Fabrication of a New Lineage of Artificial Luciferases from Natural

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Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools Sung Bae Kim, Ryo Nishihara, Daniel Citterio, and Koji Suzuki ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00081 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017

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ACS Combinatorial Science

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Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools

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Sung Bae Kim*,1, Ryo Nishihara2, Daniel Citterio2, Koji Suzuki2

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1. Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan 2. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

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* Corresponding author: [email protected]

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ACS Paragon Plus Environment


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Abstract

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The fabrication of artificial luciferases (ALucs) with unique optical properties can have

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a fundamental impact on bioassays and molecular imaging. In this study, we developed

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a new lineage of ALucs with unique substrate preferences by extracting consensus

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amino acids from the alignment of 25 copepod luciferase sequences available in natural

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luciferase pools. The primary sequence was first created with a sequence logo generator

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resulting in a total of 11 sibling sequences. Phylogenetic analysis shows that the newly

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fabricated ALucs form an independent branch, genetically isolated from the natural

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luciferases and from a prior series of ALucs produced by our laboratory using a smaller

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basis set. The new lineage of ALucs were strongly luminescent in living mammalian

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cells with specific substrate selectivity to native coelenterazine.

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comparison of the C-terminal sequences of new ALucs reveals that some amino acids in

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the C-terminal ends are greatly influential on the optical intensities, but limited in the

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color variance. The success of this approach guides on how to engineer and

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functionalize marine luciferases for bioluminescence imaging and assays.

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Key words: Artificial luciferase, bioluminescence, frequency analysis, coelenterazine,

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copepod luciferase

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A single-residue-level

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Introduction Natural beetle and marine luciferases have been established from a large variety 1 2

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of insects, marine organisms, and prokaryotes

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new luciferases from light-emitting organisms in nature has been considered as the only

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method to expand the reporter pool. These are of course the result of Darwinian

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evolution3 under selection pressures that are not well suited to the needs of biomolecular

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imaging4. Natural luciferases are therefore generally poor in optical intensities and

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stability, shortcomings that have been addressed by directed evolution in the laboratory5.

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However, such random mutagenesis approaches are often slow and tedious, and

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crystallographic information on natural beetle and marine luciferases, which is essential

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for site-directed mutagenesis, is rare 6.

. Conventionally, the establishment of

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As an alternative approach, we recently showed a new method of creating

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artificial luciferases (ALucs) by extracting the consensus amino acids from the

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sequences of natural copepod luciferases available in the National Center for

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Biotechnology Information (NCBI) database 7. This strategy is based on the premise

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that the frequently occurring amino acids at a given position have a larger

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thermostabilizing effect compared with less frequent amino acids. This approach was

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originally developed for finding effective mutation sites and thus is called “consensus

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sequence-driven mutagenesis strategy” (CSMS) 8 9. We applied the basic concept to the

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fabrication of whole sequences of consensus amino acids, and here we extend the

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approach to a larger collection of 25 copepod luciferase homologs 10.

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Results and Discussion

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As shown in Fig. 1, two primary sequences, ALuc41 and ALuc42, were created by

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extracting frequently occurring amino acids from the alignment of 25 copepod

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luciferase sequences (Supporting Information Figure 1C,D) with the help of the Web

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software WebLogo version 2.8.2 (http://weblogo.berkeley.edu/logo.cgi) 11. The newly

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fabricated ALucs were solely derived from the 25 new copepod sequences in the public

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database; the sequences of our previously-developed ALucs7 were not included. As the

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primary sequence ALuc41 consists of variable N-terminal and two conserved domains,

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it was aligned in three rows. The aligned sequence was further modified, (i) to increase

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cases of the consensus amino acids between the N- and C-terminal domains, because we

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empirically know that the higher homology allows the stronger bioluminescence

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intensities

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of ALuc41 for generating the sibling sequences (ALuc43 - ALuc51). The new artificial

