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WO2012091101A1 - Système de sonde bioluminescente de type à multireconnaissance - Google Patents

Système de sonde bioluminescente de type à multireconnaissance Download PDF

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WO2012091101A1
WO2012091101A1 PCT/JP2011/080419 JP2011080419W WO2012091101A1 WO 2012091101 A1 WO2012091101 A1 WO 2012091101A1 JP 2011080419 W JP2011080419 W JP 2011080419W WO 2012091101 A1 WO2012091101 A1 WO 2012091101A1
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probe
ligand
transcription factor
receptor
target cell
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Japanese (ja)
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誠培 金
鳥村　政基
康浩 竹中
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National Institute of Advanced Industrial Science and Technology AIST
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase

Definitions

  • the present invention relates to the development of a bioluminescent probe system having multiple recognition ability and its application.
  • Biological analysis of biological and environmental samples is an indispensable means for early diagnosis of diseases and identification of environmental pollutants.
  • biological samples such as urine, feces, sputum, saliva, nasal discharge, and sweat.
  • biological samples agricultural and livestock waste, marine waste, soil, seabed soil, river water, seawater, tap water, suspended dust, etc. can be assumed.
  • concentration of the physiologically active substance contained in such a sample is generally low, it must be dependent on sample concentration and expensive instrumental analysis. For the same reason, on-site analysis was very difficult.
  • various physiologically active substances can be identified from urine or saliva. For example, urinary stress hormone analysis is useful as an index of stress response in a living body.
  • Non-patent Document 1 Non-patent Document 1
  • the center of research in many medical / pharmaceutical fields can be said to be an important issue to identify and analyze factors that are indicators of disease diagnosis for biological samples.
  • current diagnostic methods that are actually used in diagnostic sites such as hospitals are based on immunostaining with a narrow responsive concentration range while requiring a long work.
  • Patent Document 1 Non-Patent Document 4
  • Patent Document 4 Molecular imaging probes based on the technique of circular permutation of gene sequences of luminescent enzymes have also been developed
  • Patent Document 6 Non-Patent Document 6
  • the present inventors have developed (5) a molecular strain sensor using as an index the change in enzyme activity caused by artificially straining the luminescent enzyme, and is currently applying for a patent (Japanese Patent Application No. 2009-200413, Japanese Patent Application No. 2010-018759).
  • Non-patent Document 10 since the number of cells that can be analyzed at one time is limited in a fluorescence microscope, the results obtained therefrom are qualitative rather than quantitative (Non-patent Document 10). In the case of the conventional bioluminescent method, such a problem hardly occurs (Non-Patent Documents 10 to 14). On the other hand, the light is weaker than fluorescence, and there is a problem that a substrate is always required. It was.
  • the single-molecule type bioluminescent probe developed by the present inventors is a molecule that satisfies all detection sensitivity, selectivity and S / N ratio for application to biological samples, despite its innovative molecular design. It was difficult to provide a probe.
  • bioassay methods for detecting protein-protein interactions are classified according to their measurement forms: (1) “genomic assay” (eg, reporter gene assay, two-hybrid assay, etc.) ) And (2) “nongenomic assay” (eg, protein splicing method (PSA), protein complementation method (PCA), single molecule bioluminescent probe (IMF), etc.) It is divided roughly into.
  • the advantages of ⁇ assay with reporter protein expression '' are: (a) simple principle, broad versatility; (b) accumulation of reporter protein; Points that can be expected are taken up.
  • the feature of the “measurement system without reporter protein expression” is that (a) the reporter protein is expressed in advance (that is, the reporter is part of the probe), but the response time is fast. (B) The light emission signal itself is weak because it does not go through the accumulation process of the reporter protein, and (c) precise molecular design is necessary to capture the weak signal.
  • the present inventors have previously studied various modification positions of marine animal-derived luminescent enzymes such as Gaussia luciferase (GLuc) in order to obtain modified forms with enhanced luminescence intensity and stability. Thus, it was possible to provide a stable ultra-bright bioluminescent enzyme exhibiting strong luminescence and its gene (Japanese Patent Application No. 2009-101025).
  • the present invention provides high detection when applied to a biological sample in a bioanalytical method for examining a molecular action occurring in a cell (eg, induction of protein activity, structural change, interaction between protein and protein, etc. by ligand stimulation). It is intended to provide a bioanalytical method having a high S / N ratio and a fast response time with sensitivity and high selectivity.
  • the disadvantages are minimized while taking advantage of both the conventional “genomic assay” and “nongenomic assay”.
  • the aim was to develop a method that would limit it to the limit.
  • the present inventors pay attention to a reporter protein (eg, luminescent enzyme or fluorescent protein) gene located downstream of a transcription factor response element in a genomic assay such as a conventional reporter gene assay.
  • a reporter protein eg, luminescent enzyme or fluorescent protein
  • I came up with a specific single-molecule bioluminescent probe that can recognize the same ligand as the transcription factor. As a result, the S / N ratio could be dramatically increased.
  • the assay system of the present invention since a high S / N ratio can be achieved by mounting a probe having a ligand recognizing ability instead of a reporter protein, it is a small amount of “a transcription factor inherent in living cells”. However, it is necessary and sufficient for the operation of the probe system. As a result, the conventional “plasmid expressing the transcription factor” can be omitted (since the need for introducing multiple plasmids has been eliminated), and the efficiency of the assay system has been simplified.
  • a single-molecule bioluminescent probe expression vector containing a transcription factor (eg, nuclear receptor) gene into a host cell and allowing the probe to be expressed;
  • a transcription factor eg, nuclear receptor
  • secondary sensing secondary response
  • the same ligand binds to the expressed bioluminescent probe, induces structural change in the probe, and is divided into its N-terminal side and C-terminal side.
