and the substrate of marine luciferases (Suppl. Figure 1 in the Supporting Information). This comparison reveals that GFP variants embed the chromophore inside the molecular backbone, whereas marine luciferases recruit the exogenous luciferin as the choromophore. If so, the hydrophilic interface in the luciferase should provide a favorable platform for the substrate recruitment. The first clue for the core region of GLuc as a modification target was taken from a hydrophobicity search, provided by the scale of Kyte and Doolittle (a free Web service for the hydrophobicity search, http://us.expasy.org/tools; Suppl. Figure 2C in the Supporting Information). The search revealed an extremely hydrophilic region in the center of the sequence. This characteristic region is interestingly highly conserved among marine luciferases (Suppl. Figure 2A in the Supporting Information), and it also comprises a drastic interface between highly hydrophilic and hydrophobic amino acid sequences. This hydrophilic region comprises optimal dissection sites of luciferases according to our papers and others.17,18,13,19

Furthermore, we approximately assumed the putative core region through our previous data on random, consecutive fragmentations of GLuc into two parts and the consequent reconstitution triggered by the conformation change of Xenopus calmodulin (CaM) (Suppl. Figure 2D in the Supporting Information).13 This consecutive fragmentation study revealed that a dramatic light resurrection appears near the dissection points G92/R93 and G99/D100, where some GLuc fusions (dissection sites no. 4, no. 5, and no. 6) suddenly tolerate an insertion of an exogenous protein, CaM. It may be reasonable to interpret that the insertion to sites no. 4, no. 5, and no. 6 no longer hampers the GLuc activities suggesting existence of the structural interface near the putative core region of GLuc. This putative core region is interestingly superimposed with the most hydrophilic region in the scale of Kyte and Doolittle,12 to which the substrate may favorably access (Suppl. Figure 2C in the Supporting Information). The putative core region found by the fragmentation was sitedirectly mutated under the protocol of “Quickchange.” The candidate amino acids are also referred to in the mutagenesis study on fluorescent proteins.7 In specific, the candidate amino acids in the region were carefully substituted with a new one in the following categories: (i) enhancing hydrophobicity, (ii) exerting a “conservative amino acid change,” (iii) admitting a π π stacking between phenyl groups, and (iv) constructing hydrogen binding. The initial round of the site-directed mutagenesis studies revealed that the amino acids, F89, I90, H95, Y97, I108, significantly alter the bioluminescence intensity and/or enzymatic stability. On the basis of the consequence, the second round of mutagenesis was conducted to generate multisite mutants. In total, 64 mutants of GLuc were generated on the basis of the strategy. The two rounds of mutagenesis hit several potent variants, 8990 (meaning F89W/I90L), 90115 (I90L/I115L), and Monsta (F89W/I90L/ H95E/Y97W). I90L and 8990 emit 6- and 10-times stronger bioluminescence, respectively, than intact GLuc in COS-7 cells (Figure 1). This mutagenesis study revealed that (i) I90 is a key mutation site for superluminescence of GLuc and (ii) combining I90L with other mutation points creates additionally enhanced bioluminescence and/or red shifts. Red-Shifted Variants of GLuc. Figure 1, inset A shows that the bioluminescence spectra are red-shifted by the present 8737

