WO2015025862A1 - 異種核酸プローブを用いた修飾核酸塩基の測定方法およびキット - Google Patents
異種核酸プローブを用いた修飾核酸塩基の測定方法およびキット Download PDFInfo
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- WO2015025862A1 WO2015025862A1 PCT/JP2014/071703 JP2014071703W WO2015025862A1 WO 2015025862 A1 WO2015025862 A1 WO 2015025862A1 JP 2014071703 W JP2014071703 W JP 2014071703W WO 2015025862 A1 WO2015025862 A1 WO 2015025862A1
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6804—Nucleic acid analysis using immunogens
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- 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
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- 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
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Definitions
- nucleic acids eg, DNA, RNA
- nucleic acids eg, DNA, RNA
- many techniques have been reported so far for detecting nucleobases into which a substance such as biotin has been artificially introduced by an immunoassay using an antibody.
- a technique for detecting a naturally occurring modified nucleobase (eg, methylcytosine, hydroxylmethylcytosine) by immunoassay has been reported (Patent Document 1, and Non-Patent Documents 1 and 2).
- a method for measuring a modified nucleobase comprising: (1) incubating the nucleic acid sample and the heterologous nucleic acid probe in a solution; and (2) measuring the modified nucleobase in the solution obtained in (1) using an antibody against the modified nucleobase.
- the modified nucleobase in the target nucleic acid can be measured with high sensitivity by reducing the background value of the detection signal.
- FIG. 1 is a diagram showing an example of an outline of measurement of a modified nucleobase in a target nucleic acid according to the method of the present invention.
- RN a nucleotide residue having a modified nucleobase
- N a nucleotide residue having a nucleobase
- R a substituent that the nucleobase has.
- FIG. 2 shows signal values (background values) measured in measurement under conditions of a target nucleic acid (-) containing a modified nucleobase, a heterologous or homologous nucleic acid probe (+), and an antibody against the modified nucleobase (+). ).
- Target nucleic acid DNA; nucleic acid probe: heterologous nucleic acid probe (normal RNA or 2'-O-methylated RNA) or homologous nucleic acid probe (DNA).
- FIG. 3 is a diagram showing signal-to-noise ratio (S / N) calculated in measurement of modified nucleobases at various concentrations using a nucleic acid probe. The target nucleic acid and nucleic acid probe used are the same as in FIG.
- Target nucleic acid DNA; nucleic acid probe: heterologous nucleic acid probe (normal RNA or 2'-O-methylated RNA) or homologous nucleic acid probe (DNA).
- FIG. 7 is a diagram showing signal-to-noise ratio (S / N) calculated in measurement of modified nucleobases at various concentrations using a nucleic acid probe.
- the target nucleic acid and nucleic acid probe used are the same as in FIG.
- FIG. 8 shows signal values (background values) measured in measurement under conditions of target nucleic acid ( ⁇ ) containing a modified nucleobase, heterologous or homologous nucleic acid probe (+), and antibody against modified nucleobase (+). ).
- Target nucleic acid DNA; nucleic acid probe: heterologous nucleic acid probe (2'-O-methylated RNA) or homologous nucleic acid probe (DNA).
- FIG. 9 is a diagram showing the signal-to-noise ratio (S / N) calculated in the measurement of modified nucleobases at various concentrations using a nucleic acid probe.
- the target nucleic acid and nucleic acid probe used are the same as in FIG.
- FIG. 10 shows signal values (background values) measured in measurement under conditions of a target nucleic acid (-) containing a modified nucleobase, a heterologous or homologous nucleic acid probe (+), and an antibody against the modified nucleobase (+). ).
- Target nucleic acid DNA; nucleic acid probe: heterologous nucleic acid probe (2'-O-methylated RNA) or homologous nucleic acid probe (DNA).
- FIG. 13 is a diagram showing the signal-to-noise ratio (S / N) calculated in the measurement of modified nucleobases at various concentrations using a nucleic acid probe. The target nucleic acid and nucleic acid probe used are the same as in FIG.
- the present invention provides a method for measuring modified nucleobases.
- the method of the present invention includes: (1) incubating the nucleic acid sample and the heterologous nucleic acid probe in a solution; and (2) measuring the modified nucleobase in the solution obtained in (1) using an antibody against the modified nucleobase.
- the nucleic acid sample is a sample containing a target nucleic acid containing a modified nucleobase, or a sample suspected of containing the target nucleic acid.
- the nucleic acid sample may also be a biological sample from an organism, or an environmental sample or the like. Examples of organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like. , Microorganisms, plants, fungi, and fish.
- the target nucleic acid is DNA or RNA, but DNA is preferred.
- the target nucleic acid is also a coding or non-coding region of DNA (eg, a transcriptional regulatory region).
- the number of nucleotide residues constituting the target nucleic acid is not particularly limited as long as it can hybridize with a heterologous nucleic acid probe, but is, for example, 10 or more, preferably 15 or more, more preferably 20 It may be more than one.
- the number of nucleotides constituting the target nucleic acid is not particularly limited, and may be any number that may be generated by fragmentation processing of genomic DNA, for example.
- the modified nucleobase is modified with respect to a normal nucleobase selected from the group consisting of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).
- a nucleobase having a structure includes, for example, adenine (A), guanine (G), cytosine (C) and thymine (T) when the target nucleic acid is DNA. It is done.
- the modified nucleobase is not particularly limited as long as it can exist in nature.
- Administrative Instructions under the Patent Cooperation Treaty (enforced on January 1, 2009), Annex C, Appendix 2, Table 2: Examples thereof include modified nucleobases possessed by modified nucleotides described in Modified Nucleotides.
- the modified nucleotides described in this document may be the same as the modified nucleotides described in Appendix 2, Table 2: Modified Base Table of the above guidelines. Accordingly, reference can also be made to the above guidelines for modified nucleobases.
- RNA probe examples include a normal RNA probe composed of a natural ribonucleotide having a hydroxyl group at the 2 ′ position, and a modified RNA probe composed of a ribonucleotide modified with a hydroxyl group at the 2 ′ position.
- a ribonuclease resistant RNA probe may be used as the modified RNA probe.
- modified RNA probes include 2′-O-alkylated RNA probes. The 2′-O-alkylated RNA probe is preferably a 2′-O—C 1 -C 6 alkylated RNA probe.