  • a bioluminescent system for the recognition of test ligands including The bioluminescence system is a system that simultaneously induces primary sensing (primary response) and secondary sensing (secondary response) to the same stimulus (ligand) (hereinafter, “multi-molecule recognition bioluminescence system”).
  • the bioluminescence system of the present invention once the expression of the bioluminescent probe is initiated from the plasmid, a part of the transcription factor (ie, “ligand binding domain”) inherent in the probe is used. ) Binds to the “transcription factor responsive element” and further expresses itself (FIG. 1) (so-called “self-amplification”) to induce a stronger luminescence response, “self-amplification type” luminescence. It can be said that it is a system.
  • ligand binding domain (LBD) designed so as to have the ability to recognize a ligand in a single molecule probe. It was discovered that there was a problem that hindered the organic linkage between components, and this problem was overcome by improving the molecular design. Specifically, all conventional single-molecule and bimolecular bioluminescent probes use a “ligand binding domain” which is a partial fragment of a transcription factor.
  • GR LBD glucocorticoid receptor
  • its ligand binding domain is sequence information of full-length human GR (GenBank / Based on P04150)
  • the LBD region amino acid numbers 527-777
  • the amino acid sequence up to 527-777 the N-terminal and C-terminal are greatly separated.
  • the present inventor considers that the “opening between the N-terminal and C-terminal” of the ligand binding domain in such a single-molecule probe affects the performance of the probe, and in addition, the “hinge region (HR) and DNA of the GR Considering the importance of “molecular structure of binding domain (DBD)”, we tried to combine various HR and GR LBD molecular structures. As a result, by extending the N terminus of GR LBD to 486, (1) the distance between the N terminus and C terminus of the molecule can be made adjacent, and (2) the molecular structure change of GR LBD contributes to the optimal recovery of bioluminescence. I was connected. Obtaining the above knowledge, the present invention has been completed.
  • a method for measuring the responsiveness of a target cell to a test ligand comprising the following steps (1) to (4); (1) including a base sequence encoding each region of a luminescent enzyme divided into an N-terminal side and a C-terminal side, together with a base sequence encoding a “ligand binding domain” derived from a transcription factor that recognizes the test ligand, A step of previously introducing into the target cell an expression vector in which a gene encoding a monomolecular or bimolecular bioluminescent probe is inserted downstream of a transcription factor responsive element capable of binding to the transcription factor; (2) In response to stimulation of the test ligand, an endogenous transcription factor in the target cell is activated and binds to a transcription factor responsive element upstream of the gene encoding the bioluminescent probe, so that A step (primary response) in which a bioluminescent probe is expressed, (3) The same test ligand was bound to the ligand
  • a process of light emission by recombination of a luminescent enzyme (secondary response), (4) A step of qualitatively or quantitatively measuring the luminescence intensity from the bioluminescent probe in step (3).
  • the “ligand binding domain” in the bioluminescent probe expressed in the step (2) binds to a transcription factor response element upstream of the gene encoding the bioluminescent probe in the expression vector to express the bioluminescent probe.
  • the method of the above-mentioned [1] which comprises a step (“self-amplification”).
  • the method for measuring the responsiveness of a target cell to a test ligand is a method for measuring the intensity of activity of a test ligand given to the target cell as an external stimulus [1] Or the method as described in [2].
  • the method according to [1] or [2] above, wherein the method for measuring the responsiveness of the target cell to the test ligand is a method for measuring the response ability of the target cell to the ligand.
  • the method for measuring the responsiveness of a target cell to a test ligand is a method for screening whether the test substance is an activity inducer for an endogenous transcription factor of the target cell, The method according to [1] or [2].
  • the nuclear receptor is a glucocorticoid receptor (GR), an androgen receptor (AR), an estrogen receptor (ER), a human mineralocorticoid receptor (MR), a human luteinizing hormone receptor (PR), Any selected from vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR), peroxisome proliferator-activated receptor (PPAR), nuclear factor ⁇ B (NF- ⁇ B)
  • the nuclear receptor is a glucocorticoid receptor (GR)
  • the test ligand is a glucocorticoid
  • the response to the test ligand to be measured is a level of stress on the target cell or living body.
  • An expression vector to be introduced into the target cell in order to measure the responsiveness of the target cell to the test ligand One molecule comprising a base sequence encoding each region of the luminescent enzyme fragment divided into the N-terminal side and the C-terminal side, together with a base sequence encoding a “ligand binding domain” derived from a transcription factor that recognizes the test ligand
  • a gene encoding a type or bimolecular bioluminescent probe is inserted downstream of a transcription factor responsive element capable of binding to the transcription factor,
  • the endogenous transcription factor in the target cell is activated in response to the test ligand stimulation and binds to the transcription factor response element in the expression vector, thereby starting transcription of the bioluminescent probe gene downstream thereof.
  • a base sequence encoding a “ligand binding domain” derived from a transcription factor in a gene encoding the bioluminescent probe is further upstream of the N-terminal side thereof,
  • a target cell transformed with an expression vector containing a gene encoding the bioluminescent probe described in [9] or [10], and activated by stimulation of a test ligand to the cell
  • a bioluminescent probe By binding an endogenous transcription factor in the cell to a transcription factor responsive element in the vector, a bioluminescent probe can be expressed downstream thereof, and the responsiveness of the cell to a test ligand can be measured. Possible transformed target cells.
  • the same ligand stimulation is discriminated by two types of on / off systems, and the luminescence intensity is weak. Since self-amplification with time, the background effect based on intracellular basal metabolism could be minimized, and a high S / N ratio could be achieved.
  • the system simultaneously has the ability to discriminate the presence or absence of “protein expression ability” in a genomic assay and the “instantaneous signal discrimination ability” of a nongenomic assay. As a result, it was possible to provide an assay system capable of sensing stimuli in many ways in the presence of specific external stimuli.
  • the distance between the N-terminal and C-terminal in the probe molecule can be made adjacent.
  • the bioluminescence was enhanced by changing the molecular structure without inhibiting the organic linkage between each component of the probe.
