WO2018084607A1 - Composition de détection de biothiols comprenant une protéine de régulation redox - Google Patents

Composition de détection de biothiols comprenant une protéine de régulation redox Download PDF

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WO2018084607A1
WO2018084607A1 PCT/KR2017/012344 KR2017012344W WO2018084607A1 WO 2018084607 A1 WO2018084607 A1 WO 2018084607A1 KR 2017012344 W KR2017012344 W KR 2017012344W WO 2018084607 A1 WO2018084607 A1 WO 2018084607A1
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biothiol
dna
ohrr
protein
composition
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PCT/KR2017/012344
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Korean (ko)
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김영필
이진오
이진원
양윤모
김태욱
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한양대학교 산학협력단
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Priority claimed from KR1020170144833A external-priority patent/KR102451039B1/ko
Application filed by 한양대학교 산학협력단 filed Critical 한양대학교 산학협력단
Priority to CN201780082078.4A priority Critical patent/CN110168367A/zh
Priority to US16/347,321 priority patent/US20190331690A1/en
Publication of WO2018084607A1 publication Critical patent/WO2018084607A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a biothiol detection composition
  • a biothiol detection composition comprising a redox control protein, a biothiol detection method using the composition, and a biosensor / kit for biothiol detection.
  • Biothiol is a low molecular weight (LMW) thiol, unlike the thiols such as Cysteine, which are present in proteins, to regulate oxidative stress resistance and physiological activity in vivo in all living things, from bacteria to humans. Perform important functions. These biothiols are very sensitive to redox reactions, so they switch to functional groups such as SOH, SO 2 H, SNO, and SS, and act as a control switch of biomolecule activity. It is also widely detected in major body fluids such as tears.
  • LMW low molecular weight
  • Biothiols present in body fluids include cysteine (Cys), homocysteine (Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), ⁇ -Glutamylcysteine ( ⁇ -GluCys), Cysteinylglycine (CysGly), N-acetylcysteine (N-AC), Coenzyme A (CoA), Coenzyme B ( Coenzyme B, CoB), Coenzyme M (CoM), bacillithiol (BacT), mycothiol (MyT), ergothioneine (ERT), trypanothione (TrT) Etc.
  • cysteine Cys
  • Hcy homocysteine
  • GSH glutathione
  • NAC N-acetylcysteine
  • CA cysteamine
  • ⁇ -GluCys Cysteinylglycine
  • N-AC N-acet
  • Non-Patent Documents 1 and 2 changes in the concentrations of Cys, Hcy, and GSH in vivo are closely related to various kinds of diseases and reported to exist in different concentration ranges [Non-Patent Documents 1 and 2].
  • the total concentrations of Hcy, Cys, and GSH (total amounts oxidized and reduced) in the plasma are present at concentrations of 6-20 ⁇ M, 150-350 ⁇ M and 4-10 ⁇ M, respectively, but high oxidative conditions in plasma Hcy and Cys are present in almost oxidized form, so the free form is extremely small, less than about 0.2 ⁇ M for Hcy, and less than about 10 ⁇ M for Cys.
  • biothiol measurement method antibody-based immunoassays, which are currently sold in kit form, are widely used. However, all of the used antibodies do not directly recognize free biothiols, but instead oxidize the biothiols to bind proteins. It recognizes the form (such as serum albumin for blood). Thus, there is a limitation in that dynamic changes in free biothiol and total biothiol cannot be detected.
  • the measurement of free biothiol is a chemical sensor based on chemical bonding. Fluorescence induced by binding to biothiol by using variants of fluorescent dyes such as rhodamine, fluorescein, BODIPY, cyanine, flavone, and coumarin It is based on the change measurement [nonpatent literature 8]. Although these methods react rapidly with free biothiol, most of the materials that induce such fluorescence changes are synthetic compounds based on benzene ring variants and functional groups, which have very low solubility, are vulnerable to pH changes, and are measured using the -SH group of free thiol.
  • Non-Patent Document 1 Persichilli, S., Gervasoni, J., Castagnola, M., Zuppi, C. & Zappacosta, B. A Reversed-Phase HPLC Fluorimetric Method for Simultaneous Determination of Homocysteine-Related Thiols in Different Body Fluids. Labmedicine 42, 657-662 (2011)
  • Non-Patent Document 2 Fiskerstrand, T., Refsum, H., Kvalheim, G. & Ueland, P. M. Homocysteine and Other Thiols in Plasma and Urine-Automated-Determination and Sample Stability. Clin Chem 39, 263-271 (1993))
  • Non-Patent Document 3 Seshadri, S. et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl J Med 346, 476-483, (2002)
  • Non-Patent Document 4 Refsum, H., Ueland, P. M., Nygard, O. & Vollset, S. E. Homocysteine and cardiovascular disease. Annu Rev Med 49, 31-62 (1998)
  • Non-Patent Document 5 Korean Patent Document 5
  • Non-Patent Document 6 El-Khairy, L., Ueland, P. M., Refsum, H., Graham, I. M. & Vollset, S. E. in Circulation Vol. 103 2544-2549 (2001).