- the number of nucleotide residues constituting the heterologous nucleic acid probe is not particularly limited as long as the length is sufficient for hybridization with the target nucleic acid, but is, for example, 10 or more, preferably 15 It may be more than 20, more preferably more than 20.
- the number of nucleotides constituting the heterologous nucleic acid probe may also be, for example, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, or 30 or less.
- the GC content of the target nucleic acid is not particularly limited, and may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 60% or more.
- the GC content of the target nucleic acid may also be 90% or less, 80% or less, or 70% or less, for example.
- Such a heterologous nucleic acid probe can be prepared, for example, by a probe synthesis method known in the art.
- the modified nucleobase cannot be detected, but it can be determined that the modified nucleobase is not present in the nucleic acid sample. If the nucleic acid sample contains a target nucleic acid that does not contain a modified nucleobase (in other words, a target nucleic acid that contains only an unmodified nucleobase), the modified nucleobase is obtained by incubating the nucleic acid sample and the heterologous nucleic acid probe in solution.
- the target nucleic acid containing the modified nucleobase and the heterologous nucleic acid probe react with each other by incubating the nucleic acid sample and the heterologous nucleic acid probe in solution.
- a heterologous nucleic acid hybrid composed of heterologous nucleic acid probes is formed.
- the presence of the modified nucleobase can be determined, and the modified nucleobase can also be quantified.
- the heterologous nucleic acid hybrid may have a single-stranded structure of the target nucleic acid in either the 5 ′ terminal region or the 3 ′ terminal region in addition to the double-stranded structure [eg, (b) to ( e)], and may have a single-stranded structure of the target nucleic acid in both the 5 ′ end region and the 3 ′ end region [eg, (i)].
- the number of nucleotide residues of the target nucleic acid and the heterologous nucleic acid probe corresponding to the double-stranded structure portion allows hybridization with the target nucleic acid.
- it may be, for example, 10 or more, preferably 15 or more, more preferably 20 or more.
- the number of nucleotide residues of the target nucleic acid and the heterologous nucleic acid probe corresponding to the double-stranded structure part is, for example, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, or 30 or less. May be.
- the GC content in the double-stranded structure is not particularly limited, and may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 60% or more.
- the GC content in the double-stranded structure part may also be 90% or less, 80% or less, or 70% or less, for example.
- the number of nucleotide residues of the target nucleic acid and the heterologous nucleic acid probe corresponding to the single-stranded structural part of the 5′-terminal region or 3′-terminal region is 1 or more, but not particularly limited, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, or 50 or more.
- Such a number is not particularly limited, and may be, for example, 10,000 or less, 5000 or less, 2000 or less, 1000 or less, 500 or less, 200 or 100 or less.
- a heterologous nucleic acid probe may be designed to form such a single-stranded structural moiety in the 5 'or 3' end region in a heterologous nucleic acid hybrid.
- the heterologous nucleic acid probe may be designed such that an unpaired portion of the modified nucleobase is formed in the double-stranded structure portion of the heterologous nucleic acid hybrid.
- the unpaired portion of the modified nucleobase can be introduced to facilitate detection of the modified nucleobase by the antibody.
- a heterologous nucleic acid probe having a nucleotide sequence that is not completely complementary to a target nucleic acid in a double-stranded structure portion may be used.
- nucleotide residues including a nucleotide residue having a modified nucleobase in the target nucleic acid are non-complementary Good [eg, (I'-2) in Table 2].
- Such a design is possible if the position of the nucleotide residue having the modified nucleobase in the target nucleic acid to be measured is known.
- the heterologous nucleic acid probe may be designed such that the single stranded structural portion of the heterologous nucleic acid hybrid (target nucleic acid) contains the modified nucleobase in such a manner.
- the number in the single-stranded structure portion of the nucleotide residue having a modified nucleobase is not particularly limited as long as it is 1 or more as described above.
- the heterologous nucleic acid probe has a modified nucleobase in the single-stranded structure portion of the heterologous nucleic acid hybrid, and further includes an unpaired portion of the modified nucleobase in the double-stranded structure portion of the heterologous nucleic acid hybrid as described above. It may be designed to be formed. Such a design is possible if the position of the nucleotide residue having the modified nucleobase in the target nucleic acid to be measured is known.
- the method of the present invention may further include adding a heterologous nucleic acid probe to a solution containing the nucleic acid sample to prepare a solution containing both the nucleic acid sample and the heterologous nucleic acid probe.
- the heterologous nucleic acid probe can be added to the nucleic acid sample in solid form or as a solution.
- the concentration of the target nucleic acid in the solution is not particularly limited as long as it can be detected by the method of the present invention. 0.01 nM or more, preferably 0.1 nM or more, more preferably 1 nM or more, even more preferably 5 nM or more, and particularly preferably 10 nM or more.
- the concentration of the target nucleic acid in the solution may also be, for example, 1 M or less, 100 mM or less, 10 mM or less, 1 mM or less, 100 ⁇ M or less, 10 ⁇ M or less, or 1 ⁇ M or less.
- the method of the present invention has been confirmed to have a particularly excellent effect when the concentration of the target nucleic acid in the solution is 1 nM or higher (see Example 1), so that the concentration of the target nucleic acid is 1 nM or higher. It is also preferable.
- the target nucleic acid concentration in the nucleic acid sample is often unknown, so it may be difficult to set the exact target nucleic acid concentration. However, depending on the type of nucleic acid sample, experience the concentration of the target nucleic acid that can be contained in the nucleic acid sample.
- the target nucleic acid may be a target nucleic acid that may contain two or more modified nucleobases.
- the number of modified nucleobases that may be contained in the target nucleic acid is not particularly limited as long as it is 2 or more. For example, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 (Eg, 2, 3, 4 or 5).
- the number of modified nucleobases contained in the target nucleic acid is plural, even if the target nucleic acid in the solution used for hybridization between the target nucleic acid and the heterologous nucleic acid probe is at a very low concentration (eg, 0.1 nM or more) It has been confirmed that the modified nucleic acid can be measured with high sensitivity.
- the methods of the invention can use heterologous nucleic acid probes designed to hybridize with target nucleic acids that may contain two or more modified nucleobases. Such a design is possible if the number of nucleobases that may be modified in the target nucleic acid to be measured is known.
- the modified nucleobase is measured using an antibody against the modified nucleobase in a solution containing the heterologous nucleic acid hybrid.
- the solution obtained in the step (1) may be used as it is.
