WO2015012060A1 - Capteur pour analyse de cible, dispositif d'analyse de cible et méthode d'analyse de cible les mettant en oeuvre - Google Patents

Capteur pour analyse de cible, dispositif d'analyse de cible et méthode d'analyse de cible les mettant en oeuvre Download PDF

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WO2015012060A1
WO2015012060A1 PCT/JP2014/067126 JP2014067126W WO2015012060A1 WO 2015012060 A1 WO2015012060 A1 WO 2015012060A1 JP 2014067126 W JP2014067126 W JP 2014067126W WO 2015012060 A1 WO2015012060 A1 WO 2015012060A1
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nucleic acid
region
sensor
catalytic
acid region
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PCT/JP2014/067126
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English (en)
Japanese (ja)
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金子　直人
克紀 堀井
穣 秋冨
巌 和賀
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Ｎｅｃソリューションイノベータ株式会社
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Priority to JP2015528199A priority Critical patent/JP6218250B2/ja
Publication of WO2015012060A1 publication Critical patent/WO2015012060A1/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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to a target analysis sensor, a target analysis device, and a target analysis method using the same.
  • Detecting targets is required in various fields such as clinical medicine, food, and environment.
  • the detection of the target generally uses interaction with the target, and among them, a technique using an antibody that specifically binds to the target is widely used.
  • a target is bound to an antibody labeled with an oxidoreductase such as peroxidase.
  • an oxidoreductase such as peroxidase.
  • a chromogenic substrate a chromogenic reaction is performed by the enzyme in the labeled antibody, and the color development is detected.
  • the analysis of the target for example, qualitative analysis and quantitative analysis is performed indirectly.
  • nucleic acid aptamers since the aptamer can be obtained in vitro, for example, aptamers can be obtained for toxic targets and low molecular targets.
  • DNAzyme which exhibits the same catalytic activity as peroxidase.
  • the DNAzyme is a DNA that generally has a guanine-rich structural motif, takes a G-quartet structure, and binds to hemin to form a complex, thereby generating a catalytic function similar to that of peroxidase.
  • Non-Patent Document 1 a single-stranded nucleic acid sensor in which an aptamer that is a single-stranded nucleic acid molecule and an end of DNAzyme that is a single-stranded nucleic acid molecule are linked is used.
  • Non-Patent Document 1 a single-stranded nucleic acid sensor in which an aptamer that is a single-stranded nucleic acid molecule and an end of DNAzyme that is a single-stranded nucleic acid molecule are linked.
  • an object of the present invention is to provide a new target analysis sensor, a target analysis device using the same, and a target analysis method.
  • the target analysis sensor of the present invention includes a single-stranded nucleic acid molecule, and the single-stranded nucleic acid molecule binds to the first catalytic nucleic acid region (D1), the second catalytic nucleic acid region (D2), and the target. (Ap), having the first catalytic nucleic acid region (D1) on one end side of the binding nucleic acid region (Ap), and the second catalytic nucleic acid region on the other end side of the binding nucleic acid (Ap).
  • the catalytic function of the first catalytic nucleic acid region (D1) and the second catalytic nucleic acid region (D2) is inhibited, and in the presence of the target, the binding nucleic acid region (Ap)
  • the catalytic function is caused by G-quartet formation between the first catalytic nucleic acid region (D1) and the second catalytic nucleic acid region (D2) by the contact of the target with the target.
  • the target analysis device of the present invention includes a base material, a target analysis sensor, and a detection unit.
  • the sensor and the detection unit are arranged on the base material, and the sensor is the target analysis sensor of the present invention.
  • the detection unit is a detection unit for detecting a catalyst function of the first catalytic function region (D1) and the second catalytic nucleic acid region (D2) in the sensor.
  • the reagent for target analysis of the present invention includes the sensor for target analysis of the present invention.
  • the target analysis method of the present invention includes a contact step of bringing a sample into contact with the target analysis sensor of the present invention, and the first catalyst nucleic acid region (D1) and the second catalyst nucleic acid region (D2) of the target analysis sensor. And a detection step of detecting a target in the sample by detecting the catalytic function.
  • the target analysis sensor of the present invention a target can be analyzed simply and efficiently with an excellent S / N ratio. For this reason, the present invention can be said to be an extremely useful technique for research and examination in various fields such as clinical medicine, food, and environment.
  • FIG. 1 is a diagram showing an outline of ON / OFF of the catalytic function of the single-stranded nucleic acid molecule in the target analysis sensor of the present invention.
  • FIG. 2 is a schematic diagram of a G-quartet structure formed by the first catalytic nucleic acid region (D1) and the second catalytic nucleic acid region (D2) in the target analysis sensor of the present invention.
  • FIG. 3 is a graph showing light emission intensity (RLU) in melamine detection using the target analysis sensor of Example 1 of the present invention.
  • FIG. 4 is a graph showing luminescence intensity (RLU) in melamine detection using the target analysis sensor of Example 2 of the present invention.
  • FIG. 1 is a diagram showing an outline of ON / OFF of the catalytic function of the single-stranded nucleic acid molecule in the target analysis sensor of the present invention.
  • FIG. 2 is a schematic diagram of a G-quartet structure formed by the first catalytic nucleic acid region (D
  • FIG. 5 is a graph showing luminescence intensity (RLU) in melamine detection using the target analysis sensor of Example 3 of the present invention.
  • FIG. 6 is a graph showing luminescence intensity (RLU) in melamine detection using the target analysis sensor of Example 4 of the present invention.