  • Non-Patent Document 7 Andersson, A., Lindgren, A., Arnadottir, M., Prytz, H. & Hultberg, B. Thiols as a measure of plasma redox status in healthy subjects and in patients with renal or liver failure. Clin Chem 45, 1084-1086 (1999).
  • Non-Patent Document 8 Jung, H. S., Chen, X. Q., Kim, J. S. & Yoon, J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem Soc Rev 42, 6019-6031, doi: Doi 10.1039 / C3cs60024f (2013).
  • the present inventors have made a research effort to solve the problem of the conventional detection method of the biothiol, it is possible to detect a low molecular weight biothiol using a redox-regulating protein, free biothiol and total Simultaneous measurement of (Total) biothiol was possible, and the present invention was completed by developing a composition for detecting biothiol which is excellent in terms of rapidity and storage stability as well as improved sensitivity by detecting even a small amount.
  • an object of the present invention is to provide a composition for detecting biothiol, which comprises a redox-regulating protein.
  • Another object of the present invention is to provide a method for detecting biothiol using the composition.
  • the present invention has another object to provide a biosensor using the composition.
  • the present invention has another object to provide a biochip using the composition.
  • the present invention provides a composition for detecting biothiol comprising a redox-regulating protein.
  • the present invention provides a method for detecting biothiol using the composition.
  • the present invention provides a biosensor using the composition.
  • the present invention provides a biochip using the composition.
  • the composition for detecting biothiol according to the present invention can rapidly measure the biothiol in free form in the body fluid.
  • the relative content ratio and change of total and free biothiol in body fluids can be detected in real time, making it possible to use biothiol as a major indicator of disease, thereby enabling prediction and warning of various diseases.
  • various redox stress changes associated with major diseases can be explained by the amount of change in biothiol, which can provide important technical, economic and social values for the identification and diagnosis of disease pathogenesis in the future.
  • 1 is a schematic diagram showing the principle that OhrR and dsDNA are dissociated by biothiol.
  • 3 is a result of confirming the binding of the OhrR protein and the target DNA using the electrophoresis method.
  • FIG. 9 is a schematic diagram showing a biothiol detection process according to an embodiment of the present invention.
  • FIG. 10 shows fluorescence measurement results according to the biothiol detection method of FIG. 9.
  • FIG. 11 is a chemiluminescence measurement result according to the biothiol detection method of FIG. 9.
  • 12 is a schematic diagram showing signal amplification of DNA.
  • Figure 13a shows the chemiluminescence measurement results for confirming the DNA signal amplification of Figure 12.
  • Figure 13b shows the results of electrophoresis to confirm the signal amplification of the DNA of Figure 12.
  • FIG. 14 is a schematic diagram of a biochip configuration for measuring biothiol using binding between OhrR and DNA.
  • FIG. 15 is a result of detecting biothiol using the biochip of FIG. 14.
  • FIG. 16 is a schematic diagram of the biosensor in strip form of FIG. 9.
  • a biothiol detection composition comprising a redox-regulating protein and a biothiol detection method using the same are included.
  • a biothiol detecting composition in addition to the redox control protein, further comprises a DNA bound to the redox control protein and a biothiol detection method using the composition thereof.
  • redox-regulating protein refers to all proteins whose activity is regulated by the oxidation and reduction of proteins, typically OhrR present in some bacteria such as B. subtilis . (organic hydroperoxide regulator), PerR (Peroxide regulator), and OxyR (Oxygen regulator) present in some bacteria, including E. coli .
  • protein engineering may be used to modify redox regulatory protein active site amino acids, or to screen for orthologue proteins present in other types of organisms, including redox regulatory proteins that react more selectively with specific biothiols. can do.
  • the redox regulatory protein may include a variant capable of controlling binding affinity between biothiol and DNA or a protein in which a labeled protein is conjugated.
  • the conjugate form may include a fluorescent protein-OhrR, a light emitting protein-OhrR, a FLAG-OhrR, His6-OhrR, GSH-OhrR, Biotin-OhrR, and the like.
  • biothiol refers to a low molecular weight thiol having a molecular weight of 10 Da to 1,000 Da or less (preferably 10 Da to 500 Da), specifically cysteine (Cys), homocysteine (homocysteine, Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), ⁇ -glutamylcysteine ( ⁇ -GluCys), cysteinylglycine ( cysteinylglycine, CysGly), N-acetylcysteine (N-AC), Coenzyme A (CoA), Coenzyme B (Coenzyme B, CoB), Coenzyme M (Coenzyme M, CoM), bacillithiol , BacT), mycothiol (MyT), ergothioneine (ERT) and trypanothion (trypanothione, TrT) may be one or more selected from the group, but
  • biothiol can be used for various diseases related to cardiovascular disease, neurorodegenerative disease, cancer, cancer, kidney dysfunction, diabetes, diabetes mellitus, or bacterial and viral infections.