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Analytical Chemistry mutagenesis. The maximal intensities (λmax) of I90L and Monsta exhibited 16 and 33 nm red-shifted spectra, respectively, in the presence of the natural substrate, CTZ, when compared with that of intact GLuc. Their percentages of emitted photons greater than 600 nm (%600) were 1.5 and 1.7%, respectively, whereas %600 of intact GLuc was merely 0.2% (Supplementary Table 1 in the Supporting Information). Optical Intensities of GLuc Variants. Among the variants, the mutants, I90L, 8990, and 90115 emitted extremely bright bioluminescence even by multiple injection of 0.5 mM CTZ, dissolved in a Renilla luciferase assay buffer carrying porcine gelatin and thiourea15 (E290B Promega; Figure 1, inset D). Most of the following experiments were conducted using this assay buffer unless otherwise designated. We also noted that artificial components, such as gelatin and thiourea, cannot be optimally supplied in live cell lines or in vivo studies using mice. Thus, a prolonged bioluminescence in the absence of the additives was similarly examined with native CTZ dissolved in a simple phosphate buffered saline (PBS; 0.02 M, pH 7.2) (Suppl. Figure 3A in the Supporting Information). The time course of the bioluminescence intensities revealed that I90L and I90V sustained ∼70% of their initial intensities even 10 min after CTZ injection, whereas intact GLuc and F89W merely kept 30% of the original intensities in the same condition. A corresponding optical stability in I90L and I90V was observed with a microplate reader equipped with an automatic substrate injector (data not shown). The expression level of GLuc variants was invariant according to the Western blot analysis (Figure 1, inset E). An evaluation on the absolute transfection efficiency revealed that approximately 10% of COS-7 cells are transfected in a dish (Figure 4B, inset). In Vivo Imaging Using the GLuc Variants. The evidenced optical properties of the GLuc variants convinced us of a successful molecular imaging in bioassays and living subjects. For demonstration of the improved tissue permeability of the bioluminescence, equivalent amounts of COS-7 cells expressing intact GLuc or 8990 (∼1  105 cells) were implanted into the subcutaneous layer on the back of anesthetized BALB/c nude mice (Figure 2A). The comparison revealed that the skin site implanted with COS-7 cells carrying 8990 exhibited ∼7 times stronger bioluminescence intensities than those with original GLuc. Furthermore, metastases of murine B16 melanoma to the organs of BALB/c nude mice were also illuminated (Figure 2B). The optical imaging represented the localization of metastatic cells in the lung, where the melanoma carrying I90L exhibited approximately 5 times stronger optical signature than that with intact GLuc. A detailed histological analysis also revealed that the implanted melanoma is dominantly retained in the lung and the uterus (Figure 2B, inset A and Supplementary Table 3 in the Supporting Information). Enzymatic Properties of GLuc Variants. For reasoning of the enhanced optical properties, enzymatic characteristics of the GLuc variants were extensively determined in vitro (Supplementary Table 1 in the Supporting Information). The Ni2+-affinity columnpurified variants, GLuc, I90L, 8990, and 819096, were loaded in a bioluminescence microplate reader (Mithras LB 940, Berthold) equipped with an automatic substrate injector for determining Michaelis Menten constants (KM), turnover rates, maximal rates (vmax), and QYs. The studies revealed that the GLuc variants in the presence of the Promega-buffered CTZ exhibited (i) ∼2.5 5 times faster turnover rates, for example, 1.1  1022 photons/s/mol and (ii) a maximum of 7% enhanced QYs, when compared with intact GLuc.

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On the other hand, the KM values were almost invariant before and after mutagenesis. Both the improved QYs and turnover rates rationalize the superluminescence of the GLuc variants. GLuc Variants As an Excellent Optical Signature for Bioassays. The marine luciferases with advanced optical properties should be utilized as an ideal optical signature in a broad range of bioanalysis studies. The GLuc variants and RLuc8.6-535 were also utilized in a mammalian two-hybrid assay, determining estrogenicities of the ligands (Figure 3A). The Bait and Prey proteins in this assay were the ligand-binding domain of human estrogen receptor (ER LBD) and the SH2 domain of protooncogene v-Src (Src SH2), respectively. We previously demonstrated that this binding model is effective for determining bioactive small molecules.16 The new two-hybrid system in COS-7 cells efficiently elevated the bioluminescence intensities in response to 12 h of stimulation of 17β-estradiol (E2), evidencing an ER LBD SH2 binding (Figure 3A). The absolute intensities per 1 μg of cell lysate with 8990 were ∼11.4 times stronger than those with RLuc8.6-535, which is the most advanced variant of RLuc until date. The signalto-noise (S/N) ratios with 8990 and RLuc8.6-535 were 9.4 and 5.0, respectively. This comparison revealed that 8990 is a superior optical signature to RLuc8.6-535 in both intensities and S/N ratios upon application in a two-hybrid assay. The common problem of a reporter-gene assay and a two-hybrid assay is the long stimulation time until accumulation of the reporter protein. This superluminescent feature of GLuc variants contributed to a large reduction of the stimulation time, where 3 h was adequate to discriminate the estrogenicity of E2 (Suppl. Figure 4C in the Supporting Information). Furthermore, the advantages of the GLuc variants were extensively demonstrated with a single-chain probe consisting of the ligand-binding domain of glucocorticoid receptor (GR LBD) and an LXXLL motif of the coactivator, GRIP1 (Figure 3B). For constructing the probe, 8990 was dissected into two fragments, inside of which the LXXLL-linked GR LBD was genetically inserted. The single-chain probe, named 8990N, efficiently visualized the intramolecular interaction between “stress hormone” (cortisol)activated GR LBD and the LXXLL motif in living cells. The bioluminescence intensities of 8990N were ∼5 times stronger than those of a conventional single-chain probe carrying intact GLuc, named SIMGR3.26 This study demonstrates the enhanced optical performance of 8990N upon measuring “stress hormone” levels in vitro. We also directly compared the optical intensities of GLuc and I90L with those of previously reported RLuc8.6-535 and M43I (Figure 4B and Supplementary Table 2 in the Supporting Information). The results showed that I90L emits 21 times brighter bioluminescence than RLuc8.6-535, which is currently the most advanced marine luciferase. The optical image corresponding to the relative bioluminescence intensities was also taken independently using LAS-4000 (FujiFilm) (Figure 4C). Fabrication of Competent Variants of MLuc, MpLuc1, and RLuc. To demonstrate general validity of the present methodology on marine luciferases, the amino acids in MLuc and MpLuc1 were correspondingly manipulated on the basis of the amino acid alignment table and the obtained knowledge during engineering GLuc variants (Figure 5 and Suppl. Figure 2A in the Supporting Information). The found mutation strategy in GLuc was surprisingly effective even to these marine luciferases, MLuc and MpLuc1. A mutation to MLuc induced a maximum of 3.5 times prolonged emission half-life, and a mutation to MpLuc1 generated 5.4 times 8738