  • FIG. 7 is a graph showing light emission intensity (RLU) in melamine detection using the target analysis sensor of Example 5 of the present invention.
  • FIG. 8 is a graph showing light emission intensity (RLU) in IgE detection using the target analysis sensor of Example 6 of the present invention.
  • the sensor for target analysis of the present invention includes a single-stranded nucleic acid molecule as described above, and the single-stranded nucleic acid molecule includes the first catalytic nucleic acid region (D1) and the second catalytic nucleic acid region (D2).
  • a binding nucleic acid region (Ap) that binds to the target, and has the first catalytic nucleic acid region (D1) on one end side of the binding nucleic acid region (Ap), and the other of the binding nucleic acid regions (Ap) Having the second catalytic nucleic acid region (D2) on the terminal side, and in the absence of the target, the catalytic function of the first catalytic nucleic acid region (D1) and the second catalytic nucleic acid region (D2) is inhibited, and the target In the presence, contact of the target with the binding nucleic acid region (Ap) causes a catalytic function by G-quartet formation between the first catalytic nucleic acid region (D1) and the second catalytic nucleic acid region (D2). That features To.
  • the target analysis sensor of the present invention is also referred to as a sensor, and the nucleic acid region is also referred to as a region.
  • the single-stranded nucleic acid molecule in the present invention can also be referred to as a single-stranded nucleic acid element, for example.
  • the first catalyst region (D1) and the second catalyst region (D2) are paired to form a G-quartet structure (also referred to as G-tetrad), thereby generating a catalyst function. It is a double-stranded (also referred to as split) catalytic nucleic acid molecule.
  • the G-quartet has, for example, a surface structure in which G (guanine) is a tetramer.
  • the G-quartet structure may be, for example, a parallel type or an anti-parallel type, and is preferably a parallel type. In the sensor of the present invention, the number of G-quartet structures is not particularly limited.
  • the number of G-quartet structures may be one surface or a plurality of two or more surfaces. It is preferable to form a structure (referred to as G-quadruplex).
  • the first catalyst region (D1) and the second catalyst region (D2) may be any sequence that forms the G-quartet structure, and more preferably, a guanine quadruplex structure is formed. It is an array to do.
  • the catalytic function is not particularly limited, and is, for example, a catalytic function of a redox reaction.
  • the oxidation-reduction reaction may be a reaction that causes transfer of electrons between two substrates in the process of generating a product from the substrates, for example.
  • the kind of the redox reaction is not particularly limited.
  • the catalytic function of the oxidation-reduction reaction includes, for example, the same activity as an enzyme, and specifically includes, for example, the same activity as peroxidase (hereinafter referred to as “peroxidase-like activity”). Examples of the peroxidase activity include horseradish peroxidase (HRP) activity.
  • the first catalytic region (D1) and the second catalytic region (D2) can be called DNA enzyme or DNAzyme in the case of a DNA sequence, and can be called RNA enzyme or RNAzyme in the case of an RNA sequence. it can.
  • the catalytic function of the first catalyst region (D1) and the second catalyst region (D2) is controlled to be ON-OFF depending on the presence or absence of a target. Is done. Note that the present invention is not limited to this mechanism.
  • nucleic acid sequences are considered to be thermodynamically fluctuating between structures that can be formed, and the abundance of relatively stable ones is considered to be high.
  • binding nucleic acid molecules such as aptamers generally change to a more stable three-dimensional structure having a stem-loop structure upon contact with the target and bind to the target in the presence of the target.
  • catalytic nucleic acid molecules such as DNAzyme are generally known to generate catalytic activity by a stable three-dimensional structure such as a G-quartet structure.
  • the sensor of the present invention forms a G-quartet structure as a pair, and the first catalyst region (D1) and the second catalyst region (D2) that generate catalytic activity are coupled to each other.
  • first catalyst region (D1) and the second catalyst region (D2) are spaced apart from each other, the first catalyst region (D1) and the first catalyst region (D1) and the second catalyst region (D2) are disposed in the absence of a target.
  • the formation of a G-quartet structure is inhibited between the two catalyst regions (D2), and as a result, the catalytic function of the first catalyst region (D1) and the second catalyst region (D2) is inhibited ( Switch-OFF).
  • the structure in this state is also called an inactive type.
  • the three-dimensional structure of the binding region (Ap) changes to a more stable structure having a stem-loop structure by the contact of the target with the binding region (Ap).
  • the first catalyst region (D1) and the second catalyst region (D2) come close to each other, and the first catalyst region (D1) and the second catalyst region A G-quartet structure is formed between the region (D2) and, as a result, a catalytic function is generated by the first catalyst region (D1) and the second catalyst region (D2) (switch-ON).
  • the structure in this state is also called active form.
  • the catalytic function is not generated in the absence of the target, and the catalytic function is generated only in the presence of the target, so that target analysis such as qualitative or quantitative analysis is possible.
  • the present invention uses a double-stranded catalyst nucleic acid molecule, and connects the first catalyst region (D1) and the second catalyst region (D2) via the binding region (Ap). It is arranged. For this reason, for example, it is not necessary to set conditions for each type of aptamer, and a desired aptamer can be set as the binding region (Ap).
  • region (D2) should just be arrange
  • the first catalyst region (D1) is disposed on the 5 ′ side of the binding region (Ap)
  • the second catalyst region (D2) is disposed on the three side of the binding region (Ap). An example is shown.