- another solution is added and / or another solution is added. Exchange to a solution may be performed.
- Such exchange can be performed, for example, by adding the solution obtained in step (1) to the solid phase, immobilizing the heterologous nucleic acid hybrid that can be contained in the solution to the solid phase, and then removing the solution from the solid phase, If necessary, after washing with a washing solution, another solution (eg, a solution containing an antibody against a modified nucleobase) can be added.
- the solution used for the measurement is not particularly limited as long as it is a solution suitable for the antigen / antibody reaction.
- Measurement can be performed by immunological techniques.
- immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples include immunochromatography, luminescence immunoassay, spin immunoassay, western blot, and latex agglutination.
- EIA enzyme immunoassay
- RIA radioimmunoassay
- FFA fluorescence immunoassay
- Examples include immunochromatography, luminescence immunoassay, spin immunoassay, western blot, and latex agglutination.
- the antibody fragment is a part of a full-length antibody, and examples thereof include F (ab ′) 2 , Fab ′, Fab, and Fv.
- the antibody against the modified nucleobase may also be a modified antibody such as a single chain antibody.
- the antibody against the modified nucleobase may be further used as a primary antibody in an immunoassay such as ELISA, and in this case, a secondary antibody is used in combination.
- An antibody against a modified nucleobase is a nucleoside having a modified nucleobase (a structural unit composed of a modified nucleobase and 2′-deoxyribose or ribose), or a nucleotide having a modified nucleobase (modified nucleobase, 2′-deoxyribose). Or a structural unit composed of ribose and phosphate), or two or more nucleotides including a nucleotide having a modified nucleobase (eg, an oligonucleotide composed of 2 to 5 nucleotides). Good.
- an antibody against a modified nucleobase when the target nucleic acid is RNA for example, 1 ′
- an antibody against a nucleoside having a modified nucleobase selected from the group consisting of modified adenosine, modified guanosine, modified cytidine and modified uridine e.g., 1 ′
- an antibody against a ribonucleotide having a modified nucleobase selected from the group consisting of modified adenosine 5'-phosphate, modified guanosine 5'-phosphate, modified cytidine 5'-phosphate and modified uridine 5'-phosphate e.g., 3'
- An antibody against two or more ribonucleotides including the ribonucleotide having a modified nucleobase can be mentioned.
- An antibody against a modified nucleobase is, for example, a complex of a nucleoside having a modified nucleobase, a nucleotide having a modified nucleobase, or two or more nucleotides including a nucleotide having a modified nucleobase and a carrier protein (eg, BSA, KLH).
- a carrier protein eg, BSA, KLH.
- a polyclonal antibody against a modified nucleobase is used about 2 to 4 times every 2 to 3 weeks subcutaneously or intraperitoneally in an animal together with a commercially available adjuvant (eg, complete or incomplete Freund's adjuvant) using the above complex as an antigen. It is obtained by collecting whole blood about 3 to about 10 days after the final immunization and purifying the antiserum.
- adjuvant eg, complete or incomplete Freund's adjuvant
- animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, cows, guinea pigs, and hamsters.
- a monoclonal antibody against the modified nucleobase can be produced, for example, by a cell fusion method.
- the above complex is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected.
- the leukocytes and myeloma cells eg, NS-1) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor.
- Examples of cell fusion include a PEG method and a voltage pulse method.
- a hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method.
- the hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.
- the monoclonal antibody may be any isotype such as IgG, IgM, IgA, and IgE.
- monoclonal antibody production methods include phage display (Ulman et al., Proc. Natl. Acad. Sci. USA, 90, 1184-89 (1993)), ADLib system (International Publication No. 2004 / Since in vitro methods such as No. 016444) are also known, such methods may be used for the production of antibodies against modified nucleobases.
- the labeling substance examples include enzymes (eg, peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase), affinity substances (eg, streptavidin, biotin), fluorescent substances or proteins (eg, fluorescein, fluorescein isothiocyanate, rhodamine, green) Fluorescent proteins, red fluorescent proteins), luminescent substances (eg, luciferin, aequorin), radioactive substances (eg, 3 H, 14 C, 32 P, 35 S, 125 I).
- enzymes eg, peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase
- affinity substances eg, streptavidin, biotin
- fluorescent substances or proteins eg, fluorescein, fluorescein isothiocyanate, rhodamine, green Fluorescent proteins, red fluorescent proteins), luminescent substances (eg, luci
- the measurement of the modified nucleobase by the antibody against the modified nucleobase is performed qualitatively or quantitatively, and the presence or amount of the modified nucleobase can be evaluated.
- the measurement of the modified nucleobase is intended not only to measure the modified nucleobase itself but also to measure the target nucleic acid containing the modified nucleobase.
- the measurement of the amount of the modified nucleobase may be performed together with the measurement of the background value, for example. Specifically, it may be done by: (2-1 ′) measuring the signal value by assaying with an antibody against the modified nucleobase in the solution obtained in the above step (1); (2-2 ′) assaying with an antibody against the modified nucleobase in a solution not containing the target nucleic acid containing the modified nucleobase and containing the heterologous nucleic acid probe, and measuring the background value; (2-3 ′) correcting the signal value with the background value to obtain a corrected signal value; and (2-4 ′) evaluating the amount of modified nucleobase based on the corrected signal value.
- the amount of modified nucleobase may be measured using a standard. Specifically, it may be done by: (2-1 ′′) measuring the signal value by assaying with an antibody against the modified nucleobase in the solution obtained in the above step (1); (2-2 ′′) measuring a calibration value by assaying with an antibody against the modified nucleobase in a solution containing the target nucleic acid (standard) containing the modified nucleobase and a heterologous nucleic acid probe; and (2-3 ′′) To verify the amount of modified nucleobase by comparing the signal value with a calibration value.
- the measurement using the standard may be used in combination with the measurement of the background value.
- the methods of the invention may be performed by ELISA.
- the method of the invention by ELISA may be performed as follows: (I) A heterogeneous nucleic acid sample comprising a target nucleic acid containing a modified nucleobase and a heterologous nucleic acid probe labeled with a first affinity substance are incubated in a solution to form a heterogeneous mixture composed of the target nucleic acid and the heterologous nucleic acid probe.