  • a cell-specific transcription factor is activated and binds to a promoter to initiate probe expression (primary sensing; the absolute amount of the probe increases).
  • the expressed probe senses the ligand again (secondary sensing).
  • Luc-N the N-terminal fragment of lu-ciferase
  • GR HLBD the hinge and ligand binding domain of glucocorti-coid receptor
  • TF transcription factor
  • GR glucocorticoid receptor.
  • Molecular model of transcription factor-DNA binding (example). Dark gray indicates ⁇ -helix number 12, and the arrow corresponds to the joint of the transcription factor.
  • the thumbprint indicates the cleavage position in the DNA binding domain (DBD).
  • Light gray helix indicates DNA.
  • the thumbprint indicates the extended position. This thumbprint region corresponds to the N-terminus of the GRLBD fragment.
  • a typical construct of the probe system Instead of a reporter downstream of the promoter, a single-molecule bioluminescent probe is mounted. The mounted single molecule bioluminescent probe is characterized in that the N-terminal side of GR LBD is extended as compared with the conventional type. Ligand responsiveness of multiple recognition probe system. (A) Only the primary response (white bar) or the secondary response (light gray) does not cause a sufficient contrast of the luminescence value for the same stimulus, but the combination of the two results in a synergistic effect of the S / N ratio.
  • Each construct was constructed by changing the length at the N-terminal side of GR LBD, and was named mGG1, mGG2, and mGG3 in the order of the length. Verification of ligand responsiveness of COS-7 cells equipped with probe systems from pmGG1 to pmGG3. When the luminescence values of pmGG2 and pmGG3 were compared with pmGG1, a significant increase in bioluminescence was shown, indicating that the method of extending the N-terminal side is effective. The gray bar indicates the bioluminescence intensity after 24 hours of ligand stimulation. On the other hand, the black bar shows an increase in bioluminescence intensity after 48 hours of ligand stimulation. Verification of cotisol selectivity in the present probe system using cSimgr4 shown in FIG.
  • the inset shows the operating principle of the probe of the present application.
  • the glucocorticoid receptor GR LBD
  • GR LBD glucocorticoid receptor
  • the halved Gaussia luciferase fragment re-synthesizes and emits light.
  • cSimgr4 The control site shows a quantitative increase in luminescence for stress hormones.
  • the volunteer's 2-hour saliva sample showed bioluminescence reflecting the stress hormone.
  • the greatest feature of the “multiple molecule recognition bioluminescence system” of the present invention is that primary sensing (primary response) and secondary sensing ( Secondary response) occurs simultaneously.
  • primary sensing primary response
  • Secondary response secondary sensing
  • a single molecule type bioluminescent probe gene having a ligand recognition ability is arranged instead of a reporter protein gene arranged downstream of the transcription factor response element.
  • the following molecular recognition system is activated by introducing a probe expression plasmid into a host cell: "A small amount of transcription factor inherent in the cell” or "A small amount of single molecule bioluminescent probe expressed by the basic metabolism of the cell”
  • a small amount of a single molecule bioluminescent probe is expressed (primary sensing: primary response)
  • the expressed luminescent probe is the same again
  • a system that emits light by recognizing external stimuli (secondary sensing: secondary response) (FIG. 1).
  • the overlapping of two On / Off systems based on the same stimulus results in an improved S / N ratio even with a weak external stimulus.
  • the “multiple molecule recognition bioluminescence system” of the present invention is (I) introducing a single-molecule bioluminescent probe expression vector containing a transcription factor (eg, nuclear receptor) gene into a host cell and allowing the probe to be expressed; (Ii) a step of expressing a single molecule bioluminescent probe by acting a transcription factor bound to a ligand as primary sensing (primary response) in response to external ligand stimulation; (Iii) Next, as secondary sensing (secondary response), the same ligand binds to the expressed bioluminescent probe, induces structural change in the probe, and is divided into its N-terminal side and C-terminal side.
  • a transcription factor eg, nuclear receptor
  • a process in which recombination of the luminescent enzyme occurs and emits a luminescent signal can be applied to a “method for measuring the responsiveness of a target cell to a test ligand”. Further, each step can be described in detail as follows.
  • a base sequence encoding each region of a luminescent enzyme divided into an N-terminal side and a C-terminal side, together with a base sequence encoding a “ligand binding domain” derived from a transcription factor that recognizes the target test ligand A step of preparing an expression vector in which a chimeric DNA encoding a monomolecular or bimolecular bioluminescent probe is inserted downstream of a transcription factor responsive element capable of binding to the transcription factor; (2) introducing the expression vector prepared in step (1) into the target cell; (3) a step in which an endogenous transcription factor in the cell is activated in response to the test ligand stimulation to the target cell; (4) a step in which the transcription factor binds to a transcription factor responsive element upstream of a gene encoding the bioluminescence probe, thereby expressing a downstream bioluminescence probe; (5) When the same test ligand binds to the ligand binding domain in the bioluminescent probe, the structure of the ligand binding domain changes and
  • a process in which the N-terminal side and C-terminal region of the luminescent enzyme approach and emit light (6) A step of qualitatively or quantitatively measuring the luminescence intensity from the bioluminescent probe in step (5).
  • a part of the “ligand binding domain” in the expressed bioluminescence probe is inserted upstream of the gene encoding the bioluminescence probe.
  • the bioluminescence probe is expressed by binding to the “transcription factor responsive element” (FIG.
  • the light emitting system of the present invention also has the aspect of a “self-amplifying” light emitting system.
  • the “multi-molecule recognition bioluminescence system” of the present invention is not only characterized by the ability to determine the presence or absence of “protein expression ability”, which is a characteristic of the conventional genomic assay, but also by a conventional nongenomic assay (nongenomic assay). assay) has the “instantaneous signal discrimination ability” at the same time, can achieve a sufficient S / N ratio for measurement and diagnosis, and has a low risk of dummy response. ⁇ Suitable for diagnostic systems.