  • the single-stranded nucleic acid molecule may be directly or indirectly linked between the first catalytic region (D1) and the binding region (Ap), or the second The catalyst region (D2) and the binding region (Ap) may be directly or indirectly connected.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded
  • the indirect connection is, for example, 3 of one region.
  • the linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, and is preferably the former.
  • the single-stranded nucleic acid molecule has the nucleic acid linker region (first linker region (L1)) between the first catalytic region (D1) and the binding region (Ap),
  • the nucleic acid linker region (second linker region (L2)) is preferably provided between the second catalyst region (D2) and the binding region (Ap).
  • the first linker region (L1) and the second linker region (L2) may be either one or preferably both. When both the first linker region (L1) and the second linker region (L2) are included, the lengths may be the same or different.
  • the length of the linker region is not particularly limited, and the lower limit is, for example, 1, 3, 5, 7, 9 bases, and the upper limit is, for example, 20, 15, 10 bases.
  • the base sequence from the 5 ′ end side of the first linker region (L1) and the base sequence from the 3 ′ end side of the second linker region (L2) may be non-complementary to each other, for example. preferable.
  • the base sequence from the 5 ′ end of the first linker region (L1) and the base sequence from the 3 ′ end of the second linker region (L2) are aligned and the single-stranded nucleic acid It can also be said that the region forms an internal loop in the molecule.
  • the region (D2) for example, the distance between the first catalyst region (D1) and the second catalyst region (D2) can be sufficiently maintained. For this reason, for example, the formation of a G-quartet structure by the first catalyst region (D1) and the second catalyst region (D2) in the absence of the target is sufficiently suppressed, and in the absence of the target, The background based on the catalytic function can be sufficiently reduced.
  • FIG. 1 is a schematic diagram showing ON / OFF of the catalytic function in the single-stranded nucleic acid molecule. As shown on the left of FIG. 1, in the absence of a target, the single-stranded nucleic acid molecule forms a G-quartet structure between the first catalyst region (D1) and the second catalyst region (D2). Is inactivated.
  • the catalyst region (D2) approaches and becomes an active type in which a G-quartet structure is formed between them.
  • W in the formula is, for example, from the 5 ′ side
  • W in the formula is, for example, from the 5 ′ side
  • W in the formula is, for example, from the 5 ′ side
  • W in the formula is, for example, from the 5 ′ side
  • W in the formula is, for example, From the 5 ′ side, the first linker region (L1), the binding region (Ap), and the second linker region (L2) are provided in this order.
  • the single-stranded nucleic acid molecule represented by D1-W-D2 is represented by, for example, D1-L1-Ap-D2, D1-Ap-L2-D2, or D1-L1-Ap-L2-D2, respectively. be able to.
  • the first catalyst region (D1) and the second catalyst region (D2) are complementary to each other at the ends opposite to the position of the binding region (Ap). It is preferable to have a typical sequence. Specifically, for example, when the first catalyst region (D1) is disposed on the 5 ′ side of the binding region (Ap), the first catalyst region (D1) and the second catalyst region (D2). Preferably has a sequence complementary to each other at the 5 ′ end of the first catalyst region (D1) and the 3 ′ end of the second catalyst region (D2).
  • the first catalyst region (D1) and the second catalyst region (D2) are: It is preferable that the 3 ′ end of the first catalyst region (D1) and the 5 ′ end of the second catalyst region (D2) have sequences complementary to each other. As described above, the first catalyst region (D1) and the second catalyst region (D2) have the complementary sequences at the respective ends, so that the stem structure is formed between the sequences by intramolecular annealing. Formation is possible.
  • the single-stranded nucleic acid molecule can be represented by D1-W-D2, and specifically can be represented by the following formula (I).
  • 5 'side sequence (N) n1 -GGG- (N) n2 - (N) n3 - is the sequence of the first catalyst zone (D1) (d1), 3 ' end
  • the sequence-(N) m3- (N) m2 -GGG- (N) m1 is the sequence (d2) of the second catalyst region (D2)
  • W is the first catalyst region (D1) and the A region between the second catalyst region (D2) and including the binding region (Ap)
  • N represents a base
  • n1, n2 and n3 and m1, m2 and m3 each represent a repeat of the base N Indicates the number.
  • the formula (I) shows a state in which the first catalyst region (D1) and the second catalyst region (D2) are aligned in the molecule in the single-stranded nucleic acid molecule.
  • FIG. 4 is a schematic diagram for illustrating an arrangement relationship between a region (D1) and the second catalyst region (D2).
  • the first catalyst region (D1) and the second catalyst region (D2) However, taking this state is not limited.
  • (N) n1 and (N) m1 satisfy the following condition (1)
  • (N) n2 and (N) m2 preferably satisfy the following condition (2)
  • (N) n3 and (N) m3 preferably satisfy the following condition (3).
  • (N) n2 and (N) m2 are such that the base sequence from the 5 ′ end of (N) n2 and the base sequence from the 3 ′ end of (N) m2 are non-complementary to each other, m2 is a positive integer, and may be the same or different.
  • N and (N) m3 are those in which n3 and m3 are 3 or 4, respectively, and may be the same or different, have three bases G, and when n3 or m3 is 4, (N) In n3 and (N) m3 , the second or third base is a base H other than G.
  • the condition (1) is a condition of (N) n1 at the 5 ′ end and (N) m1 at the 3 ′ end when the first catalyst region (D1) and the second catalyst region (D2) are aligned. It is.