  • a marker it is an indicator that can detect abnormal biological reactions early.
  • the present invention also includes a biothiol detection composition comprising a redox control protein and a DNA bound to the redox control protein and a biothiol detection method using the composition.
  • biothiol can be detected using the principle of binding / dissociation of redox regulatory protein and DNA bound to redox regulatory protein.
  • the DNA may be, for example, one represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 used in the following Examples, and may be any compound that binds to a redox regulatory protein.
  • FIG. 1 The detection principle of such a biothiol is shown in FIG. 1 as an embodiment.
  • OhrR a representative example of the redox control protein, is an organic perperoxide (ROOH) sensor in bacteria.
  • OhrR is a homodimer and has one cysteine residue per monomer. In the state where the cysteine residue is reduced (-SH), OhrR maintains the form of binding to DNA (complex of OhrR and DNA), and OhrR is rapidly oxidized in the presence of organic peroxide (-SOH).
  • the oxidation is OhrR while maintaining engagement with the DNA, the environment in which the present bio-thiol to quickly react with the bio-thiol is released from the DNA (typically bio-thiol
  • Dissociation rates vary depending on the concentration and type of biothiol.
  • OhrR forms sulfenamide (-SN-) at a relatively slow rate by ROOH and slowly dissociates from DNA (t 1/2 ⁇ 10 min).
  • DNA bound to the redox regulatory protein may be coupled to a fluorescence factor or DNA-based enzyme (DNAzyme) to enable fluorescence, chemiluminescence, and absorption detection, or amplify DNA sequences to improve reaction sensitivity.
  • DNAzyme DNA-based enzyme
  • the specific kind of the fluorescent factor is not particularly limited, and examples thereof include rhodaman and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, and erythrosine. At least one selected from the group consisting of derivatives thereof, eosin and derivatives thereof, and 4-acetamido-4'-isothiocyanatostilben-2,2'disulfonic acid. More specifically illustrating the fluorescent material that can be used in the present invention is as follows.
  • Rhodamine and its derivatives include 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B sulfonyl chloride, rhodamine (Rhodamine B, rhodamine 123, Rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives (Texas Red) of sulforhodamine 101, N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547 and Alexa-647, etc. May be mentioned;
  • Pyrene and its derivatives include pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron® Brilliant Red 3B-A) and the like;
  • Erythrosine and its derivatives include erythrosin B, erythrosin isothiocyanate and ethidium;
  • Eosin and Eosin isothiocyanate etc. are mentioned as Eosin and its derivatives ;
  • the DNA-based enzyme is a peroxidase-mimicking DNA enzyme having peroxidase characteristics among various DNA-based enzymes (Wang Li et al. Insight into G-quadruplex-hemin DNAzyme / RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity.
  • Nucleic Acids Research 44 (15); 7373-7384 (2016)] and RNA-sequencing DNA enzymes (RNA-cleaving DNA enzymes) [Meng Liu, Dingran Chang, and Yingfu Li. Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes, Accounts of Chemical Research, 50; 2273-2283 (2017)] may be, but is not limited to having one or more sequences selected from the group consisting of.
  • the signal amplification method binds a short DNA sequence to one end of the redox regulatory protein binding DNA (a single strand DNA template capable of DNA amplification) and additionally reacts Hairpin1 (HP1) and Hairpin 2 (HP2). DNA sequences are amplified without the use of PCR. DNA sequence and length, and the type of hairpin is not limited to those described in the following Examples (Table 1).
  • a tag including a biotin group, an alkyne group, an azide group, a thiol group, an amine group, etc. is attached to the 5 'end or 3' end of the DNA sequence to separate or bind DNA only on beads, nanoparticles, and chip surfaces. Can be.
  • biothiol detection means measuring biothiol using redox control.
  • the biothiol measurement may include gel electrophoresis, fluorescence anisotropy, matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, surface plasmon resonance (SPR) , At least one selected from the group consisting of interferometry and bead measurement.
  • SPR surface plasmon resonance
  • the binding of redox regulator protein and biothiol can be quantitatively analyzed according to the type of biothiol, and the amount of free biothiol or total biothiol can be analyzed.
  • a biothiol can be detected by fluorescence or chemical luminescence by connecting a fluorescent label or a DNA-based enzyme to a DNA binding to a redox regulatory protein.
  • the redox regulatory protein may be combined with a FLAG tag, His6 tag, GSH tag, biotin tag, and the like, and the bound protein may be affinity beads (FLAG affinity beads, NTA-beads, glutathione beads, avidin). Series of beads, etc.) to detect biothiol.
  • composition for detecting biothiol according to the present invention can detect free biothiol and total biothiol in free form or detect free biothiol and total biothiol at the same time.