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and (ii) to introduce multiply mutated amino acids at the C-terminal end

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sequences were found to be considerable different from those of any existing marine

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luciferases according to the program BLASTP 2.5.1+ (NCBI BLAST). For example, the

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closest sequence was that of Pleuromamma xiphias, the maximal identity of which was

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approximately 77% of ALuc41 according to the program BLASTP 2.5.1+ in the NCBI

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BLAST (Fig. 1(B), inset a). The phylogenetic tree showed that the newly fabricated

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artificial luciferase 40 (ALuc40) series formed an independent branch, which was

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isolated even from precedent ALuc series (Fig. 1(B)).

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Figure 1. Fabrication of a primary sequence of new artificial luciferases from an

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alignment of natural copepod luciferases in public databases (NCBI BLAST and SIB

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BLAST). (A) The frequently occurring amino acids were extracted from the alignment

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with

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(http://weblogo.berkeley.edu/logo.cgi) 11. (B) An unrooted phylogenetic tree according

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to CLUSTALW version 2.1. The blue and red circles mark the relative positions of

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existing and new artificial luciferases, respectively. Inset a shows the identity ranking of

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existing marine luciferases compared with ALuc41. The identity ranking was

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determined with the program BLASTP 2.5.1+ in the NCBI BLAST. (C) The relative

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optical intensities of ALuc41–45 compared with conventional luciferases (n=5). The

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optical image in pseudo-color was developed with nCTZ.

the

help

of

the

Web

software

WebLogo

version

2.8.2

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ALuc40s survive in mammalian cells and emit strong bioluminescence. Each new

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ALuc sequence was expressed in COS-7 cells using the same plasmid vector and

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induction marker. Explain what these were. The relative optical intensities were

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evaluated in the presence of

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coelenterazine (nCTZ) directly in cell lysates without correction for protein expression

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levels (Fig. 1C). Thus, this assay identifies the best candidates for practical use under

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these conditions, not necessarily the most active among the isolated luciferase variants.

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The results showed the new sequences to mediate the production of stronger optical

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signals than the conventional marine luciferases used for comparison, GLuc and

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RLuc8.6-535, which were chosen because they are the most frequently used marine

the Promega’s assay buffer dissolving 0.1 mM native

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luciferases among researchers.

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6.2-fold (±1.2 s.d.) more intense than from GLuc.

For example, the optical signal from ALuc45 was

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Differences among the artificial luciferases were revealing. As shown in Fig. 1(C),

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ALuc43 and ALuc45 gave approximately 7- and 15-fold stronger bioluminescence than

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ALuc41, respectively. Only one amino acid distinguishes ALuc41 (187T) from ALuc 45

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(187N), and only one amino acid differentiates ALuc43 (183T) and ALuc44 (185Q)

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from ALuc41 (183K, 185V). Assuming that luciferase expression is comparable in these

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tests, which seems likely given the modest differences in sequence, these dramatic

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differences in light intensity suggests that positions 183, 185, and 187 at the C-terminal

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end of ALuc41 are key sites for enzymatic activity.

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The C-terminal end of ALuc41 dominates the optical intensities. The X-ray crystal

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structure of the coelenterazine-binding protein (CBP; PDB accession numbers 2hps and

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2hq8)12 shows the C-terminal end to be close to the C6 position of the bound

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light-generating substrate nCTZ (Fig. 2(A)). This, along with the sequence comparisons

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discussed above, suggested that further modifications of C-terminal domain residues

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could be informative (Fig. 2).

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Additional derivatives of ALuc45 were created by single mutations among residues

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183-192 (Figure 2Ab). Among these candidates, the three brightest appeared in the

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intensity order ALuc49 ≈ ALuc50 > ALuc45 (Fig. 2B). The optical stabilities of the new

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ALucs were examined by comparing the optical intensities at 5 minutes after nCTZ

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injection with those at immediately after nCTZ injection (Fig. 2(B), inset a). ALuc51

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and ALuc48 maintained 49% and 40% of their initial optical intensities after 5 minutes.