- Forming a nucleic acid hybrid Forming a nucleic acid hybrid; (Ii) immobilizing the heterologous nucleic acid hybrid to a solid phase treated with a second affinity substance; (Iii) reacting a primary antibody against the modified nucleobase with a heterologous nucleic acid hybrid immobilized on a solid phase to obtain a primary complex of the primary antibody and the heterologous nucleic acid hybrid; (Iv) reacting a secondary antibody labeled with a labeling substance with the primary complex to obtain a secondary complex of the secondary antibody and the primary antibody; and (v) 2 in the secondary complex.
- the first affinity substance and the second affinity substance are: , Used in a combination having affinity for each other (eg, a combination of biotin and streptavidin).
- a nucleic acid sample containing a target nucleic acid containing a modified nucleobase and a heterologous nucleic acid probe immobilized on a solid phase are used as a solution.
- the present invention also provides a kit for measuring a modified nucleobase.
- the kit of the present invention includes, for example: (I) a heterologous nucleic acid probe; and (II) an antibody against a modified nucleobase.
- the heterologous nucleic acid probe and the modified nucleobase are as described above.
- the heterologous nucleic acid probe may be labeled with an affinity substance
- the antibody against the modified nucleobase may be labeled with a labeling substance.
- the kit of the present invention comprises an affinity substance, a labeling substance, a secondary antibody, a detection reagent for the secondary antibody (eg, a substrate for the enzyme when the secondary antibody is labeled with an enzyme), a solid phase, etc.
- kit of the present invention may also contain a modified nucleobase preparation or a target nucleic acid preparation containing the modified nucleobase as a solution or as a powder.
- the kit of the present invention includes each component in an isolated form or a mixed form.
- each component may be provided in a form accommodated in a different container (eg, tube, plate).
- the kit of the present invention may be provided in the form of a device. Specifically, all of the components may be provided in a form housed in the device. Alternatively, some of the components may be provided in a form housed in the device, and the rest may be provided in a form that is not housed in the device (eg, a form housed in a different container). In this case, components that are not contained in the device may be used by being injected into the device during measurement of the target substance.
- Example 1 Use of heterologous nucleic acid probes in the measurement of modified nucleobases (1)
- the nucleotide sequence of the target nucleic acid (DNA) is 5′-TTGCCGCGCGTC [C] GTCCTGTTGAACTTC-3 ′ (SEQ ID NO: 1: [C] indicates 5-methylcytosine), the nucleotide sequence of the heterologous nucleic acid probe for capturing the target nucleic acid Is 5′-GAAGUCAACAGGACGGACGCCGCCAA-3 ′ (SEQ ID NO: 2, the main chain of the nucleic acid is normal RNA or 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin), which is artificially synthesized by Hokkaido System Science Each was used.
- the heterologous nucleic acid probe was designed such that when a heterologous nucleic acid hybrid with the target nucleic acid was formed, an unpaired portion was formed at the modified nucleobase [C] in the double-stranded structure portion of the heterologous nucleic acid hybrid.
- Measurement of the modified nucleobase using a heterologous nucleic acid probe was performed as follows. First, a target nucleic acid containing 5-methylcytosine (1 pmol, 0.1 pmol, or 0.01 pmol) and a heterologous nucleic acid probe (5 pmol) were dissolved in 100 ⁇ L of hybridization buffer (4 ⁇ SSC, 0.3% Tween 20).
- the concentration of the target nucleic acid is 10 nM, 1 nM, or 0.1 nM, respectively, and the concentration of the heterologous nucleic acid probe is 50 nM).
- a heterologous nucleic acid hybrid composed of probes was formed.
- a solution not containing the target nucleic acid was also prepared and the same operation was performed. 100 ⁇ L of the solution after hybridization reaction was added to a streptavidin-coated plate (Thermo Scientific) and reacted at 37 ° C. for 30 minutes to immobilize the heterologous nucleic acid hybrid on the streptavidin plate.
- This antibody is not limited to 5-methylcytosine but also the main chain structure of DNA (eg, deoxyribose moiety and 100 ⁇ L of an antibody that recognizes at least a part of a structure composed of repeating units of a phosphate moiety) was added and reacted at 37 ° C. for 1 hour.
- the plate was washed 3 times with 300 ⁇ L of PBS-T, and 100 ⁇ L of 250 ng / mL peroxidase-labeled anti-IgG antibody (manufactured by Thermo Scientific) was added and reacted at 37 ° C. for 30 minutes. After washing 3 times with 300 ⁇ L PBS-T, 100 ⁇ L each of 3,3 ′, 5,5′-tetramethylbenzidine was added and allowed to react for 15 minutes at light-shielded room temperature. Thereafter, 100 ⁇ L of 2N hydrochloric acid solution was added, and the absorbance at 450 nm was measured with a microplate reader (Arvo manufactured by PerkinElmer).
- a heterologous nucleic acid probe whose main chain is normal RNA or 2′-O-methylated RNA a homologous nucleic acid probe whose main chain is DNA (SEQ ID NO: 3: 5′-GAAGTCAACAGGACGACGCCGCGCAA-3 ′) was used. Except for the above, the target nucleic acid was measured by the same method. As a result, the heterologous nucleic acid probe decreased the background value of the detection signal (Table 4, FIG. 2) and improved the S / N value at each concentration of the target nucleic acid as compared to the homologous nucleic acid probe (Table). 5, FIG. 3).
- a heterologous nucleic acid probe with respect to the target nucleic acid is effective in improving the detection sensitivity in the measurement of the target nucleic acid containing the modified nucleobase with an antibody.
- the amount of the target nucleic acid is 0.1 pmol or more (that is, when the concentration of the target nucleic acid in the solution is 1 nM or more and hybridized with a heterologous nucleic acid probe to form a heterologous nucleic acid hybrid)
- the homologous nucleic acid A particularly excellent effect of the heterologous nucleic acid probe on the probe was confirmed (Table 5, FIG. 3).
- Example 2 Examination of differences in antibody clones Instead of the anti-methylcytosine antibody (Nippon Gene, Clone33D3) used in Example 1, 100 ng / mL anti-methylcytosine antibody (Millipore, Clone33D3), 100 ng / mL The test was performed in the same manner as in Example 1, except that an anti-methylcytosine antibody (Millipore, Clone162 33 D3) and a 10 ng / mL anti-methylcytosine antibody (ZYMO RESEARCH, Clone10G4) were used. The reason for adopting different concentrations for each antibody is that the activity of each antibody is different and the detection signal changes greatly, so that the detection signal of the same degree is adjusted.