  • the composition when detecting the total biothiol, further comprises a reducing agent. Since OhrR binds only to the reduced free biothiol, in order to detect total biothiol in the sample, the total biothiol can be detected by OhrR by rapidly reducing the oxidized biothiol in the sample using a reducing agent.
  • the reducing agent is one or more selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (2-mercaptoenthanol), and TCEP (tris (2-carboxyethyl) phosphine), but is not limited thereto.
  • the present invention also includes a biothiol detection biochip comprising the composition.
  • the biochip is reacted with a redox regulatory protein on a plate onto which the DNA is immobilized to form a complex with a DNA capable of binding to a redox regulatory protein, as shown in FIG. 14.
  • the redox regulatory protein bound to the affinity tag is used to measure the detection signal by reducing the redox regulatory protein from the DNA attached to the surface of the biochip in the presence of biothiol. .
  • the present invention also includes a biosensor for detecting a biothiol comprising the composition.
  • a biosensor for detecting a biothiol in a strip form is included.
  • a sample introduction part including a fixation site capable of binding to a complex of a redox control protein and a DNA bound to the protein, the sample introduction part being configured to introduce a sample and the complex into a mixture;
  • a measurement unit configured to move the dissociated DNA to measure biothiol
  • biosensor for detecting biothiol comprising a.
  • the fixation site may be an antibody (eg FLAG tag-OhrR, Anti-FLAG antibody, anti-His6 antibody, etc.) or a receptor (eg His6 tag-OhrR, NTA or Biotin-OhrR, Strepavidin (including avidin, NeutrAvidin), etc.). It may be combined.
  • an antibody eg FLAG tag-OhrR, Anti-FLAG antibody, anti-His6 antibody, etc.
  • a receptor eg His6 tag-OhrR, NTA or Biotin-OhrR, Strepavidin (including avidin, NeutrAvidin), etc.
  • the biosensor for detecting a biothiol in the form of a strip is, for example, as shown in FIG. 16.
  • the oxidized / reduced amount of low molecular weight biothiol present in the blood can be measured relatively.
  • the sample introduction part is a fixed site capable of binding to the OhrR-dsDNA complex, and immobilizes an antibody (e.g., an anti-FLAG antibody or an anti-His6 antibody) or an affinity receptor (e.g., an avidin family, NTA, etc.) Plasma)
  • OhrR-dsDNA is mixed and dropped in the sample introduction, the mixed solution flows to the right by the chromatographic principle to pass through the fixed site, the OhrR-dsDNA is bound to the fixed site without moving anymore.
  • dsDNA bound to OhrR is rapidly dissociated in the presence of biothiol in the sample, and only dsDNA is moved to the right side, and it is detected by chemiluminescence by reacting with the substrate by DNAzyme in dsDNA at the right measuring part of the sensor.
  • biothiol using the redox control protein according to the present invention has the following differentiation and superiority compared to the conventional biothiol detection method.
  • the sensitivity is significantly improved and the sample volume to be measured can be reduced to a minimum. Since monomers of OhrR are 1: 1 (molar ratio) with biothiol, reaction sensitivity can be improved by measuring proteins (eg, OhrR) of macromolecules that react with biothiol (eg, OhrR and OhrR + bio). Mass spectrometry of thiols).
  • proteins eg, OhrR
  • the signal and amplification factors are introduced into the DNA site that binds to the protein and the DNA signal is controlled to be controlled by the binding or dissociation process with the protein, compared with the conventional methods (chromatography, immunoassay, chemical sensor-based analysis) High sensitivity can be obtained.
  • the OhrR protein it is possible to measure not only the whole biothiol but also the free form of the biothiol using only 1-2 ⁇ L of blood, thereby reducing the amount of sample to be analyzed.
  • OhrR a redox control protein
  • a redox control protein can be expressed in large quantities and can be stored for a long time because it is not easily degraded at room temperature in a relatively small size (17 kD).
  • oxidizing conditions eg oxygen and hydrogen peroxide
  • -9 M or less is so high that it is not easily dissociated under normal conditions, so the background signal can be kept very low when using protein-DNA binding.
  • OhrR a redox regulator protein, reacts only with low molecular weight biothiol in the presence of organic oxides and does not react with thiol groups present in macromolecular protein, so that only low molecular weight biothiol can be detected.
  • free biothiol and total biothiol can be measured simultaneously.
  • Redox proteins can be used for rapid detection.
  • the reaction time can be significantly reduced.
  • the second reaction rate between the Cys residues and the peroxide is from about 10 4 - 10 5 M -1 Cys residues present in or as a common protein s -1 glass Bio-thiol Tens of thousands or hundreds of thousands of times faster than the peroxide reaction rate (about 1 M -1 s -1 ), and the secondary reaction rate of biothiol and redox regulatory protein was about 10 3 M -1 s -1 . -2 -10 1 M -1 s -1 ) thousands of times faster.