  • the base sequence from the 5 ′ end of (N) n1 and the base sequence from the 3 ′ end of (N) m1 are complementary to each other and have the same length. . Since (N) n1 and (N) m1 are complementary sequences of the same length, they can be said to be stem regions that form stems in an aligned state.
  • N1 and m1 may be the same 0 or a positive integer, for example, 0, 1 to 10, 1, 2, or 3, respectively.
  • the condition (2) is a condition of (N) n2 and (N) m2 when the first catalyst region (D1) and the second catalyst region (D2) are aligned.
  • the base sequence of (N) n2 and the base sequence of (N) m2 are non-complementary to each other, and n2 and m2 may have the same length or different lengths. Since (N) n2 and (N) m2 are non-complementary sequences, they can be said to be regions that form an inner loop in an aligned state.
  • N2 and m2 are positive integers, for example, 1 to 10 respectively, preferably 1 or 2.
  • n2 and m2 may be the same or different.
  • n2 m2, n2> m2, and n2 ⁇ m2, and preferably n2> m2 and n2 ⁇ m2.
  • the condition (3) is a condition of (N) n3 and (N) m3 when the first catalyst region (D1) and the second catalyst region (D2) are aligned.
  • the base sequence of (N) n3 and the base sequence of (N) m3 are 3 or 4 base length sequences having 3 bases G, and the same or different May be.
  • n3 or m3 is 4,
  • (N) n3 and (N) m3 are bases H other than G in the second or third base.
  • Examples of the base H that is a base other than G include A, C, T, and U, and preferably A, C, or T.
  • condition (3) include the following conditions (3-1), (3-2), and (3-3).
  • Condition (3-1) Among (N) n3 and (N) m3 , the sequence from one 5 ′ side is GHGG, and the sequence from the other 5 ′ side is GGG.
  • Condition (3-2) Among (N) n3 and (N) m3 , the sequence from one 5 ′ side is GGHG, and the sequence from the other 5 ′ side is GGG.
  • Condition (3-3) Both (N) n3 and (N) m3 sequences are GGG.
  • the length of the first catalyst region (D1) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, The length is 10 bases, and the range is, for example, 7 to 30 bases, 7 to 20 bases, 7 to 10 bases.
  • the length of the second catalyst region (D2) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, The length is 10 bases, and the range is, for example, 7 to 30 bases, 7 to 20 bases, 7 to 10 bases.
  • the lengths of the first catalyst region (D1) and the second catalyst region (D2) may be the same or different.
  • W means a region between the sequence (d1) and the sequence (d2) in the single-stranded nucleic acid molecule, and is a lower case region on the 5 ′ end side and the 3 ′ end side
  • the underlined regions on the 5 ′ side and 3 ′ side represent (N) n2 and (N) m2 , respectively, and are underlined on the 5 ′ side and 3 ′ side, respectively.
  • Regions between the partial region and W indicate (N) n3 and (N) m3 , respectively.
  • stem regions (N) n1 and (N) m1 are changed to 0-3 base lengths, and internal loop regions (N) n2 and (N) m2 are converted to AC (N) n3 and (N) m3, which are the G region, are set to 3 base lengths of GGG and 4 base lengths of GTGG, and W is not limited.
  • Combinations 25-48 in Table 2 show that (N) n1 and (N) m1 that are stem regions are one base length of A and one base length of T and (N) n2 and (N) m2 that are internal loop regions Is changed to 1 or 2 base length, and (N) n3 and (N) m3, which are G regions, are set to 4 types of 4 base lengths of GAGG, GGAG, GCGG and GTGG and 3 base lengths of GGG And W is not limited.
  • the combination 49 in Table 2 shows (N) n1 and (N) m1 that are stem regions, 2 base lengths of CA and 2 bases of TG, and (N) n2 and (N) m2 that are internal loop regions, This is a sequence in which (N) n3 and (N) m3, which are 1 base length of T and A and G region, are set to 4 base lengths of GAGG and 3 base lengths of GGG, and W is not limited.
  • FIG. 2 is a schematic diagram of a G-quartet structure formed between the first catalyst region (D1) and the second catalyst region (D2) in the single-stranded nucleic acid molecule of the combination 24 (SEQ ID NO: 24). is there.
  • a guanine quadruplex in which G-quartets are three-sided is formed between G in the first catalyst region (D1) and G in the second catalyst region (D2). Note that the present invention is not limited to this example.
  • the target is not particularly limited, and any target can be selected. And according to the arbitrary target, a binding nucleic acid molecule that binds to the target may be used as the binding region (Ap).
  • the target is not particularly limited, and examples thereof include low molecular weight compounds, microorganisms, viruses, food allergens, agricultural chemicals, mold poisons, and antibodies.
  • Examples of the low molecular weight compound include melamine, antibiotics, agricultural chemicals, and environmental hormones.
  • Examples of the microorganism include Salmonella, Listeria, Escherichia coli, and mold, and examples of the virus include norovirus.
  • the length of the binding region (Ap) is not particularly limited, and the lower limit is, for example, 12 base length, 15 base length, 18 base length, and the upper limit is, for example, 140 base length, 80 base length, 60 bases
  • the range is, for example, 12 to 140 bases long, 15 to 80 bases long, 18 to 60 bases long.
  • the phrase “the other sequence is complementary to a certain sequence” means, for example, a sequence that can be annealed between the two.
  • the annealing is also referred to as stem formation.
  • complementary means, for example, complementarity when two kinds of sequences are aligned is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or less, 99% or more, 100%, ie fully complementary.