  • the present invention when “to measure the responsiveness of a target cell to a test ligand”, for example, (1) When qualitatively or quantitatively measuring the strength of a test ligand (external stimulus) against an endogenous transcription factor of a target cell, (2) When qualitatively or quantitatively measuring the strength of the response ability of the target cell to the ligand, (3) In the screening method for determining whether a test substance is a ligand candidate substance for an endogenous transcription factor of a target cell, there may be a case where the intensity of stimulation from the test substance is measured qualitatively or quantitatively.
  • the present invention encompasses both cases.
  • a typical example is a case where the amount of stress applied to a eukaryotic cell or a mouse individual is measured by applying it to a transcription factor GR having a glucocorticoid called a stress hormone as a ligand.
  • transcription factors are the characteristic of undergoing dramatic internal and external changes upon stimulation with external ligands, and are activated upon stimulation and complex with coupling factors into the nucleus. To cause transcriptional activity by binding to a transcription factor response element in DNA. Transcription factors that function in this way are androgen receptor (AR), female hormone receptor (ER), progesterone receptor (PR), glucocorticoid receptor (glucocorticoid receptor).
  • AR androgen receptor
  • ER female hormone receptor
  • PR progesterone receptor
  • glucocorticoid receptor glucocorticoid receptor
  • a substance that can specifically bind to the “ligand binding domain” of the transcription factor of the present invention and change its function is a “ligand”, and various hormones such as male hormones refer to ligands inherent in the living body.
  • the subject of the assay of the present invention includes antagonists and agonists of these original ligands.
  • a “ligand binding domain” fragment of a “transcription factor” (eg, a nuclear receptor) that recognizes an external stimulus (ligand) is a monomolecular bioluminescent probe or a bimolecular organism.
  • the light emitting probe is incorporated as a partial component.
  • An expression vector into which a gene encoding this probe has been inserted is introduced into the cell. Later, when the luminescent probe is expressed, a transcription factor incorporated as a part of the probe reacts to an external stimulus to enable secondary sensing.
  • the base sequence and amino acid sequence of each transcription factor are known and can be obtained from an existing database (eg, http://www.ncbi.nlm.nih.gov/) as described later.
  • Bioluminescence probe used in the present invention and expression vector containing the gene thereof A bimolecular bioluminescence probe (Patent Documents 1 and 2) together with a single molecule probe among various bioluminescence probes that have been developed by the present inventors. 3, Non-Patent Documents 4, 5, 6, 10, and 14).
  • Patent Documents 1 and 2 A bimolecular bioluminescence probe (Patent Documents 1 and 2) together with a single molecule probe among various bioluminescence probes that have been developed by the present inventors. 3, Non-Patent Documents 4, 5, 6, 10, and 14).
  • a typical single molecule probe will be described as an example.
  • An expression vector into which a base sequence encoding the single-molecule probe is inserted is introduced into a host cell.
  • the amino acid sequences of these single-molecule probes include a sequence containing a “ligand binding domain” sequence of a transcription factor for recognizing external stimuli (ligands), a transcription factor response sequence, an N-terminal region and C It contains a luminescent enzyme (LE) sequence divided into the terminal region.
  • a known mammalian cell expression vector can be used as an expression vector encoding the luminescent probe. When expression is desired in a specific tissue in an individual organism, a known tissue-specific promoter sequence and plasmid are used. May be.
  • the probe expression vector can be introduced into cells by known transfection methods such as microinjection and electroporation.
  • an expression vector containing a gene encoding a bioluminescent probe may be a vector having a known eukaryotic or prokaryotic control sequence in accordance with the host cell.
  • the terms “transformation” and “transformed cell” typically refer to the case where the gene in the expression vector is integrated into the genome of the cell and stably expressed. It may be a case of transient expression in which the expression vector is not integrated into the genome of the introduced cell.
  • the outline about the component of the bioluminescent probe of this invention is demonstrated.
  • Bioluminescent enzyme used in the bioluminescent probe of the present invention As the luminescent enzyme (LE) of the present invention, any luminescent enzyme or a modified form thereof can be used as long as it can be divided into two. However, typically, Gaussia luciferase (GLuc), firefly luciferase (FLuc), Renilla luciferase ( Renilla luciferase (RLuc) and Click Beetle luciferase (CBLuc). A high-brightness Gaussia luciferase variant (PCT / JP2010 / 052511) is particularly preferred.
  • Typical luminescent enzymes have known amino acid sequences and base sequences of genes (cDNA) (for example, FLuc is GenBank / AB062786 etc., CBLuc is GenBank / AY258592.1 etc.), and is known based on these sequence information DNA can be obtained by this method.
  • the position to divide into two is also known, and part of the sequence of both of them overlaps as long as the function of emitting light is lost when the N-terminal fragment (N-LE) and the C-terminal fragment (C-LE) are combined. Or deleted.
  • the “ligand binding domain” in the present invention is a partial region of a transcription factor, and can change the protein structure in response to stimuli (ligands) inside and outside the cell. As a result, other proteins or coupling factors (coactivator) and It binds and causes transcriptional activity.
  • the following example corresponds to a partial region of the transcription factor.
  • the ligand binding domain (GR LBD) in the glucocorticoid receptor (GR) is based on the sequence information (GenBank / NM_000176) of the full-length human GR and the nucleotide sequence corresponding to the LBD region (amino acid numbers 527-777) is gene It can be prepared engineeringly or by PCR synthesis.
  • androgen receptor (AR) LBD can prepare its LBD region (amino acid number: 672-910AA) based on the sequence information (GenBank / NM_000044) of full-length human AR.
  • the body (estrogen receptor; ER) LBD can prepare its LBD region (amino acid number 305-550) based on the sequence information of full-length human ER (GenBank / P03372), and androgen receptor LBD (LBD of androgen receptor; AR LBD) ) May be prepared based on the sequence information of full-length human AR (GenBank / AF162704) and its LBD region (amino acid numbers 672-910).