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Analytical Chemistry enhanced bioluminescence intensities, when compared with the intact luciferases (Figure 5A). The optical stabilities were remarkably improved (Figure 5B). A corresponding feature was observed with RLuc (Suppl. Figure 3D in the Supporting Information).

’ DISCUSSION In the present study, we suggest a new method to semirationally determine the suitable core region in marine luciferases as an engineering target. We speculated that (i) the chemical structural similarity between the chromophore of GFPs and the substrate of marine luciferases is a hint for luciferase engineering; (ii) GFP variants embed the chromophore inside the molecular backbone, whereas marine luciferases recruit the exogenous luciferin as the choromophore. Thus, the hydrophilic interface in the luciferase should provide a favorable platform for the substrate recruitment. We searched the second clue for narrowing the core region using a consecutive fragmentation of GLuc (Suppl. Figure 2D in the Supporting Information). The found, putative core region is interestingly superimposed with the most hydrophilic region in the scale of Kyte and Doolittle,12 to which the substrate may favorably access (Suppl. Figure 2C in the Supporting Information). A site-directed mutagenesis to the above-narrowed core region was conducted to improve the enzymatic properties of GLuc variants. The effective amino acids for modifying GFP such as F, W, Y, and L were correspondingly chosen for the mutagenesis of GLuc. This view is applicable for other marine luciferases. The above-mentioned approach may be disputable in point that a hydrophilic loop of the host luciferase may hit as falsepositive information. However, this approach provides a laboreffective breakthrough in the absence of the structural information. The following references show that the hydrophilic region practically directs an approximate active site region: i.e., (i) The active site consisting of “STG” motifs of FLuc is actually close to or inside the hydrophilic loop region (e.g., 420 422 AA).20 (ii) The known active site of RLuc comprises several motifs in the hydrophilic region (156 163 AA and 220 224 AA).21 The red-shifted emission peak of Monsta (λmax = 503 nm) is still near the major hemoglobin absorption peaks.22 This limitation may be relieved by appropriate selection of the substrate as marine luciferases recruit the exogenous luciferin as the chromophore (our premise). This view is supported by the RLuc experiment comparing CTZ and CTZ-v as the substrate.21 We understand that a remarkably red-shifted spectrum was contributed by the extended π-conjugation of CTZ-v. Monsta carries four mutation sites, F89W, I90L, H95E, and Y97W. We consider that the mutation sites synergically contribute to the red shifts of the spectrum. Of the mutation sites, the contribution of I90L to the optical intensity is remarkable. Most of the chemical properties of isoleucine (I) and leucine (L), such as pKa, isoelectric point, and nonpolarity, are equivalent except for the hydropathy index, 4.5 and 3.8. This small variance exerts a dramatic increase of the optical intensities of I90L and 8990. This result suggests that F89 and I90 are key amino acids in the hydrophilic platform of GLuc. They are probably substrateinteracting amino acids. The red shift of Y97W may be contributed by the π π stacking between the side chains of the substrate and tryptophan. For reasoning, the enhanced optical properties and enzymatic characteristics of the GLuc variants were extensively determined in vitro using the Ni2+-affinity column (Supplementary Table 1 in the Supporting Information). The studies revealed that the GLuc