  • the redox regulatory protein which senses the biothiol, is immediately dissociated from the DNA (50% dissociation time, t 1/2 ⁇ 0.5 min), thereby reacting with the sample within a few minutes to rapidly induce a resultant signal.
  • Fluorescence anisotropy was used to measure real-time DNA binding activity of OhrR.
  • Buffer 20 mM Tris (pH 8.0) 150 mM NaCl, 5% Glycerol (vol / vol)
  • Measurement time every 10s measurement
  • Measurement conditions ex 492 nm; slit width 15 nm, em 520 nm; slit width 20 nm, integration time 1 s
  • OhrR sequence Direct cloning in B. subtilis strains. MENKFDHMKLENQLCFLLYASSREMTKQYKPLLDKLNITYPQYLALLLLWEHETLTVKKM GEQLYLDSGTLTPMLKRMEQQGLITRKRSEEDERSVLISLTEDGALLKEKAVDIPGTILGLSKQSGEDLKQLKSALYTLL ETLHQKN (SEQ ID NO: 3)
  • OhrR and fluorescence (6FAM, 6-carboxyfluorescein) labeled OhrR binding DNA was dissolved in 3 mL of buffer using a LS55 luminescence spectrometer (PerkinElmer).
  • Anisotropy Asnis
  • Anisotropy increases, and when it dissociates with DNA, Anisotropy decreases.
  • three representative biothiols, Cys (Cysteine), Hcy (Homocysteine), and GSH (Glutathione) were treated at various concentrations (0, 1, 2, 4, 8, 16, 32, 64 ⁇ M).
  • CHP cumene hydroperoxide
  • one of the representative organic peroxides at 300 sec.
  • Figure 2 confirms that the higher the biothiol concentration, the faster the DNA dissociation rate of OhrR. 2 shows anisotropy value when OhrR dissociates with DNA depending on the concentration of biothiol. The time it takes to decrease from OhrR to DNA Time to dissociate).
  • Fluorescent probe (FAM) -coupled double strand DNA (200 nM) [SEQ ID NOs: 1 and 2] and OhrR were mixed at the concentrations shown in FIG. 3 and reacted at room temperature for 30 minutes, followed by polyacrylamide gel (7%).
  • electrophoresis 25 mA, 30 min
  • the dsDNA band was measured with a fluorescence measuring instrument (Model KIF-300, Korea Lab Tech, Korea). It can be seen that the fluorescence band of dsDNA shifted upward from the concentration of OhrR of about 1.6 ⁇ M (about 8 times the concentration of DNA).
  • PAGE electrophoresis is not a result of measuring protein-DNA binding in real time, and a large amount of protein is required for OhrR-to-DNA binding (hence the actual binding constant It is not possible to measure with this method.) It can be used as a method to easily check the coupling without a specific device.
  • Example 3 without fluorescent label DNA and OhrR The bond between proteins In biothiol The process of being dissociated by At photorefractive index Measured experiment
  • a device capable of measuring biolayer interferometry (Blitz, Fortebio, USA) was used to measure the degree of binding between DNA and protein on the optical fiber surface.
  • binding buffer, washing buffer TBS (Tris 20 mM, NaCl 150 mM)
  • biotin-coupled double strand DNA [SEQ ID NOS: 1 and 2] was dissolved in the TBS buffer at the above concentration and flowed for 120 seconds on the streptavidin-coated optical fiber, and then washed with a buffer and washed with OhrR protein TBS buffer. At 120 ° C., the solution was further flowed for 120 seconds to associate with double strand DNA bound to an optical sensor. Even after the association was completed, dissociation of the DNA-OhrR complex was confirmed by additionally flowing only the buffer for 120 seconds.
  • Biolayer interferometry measurements inevitably involve changes in the refractive index of the optical sensor when the biomaterial (DNA or OhrR) is bound to the optical sensor surface (refractive index is positively correlated with the concentration of the biomaterial). It applies the principle of converting the change value into thickness.
  • OhrR was strongly bound to dsDNA to maintain a binding thickness value of about 4.3 nm (minus the value of the dissociation equilibrium minus dsDNA binding equilibrium), and similarly, after mixing only one of CHP or biothiol with OhrR protein Even when spilled into DNA, it can be seen that OhrR binds to DNA effectively.
  • This method can be used as a method to effectively compare the reactivity to various types and concentrations of biothiol without separately labeling dsDNA or OhrR with phosphors on the optical sensor surface.
  • MALDI-TOF MS (Maltitop Mass Spectrometer, Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometer) was used to confirm the quantitative analysis of the binding of OhrR to biothiol.
  • Biothiol (Cys, Hcy, GSH) was added to 1 mL of buffer (20 mM Tris (pH 8.0) 100 mM NaCl, 5% Glycerol) in 300 nM OhrR (0, 1, 2, 4, 8, 16). , 32, 64 ⁇ M). After reacting 3 ⁇ M CHP for 2 minutes to each, 110 ⁇ L of 100% TCA was used to stop the oxidation reaction and precipitated OhrR. In order to alkylate the precipitated OhrR, 50 mM Iodoacetamide was treated to block the reduced cysteine residue of OhrR.