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GLuc showed the poorest optical stability of 16% in the same experimental conditions.

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Figure 2. The significance of the C-terminal end of ALuc41–51, promoted by the super

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2-dimensional molecular structure of ALuc30. (A) A super 2-dimensional molecular

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structure of ALuc30. Inset a shows the C-terminal end of ALuc30; inset b aligns the

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C-terminal sequences of ALuc41–51. (B) The bioluminescence image of ALuc41–51

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compared with conventional marine luciferases. Inset a shows the absolute optical

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intensities normalized by integration time (sec) and light-emitting area (mm2). The

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percentages in the bar graphs denote the remaining optical intensities after 5 minutes

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(n=3).

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Substrate selectivity. The relative substrate preference of the new lineage of ALucs

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was determined with the use of various coelenterazine analogues (Fig. 3;

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Supplementary Fig. 2). Most analogues were not accommodated by these luciferase

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variants, except nCTZ (Supplementary Fig. 2). The previously reported ALuc16 and

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ALuc30 were able to use both nCTZ and CTZh, whereas the new lineage of ALucs

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presented a high preference for nCTZ (Fig. 3). The mostly biased optical intensities

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were observed with ALuc47; i.e., the intensity of the nCTZ–ALuc47 pair was found to

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be 217-fold brighter than that of CTZh–ALuc47. This type of substrate preference may

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reflect changes in the binding or turnover properties of the new ALucs or may reflect

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established preferences of copepod enzymes, as the relative brightness of the natural

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enzymes with various CTZ analogues has not been established.

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The emission wavelengths of the new luciferases were unchanged, remaining

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in the 487-500 nm range (Fig. 3B). In the case of ALuc42, the percentage of red light

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emitted (longer than 600 nm) was approximately 4% of the total. Substitution of T187

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with N (ALuc45), Y (ALuc49), F (ALuc50), or W (ALuc51) gave rise to no change in

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emission wavelength, in spite of the additional opportunity provided by these mutations

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to create additional π–π stacking or hydrogen bonds with the substrate.

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Figure 3. Substrate-driven properties of the bioluminescence spectra of ALucs. (A) The

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peak heights of the bioluminescence spectra of various luciferases according to nCTZ

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(blue bars) and CTZh (red bars). The numbers on the bars indicate the fold intensities of

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each luciferase with nCTZ compared with CTZh. The red asterisk “*” denotes the

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maximal fold intensity difference with nCTZ and CTZh. The yellow shaded area shows

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the spectral heights of previously reported ALucs. Inset a presents a comparison of the

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chemical structures of nCTZ and CTZh. (B) Comparison of the bioluminescence spectra

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of ALuc16, 42, and ALuc47 according to nCTZ (dotted line) and CTZh (solid line). The

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pink shaded area indicates wavelengths longer than 600 nm.

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Live cell bioluminescence imaging with conventional and newly established ALucs.

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The live cell images of conventional and newly established ALucs were determined in

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COS-7 cells grown in 6-channel microslides (Fig. 4). NanoLuc and RLuc8.6-535 have

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been previously reported as the brightest among marine luciferases

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live cell images were compared with those of ALuc49 (Fig. 4(A)). The results showed

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the expected substrate selectivity: furimazine and 6piOH-CTZ selectively illuminated

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NanoLuc and RLuc8.6-535, respectively, whereas ALuc49-containing cells were

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illuminated in the presence of nCTZ.

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. Thus, their

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187 188

Figure 4. (A) Live cell bioluminescence imaging with conventional and newly

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established ALucs. The optical image was taken with a CCD camera equipped with the

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LAS-4000 system (Fujifilm) after simultaneous injection of furimazine, nCTZ, or 11



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6piOH-CTZ with the use of a multichannel micropipette. Inset a shows the optical

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profile of the microslide containing COS-7 cells after the addition of nCTZ. (B) The

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absolute optical intensities of the live cells on the microslides after the addition of

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furimazine, nCTZ, or 6piOH-CTZ.