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variants in the presence of the Promega-buffered CTZ exhibited (i) ∼2.5 5 times faster turnover rates, for example, 1.1  1022 photons/s/mol, than native GLuc and (ii) a maximum of 7% enhanced QYs, when compared with intact GLuc. On the other hand, the KM values were almost invariant before and after mutagenesis. These results should be interpreted as follows: (i) both the improved QYs and turnover rates rationalize the superluminescence of the GLuc variants. (ii) Between the two parameters, QYs and turnover rates, turnover rates of our variants dominantly contribute to the improvement of the optical intensities. Furthermore, we consider that (iii) the above-mentioned efforts including mutagenesis contributed to favorable access of the substrate to the putative core region of GLuc variants, as shown in the increase in the turnover rates (photons/s/mol). Meanwhile, the rapid turnover rates of GLuc variants are probable to cause an immediate consumption of nearby CTZ and O2 and thus may cause a substrateanoxia condition upon light emission. Thus, we chose native GLuc and I90L exhibiting similar turnover rates in the in vivo comparison (Figure 2B and Supplementary Table 1 in the Supporting Information). The measured improvements in the kinetic constants are not as much as the amplified magnitude of the optical intensities in I90L and 8990 (Supplementary Table 1 in the Supporting Information). It may be caused by (i) denaturing of the purified GLuc variants, (ii) reaction conditions (pH, buffer ingredients, etc.), (iii) an artificial effect of the His-tag to the activity, and (iv) the reducing conditions of Escherichia coli causing misfolding of the variants. I90L exhibited unique features in Km and full width at half maximum (FWHM), compared to other mutants (Suppl. Figure 1 in the Supporting Information). The Km value of I90L is relatively high, meaning a lower binding affinity of the substrate to I90L. The standard deviation (SD) of the Km value is relatively large, 0.2. Meanwhile, FWHM of I90L shows the largest bandwidth among single mutants. Turnover rates and QY of I90L are clearly increased, but not as much as 5 times reported by the mammalian cell-based study. These confusing kinetic data suggest that mutation of I90 to leucine (L) may modulate the structural, enzymatic stability of GLuc, especially in E. coli expression and column-purified in vitro conditions. We suspect that the relatively large standard deviation of column-purified I90L in vitro may be correlated with the band broadening of the spectrum and the lower binding affinity to the substrate. It is interesting to compare bioluminescence-based assays with fluorescence-based ones. It is known that bioluminescence-based assays yield relatively lower light intensities than fluorescencebased ones due to the slower rate creating excited states, although they can deliver 10- to 1 000-fold higher assay sensitivity than fluorescence assays.23 This view indicates that the present GLuc variants should be an excellent alternative to compensate the intrinsic drawback of bioassays using marine luciferases. In practice, the GLuc variants were found to remarkably improve the sensorial properties of bioassays including a mammalian 2-hybrid assay and a single-chain probe (Figure 3). The GLuc variants with advanced optical properties may be utilized as an ideal optical signature in a broad range of bioanalysis studies. A broad validity of the present strategy for other marine luciferases is another key feature for bioassays. In the present study, the general validity was demonstrated with Metridia longa luciferase (MLuc), Metridia pacifica luciferase 1 (MpLuc1), and 8739

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Analytical Chemistry Renilla reniformis luciferase (RLuc), respectively (Figure 5 and Suppl. Figure 3D in the Supporting Information). The secretion luciferases, GLuc, MLuc and MpLuc1, were originally cloned from marine copepod and highly conserved each other in the amino acid sequence (Suppl. Figure 2A in the Supporting Information).24 On the other hand, RLuc phylogenetically differs from the marine copepod luciferases, although they share the common substrate, coelenterazine, for light emission. The alignment of amino acid sequences of the luciferases in Suppl. Figure 2A in the Supporting Information highlights the equivalent mutation sites as those of GLuc. The deduced mutation sites are found to greatly enhance optical intensities and stability of MpLuc1, MLuc, and RLuc (Figure 5). Marine luciferases sharing the common substrate, coelenterazine, may be laboreffectively modified with the same approach as the present study. Prolonged bioluminescence intensities of marine luciferases are another key feature for ensuring the fidelity of the optical signature in bioassays. The optical intensities of native GLuc, MpLuc1, and MLuc are rapidly declined after addition of the substrates. This common feature of copepod luciferases was highly relived by the present mutagenesis, e.g., a single mutation of I114 in MpLuc1 to leucine (L) greatly enhanced the optical intensity and stability (Figure 5). This feature was also observed in cases of I90L of GLuc (Suppl. Figure 3A in the Supporting Information) and MLuc4 (Figure 5). In general, prolonged bioluminescence may be achieved by two cases: (i) One is simply because of a slow depletion of the substrate in the enzyme reaction; (ii) the other is because of the improved enzymatic robustness of the mutants. The above results support the later case. Rees et al. previously speculated that the luminescent substrates of the luminous reactions are the evolutionary core of most luminescent systems.25 We support this speculation in point that marine luciferases are conserved with respect to the specific substrates rather than the phylogenetic classification. This feature of marine luciferases may inspire other researchers to modify the marine luciferases in a new scheme. Taken together, we have introduced a unique strategy for creating superluminescent variants of marine luciferases with prolonged bioluminescence. This creation and utilization of the superluminescent variants of marine luciferases as an optical signature in vitro and in vivo provides a wide variety of applications for screening pharmacological or toxicological compounds and testing them in bioassays and living animals.