  • the total length of the single-stranded nucleic acid molecule is not particularly limited, and the lower limit is, for example, 25 base length, 30 base length, 35 base length, and the upper limit is, for example, 200 base length, 100 base length, 80 base length, the range is 25-200 base length, 30-100 base length, 35-80 base length, for example.
  • the sensor of the present invention may be, for example, a molecule having the single-stranded nucleic acid molecule or a molecule composed of the single-stranded nucleic acid molecule.
  • the sensor of the present invention is a molecule containing a nucleotide residue, and may be, for example, a molecule consisting only of a nucleotide residue or a molecule containing a nucleotide residue.
  • the nucleotide is, for example, ribonucleotide, deoxyribonucleotide and derivatives thereof.
  • the sensor may be, for example, DNA containing deoxyribonucleotide and / or a derivative thereof, RNA containing ribonucleotide and / or a derivative thereof, or a chimera (DNA / RNA) containing the former and the latter But you can.
  • the sensor is preferably DNA.
  • the nucleotide may contain, for example, either a natural base (non-artificial base) or a non-natural base (artificial base) as a base.
  • a natural base include A, C, G, T, U, and modified bases thereof.
  • the modification include methylation, fluorination, amination, and thiolation.
  • the unnatural base include 2′-fluoropyrimidine, 2′-O-methylpyrimidine and the like. Specific examples include 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, Examples include 2-thiouracil.
  • the nucleotide may be, for example, a modified nucleotide, and the modified nucleotide is, for example, a 2′-methylated-uracil nucleotide residue, 2′-methylated-cytosine nucleotide residue, 2′-fluorinated-uracil nucleotide. Residue, 2′-fluorinated-cytosine nucleotide residue, 2′-aminated-uracil nucleotide residue, 2′-aminated-cytosine nucleotide residue, 2′-thiolated-uracil nucleotide residue, 2′- Thio-cytosine nucleotide residues and the like.
  • the target analysis sensor may include non-nucleotides such as PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid), for example.
  • Some catalytic nucleic acids having a G-quartet structure exhibit higher catalytic activity, for example, by forming a complex with porphyrin. Therefore, in the sensor of the present invention, it is preferable that the catalytic function of the first catalyst region (D1) and the second catalyst region (D2) by the G-quartet structure is detected in the presence of porphyrin, for example.
  • the porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and derivatives thereof.
  • examples of the derivatives include substituted porphyrins and metal porphyrins complexed with metal elements.
  • Examples of the substituted porphyrin include N-methylmesoporphyrin.
  • Examples of the metal porphyrin include hemin, which is a trivalent iron complex.
  • the porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
  • the sensor of the present invention may be used in a free state or in an immobilized state, for example.
  • the sensor can be fixed to the substrate and used as a device.
  • the substrate include substrates such as plates, sheets, films, and swabs; containers such as well plates and tubes; beads, particles, filters, and the like.
  • the sensor may be immobilized at either the 5 'end or the 3' end.
  • the immobilization method is not particularly limited, and examples thereof include chemical bonding.
  • examples thereof include chemical bonding.
  • streptavidin or avidin is bound to one of the base material and the sensor, biotin is bound to the other, and immobilization is performed using the binding between the former and the latter. can give.
  • the immobilization method for example, other known nucleic acid immobilization methods can be adopted.
  • the method include a method using photolithography, and specific examples thereof can be referred to US Pat. No. 5,424,186.
  • the immobilization method include a method of synthesizing the sensor on the base material. As this method, for example, a so-called spot method can be mentioned.
  • US Pat. No. 5,807,522, Japanese Patent Publication No. 10-503841 and the like can be referred to.
  • the sensor may be fixed directly or indirectly to the base material, for example.
  • the sensor may be immobilized on the base material via a linker for immobilization.
  • the linker may be, for example, a nucleic acid sequence or a non-nucleic acid sequence.
  • the method of using the sensor of the present invention is not particularly limited, and can be used for the target analysis method of the present invention as follows.
  • the analysis method of the present invention is a target analysis method, a contact step of bringing a sample into contact with the sensor of the present invention, and the first catalyst region (D1) and the second of the sensor. It includes a detection step of detecting a target in the sample by detecting a catalyst function due to the catalyst region (D2).
  • the sample is not particularly limited.
  • the sample may be, for example, a sample including a target or a sample in which it is unknown whether or not the target is contained.
  • the sample is preferably a liquid sample, for example.
  • the analyte when the analyte is a liquid, the analyte may be used as it is as a sample, or a diluted solution mixed in a solvent may be used as a sample.
  • the analyte is, for example, a solid or a powder, a mixed solution mixed with a solvent, a suspension suspended in a solvent, or the like may be used as a sample.
  • the solvent is not particularly limited, and examples thereof include water and a buffer solution. Examples of the specimen include specimens collected from living organisms, soil, seawater, river water, sewage, food and drink, purified water, air, and the like.
  • the sensor of the present invention When using the sensor of the present invention in a free state, for example, it is preferable to contact the sensor and the sample in the container. Moreover, when using the sensor of this invention in the state fixed to the said base material, the said sample can be made to contact the said sensor on the said base material, for example.
  • the detection step for example, it is preferable to detect a signal generated by a catalytic function of the first catalyst region (D1) and the second catalyst region (D2).
  • the signal include an optical signal and an electrochemical signal.
  • the optical signal include a color development signal, a luminescence signal, and a fluorescence signal.