- Example 3 Use of heterologous nucleic acid probes in the measurement of modified nucleobases
- the nucleotide sequence of the target nucleic acid (DNA) is 5′-AATCAG [C] GGGAGCTCTTTCTTTGCGCGGGCGTCCGTCCTGTTGACTTC-3 ′ (SEQ ID NO: 4: [C] indicates 5-methylcytosine)
- the nucleotide sequence of the heterologous nucleic acid probe for capturing the target nucleic acid Is 5′-GAAGUCAACAGGACGGACGCCGCCAA-3 ′
- SEQ ID NO: 2 the main chain of the nucleic acid is normal RNA or 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin
- the heterologous nucleic acid probe was designed such that when a heterologous nucleic acid hybrid with the target nucleic acid was formed, the modified nucleobase [C] was present in the single-stranded structure portion of the heterologous nucleic acid hybrid.
- the measurement of the modified nucleobase using the heterologous nucleic acid probe was performed in the same manner as in Example 1 except that a different target nucleic acid was used as described above.
- a homologous nucleic acid probe whose main chain is DNA SEQ ID NO: 3: 5′-GAAGTCAACAGGACGACGCCGCGCAA-3 ′
- a heterologous nucleic acid probe designed so that an unpaired part is formed with the modified nucleobase [C] in the double-stranded structure part of the heterologous nucleic acid hybrid.
- a heterologous nucleic acid probe designed such that a modified nucleobase [C] is present in the single-stranded structure portion of the heterologous nucleic acid hybrid is also shown to be effective. It was done.
- Example 4 Use of heterologous nucleic acid probes in the measurement of modified nucleobases (3)
- the nucleotide sequence of the target nucleic acid (DNA) is 5′-G [C] GGAGCTCTCCCT [C] GGGA [C] GGTGGCAGCCCTGAGGTGCCTGCA-3 ′ (SEQ ID NO: 5: [C] indicates 5-methylcytosine) and captures the target nucleic acid
- the nucleotide sequence of the heterologous nucleic acid probe for this is 5′-UGCAGGGACCACUCGAGGCUGCCAC-3 ′ (SEQ ID NO: 6, the main chain of the nucleic acid is 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin).
- the heterologous nucleic acid probe was designed such that when a heterologous nucleic acid hybrid with the target nucleic acid was formed, the modified nucleobase [C] was present in the single-stranded structure portion of the heterologous nucleic acid hybrid.
- the measurement of the modified nucleobase using the heterologous nucleic acid probe was performed in the same manner as in Example 1 except that different target nucleic acids and heterologous nucleic acid probes were used as described above.
- a homologous nucleic acid probe whose main chain is DNA (SEQ ID NO: 7: 5′-TGCAGGACCACTCGAGGCTGCCAC-3 ′) is used.
- the target nucleic acid was measured by the same method.
- 10 1 order for the amount of target nucleic acid also improved the detection sensitivity.
- the detection sensitivity was amplified, and thus a particularly excellent effect of the heterologous nucleic acid probe on the homologous nucleic acid probe was confirmed (Table 8, FIG. 9).
- Example 5 Use of heterologous nucleic acid probes in the measurement of modified nucleobases (4)
- the nucleotide sequence of the target nucleic acid (DNA) is 5′-T [C] GTGGTGGAACTTCTCTCCAATTTTCTAGGGGG-3 ′ (SEQ ID NO: 8: [C] indicates 5-methylcytosine), the nucleotide sequence of the heterologous nucleic acid probe for capturing the target nucleic acid Is 5′-CCCCCUAGAAAAAUUGGAGAAGUCCCACCCACAAAAAAAAA-3 ′ (SEQ ID NO: 9, the main chain of the nucleic acid is 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin), which were artificially synthesized by Hokkaido System Science, respectively.
- the heterologous nucleic acid probe was designed such that when a heterologous nucleic acid hybrid with the target nucleic acid was formed, the modified nucleobase [C] was present in the single-stranded structure portion of the heterologous nucleic acid hybrid.
- the measurement of the modified nucleobase using the heterologous nucleic acid probe was performed in the same manner as in Example 1 except that different target nucleic acids and heterologous nucleic acid probes were used as described above.
- a homologous nucleic acid probe whose main chain is DNA (SEQ ID NO: 10: 5′-CCCCCTAGAAAAATTGAGAGAAGTCCCACCCACAAAAAAAAAAA-3 ′) was used.
- the target nucleic acid was measured by the same method.
- Example 9 As a result, even when a target nucleic acid and a heterologous nucleic acid probe different from those in Example 1, Example 3 and Example 4 were used, a decrease in the background value and an improvement in the S / N value were observed (Table 9, 10 and 11). Further, when the amount of the target nucleic acid is 1 pmol or more (that is, when the concentration of the target nucleic acid in the solution is 10 nM or more and hybridized with the heterologous nucleic acid probe to form a heterologous nucleic acid hybrid), A particularly excellent effect of the heterologous nucleic acid probe was confirmed (Table 9, FIG. 11).
- Example 6 Use of heterologous nucleic acid probes in the measurement of modified nucleobases (5)
- the nucleotide sequence of the target nucleic acid (DNA) is 5′-GGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTT [C] GT-3 ′ (SEQ ID NO: 11: [C] indicates 5-methylcytosine), nucleotide sequence of the heterologous nucleic acid probe for capturing the target nucleic acid Is 5′-CACUGAACAAAAUGGCACUAUGUAAAACUGAGCCAAAAAAAAAA-3 ′ (SEQ ID NO: 12, the main chain of the nucleic acid is 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin), which were artificially synthesized by Hokkaido System Science, respectively.
- the heterologous nucleic acid probe was designed such that when a heterologous nucleic acid hybrid with the target nucleic acid was formed, the modified nucleobase [C] was present in the single-stranded structure portion of the heterologous nucleic acid hybrid.
- the measurement of the modified nucleobase using the heterologous nucleic acid probe was performed in the same manner as in Example 1 except that different target nucleic acids and heterologous nucleic acid probes were used as described above.
- a homologous nucleic acid probe whose main chain is DNA (SEQ ID NO: 13: 5′-CACTGAACAAATGGCACTAGTAAACTGAGCCAAAAAAAAA-3 ′) is used.
- the target nucleic acid was measured by the same method.