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Molecular Tension Probes. The useful features of the new lineage of ALucs were

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shown by applying the ALucs to a single-chain bioluminescent probe, referred to as a

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“bioluminescent capsule”, where a full-length ALuc is initially designed to be fixed at

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the plasma membrane (Supplementary Fig. 3). Two such probes, designated P47 and

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P49 to reflect their use of ALuc47 and ALuc49, respectively, were created. The addition

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of the apoptosis inducer staurosporine increased the optical intensities of P47 and P49

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by 50% and 28%, respectively. This is attributed to the caspase-induced release of

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full-length ALucs from the capsules.

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Conclusions

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We have previously created a series of artificial luciferases by extracting the

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frequently occurring amino acids in a bundle of aligned copepod luciferase sequences 7.

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Using a larger data pool, we created here a new lineage of ALucs that is

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phylogenetically distinctive from any existing luciferases, including previously reported

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ALucs, as shown in Fig. 1(B). In addition to brighter output of light, the new ALucs are

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selective in their use of nCTZ, which contains a para-hydroxyphenyl group at the C-2

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position of the imidazolone ring, over CTZh, which is missing the hydroxyl group at

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that site (Fig. 3). These variants do not accommodate iodine or fluorine at this position.

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These observations suggest that the small variations in amino acid composition in the

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new ALucs are localized at the luciferase binding site. In contrast, the active site of the

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old lineage of ALucs (ALuc16-34) is more promiscuous concerning functional groups at

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the C-2 imidazoline benzyl group; for example, ALuc30 activates CTZh, CTZf, and

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CTZi with high selectivity 12. The varying substrate preferences for marine luciferases

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open exciting possibilities for multiplexed assay systems, where the optical signals may

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be pinpointed by adding specific substrates. The success of this approach is doubtless

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due to the highly conserved nature of the copepod luciferases and, for such families,

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highlights the potential value of focusing on the degrees of conservation at the

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single-residue level.

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Experimental Procedures

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Design of ALucs by extracting frequently occurring amino acids from the

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alignment of copepod luciferases. Twenty-five sequences of marine planktonic

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copepod luciferases were collected from the literature,10, 15 transformed into FASTA

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format (text-based single-letter amino acid codes), and aligned to determine amino acid

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frequency

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(http://weblogo.berkeley.edu/logo.cgi)

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named “ALuc41”. ALuc41 contains two consecutive amino acids in the sequence of

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ALuc41 were further modified by embedding a His-tag and elongating the highly

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variable region in the N-terminal side, which was named “ALuc42.” A series of sibling

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sequences of ALucs was further fabricated from the prototypical sequence by

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substituting several amino acids in the C-terminal region with new ones (Supplementary

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Fig. 1).

with

the

Web

software 11

WebLogo

version

2.8.2

. The initially suggested primary sequence was

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Lastly, the endoplasmic reticulum (ER) retention signal KDEL was added at the

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C-terminal end of each new sequence instead of the N-terminal secretion peptide

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common to copepod luciferases. The average length and molecular weight (MW) of the

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newly made artificial sequences were 195.7 (±5.7 s.d.) AAs and 21.2 (±0.7 s.d.) kD,

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respectively. The average theoretical isoelectric point (pI) was 5.7 (±0.5 s.d.).

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Synthesis of cDNA constructs encoding the new lineage of artificial luciferases. The

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murine codon-optimized cDNA constructs encoding the artificially designed amino acid

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sequences (ALuc41-51) were custom-synthesized on order by Eurofins Genomics

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(Tokyo, Japan) (Supplementary Figs. 1(A)). The synthesized cDNAs were subcloned

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into pcDNA3.1(+) (Invitrogen) by using the specific restriction sites HindIII and XhoI

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for expression in mammalian cells. The overall sequence fidelity was confirmed with

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the use of a sequencing service provided by Eurofins Genomics (Tokyo, Japan).