  • the signal is preferably generated from the substrate by the catalytic function of the first catalyst region (D1), for example. Therefore, the detection step is preferably performed, for example, in the presence of a substrate corresponding to the catalytic function of the first catalyst region (D1) and the second catalyst region (D2).
  • the substrate is, for example, a substrate that generates a colored, luminescent or fluorescent product by the catalytic function, a substrate that generates a product in which the colored, luminescent or fluorescent light disappears by the catalytic function, and the catalytic function. And substrates that produce different colored, luminescent or fluorescent products.
  • the catalytic function can be detected by visually confirming, for example, the presence or absence of color development, luminescence or fluorescence, or the change or intensity of color development, luminescence or fluorescence as a signal.
  • the catalytic function can be detected by measuring the absorbance, reflectance, fluorescence intensity, and the like as signals using an optical technique. Examples of the catalytic function include the catalytic function of the oxidation-reduction reaction as described above.
  • the catalytic function is a catalytic function of the oxidation-reduction reaction
  • a substrate that can exchange electrons can be cited.
  • a product is generated from the substrate by the first catalyst region (D1) and the second catalyst region (D2), and exchange of electrons occurs in the process.
  • This electron transfer can be detected electrochemically as an electrical signal by application to an electrode, for example.
  • the electric signal can be detected by measuring the intensity of the electric signal such as an electric current.
  • the substrate is not particularly limited, and examples thereof include hydrogen peroxide, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 1,2-Phenylenediamine (OPD), 2,2′-Azinobis (3-ethylbenzothiazoline- 6-sulfonic Acid Ammonium Salt (ABTS), 3,3'-Diaminobenzidine (DAB), 3,3'-Diaminobenzidine Tetrahydrochloride Hydrate (DAB4HCl), 3-Amino-9-ethylcarbazole (AEC), 4-Chloro-1-naphthol (4C1N), 2,4,6-Tribromo-3-hydroxybenzoic Acid, 2,4-Dichlorophenol, 4-Aminoantipyrine, 4-Aminoantipyrine Hydrochloride, Luminol and the like.
  • TMB 5,5′-tetramethylbenzidine
  • OPD 1,2-Phenylenediamine
  • 2,2′-Azinobis (3-ethylbenzothiazo
  • the substrate may be supplied to the sensor in advance, for example, before contacting the sample with the sensor, or at the same time as the sample contact or after contacting the sample, You may supply to a sensor.
  • the substrate is preferably supplied to the sensor, for example, as a substrate liquid mixed with a liquid.
  • the liquid mixed with the substrate is preferably a buffer such as Tris-HCl.
  • the concentration of the substrate in the substrate solution is not particularly limited, and is, for example, 0.1 to 5 mmol / L, 0.5 to 2 mmol / L.
  • the pH of the substrate solution is, for example, 6-9, 6.8-9.
  • the reaction conditions by the catalyst region (D) are not particularly limited.
  • the temperature is, for example, 15 to 37 ° C.
  • the time is, for example, 10 to 900 seconds.
  • porphyrin may coexist in addition to the substrate.
  • Some known DNAzymes exhibit higher redox activity by forming a complex with porphyrin, for example. Therefore, also in the present invention, for example, redox activity may be detected as a complex of the first catalyst region (D1), the second catalyst region (D2) and the porphyrin in the presence of porphyrin.
  • the supply of porphyrin is not particularly limited and can be performed in the same manner as the substrate. The porphyrin is as described above.
  • a cleaning step may be further provided between the contact step and the detection step.
  • the cleaning step is, for example, a step of cleaning the sensor with a cleaning liquid after bringing the sensor into contact with the sample.
  • the washing liquid is not particularly limited, and examples thereof include aqueous solvents such as water and buffer solutions.
  • the sensor is preferably fixed to the base material as described above because the cleaning process can be easily performed.
  • the analysis device of the present invention is a target analysis device, and includes a base material, a target analysis sensor, and a detection unit, and the sensor and the detection unit are disposed on the base material,
  • the sensor is the target analysis sensor of the present invention
  • the detection unit is a detection unit that detects a catalyst function of the first catalyst region (D1) and the second catalyst region (D2) in the sensor. It is characterized by that.
  • the analysis device of the present invention is characterized by using the sensor for target analysis of the present invention, and the other configurations are not limited at all. Unless otherwise specified, the analysis device of the present invention can use, for example, the description of the target analysis sensor of the present invention.
  • the method for arranging the sensors is not particularly limited.
  • the sensor may be fixed to the base material or may not be fixed.
  • the arrangement of the sensor for example, the description of the sensor for target analysis of the present invention can be cited.
  • the arrangement part of the sensor in the base material is not particularly limited, and examples thereof include a form arranged in the detection unit.
  • the analysis device of the present invention may further have a reagent part, for example.
  • the reagent unit may be disposed in the detection unit.
  • a reagent may be arranged in advance in the reagent unit, or the reagent may be supplied at the time of use.
  • the reagent include the aforementioned substrate and the porphyrin.
  • the detection unit is a detection unit that detects the catalytic function of the first catalyst region (D1) and the second catalyst region (D2).
  • the detection unit is preferably a detection unit that detects a signal generated by a catalytic function of the first catalyst region (D1) and the second catalyst region (D2), for example.
  • the signal is, for example, a signal from a substrate due to the catalytic function of the first catalyst region (D1) and the second catalyst region (D2).
  • Examples of the signal include an optical signal and an electrochemical signal as described above.