- Example 3 Example 4 and Example 5
- a decrease in the background value and an improvement in the S / N value were observed.
- the amount of the target nucleic acid is 1 pmol or more (that is, when the concentration of the target nucleic acid in the solution is 10 nM or more and hybridized with the heterologous nucleic acid probe to form a heterologous nucleic acid hybrid)
- a particularly excellent effect of the heterologous nucleic acid probe was confirmed (Table 9, FIG. 13).
- the present invention is useful for lowering the background value and improving the S / N value regardless of the type of target nucleic acid and heterologous nucleic acid probe. It has been shown.
- Reference Example 1 Use of homologous nucleic acid probe in measurement of modified nucleobase including modified nucleobase
- the target nucleic acid was measured under the conditions described below. Other experimental conditions (eg, reaction system capacity) were the same as in Example 1.
- Target nucleic acid DNA comprising a modified nucleobase consisting of the nucleotide sequence of SEQ ID NO: 1 (10 pmol);
- Nucleic acid probe Homologous nucleic acid (DNA) probe (5 pmol) consisting of the nucleotide sequence of SEQ ID NO: 3;
- Antibody to modified nucleobase 100 ng / mL anti-methylcytosine antibody (Millipore, Clone 33D3).
- the method and kit of the present invention are useful for measuring modified nucleobases.
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Abstract
Description
〔1〕以下を含む、修飾核酸塩基の測定方法:
(1)核酸サンプルおよび異種核酸プローブを溶液中でインキュベートすること;ならびに
(2)(1)で得られた溶液において、修飾核酸塩基に対する抗体を用いて修飾核酸塩基を測定すること。
〔2〕核酸サンプルが、修飾核酸塩基を含む標的核酸を含有し、かつ工程(1)および(2)がそれぞれ(1’)および(2’)により行われる、〔1〕の方法:
(1’)修飾核酸塩基を含む標的核酸を含有する核酸サンプル、および異種核酸プローブを溶液中でインキュベートにより反応させて、当該標的核酸および異種核酸プローブから構成される異種核酸ハイブリッドを形成すること;ならびに
(2’)当該異種核酸ハイブリッドを含む溶液において、修飾核酸塩基に対する抗体を用いて修飾核酸塩基を測定すること。
〔3〕標的核酸が、2以上の修飾核酸塩基を含む可能性がある標的核酸である、〔1〕または〔2〕の方法。
〔4〕前記核酸サンプルを含有する溶液に異種核酸プローブを添加して、核酸サンプルおよび異種核酸プローブの双方を含有する溶液を調製することをさらに含む、〔1〕~〔3〕のいずれかの方法。
〔5〕前記核酸サンプルが、修飾核酸塩基を含む標的DNAを含有するサンプルである、〔1〕~〔4〕のいずれかの方法。
〔6〕異種核酸プローブがRNAプローブである、〔1〕~〔5〕のいずれかの方法。
〔7〕修飾核酸塩基を構成する核酸塩基がシトシンである、〔1〕~〔6〕のいずれかの方法。
〔8〕修飾核酸塩基がメチルシトシンである、〔1〕~〔7〕のいずれかの方法。
〔9〕異種核酸ハイブリッドの二本鎖構造部分において修飾核酸塩基の不対部分が形成されるように、異種核酸プローブが設計される、〔2〕~〔8〕のいずれかの方法。
〔10〕異種核酸ハイブリッドの一本鎖構造部分において修飾核酸塩基が存在するように、異種核酸プローブが設計される、〔2〕~〔9〕のいずれかの方法。
〔11〕修飾核酸塩基に対する抗体を用いる修飾核酸塩基の測定が、ELISAにより行われる、〔1〕~〔10〕のいずれかの方法。
〔12〕以下を含む、修飾核酸塩基の測定用キット:
(I)異種核酸プローブ;および
(II)修飾核酸塩基に対する抗体。
(1)核酸サンプルおよび異種核酸プローブを溶液中でインキュベートすること;ならびに
(2)(1)で得られた溶液において、修飾核酸塩基に対する抗体を用いて修飾核酸塩基を測定すること。
核酸サンプルが標的核酸を含まない場合、核酸サンプルおよび異種核酸プローブを溶液中でインキュベートしても、異種核酸ハイブリッドは形成されない。この場合、後述する工程(2)において、修飾核酸塩基を検出することはできないが、核酸サンプル中に修飾核酸塩基が存在しないことを判定できる。
核酸サンプルが修飾核酸塩基を含まない標的核酸(換言すれば、非修飾核酸塩基のみを含む標的核酸)を含有する場合、核酸サンプルおよび異種核酸プローブを溶液中でインキュベートすることにより、修飾核酸塩基を含まない標的核酸および異種核酸プローブが反応して、当該標的核酸および異種核酸プローブから構成される異種核酸ハイブリッドが形成される。この場合、後述する工程(2)において、修飾核酸塩基を検出することはできないが、核酸サンプル中に(標的核酸が存在するにもかかわらず)修飾核酸塩基が存在しないこと、換言すれば、標的核酸中の所定の核酸塩基が修飾されていないことを判定できる。