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’ REFERENCES (1) Hoshino, H. Expert Opin. Drug Discovery 2009, 4, 373–389. (2) Paulmurugan, R.; Gambhir, S. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15883–15888. (3) Loening, A. M.; Fenn, T. D.; Wu, A. M.; Gambhir, S. S. Protein Eng., Des. Sel. 2006, 19, 391–400. (4) Welsh, J. P.; Patel, K. G.; Manthiram, K.; Swartz, J. R. Biochem. Biophys. Res. Commun. 2009, 389, 563–568. (5) Hoshino, H.; Nakajima, Y.; Ohmiya, Y. Nat. Methods 2007, 4, 637–639. (6) Lehmann, M.; Loch, C.; Middendorf, A.; Studer, D.; Lassen, S. F.; Pasamontes, L.; van Loon, A. P. G. M.; Wyss, M. Protein Eng. 2002, 15, 403–411. (7) Tsien, R. Y. Angew. Chem., Int. Ed. 2009, 48, 5612–5626. (8) Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12501–12504. (9) Tsien, R. Y.; Zhang, J.; Campbell, R. E.; Ting, A. Y. Nat. Rev. Mol. Cell Biol. 2002, 3, 906–918. (10) Tannous, B. A.; Kim, D. E.; Fernandez, J. L.; Weissleder, R.; Breakefield, X. O. Mol. Ther. 2005, 11, 435–443. (11) Inouye, S.; Sahara, Y. Biochem. Biophys. Res. Commun. 2008, 365, 96–101. (12) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105–132. (13) Kim, S. B.; Sato, M.; Tao, H. Anal. Chem. 2009, 81, 67–74. (14) Kunkel, T. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 488–492. (15) Hawkins, E.; Centanni, J. M.; Sankbeil, J.; Wood, K. V. Method for increasing luminescence assay sensitivity. U.S. Patent 7,118,878, October 10, 2006. (16) Kim, S. B.; Umezawa, Y.; Kanno, K. A.; Tao, H. ACS Chem. Biol. 2008, 3, 359–372. (17) Kim, S. B.; Awais, M.; Sato, M.; Umezawa, Y.; Tao, H. Anal. Chem. 2007, 79, 1874–1880. (18) Kim, S. B.; Otani, Y.; Umezawa, Y.; Tao, H. Anal. Chem. 2007, 79, 4820–4826. (19) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2005, 77, 1295– 1302. (20) Conti, E.; Franks, N. P.; Brick, P. Structure 1996, 4, 287–298. (21) Loening, A. M.; Wu, A. M.; Gambhir, S. S. Nat. Methods 2007, 4, 641–643. (22) Horecker, B. L. J. Biol. Chem. 1943, 148, 173–183. (23) Fan, F.; Wood, K. V. Assay Drug Dev. Technol. 2007, 5, 127–136. (24) Takenaka, Y.; Masuda, H.; Yamaguchi, A.; Nishikawa, S.; Shigeri, Y.; Yoshida, Y.; Mizuno, H. Gene 2008, 425, 28–35. (25) Rees, J. F.; De Wergifosse, B.; Noiset, O.; Dubuisson, M.; Janssens, B.; Thompson, E. M. J. Exp. Biol. 1998, 201, 1211–1221. (26) Kim, S. B.; Sato, M.; Tao, H. Anal. Chem. 2009, 81, 3760–3768.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by a Wakate grant for S.B.K. from Japan Society for the Promotion of Science (JSPS) and a grant for M.S. from the National Institute of Biomedical Innovation, Japan. The authors also appreciate Mr. Uchida and Mr. Hasegawa (Nippon Roper) and Mr. Koga (Carl Zeiss) for their technical support. 8740

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