  • the mass signal value of OhrR-Cys was reduced in arteriosclerosis model mouse blood compared to normal mouse blood, and the mass signal value of OhrR-Cys was higher in 45 year old male smoker blood than in 25 year old non-smoker blood. It can be seen that the decrease. In other words, given that OhrR reacts with free biothiol in a fast time, it can be observed that the concentration of free biothiol (particularly free cysteine) present in the blood is relatively decreased in the mouse atherosclerosis model and in the smoker model. Can be. As such, it was confirmed that the amount of free biothiol can be effectively analyzed by mass spectrometry of OhrR even with a small amount of blood of various disease patients.
  • DTT results in the reduction of the disulfide bonds of all kinds of (oxidized) biothiols to form a free form.
  • CHP accelerates the dissociation rate of the OhrR DNA from the oxidized OhrR again with the rapidly reduced free form of biothiol, thereby measuring the total amount of biothiol (DTT).
  • cyclization is performed by internal binding between thiol molecules of DTT and thus does not participate in the reduction reaction of additional biothiol and does not react with OhrR (see left figure of FIG. 7).
  • Plasma volume 2 ⁇ L (purchased from Norma Mouse serum, Jackson ImmunoResearch)
  • Plasma collected from the mice was reacted with-/ + 1 mM DTT at RT (23-25 ° C.) for 1 hour. Thereafter, 2 ⁇ L of Plasma was reacted with 25 ⁇ M of OhrR and 50 ⁇ M of CHP for 10 minutes (total reaction at 2.5 ⁇ L). After the reaction, 1 mL of 10% TCA was treated to precipitate proteins containing OhrR. In order to prevent further oxidation of the reduced cysteine of precipitated OhrR, the alkylating agent Iodoacetamide was treated at a concentration of 50 mM to react with the reduced cysteine.
  • the reduced biothiol reacts rapidly with OhrR in the presence of CHP (within about 5 minutes) even in the presence of about 1 mM of DTT. Afterwards, biothiol can be dissociated into OhrR by DTT. It can be seen that. From this, it is preferable to measure OhrR and CHP within about 5 minutes after treatment to detect the amount of total biothiol after treatment with DTT. These conditions were analyzed by MALDI-TOF MS, and it was confirmed that the amount of free cysteine can be measured when DTT is not treated in mouse plasma and the amount of total cysteine when DTT is treated. (Right figure of FIG. 7).
  • Free cysteine and total cysteine were detected after addition of cysteine concentrations in mouse blood using MALDI-TOF MS and OhrR. That is, it was confirmed through MALDI-TOF MS analysis that the amount of biothiol analyzed by OhrR is increased when the oxidized biothiol is reduced by treating DTT with mouse serum.
  • Plasma volume 2 ⁇ L (from Norma Mouse serum, Jackson ImmunoResearch)
  • Example 9 phosphor labeling on dsDNA
  • Example 10 ssDNAzyme on dsDNA
  • the label is an example in which this experimental procedure is commonly applied and only the measuring method is different.
  • M2 FLAG affinity bead (sigma, A2220) was added to the tube by the number of samples, and then FLAG tagged OhrR (2 ⁇ M) was bound for 1 hour so as to have a total volume of 200 ⁇ L (OhrR bound to His6 tag). If so, you can use NTA-Bead).
  • OhrR binding dsDNA 100 nM, SEQ ID NO: 1 and 2) to which phosphor or DNAzyme was bound was bound to OhrR bound beads for 30 minutes.
  • FAM was combined with OhrR-binding dsDNA as a fluorescence factor and 200 ⁇ L of the final supernatant was transferred to a 96 well plate, and fluorescence was measured using a multi plate reader (Variokan, Thermo Scientific). Fluorescence measurements were made after obtaining fluorescence signal values at 525 nm emission wavelengths obtained from 480 nm excitation wavelengths, and then comparing the fluorescence values of each well with the wells containing no buffers but only buffers as reference values.
  • biothiol amount can be detected effectively.
  • TBS Tris 20 mM, NaCl 150 mM
  • DNAzyme (5'-GG GTT GGG CGG GAT GGG-3 '[SEQ ID NO: 6], synthesized by IDT)
  • NeutrAvidin ® is put into a suspension 20 ⁇ L of the coated beads (Thermo Scientific Inc., USA) was shaking 30 min at room temperature.
  • TBS Tris Buffered Saline, 20 mM Tris, NaCl 150 mM, pH 7.4
  • 180 ⁇ L of TBS buffer and 20 ⁇ L of 1 ⁇ M Hemin (Calbiochem, USA) (in TBS) solution were mixed to induce the activity of DNA enzymes.