核酸サンプルが修飾核酸塩基を含む標的核酸を含有する場合、核酸サンプルおよび異種核酸プローブを溶液中でインキュベートすることにより、修飾核酸塩基を含む標的核酸および異種核酸プローブが反応して、当該標的核酸および異種核酸プローブから構成される異種核酸ハイブリッドが形成される。この場合、後述する工程(2)において、修飾核酸塩基が存在することを判定でき、また修飾核酸塩基を定量することもできる。
溶液中の異種核酸プローブの濃度は、本発明の方法により標的核酸が検出可能である限り特に限定されないが、例えば0.01nM以上、好ましくは0.1nM以上、より好ましくは1nM以上、さらにより好ましくは5nM以上、特に好ましくは10nM以上であってもよい。溶液中の異種核酸プローブの濃度はまた、例えば1M以下、100mM以下、10mM以下、1mM以下、100μM以下、10μM以下または1μM以下であってもよい。したがって、このような濃度が達成されるように、異種核酸プローブを溶液に添加してもよい。
例えば、修飾核酸塩基に対するポリクローナル抗体は、上記複合体を抗原として、市販のアジュバント(例、完全または不完全フロイントアジュバント)とともに、動物の皮下あるいは腹腔内に2~3週間おきに2~4回程度投与し、最終免疫から約3~約10日後に全血を採取して抗血清を精製することにより取得できる。抗原を投与する動物としては、例えば、ラット、マウス、ウサギ、ヤギ、ウシ、モルモット、ハムスターなどの哺乳動物が挙げられる。
修飾核酸塩基に対するモノクローナル抗体は、例えば、細胞融合法により作製できる。例えば、上記複合体を市販のアジュバントと共にマウスに2~4回皮下または腹腔内に投与し、最終投与の約3日後に脾臓あるいはリンパ節を採取し、白血球を採取する。この白血球と骨髄腫細胞(例、NS-1)を細胞融合して該因子に対するモノクローナル抗体を産生するハイブリドーマを得る。細胞融合としては、PEG法、電圧パルス法が挙げられる。所望のモノクローナル抗体を産生するハイブリドーマは、周知のEIAまたはRIA法等を用いて抗原と特異的に結合する抗体を、培養上清中から検出することにより選択できる。モノクローナル抗体を産生するハイブリドーマの培養は、インビトロ、またはマウスもしくはラット、好ましくはマウス腹水中等のインビボで行うことができ、抗体はそれぞれハイブリドーマの培養上清および動物の腹水から取得できる。モノクローナル抗体は、IgG、IgM、IgA、IgE等のいずれのアイソタイプであってもよい。あるいは、モノクローナル抗体の作製方法としては、ファージディスプレイ法(Ulmanら,Proc.Natl.Acad.Sci.U.S.A.,90,1184-89(1993))、ADLibシステム(国際公開第2004/011644号)等のin vitro法も知られているので、修飾核酸塩基に対する抗体の作製のため、このような方法を使用してもよい。
(2-1)上記工程(1)で得られた溶液において、修飾核酸塩基に対する抗体を用いてアッセイして、シグナル値を計測すること;
(2-2)修飾核酸塩基を含む標的核酸を含有せず、かつ異種核酸プローブを含有する溶液において、修飾核酸塩基に対する抗体を用いてアッセイして、バックグランド値を計測すること;および
(2-3)シグナル値をバックグランド値と比較して、修飾核酸塩基の有無を評価すること。
修飾核酸塩基の測定において、シグナル値およびバックグランド値は、修飾核酸塩基に対する抗体または2次抗体(2次抗体が用いられる場合)に結合した標識を利用して計測される値(例、吸光度、蛍光度、発色度、および放射活性)である。
(2-1’)上記工程(1)で得られた溶液において、修飾核酸塩基に対する抗体を用いてアッセイして、シグナル値を計測すること;
(2-2’)修飾核酸塩基を含む標的核酸を含有せず、かつ異種核酸プローブを含有する溶液において、修飾核酸塩基に対する抗体を用いてアッセイして、バックグランド値を計測すること;
(2-3’)バックグランド値でシグナル値を補正して、補正シグナル値を得ること;ならびに
(2-4’)補正シグナル値に基づき、修飾核酸塩基の量を評価すること。
(2-1’’)上記工程(1)で得られた溶液において、修飾核酸塩基に対する抗体を用いてアッセイして、シグナル値を計測すること;
(2-2’’)修飾核酸塩基を含む標的核酸(標品)、かつ異種核酸プローブを含有する溶液において、修飾核酸塩基に対する抗体を用いてアッセイして、検量用値を計測すること;ならびに
(2-3’’)シグナル値を検量用値に照合して、修飾核酸塩基の量を評価すること。
標品を用いる上記測定は、バックグランド値の上記測定と併用されてもよい。
(i)修飾核酸塩基を含む標的核酸を含有する核酸サンプルおよび第1の親和性物質で標識された異種核酸プローブを溶液中でインキュベートして、当該標的核酸および当該異種核酸プローブから構成される異種核酸ハイブリッドを形成すること;
(ii)異種核酸ハイブリッドを、第2の親和性物質で処理された固相に固定すること;
(iii)修飾核酸塩基に対する1次抗体を、固相に固定された異種核酸ハイブリッドと反応させて、1次抗体と異種核酸ハイブリッドとの1次複合体を得ること;
(iv)標識物質で標識された2次抗体を1次複合体と反応させて、2次抗体と1次抗体との2次複合体を得ること;ならびに
(v)2次複合体中の2次抗体が有する標識物質を利用して、形成された異種核酸ハイブリッド(換言すれば、修飾核酸塩基)の存在および/または量を測定すること
第1の親和性物質および第2の親和性物質は、互いに親和性を有する組合せ(例、ビオチンとストレプトアビジンとの組合せ)で用いられる。なお、本発明の方法は、上記工程(i)および(ii)の代わりに、(i’)修飾核酸塩基を含む標的核酸を含有する核酸サンプル、および固相に固定された異種核酸プローブを溶液中でインキュベートして、当該標的核酸および当該異種核酸プローブから構成される異種核酸ハイブリッドを形成することを含んでいてもよい。この場合、固相に固定された異種核酸プローブを得ること(例、第1の親和性物質で標識された異種核酸プローブを、第2の親和性物質で処理された固相に加えること)をさらに含んでいてもよい。本発明の方法は、工程(iii)の前に、固相を洗浄することを含んでいてもよい。2次抗体は、1次抗体のみを認識する抗体(例、1次抗体の定常領域に結合する抗体)であってもよいが、2次複合体中の1次抗体および1次複合体の双方を認識するものであってもよい。(i)~(v)を含む本発明の方法は、本明細書中に詳細に記載された方法論にしたがって行うことができる。
(I)異種核酸プローブ;および
(II)修飾核酸塩基に対する抗体。