  • the detection unit can also be referred to as a detected unit because, for example, a signal generated in the detection unit is detected from the outside.
  • the detection unit is, for example, an optical signal detection unit, and examples include a detection unit such as absorbance, reflectance, and fluorescence intensity.
  • the detection unit has, for example, an electrode system.
  • the said detection part can be formed by arrange
  • the arrangement method of the electrodes is not particularly limited, and for example, a known method can be adopted. Specific examples include thin film forming methods such as vapor deposition, sputtering, screen printing, and plating.
  • the electrode may be disposed directly or indirectly on the substrate.
  • An indirect arrangement includes, for example, an arrangement through another member.
  • the electrode system may include, for example, a working electrode and a counter electrode, or may include a working electrode, a counter electrode, and a reference electrode.
  • the material of the electrode is not particularly limited, and examples thereof include platinum, silver, gold, and carbon.
  • Examples of the working electrode and the counter electrode include a platinum electrode, a silver electrode, a gold electrode, and a carbon electrode, and examples of the reference electrode include a silver / silver chloride electrode.
  • the silver / silver chloride electrode can be formed, for example, by laminating a silver chloride electrode on a silver electrode.
  • the senor is preferably arranged, for example, in the electrode system, and is preferably arranged in the working electrode among the electrodes.
  • the analytical device of the present invention includes the electrode system and the reagent part, for example, the reagent part is preferably disposed on the electrode system.
  • the analysis device of the present invention may include a plurality of detection units, for example.
  • the analytical device fractionates the surface of the base material into a matrix, and includes a detection unit as described above in each fractionation region.
  • the number of sensors arranged in one detection unit is not particularly limited.
  • the substrate is not particularly limited.
  • the substrate is preferably a substrate having an insulating surface, for example.
  • the substrate may be, for example, a substrate made of an insulating material, or a substrate having an insulating layer made of an insulating material on the surface.
  • the insulating material is not particularly limited, and examples thereof include known materials such as glass, ceramic, insulating plastic, and paper.
  • the insulating plastic is not particularly limited, and examples thereof include silicone resin, polyimide resin, epoxy resin, and fluorine resin.
  • the method for using the analysis device of the present invention is not particularly limited, and can be used for the target analysis method of the present invention as follows.
  • the analysis method of the present invention is a target analysis method.
  • the analysis method of the present invention is characterized by using the analysis device including the target analysis sensor of the present invention, and other conditions are not limited at all.
  • the analysis method of the present invention for example, the description of the analysis method in the target analysis sensor of the present invention can be cited.
  • the analytical reagent of the present invention includes the target analytical sensor of the present invention.
  • the analytical reagent of the present invention is characterized by including the sensor, and other configurations are not limited at all.
  • the analytical reagent of the present invention may contain, in addition to the sensor, components such as the substrate, the porphyrin, the buffer solution, and / or the substrate.
  • the analysis reagent of the present invention may be, for example, an analysis kit.
  • the sensor and the other components described above may be included and separately accommodated.
  • the sensor may be fixed to the base material or may not be fixed.
  • the analysis kit may further include instructions for use, for example.
  • Example 1 For the first catalyst region (D1) and the second catalyst region (D2), a sensor for melamine analysis in which (N) n1 and (N) m1 of the formula (I) forming the stem region are changed is manufactured. The melamine was analyzed.
  • W was the base sequence W1 represented by SEQ ID NO: 50.
  • the (N) n1 and (N) m1 used as the stem region were changed to 0-3 base lengths.
  • the underlined sequence is a melamine-binding region (Ap) having a 12-base-long poly dT (hereinafter referred to as Mel06), and the lower-case sequence on the 5 ′ side is the first linker region (L1).
  • the lower case sequence on the 3 ′ side is the second linker region (L2).
  • the first linker region (L1) and the second linker region (L2) were non-complementary to each other in an aligned state.
  • W1 SEQ ID NO: 50
  • Reagent 1 400 nmol / L nucleic acid sensor 200 nmol / L hemin 50 mmol / L Tris-HCl (pH 7.4) 20 mmol / L KCl 0.05% (w / v) Triton X-100 (Reagent 2) 5 mmol / L melamine (reagent 3) 50 ⁇ mol / L substrate 50 ⁇ mol / L H 2 O 2
  • PC DNAzyme
  • NC w / o sensor
  • FIG. 3 is a graph showing the luminescence intensity (RLU) of the reaction solution.
  • the S / N ratio is shown in Table 4.
  • the numerical value of H in the sequence name indicates the length of the poly dT in the binding region (Ap) (the same applies hereinafter).
  • the luminescence intensity with melamine addition shows a significant difference with respect to the luminescence intensity without addition of melamine.
  • the ratio (S / N) between the intensity and the luminescence intensity without addition of melamine was greatly improved as compared with PC (DNAzyme).
  • Example 2 A melamine analysis sensor was prepared and analyzed for melamine in the same manner as in Example 1 except that the length of poly dT in the binding region (Ap) to melamine was changed to 30 bases.
  • W2 is the same sequence as W1 in Example 1 except that poly dT has a length of 30 bases, and the underlined sequence has a poly T having a length of 30 bases (Ap, Mel06)
  • the lowercase sequence on the 5 ′ side is the first linker region (L1), and the lowercase sequence on the 3 ′ side is the second linker region (L2).
  • W2 (SEQ ID NO: 52) 5'-aagaa CGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCG aaaat-3 '
  • FIG. 4 is a graph showing the luminescence intensity (RLU) of the reaction solution.