  • beads were precipitated in a total of 200 ⁇ L. It was left to stand. Then, 200 ⁇ L of the mixed solution containing beads was placed in a tube, mounted on a luminometer (Model Glo-Max 20/20, Promega, USA), and then ECL reaction solution (G-healthcare, USA) A (Luminol + H 2 O 2 Solution, 50 ⁇ L) and 100 ⁇ L of a mixture of B (enhancer, 50 ⁇ L) were added and the chemiluminescence intensity was measured immediately.
  • a luminometer Model Glo-Max 20/20, Promega, USA
  • OhrR-binding dsDNA sequence is linked to the ssDNAzyme (a DNAzyme having the property of Horseradish peroxidase) by the T9 linker [SEQ ID NO: 4], and the other strand is introduced with a biotin to introduce the DNA probe (SEQ ID NO: 5).
  • ssDNAzyme a DNAzyme having the property of Horseradish peroxidase
  • T9 linker [SEQ ID NO: 4]
  • the other strand is introduced with a biotin to introduce the DNA probe (SEQ ID NO: 5).
  • OhrR protein was used in the experiment after expression / purification in Escherichia coli by combining the flag peptide (DYKDDDDK, expressed in direct strain after making recombinant DNA).
  • the DNA probe portion is rapidly dissociated by adding CHP and cysteine in the sample.
  • the dissociated DNA probe is present in the supernatant after centrifugation.
  • the supernatant is reacted with the avidin-bound bead, washed, and then combined with dsDNA and hemin in the bead-containing solution, followed by luminol reaction. This can induce strong chemiluminescence.
  • Example 11 DNA Signal amplification method and confirmation
  • OhrR + F template forward DNA
  • OhrR_R complementary reverse DNA
  • Modified OhrR-F shows the addition of a short DNA sequence for amplification (Table 1).
  • hairpin 1 and hairpin 2 are added to the final reaction product, DNA sequences are amplified and can be detected by various methods (electrophoresis, fluorescence, luminescence) [FIG. 12].
  • All DNA oligos were used after production order through IDT. 20 ⁇ L of 100 ⁇ M Modified OhrR_F and OhrR-R were mixed, and a double strand sequence was prepared by binding to OhrR by heating at 95 ° C. for 10 minutes and then slowly cooling at room temperature. Using this double strand DNA as a template, 2 ⁇ L of 5 ⁇ M template, 2 ⁇ L of 20 ⁇ M hairpin 1 and 2 ⁇ L of 20 ⁇ M hairpin 2 were reacted at room temperature for 30 minutes at a total volume of 20 ⁇ L. Thereafter, 8 ⁇ L of each sample was amplified by agarose gel electrophoresis, and the length of the DNA band was visually confirmed by UV.
  • HP1 and HP2 are single stranded and have a band size of less than 50 bp.
  • no chain reaction occurs (no change in the band position on the DNA gel).
  • H1 is first combined with the template to release the hairpin structure of H1.
  • the opened H1 sequence is then combined with the H2 sequence to make H2 an open structure.
  • it is observed at a size larger than the DNA band size of each template DNA and H1, and the remaining amount of H1 still remains below because it is relatively larger than template DNA. can see.
  • a biochip method is measured by using His6-tag of OhrR. In the presence of biothiol, OhrR is separated from dsDNA attached to the surface of biochip.
  • the OabR-immobilized Hisprobe-HRP which targets the His-tag of OhrR, binds to dsDNA immobilized in the well, resulting in the highest absorption of TMB.
  • biothiol is present with CHP, OhrR is dissociated from dsDNA immobilized on the surface of the biochip and is removed by washing. Therefore, hisprobe-HRP is combined to reduce the absorption signal of TMB reacting with HRP.
  • the biothiol was observed to have a greater effect of reducing the absorption signal than L-cysteine and Homocysteine or GSH under the same concentration conditions [FIG. 15].
  • the strip sensor illustrated in FIG. 16 may be composed of three main parts (sample introduction part, reaction part, and measuring part), and cellulose, nitrocellulose and glass fiber as membranes for fabricating the strip sensor. -fiber) membrane and the like can be used.
  • the structure and characteristics of each site are as follows.
  • Each part is composed of different membranes, but each membrane is fixed to overlap on a general OHP film (0.4 cm ⁇ 5.5 cm), and the reaction pad is placed at the bottom of the sample to keep the capillary phenomenon of the entire strip sensor constant. It is fixed to be connected to the introduction part and the measurement part pad, and the sample introduction part is positioned at the top for easy absorption.
  • Sample introduction (corresponding to the left assay site where the sample from the upper sensor is introduced): 10 ⁇ L of a solution of 1 ⁇ M (TBS buffer) complex and biothiol combining OhrR tagged with FLAG and double stand DNA (SEQ ID NOs: 1 and 2) After mixing 10 ⁇ L of the sample solution (buffer, blood, urine, etc.) containing 20 ⁇ L of this mixture and 20 ⁇ L of the buffer containing CHP solution (2 ⁇ M), a total of 40 ⁇ L solution was added to the cellulose membrane (0.4 cm ⁇ 1.5 cm). And then adsorbed, the substance dissolved in the sample moves to the sensor reaction part by the principle of chromatography.