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Determination of the sequential identity ranking and phylogenetic trees of the new

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lineage of artificial luciferases. The rooted and unrooted phylogenetic trees of the

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artificially designed ALucs and existing marine luciferases were determined by using

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CLUSTALW version 2.1 (Fig. 1(B); Supplementary Fig. 1(B)). The maximal identities

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and similarity rankings of the new ALucs were also compared with those of existing

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marine luciferases by using an alignment search tool provided by the NCBI BLAST

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(BLASTP 2.5.1+; http://www.ncbi.nlm.nih.gov/). Figure 1(B), inset a, shows the

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maximal sequential identity rankings.

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Determination of the absolute optical intensities of the new ALucs and comparison

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with conventional luciferases. The relative optical properties of ALuc41–45 were

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examined in COS-7 cells derived from African green monkey kidney fibroblasts. COS-7

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cells were grown in Dulbecco’s minimal essential medium (DMEM; high glucose)

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(Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) and

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1% penicillin/streptomycin (P/S) (Gibco) (Fig. 1(C)).

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The COS-7 cells, grown in a 96-well microplate (Nunc), were transiently transfected

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with the pcDNA3.1(+) plasmids encoding each ALuc (Supplementary Fig. 1) or the

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existing marine luciferases, i.e., Gaussia princeps luciferase (Gluc; GenBank

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AAG54095.1)16 or Renilla reniformis luciferase 8.6-535 (RLuc8.6-535)5, as internal

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references by using the lipofection reagent TransIT-LT1 (Mirus). Sixteen hours after the

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transfection, the cells in each well were lysed with 50 µL lysis buffer (E291A; Promega).

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An aliquot of the lysates (10 µL) was transferred into a fresh 96-well optical bottom

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microplate (Nunc) by using an 8-channel micropipette. The relative optical intensities in

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the plate were also determined with the use of an image analyzer (LAS-4000; Fujifilm)

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immediately after simultaneous injection of 40 µL of the RLuc assay buffer carrying

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nCTZ by using a multichannel micropipette (Fig. 1(C)).

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The absolute optical intensities shown in Fig. 2(B) were determined by applying the

280

same protocol as that in Fig. 1(C). The bioluminescence intensities were normalized by

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integration time (sec) and light-emitting area (mm2). The normalized bioluminescence

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intensities were subsequently expressed in relative luminescence units per second–area

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(RLU/sec/mm2).

284 285

Substrate specificity of the new lineage of ALucs. The substrate-specific

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bioluminescence of the new lineage of ALucs was examined with the use of various

287

coelenterazine analogues (Fig. 3; Supplementary Fig. 2). The COS-7 cells grown in a

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96-well microplate were transiently transfected with a pcDNA3.1(+) vector encoding

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one of the following marine luciferases: GLuc, RLuc8.6-535, ALuc16, ALuc30, and

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ALuc41–48. Sixteen hours after the transfection, the cells were lysed, and an aliquot of

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the lysates (20 µL) was carefully transferred to a 200-µL-volume microtube. The lysate

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in the microtube was mixed with 60 µL of the substrate solution dissolving nCTZ or

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CTZh and then was immediately transferred into the dark chamber of a precision

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spectrophotometer (AB-1850; ATTO), which can simultaneously acquire all emitted

295

photons ranging from 391 to 789 nm.