  • a human mineralocorticoid receptor (MR; GenBank / P08235) and a human luteinizing hormone receptor (progesterone receptor; PR; GenBank / P06401) ligand-binding domain can be typically used.
  • phosphorylation recognition domains such as “SH2 domains” of various kinase proteins can be used.
  • Src proto-oncogene tyrosine-protein kinase Src; GenBank / NP938033
  • SH2 domain amino acid number 150-248
  • cell proliferation / carcinogenesis etc.
  • the SH2 domain of growth factor receptor binding protein Grb2 growth factor receptor-binding protein 2
  • an LXXLL motif derived from a coactivator is typically used as a peptide sequence that can bind to GR LBD in the present invention.
  • Rip140 (GenBank / NP003480), which is a kind of co-transcription factor, or LXXLL motif (about 15 amino acids) of Src-1a (steroid receptor coactivator 1 isoform 1; GenBank / NP003734) is used.
  • LXXLL motif about 15 amino acids
  • Src-1a steroid receptor coactivator 1 isoform 1; GenBank / NP003734
  • an FXXLF motif, a WXXLF motif, or the like can also be used as a peptide sequence that can bind to GR LBD.
  • a transcription factor for example, glucocorticoid receptor (GR) has a transcriptional activity (AF2 activity) even though it is a partial fragment (Non-patent Document 15).
  • GR glucocorticoid receptor
  • AF2 activity transcriptional activity
  • Non-patent Document 15 Non-patent Document 15
  • the present inventors have made a relative comparison between the N-terminal fragment and the C-terminal fragment in the molecular model diagram (FIG. 2) of the glucocorticoid receptor (GR) prepared based on the crystal structure analysis by GR X-rays. The position was examined and attention was paid to the fact that appropriately extending the N-terminal side of the ligand-binding domain (LBD) adjoins the distance between the N-terminal and C-terminal fragments.
  • FFD glucocorticoid receptor
  • a region of 10 to 300 amino acids (AA) upstream of LBD preferably 20 to 200 AA, more preferably 40 to 160 AA is used. This region corresponds to the region (40AA) and the DNA binding domain (DBD).
  • AA amino acids
  • DBD DNA binding domain
  • GR LBD is described below, it can be similarly applied to other transcription factors.
  • the “single molecule bioluminescent probe” in which the N-terminal side of GR LBD is extended has a stronger stress hormone sensitivity than that before modification (FIG. 4).
  • the ligand binding domain is an LBD region (amino acid numbers 527-777) prepared based on the sequence information of full-length human GR (GenBank / P04150).
  • GR LBD ligand binding domain
  • the inventor pays attention to the importance of GR "hinge region (HR) and DNA binding domain (DBD) molecular structure" together with GR LBD, and combined the molecular structure of HR and GR LBD.
  • HR high-density lipoprotein
  • DBD DNA binding domain
  • the N-terminal of the GR LBD was extended to 486, (A) the distance between the N-terminal and the C-terminal of the molecule could be adjacent, and (B) the bioluminescent enzyme fragment due to the molecular structure change of the GR LBD. Between recombination and emission emission were optimized.
  • plasmids loaded with circular permutation probes containing the 486-777AA fragment of GR were named pmGG1, pmGG2 when the 407-777AA fragment was loaded, and pmGG3 when the 361-777AA fragment was loaded. .
  • the method of extending the N-terminal side of a transcription factor is the simplest and most reliable method using the Hinge region and DNA binding region (DBD) upstream of the LBD region of each transcription factor.
  • DBD DNA binding region
  • a well-known linker sequence can also be utilized regardless of DBD.
  • a known LBD of the nuclear receptor is employed.
  • phosphorylated amino acid residues and G protein-coupled receptor ligands phosphorylated amino acid binding domains, G protein-coupled receptors and the like can be employed as LBDs, respectively.
  • the LBD of the nuclear receptor for example, the androgen receptor LBD (AR LBD) is based on the sequence information of the full-length human AR (GenBank / NM_000044) and its NBD region (amino acid numbers 672-910) and its N A DNA encoding a region to which all or part of the terminal region (amino acid numbers 625 to 672) is added can be prepared and used by genetic engineering or chemical synthesis.
  • the female hormone receptor (ER) LBD is also genetically engineered for its LBD region (amino acid number 305-550) and its N-terminal side based on the sequence information (GenBank / P03372) of full-length human ER, or PCR. It can be extended by synthesis.
  • LBD of other nuclear receptors including human mineralocorticoid receptor (MR; GenBank / P08235) and human luteinizing hormone receptor (progesterone receptor; PR; GenBank / P06401). Can be used by extending the sequence.
  • Assay system to which the probe system of the present invention can be applied The probe system of the present invention can be suitably applied to various assay systems.
  • reporter gene assay and two-hybrid assay it can be used without particular limitation.
  • the reporter gene in the reporter gene assay plasmid is replaced with a gene encoding the probe of the present invention.
  • a “multiple recognition type bioluminescence assay system” can be suitably constructed by providing a promoter that recognizes a signal similar to the above probe upstream.
  • the gene encoding the single-molecule probe of the present invention is mounted in place of the normal reporter gene in the plasmid for two-hybrid assay.
  • transcription factor responsive elements can also be mounted on the “multiple recognition bioluminescence assay system” of the present invention.
  • the following abbreviations are vitamin D receptor (VDR), retinoic acid receptor (RDR), DAX-1 orphan nuclear receptor (DAX), retinoid X receptor ⁇ (RXR ⁇ ), androstane receptor (CAR), respectively.
  • PXR Pregnane X receptor
  • FXR farnesoid X receptor
  • PPAR ⁇ peroxisome proliferator-activated receptor ⁇
  • PPAR ⁇ peroxisome proliferator-activated receptor ⁇
  • the assay system of the present invention can be used for screening antagonists of transcription factors, substances having agonist activity, or analogs thereof.