  • the S / N ratio is shown in Table 5.
  • the numerical value of H in the sequence name indicates the length of the poly dT in the binding region (Ap).
  • the luminescence intensity with melamine addition showed a significant difference from the luminescence intensity without melamine addition, and further, as shown in Table 5, the luminescence with melamine addition
  • the ratio (S / N) between the intensity and the luminescence intensity without addition of melamine was greatly improved as compared with PC (DNAzyme). From these results, it was found that according to the sensor of the example, melamine can be detected with the background suppressed.
  • Example 3 For the first catalyst region (D1) and the second catalyst region (D2), (N) n2 and (N) m2 of the above formula (I) that forms an internal loop region and the above formula (G) that forms a G region ( Sensors for melamine analysis in which (N) n3 and (N) m3 in (I) were changed were prepared and analyzed for melamine.
  • W was the base sequence W1 represented by SEQ ID NO: 50.
  • (N) n1 and (N) m1 that are stem regions, 1 base length of A and 1 base length of T, and (N) n3 and (N) m3 that are G regions are GAGG, GGAG, GCGG and GTGG
  • Four types of base lengths were fixed to 3 base lengths of GGG, and (N) n2 and (N) m2 which are internal loop regions were changed to 1 or 2 base lengths.
  • FIG. 5 is a graph showing the luminescence intensity (RLU) of the reaction solution.
  • Table 7 shows the S / N ratio.
  • the numerical value of H in the sequence name indicates the length of the poly dT in the binding region (Ap).
  • the luminescence intensity with melamine addition shows a significant difference with respect to the luminescence intensity without addition of melamine.
  • the ratio (S / N) between the intensity and the luminescence intensity with no melamine added was greatly improved as compared with PC.
  • Example 4 A sensor for melamine analysis was prepared and analyzed for melamine in the same manner as in Example 3 except that the length of poly dT in the binding region (Ap) to melamine was 36 bases.
  • W was set to the following base sequence W3.
  • W3 is the same sequence as W1 in Example 1 except that poly dT has a length of 36 bases, and the underlined sequence has a poly T with a length of 36 bases (Ap, Mel06)
  • the lowercase sequence on the 5 ′ side is the first linker region (L1), and the lowercase sequence on the 3 ′ side is the second linker region (L2).
  • FIG. 6 is a graph showing the luminescence intensity (RLU) of the reaction solution.
  • the S / N ratio is shown in Table 8.
  • the numerical value of H in the sequence name indicates the length of the poly dT in the binding region (Ap).
  • the luminescence intensity with melamine addition shows a significant difference with respect to the luminescence intensity without addition of melamine.
  • the luminescence with melamine addition The ratio (S / N) between the intensity and the luminescence intensity without addition of melamine was greatly improved as compared with PC (DNAzyme). From these results, it was found that according to the sensor of the example, melamine can be detected with the background suppressed.
  • Example 5 A sensor for melamine analysis was prepared and the detection sensitivity of melamine was confirmed.
  • FIG. 7 is a graph showing the luminescence intensity (RLU) of the reaction solution. As shown in FIG. 7, as a result of using the sensor of the example, sufficient detection sensitivity was shown for a melamine concentration of 3 to 5 mmol / L.
  • Example 6 A sensor for IgE analysis in which the binding region (Ap) was changed to a binding nucleic acid molecule binding to IgE was prepared, and IgE was analyzed.
  • FIG. 8 is a graph showing the luminescence intensity (RLU) of the reaction solution.
  • RLU luminescence intensity
  • the luminescence intensity with the addition of IgE shows a significant difference from the luminescence intensity without the addition of IgE.
  • the ratio (S / N) was excellent. From these results, it was found that IgE can be detected with the background suppressed, according to the sensor of the example.
  • the target analysis sensor of the present invention a target can be analyzed simply and efficiently with an excellent S / N ratio. For this reason, the present invention can be said to be an extremely useful technique for research and examination in various fields such as clinical medicine, food, and environment.

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Abstract

L'invention concerne un nouveau capteur d'analyse de cible, ainsi qu'une méthode d'analyse de cible le mettant en oeuvre. Le capteur selon l'invention se caractérise en ce que : il comprend une molécule d'acide nucléique monocaténaire ; la molécule d'acide nucléique monocaténaire comprend une première zone d'acide nucléique catalyseur (D1), une deuxième zone d'acide nucléique catalyseur (D2), ainsi qu'une zone d'acide nucléique de liaison (Ap) qui se lie à une cible ; un côté terminal de la zone d'acide nucléique de liaison (Ap) comprend la première zone d'acide nucléique catalyseur (D1) ; l'autre côté terminal de la zone d'acide nucléique de liaison (Ap) comprend la deuxième zone d'acide nucléique catalyseur (D2) ; la fonction de catalyseur de la première zone d'acide nucléique catalyseur (D1) et de la deuxième zone d'acide nucléique catalyseur (D2) étant bloquée en l'absence de la cible ; et la fonction de catalyseur est activée par la première zone d'acide nucléique catalyseur (D1) et la deuxième zone d'acide nucléique catalyseur (D2) forme le quadruplet en G en la présence de la cible et lorsque la cible vient en contact avec la zone d'acide nucléique de liaison (Ap).
PCT/JP2014/067126 2013-07-23 2014-06-27 Capteur pour analyse de cible, dispositif d'analyse de cible et méthode d'analyse de cible les mettant en oeuvre WO2015012060A1 (fr)

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