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Similarly, the substrate specificity of the new lineage of ALucs was examined with

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the use of a larger number of CTZ analogues (Supplementary Fig. 2). COS-7 cells

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expressing ALucs were prepared by applying the same protocol as that in Fig. 3. The

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cells were lysed, and then an aliquot of the lysates (10 µL) was carefully transferred to a

300

96-well optical bottom microplate, simultaneously mixed with 40 µL of each

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coelenterazine variant (nCTZ, CTZ400, CTZh, CTZn, CTZi, CTZf, CTZcp, CTZhcp,

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CTZ fcp, and CTZip) by using a multichannel micropipette, and placed in an image

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analyzer (LAS-4000; Fujifilm). The relative optical intensities were immediately

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integrated for 30 seconds.

305 306

Live cell bioluminescence imaging with marine luciferases. The live cell images and

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optical profiles of NanoLuc (Promega), RLuc8.6-535, and ALuc49 were determined

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with the use of the COS-7 cells grown in 6-channel microslides (µ-Slide VI0.4; ibidi)

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(Fig. 4).

310

The COS-7 cells in 6-channel microslides were transiently transfected with the

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pcDNA 3.1(+) vector encoding NonoLuc, RLuc8.6-535, or ALuc49. Sixteen hours after

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the incubation, the cells in each channel were rinsed once with HBSS buffer (Gibco)

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and simultaneously bathed with 60 µL of furimazine, 6piOH-CTZ, or nCTZ dissolved

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in HBSS buffer (final concentration: 10-4 M) by using a multichannel micropipette

315

(Gilson). The microslides were immediately set in the dark chamber of the LAS-4000

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image analyzer (Fujifilm), and the corresponding optical intensities from the

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microslides were integrated for 20 sec. The optical intensities and profiles from the

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living COS-7 cells in the microslide were normalized by measured time (sec) and area

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319

(mm2); the unit was RLU/sec/mm2, and the specific image analysis software Multi

320

Gauge version 3.1 (Fujifilm) was used.

321 322 323

Construction of single-chain bioluminescent probes with ALucs. The advantages of

324

using the newly fabricated ALucs in bioassays were determined with the use of

325

single-chain bioluminescent probes (“bioluminescent capsules”) carrying full-length

326

ALuc47 or ALuc49 (Supplementary Fig. 3). The corresponding probes were named P47

327

and P49, respectively.

328

The working mechanism of bioluminescent capsules has been previously shown by 17

329

the authors

330

C-terminal end of which is linked to a membrane localization signal (MLS) through a

331

known substrate sequence of caspases (“DEVD”). The luciferase fixed in the capsule is

332

dissected by active caspase 3 and is eventually diffused freely to the cytosol. The free

333

luciferase has higher optical activities than the fixed one to the plasma membrane.

. Briefly, the capsule is designed to carry a full-length ALuc, the

334 335 336

Author contributions statements

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337

SBK and RN wrote the main manuscript, and prepared all the Figures. All authors

338

reviewed the manuscript.

339 340

Competing financial interests

341

The authors declare no competing financial interests.

342 343

Acknowledgement

344

This work was supported by grants from the Japan Society for the Promotion of

345

Science (JSPS) (grants 26288088, 15KK0029, 16K14051, 24225001, and

346

17H01215).

347 348

Supporting Information Available:

349

A specified alignment of the sequences of the newly created ALucs; Figure S1(A).

350

A rooted phylogenetic tree of the newly created ALucs and existing marine luciferases

351

according to CLUSTALW version 2.1; Figure S1(B). The aligned sequences of 25

352

copepod luciferases; Figures S1(C) and (D). Substrate-specific bioluminescence

353

intensities of the new lineage of ALucs; Figure S2.

354

with ALuc47- and ALuc49-based bioluminescent probes: Figure S3.

Illumination of caspase activities

20


HPLC spectra

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355

showing the purity of the substrates nCTZ (A) and CTZh (B); Figure S4. Relative

356

optical intensities of new ALucs in photon counts; Figure S5, Table S1, and

357

Experimental Procedure S2.

358 359

This material is available free of charge via the Internet at http://pubs.acs.org.

360

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Table of Contents Graphic

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Fabrication of a New Lineage of Artificial Luciferases from Natural

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