  • a substance having an antagonistic or agonistic activity against the endogenous transcription factor of the target cell to be screened may be referred to as “transcription factor activation inducer” or simply “activity inducer” in the present invention.
  • transcription factor activation inducer or simply “activity inducer” in the present invention.
  • a luminometer eg, Mithras LB 940, Berthold. taking measurement. If the ligand has biological activity, it induces probe expression and transcriptional activity, and the expressed probe senses the same ligand again.
  • a sensing principle is particularly effective for measurement of a weakly active ligand, and an excellent sample processing ability can be expected when measuring the activity of a large number of ligands.
  • Ligand activity may be measured according to a normal bioluminescence assay, and a conventional protocol can be applied without particular limitation.
  • Luminometers eg, MiniLumat LB 95066
  • a cell lysate is prepared by applying lysate buffer to the cells cultured on the plate, and the luminescence value after mixing with the substrate is measured immediately.
  • a ready-made bioluminescent plate reader eg, Mithras LB 940, Berthold
  • bioluminescence by the expressed probe can be instantaneously introduced into the substrate and measured for luminescence by using an automatic substrate solution injector attached to the plate reader.
  • an apparatus instead of a cell lysate, an apparatus has been developed that can measure ligand sensitivity while the cultured cells remain alive. For example, by using LUMINOVIEW (LV100) (Olympus), the bioluminescence intensity from individual cells can be measured with excellent spatial resolution.
  • test substances to be subjected to these screening methods include, for example, organic or inorganic compounds (particularly low molecular weight compounds), biologically active proteins, peptides and the like. These substances may be known or unknown in function and structure.
  • the “combinatorial chemical library” is an effective means as a test substance group for efficiently specifying a target substance.
  • the preparation and screening of combinatorial chemical libraries is well known in the art (see, eg, US Pat. Nos. 6,004,617; 5,985,365).
  • commercially available libraries for example, libraries such as US ComGenex, Russian Asinex, US Tripos, Inc., Russian ChemStar, Ltd, US 3D Pharmaceuticals, Martek Biosciences, etc.
  • so-called “high-throughput screening” can be performed by applying a combinatorial chemical library to a population of cells that express the probe.
  • Example 1 Design of an improved construct for a multi-recognition bioluminescent probe system N in a molecular model diagram (Fig. 3) of a glucocorticoid receptor (GR) prepared based on crystal structure analysis by X-ray
  • Fig. 3 molecular model diagram
  • GR glucocorticoid receptor
  • dark gray is a variable region that changes its position when GR LBD is activated.
  • the N terminal of GR LBD is connected to a sequence (DBD) for binding GR to DNA.
  • DBD sequence for binding GR to DNA.
  • Example 2 In order to construct a multi-aware bioluminescent probe system constructing step present invention of the multi-aware bioluminescent probe system of the present invention, the present inventors have single-molecule-bioluminescent probes previously developed (Patent The production method of Document 1) was followed (see FIG. 1). First, in order to prevent the possibility of the probe body being secreted, the secretion signal (1-17AA) at the N-terminus of Gaussia luciferase (GLuc) is removed, and the remaining GLuc is divided into two by genetic engineering. did.
  • a glucocorticoid receptor ligand-binding domain (GR LBD) and its response element (that is, GR LBD binding partner) are linked between two split GLuc ( Chimeric DNA) was produced. Further, a construct containing the improved type (GR LBD + positions 486 to 526 in the hinge region) synthesized in Example 1 was synthesized between GLuc divided into two (FIG. 4). The constructs were inserted into pcDNA3.1 (+), and the resulting plasmid was called pSimgr3 and the latter was called pSimgr4.
  • the construct can be multiplexed by introducing the construct into a pTL (pTransLucent) vector (manufactured by Panomics), which is a plasmid for a conventional reporter gene.
  • pTL pTransLucent
  • Panomics a plasmid for a conventional reporter gene.
  • a recognizable bioluminescent probe system can be constructed.
  • the experimental conditions such as the transformation method, cell culture method, and measurement method used in the following examples of the present invention are the same as those described in our previous paper (Non-patent Document 17). It was.
  • the COS-7 cells were introduced with the above-described expression vector carrying the single-molecule bioluminescent probe constructed in the present invention, cultured for 16 hours after transformation, stabilized, and then treated with control or stimulating hormone.
  • a predetermined amount of eg, cortisol
  • a substrate coelenterazeine
  • the luminescence intensity was measured using a spectroscopic instrument such as a luminometer.
  • Example 3 Measurement of stress response hormone activity
  • the probe system of the present invention is characterized by sensing twice for a target ligand. Therefore, the ON / OFF system is activated twice for the same signal, and the S / N ratio is improved. Therefore, in this example, in order to construct a probe system for analyzing “cortisol” activity, which is a typical stress response hormone, two analysis systems were employed and combined. One is a reporter gene assay system for measuring “stress hormone” activity, and the other is a single molecule bioluminescent probe that responds to “stress hormone”. According to the results shown in FIG. 5, (i) the reporter gene assay for measuring “stress hormone” showed an S / N ratio of about 3 times (FIG. 5; recognition 1).
  • the single-molecule bioluminescent probe showed an S / N ratio of about twice that of stress hormone (cortisol; 10 ⁇ 6 M) (FIG. 5; recognition 2).
  • a phenomenon that the S / N ratio jumps up to 8.5 times was observed.
  • the probe system employs a reporter system although it is weak, its amplification ability was confirmed by Western blot. First, using ⁇ -tubulin antibody as a house keeping protein, it was confirmed that there was no difference in the total amount of protein in the sample.
  • results shown in this example mean that primary recognition occurred as expected and the number of “single molecule bioluminescent probes” increased although it was weak. That is, the expression of “single molecule bioluminescent probe” occurred in the presence of stress hormone.