  • Reaction part (corresponding to the central gray area where the DNA movement arrow of the upper sensor of FIG. 16 is drawn): Constructed using a nitrocellulose membrane (approximately 0.4 cm x 2.5 cm) and located about 1 cm to the right of the sample introduction direction. Immobilize the anti-FLAG antibody solution (1 mg / mL, phosphorylated buffer solution) at about 1 hr by dropping about 1 ⁇ L. This site is bound by the FLAG antibody immobilized on the surface as the Flag-OhrR-dsDNA complex reacted at the sample introduction part moves. In the presence of biothiol in the sample, dsDNA dissociates from OhrR and continues to move to the right on the membrane. In the absence of biothiol, dsDNA is fixed like OhrR by the antibody and stops moving.
  • the measuring unit is a site for measuring double strand DNA dissociated from the binding and separation unit, and the signal of DNAzyme attached to the dsDNA terminal It is a site that induces.
  • the glass fiber membrane (0.4 cm ⁇ 0.5 cm) can be used to pre-dispense the Hemin solution to remain and confirm the signal response by dropping the final TMB solution or ECL solution.
  • Hemin was prepared by mixing a 5% casein solution in a buffer (40 mM Tris, 200 mM NaCl, 50 mM KCl, and 20 mM MgCl 2 ) solution, and then sufficiently absorbed 100 ⁇ L into a glass fiber membrane and dried at 55 ° C. for 30 minutes. Prepare. After sufficient dissociation of the dsDNA in the reaction part, 10 ⁇ L of the ECL solution and the TMB reaction solution are added for the final signal analysis to induce the reaction. The ECL solution can be analyzed by imaging as soon as the reaction solution is added with the chemiluminescence spectrometer, and the TMB reaction results can be analyzed about 30 minutes after the solution is added to the holder equipped with a mobile phone or digital camera.
  • a buffer 40 mM Tris, 200 mM NaCl, 50 mM KCl, and 20 mM MgCl 2
  • the same sample is divided into two equal volumes, one is treated with DNA-OhrR complex after treatment with DTT for 1 hour or more, and the other is directly reacted with DNA-OhrR complex without DTT addition. That is, the following items can be determined from the same two analysis results.
  • Sample 1 and Sample 2 are analyzed by classifying each sample into characteristics, by disease type, by disease stage, by age, and by gender, and then comparing them.

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Abstract

La présente invention concerne une composition de détection de biothiols comprenant une protéine de régulation redox, un procédé de détection de biothiols à l'aide de ladite composition de détection de biothiols et un biocapteur/kit de détection de biothiols. La présente invention mesure rapidement des biothiols libres dans des fluides corporels. De plus, des rapports de contenu relatifs et des changements de biothiols totaux dans des fluides corporels peuvent être détectés en temps réel, ce qui permet à des biothiols d'être disponibles en tant qu'indices principaux de maladies à travers lesquels la prédiction et l'avertissement peuvent être effectués contre diverses maladies. En outre, étant donné que divers changements de stress redox associés aux maladies principales peuvent être révélés par des variations de biothiols, la présente invention peut fournir des valeurs importantes techniques, économiques et sociales pour l'étude de mécanismes de pathogenèse et le diagnostic de maladies à l'avenir.
PCT/KR2017/012344 2016-11-04 2017-11-02 Composition de détection de biothiols comprenant une protéine de régulation redox WO2018084607A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
JP4288334B2 (ja) * 2002-09-26 2009-07-01 株式会社産学連携機構九州 ホモシステインの測定方法及び測定用試薬
KR101535750B1 (ko) * 2014-08-11 2015-07-09 경북대학교 산학협력단 화학 센서

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JP4288334B2 (ja) * 2002-09-26 2009-07-01 株式会社産学連携機構九州 ホモシステインの測定方法及び測定用試薬
KR101535750B1 (ko) * 2014-08-11 2015-07-09 경북대학교 산학협력단 화학 센서

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PARK, K. S. ET AL.: "A Label-free Method for Detecting Biological Thiols Based on Blocking of Hg2+-Quenching of Fluorescent Gold Nanoclusters", BIOSENSORS AND BIOELECTRONICS, (ELECTRONIC PUBLISHING, vol. 45, 4 February 2013 (2013-02-04), pages 65 - 69, XP055482543 *
POOLE, L. B.: "The Basics of Thiols and Cysteines in Redox Biology and Chemistry", FREE RADICAL BIOLOGY AND MEDICINE, (ELECTRONIC PUBLISHING, vol. 80, 27 November 2014 (2014-11-27), pages 148 - 157, XP055482538 *
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