  • a synergistic effect with a high S / N ratio could be observed by overlapping two weak S / N ratios.
  • Example 4 Verification of ligand selectivity of the multi-recognition bioluminescence probe
  • the left side of FIG. 6 shows a response result to a “multiple recognition type bioluminescent probe system” equipped with a conventional single molecule type bioluminescent probe (SimgrIII; probe expressed from pSimgr3) prepared as a control.
  • SimgrIII single molecule type bioluminescent probe
  • the ligand selectivity of the “multi-recognition bioluminescence probe system” equipped with the single molecule bioluminescence probe according to the present invention (SimgrIV; a probe expressed from pSimgr3) shown on the right side of FIG. 6 was compared.
  • SimgrIII when SimgrIII was installed, the absolute value of bioluminescence was low and the selectivity for stress hormone (cortisol) was only 3-4 times that of the control, whereas when SimgrIV was installed, The absolute value of the luminescence was high, and relative ligand selectivity and S / N ratio improvement could be observed.
  • the device of the present invention can be suitably applied to the construction of a “multiple recognition type bioluminescent probe system”, and multiple recognition type organisms can be similarly applied to other transcription factors. It shows that a luminescent probe system can be constructed.
  • the ligand responsiveness of each probe system is measured by measuring the change in luminescence value over time before and after substrate introduction after introducing the substrate 20 minutes after ligand (cortisol) stimulation. The change reflected the condition with and without the ligand, and the intensity also reflected the numerical value shown in the bar graph of FIG.
  • Example 6 Construction of “Multiple Recognition Bioluminescence Probe System” Using Male Hormone Receptor (AR)
  • the stress hormone receptor GR
  • FIG. 8A a “multi-recognition bioluminescent probe system” in which a construct in which the invention was devised was synthesized and introduced into a reporter gene expression vector (pTL) similar to that in GR was prepared. Constructed (probe expressed from pSimar4, Simar IV).
  • the amino acid sequence of the male hormone receptor (AR) incorporated into the probe was from 625 amino acids to 910 amino acids.
  • a “multi-recognition bioluminescent probe system” in which a conventional single molecule bioluminescent probe (SimarIII expressed from pSimar3) was introduced into the same expression vector was prepared.
  • a significant increase in bioluminescence intensity was observed when pSimar4 was introduced (FIG. 8B).
  • Example 7 Verification of time-dependent changes in ligand responsiveness of SimarIV
  • the probe system expressing SimarIV was introduced into COS-7 cells, and male hormone (DHT) 10 -6 M was used. After stimulation for 20 minutes, a cell lysate was prepared, and changes in bioluminescence intensity before and after substrate introduction were measured with Mithras LB 940 (Berthold) (FIG. 9).
  • DHT male hormone
  • FIG. 9 the bioluminescence intensity varied depending on the presence or absence of ligand stimulation performed in advance.
  • male hormone (DHT) 10 ⁇ 6 M stimulation it shows stronger bioluminescence intensity, and it can be seen that the change over time is significantly different from that without male hormone stimulation.
  • cPresso-C1 and Simgr3 Conventional probes (cPresso-C1 and Simgr3) developed by the present inventors were also introduced into COS-7 cells, and the ligand responsiveness was verified under the same conditions (FIG. 10B).
  • the cPresso series such as cPresso-C1 used here is a stress hormone probe having a GR LBD previously developed by the present inventors (Non-Patent Document 16), and circular permutation is applied to the bioluminescent enzyme. It is shown that.
  • the detection sensitivity of cSimgr4 developed by the present invention was improved about 10 times compared with cPresso-C1, which was the most excellent probe in the past (MiniLumat LB 9506 (Berthold) of luminometer). Measurement).
  • This detection sensitivity starts to respond from the stress hormone (cortisol) 10 -9 M, and shows a linear response to around 10 -6 M.
  • This region is a concentration range in which stress hormones can be measured directly from human saliva and urine samples. That is, the concentration range allows sufficient hormone measurement from a biological sample without concentration.
  • *, **, and *** in FIG. 10B are statistical significance or p values, and the confidence range between two data groups by two tail determination is indicated by *. That is, * represents p ⁇ 0.5, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, and the cortisol concentrations indicated as “saliva” and “serum & urine” are the saliva and plasma (serum) of healthy individuals. ), The concentration of stress hormone in urine.
  • the probe is characterized by (i) a probe incorporated into the reporter gene assay, (ii) a circular permutation of the bioluminescent enzyme, and (iii) an LXXLL motif and a GR fragment linked to both ends. (Iv) It is expressed by a weak promoter (TA). At this time, the inserted GR fragment is characterized in that the N-terminal side is extended.
  • the prototype of the mounted LBD with the N-terminal side extended is called mGG1, and those with the N-terminal side extended are named mGG2 and mGG3, respectively.
  • Example 10 Verification of ligand responsiveness of the probe system of the present invention
  • the ligand responsiveness of the bioluminescent probe prepared in Example 9 was verified under the condition that there was no stress hormone (Fig. 12).
  • the probe system was introduced into COS-7 cells, stress hormone stimulation (cortisol 10 -6 M) was applied soon, and the increase in bioluminescence intensity after 24 hours and 48 hours was measured with MiniLumat LB 9506 (Berthold) .
  • stress hormone stimulation cortisol 10 -6 M
  • Example 11 Monitoring of salivary stress hormone using the probe system of the present invention
  • the probe (cSimgr4) constructed in Example 8 is the optimum probe of the present invention in view of the results of the above-mentioned Examples.
  • the verification experiment using the biological samples of Examples 11-12 below was performed using cSimgr4.
  • transformed cells were prepared under the conditions shown in Non-Patent Document 17 in order to verify the stress hormone (cotisol) selectivity. Specifically, COS-7 cells were cultured in a 96-well plate, and pcDNA3.1 (+) vector encoding cSimgr4 was introduced into the cells.
  • the molecular probe (cSimgr4) was used to monitor the upper and lower levels of the stress hormone concentration in a normal person throughout the day (FIGS. 14, 15, and 16). Specifically, 200 mL of saliva samples were collected every 2 hours from 3 adult volunteers (L: female 33 years old, T: male 37 years old, K: male 39 years old) over 2 days.
  • COS-7 cells expressing cSimgr4 were cultured in a 96-well plate, and further cultured for 16 hours after transfection.
  • the left part of this 96-well plate is set as the control area, and cortisol of known concentration (cortisol 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M , 10 -11 M, or vehicle).
  • cortisol 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M , 10 -11 M, or vehicle cortisol 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M , 10 -11 M, or vehicle.
  • the same volume of the above saliva sample was added to the remaining part on the right, and reacted in a CO 2 incubator for 20 minutes.
  • the substrate was added to the well of the plate using a multi-channel pipette, and the plate was placed in a luminescence detection apparatus (LAS-4000) to simultaneously detect the fermentation amount (FIG. 14).
  • LAS-4000 luminescence detection apparatus
  • the increase in the amount of luminescence in the control area made a very beautiful rising curve.
  • different bioluminescence value changes were shown for each person depending on the concentration of stress hormone in saliva (FIG. 15). From the luminescence values shown in FIG. 15, it was possible to monitor the increase and decrease of the stress level of each volunteer for two days (FIG. 16).
  • Example 12 Comparison of the stress hormone value of the present probe and the conventional ELISA method for the same saliva sample
  • the measurement was performed using the probe system of the present application and the ELISA method, and the obtained numerical values were compared (FIGS. 17 and 18).
  • the method for collecting saliva was performed in the same manner as in Example 11.
  • Transformed cells were also prepared in the same manner as in Example 11.
  • the stress hormone detection principle of the probe of the present application is to prepare cells expressing the probe and fill the cells with saliva during detection. As a result, the stress hormone in saliva is extracted into the cell and binds to the probe waiting in the cell. As a result, the bioluminescence intensity increases (FIG. 17).
  • Sequence number 1 Simgr3 Sequence number 2: Simgr4 Sequence number 3: Simar3 Sequence number 4: Simar4 Sequence number 5: Glucocorticoid receptor response element (GRE) Sequence number 6: GRE consensus sequence SEQ ID NO: 7: Androgen receptor response element (ARE) Sequence number 8: Progesterone receptor response element (PRE) SEQ ID NO: 9: Estrogen receptor response element (ERE) Sequence number 10: ERE consensus sequence Sequence number 11: serum response element (SRE) Sequence number 12: heat-shock response element (HSE) SEQ ID NO: 13: cAMP response element (CRE) Sequence number 14: CRE consensus sequence (SEQ ID NO: 15: TPA response element TRE) Sequence number 16: TRE consensus sequence: SEQ ID NO: 17: p53 response element Sequence number 18: E2F response element SEQ ID NO: 19: erythrocyte GATA response element SEQ ID NO: 20: NF-

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Abstract

La présente invention concerne un « système bioluminescent de type à multiple reconnaissance moléculaire », dans lequel on transforme d'abord une cellule cible à l'aide d'un vecteur d'expression pour une sonde bioluminescente de type à molécule unique contenant un gène d'un facteur de transcription tel qu'un récepteur nucléaire, et on mesure ensuite « une réponse primaire » dans laquelle l'action d'un facteur de transcription endogène est induite en réponse à la stimulation par un ligand d'intérêt pour exprimer la sonde bioluminescente, puis on mesure l'émission de lumière (« une réponse secondaire ») dans laquelle ce même ligand est lié à la sonde bioluminescente ainsi exprimée pour induire le changement structural dans la sonde de sorte qu'une partie N-terminale et une partie C-terminale d'une enzyme de visualisation, qui a été séparée en ladite partie N-terminale et ladite partie C-terminale, peuvent à nouveau être liées ensemble. Le système peut être appliqué à un échantillon biologique pour servir de procédé d'analyse biologique permettant d'examiner une action d'une molécule dans une cellule, et peut présenter des propriétés avantageuses, à savoir une sensibilité élevée de détection, une sélectivité élevée, un rapport S/B élevé et un temps de réponse rapide.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009034059A (ja) * 2007-08-02 2009-02-19 National Institute Of Advanced Industrial & Technology 多色生物発光可視化プローブセット、又は一分子型多色生物発光可視化プローブ
JP2009261336A (ja) * 2008-04-25 2009-11-12 National Institute Of Advanced Industrial & Technology 一分子型プローブ及びその利用
WO2010119721A1 (fr) * 2009-04-17 2010-10-21 独立行政法人産業技術総合研究所 Enzyme bioluminescente artificielle stable ayant une luminosité super-élevée

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009034059A (ja) * 2007-08-02 2009-02-19 National Institute Of Advanced Industrial & Technology 多色生物発光可視化プローブセット、又は一分子型多色生物発光可視化プローブ
JP2009261336A (ja) * 2008-04-25 2009-11-12 National Institute Of Advanced Industrial & Technology 一分子型プローブ及びその利用
WO2010119721A1 (fr) * 2009-04-17 2010-10-21 独立行政法人産業技術総合研究所 Enzyme bioluminescente artificielle stable ayant une luminosité super-élevée

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Title
KIM S.B. ET AL.: "A Bioluminescent Probe for Salivary Cortisol", BIOCONJUGATE CHEMISTRY, vol. 22, no. 9, August 2011 (2011-08-01), pages 1835 - 1841 *
SUNG BAE KIM ET AL.: "Molecular Imaging Probes Based on Bioluminescence and Fluorescence", JOURNAL OF JAPAN SOCIETY FOR ANALYTICAL CHEMISTRY, vol. 58, no. 6, 2009, pages 435 - 446 *

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