異種核酸プローブおよび修飾核酸塩基は、上述したとおりである。例えば、異種核酸プローブは、親和性物質で標識されていてもよく、修飾核酸塩基に対する抗体は、標識物質で標識されていてもよい。本発明のキットは、親和性物質、標識物質、2次抗体、2次抗体の検出試薬(例、2次抗体が酵素で標識されている場合には、その酵素の基質)および固相等の上述したような構成成分をさらに含んでいてもよい。固相は、親和性物質で処理されていてもよい。本発明のキットはまた、修飾核酸塩基の標品、または修飾核酸塩基を含む標的核酸の標品を、溶液としてまたは粉末として含んでいてもよい。
標的核酸(DNA)のヌクレオチド配列は5’-TTGCGCGGCGTC[C]GTCCTGTTGACTTC-3’(配列番号1:[C]は5-メチルシトシンを示す)、標的核酸を捕捉するための異種核酸プローブのヌクレオチド配列は5’-GAAGUCAACAGGACGACGCCGCGCAA-3’(配列番号2、核酸の主鎖はノーマルRNAまたは2’-O-メチル化RNA、5’末端はビオチン標識)であり、北海道システムサイエンス社により人工合成したものをそれぞれ使用した。異種核酸プローブは、標的核酸との異種核酸ハイブリッドが形成されたとき、異種核酸ハイブリッドの二本鎖構造部分中の修飾核酸塩基[C]で不対部分が形成されるように設計した。
異種核酸プローブを用いた修飾核酸塩基の測定は、以下のとおり行った。まず、5-メチルシトシンを含む標的核酸(1pmol、0.1pmol、または0.01pmol)と異種核酸プローブ(5pmol)をハイブリダイゼーションBuffer(4×SSC、0.3%Tween20)100μL中に溶解させた後(核酸サンプル溶液中、標的核酸の濃度はそれぞれ10nM、1nM、または0.1nMであり、異種核酸プローブの濃度は50nMである)、60℃で2時間反応させることで、標的核酸と異種核酸プローブから構成される異種核酸ハイブリッドを形成させた。また、標的核酸を含まない溶液も調製し、同様の操作を行った。ハイブリダイゼーション反応後の溶液を、ストレプトアビジンでコートされたプレート(Thermo Scientific社製)に100μL加え、37℃で30分反応させることで、ストレプトアビジンプレート上に異種核酸ハイブリッドを固定化した。300μLのPBS-Tで2回洗浄し、50ng/mLの抗メチルシトシン抗体(ニッポンジーン社製、Clone33D3。本抗体は、5-メチルシトシンのみならず、DNAの主鎖構造(例、デオキシリボース部分およびリン酸部分の繰り返し単位から構成される構造)の少なくとも一部を認識する抗体である)を100μLずつ加え、37℃で1時間反応させた。300μLのPBS-Tで3回洗浄し、250ng/mLのペルオキシダーゼ標識抗IgG抗体(Thermo Scientific社製)を100μLずつ加え、37℃で30分反応させた。300μLのPBS-Tで3回洗浄した後、3,3’,5,5’-Tetramethylbenzidineを100μLずつ加え、遮光室温で15分反応させた。その後、2N塩酸溶液を100μLずつ加え、マイクロプレートリーダー(PerkinElmer社製Arvo)により450nmの吸光度を測定した。
また、主鎖がノーマルRNAまたは2’-O-メチル化RNAである異種核酸プローブの代わりに、主鎖がDNAである同種核酸プローブ(配列番号3:5’-GAAGTCAACAGGACGACGCCGCGCAA-3’)を用いた以外は、同様の方法で標的核酸を測定した。
その結果、異種核酸プローブは、同種核酸プローブに比し、検出シグナルのバックグラント値を低下させ(表4、図2)、また、各濃度の標的核酸においてS/N値を向上させた(表5、図3)。このことは、修飾核酸塩基を含む標的核酸の抗体による測定における検出感度の向上には、当該標的核酸に対して異種の核酸プローブの併用が有効であることを示す。特に、標的核酸の量が0.1pmol以上であるとき(即ち、溶液中の標的核酸の濃度を1nM以上にして異種核酸プローブとハイブリダイズさせて、異種核酸ハイブリッドを形成させたとき)、同種核酸プローブに対する異種核酸プローブの特に優れた効果が確認された(表5、図3)。
実施例1で用いた抗メチルシトシン抗体(ニッポンジーン社製、Clone33D3)の代わりに、100ng/mL抗メチルシトシン抗体(Millipore社製、Clone33D3)、100ng/mL抗メチルシトシン抗体(Millipore社製、Clone162 33 D3)、10ng/mL抗メチルシトシン抗体(ZYMO RESEARCH社製、Clone10G4)を用いた以外は、実施例1と同様の方法で試験した。なお、各抗体について異なる濃度を採用した理由は、各抗体の活性が異なり、検出シグナルが大きく変わってしまうことから、同程度の検出シグナルが得られるように調整したためである。
標的核酸(DNA)のヌクレオチド配列は5’-AATCAG[C]GGGAGCTCTTTCTTTGCGCGGCGTCCGTCCTGTTGACTTC-3’(配列番号4:[C]は5-メチルシトシンを示す)、標的核酸を捕捉するための異種核酸プローブのヌクレオチド配列は5’-GAAGUCAACAGGACGACGCCGCGCAA-3’(配列番号2、核酸の主鎖はノーマルRNAまたは2’-O-メチル化RNA、5’末端はビオチン標識)であり、北海道システムサイエンス社により人工合成したものをそれぞれ使用した。異種核酸プローブは、標的核酸との異種核酸ハイブリッドが形成されたとき、異種核酸ハイブリッドの一本鎖構造部分において修飾核酸塩基[C]が存在するように設計した。
異種核酸プローブを用いた修飾核酸塩基の測定は、上述のとおり異なる標的核酸を用いた以外は、実施例1と同様の方法で行った。
また、主鎖がノーマルRNAまたは2’-O-メチル化RNAである異種核酸プローブの代わりに、主鎖がDNAである同種核酸プローブ(配列番号3:5’-GAAGTCAACAGGACGACGCCGCGCAA-3’)を用いた以外は、同様の方法で標的核酸を測定した。
標的核酸(DNA)のヌクレオチド配列は5’-G[C]GGAGCTCTCCCT[C]GGGA[C]GGTGGCAGCCTCGAGTGGTCCTGCA-3’(配列番号5:[C]は5-メチルシトシンを示す)、標的核酸を捕捉するための異種核酸プローブのヌクレオチド配列は5’-UGCAGGACCACUCGAGGCUGCCAC-3’(配列番号6、核酸の主鎖は2’-O-メチル化RNA、5’末端はビオチン標識)であり、北海道システムサイエンス社により人工合成したものをそれぞれ使用した。異種核酸プローブは、標的核酸との異種核酸ハイブリッドが形成されたとき、異種核酸ハイブリッドの一本鎖構造部分において修飾核酸塩基[C]が存在するように設計した。
異種核酸プローブを用いた修飾核酸塩基の測定は、上述のとおり異なる標的核酸および異種核酸プローブを用いた以外は、実施例1と同様の方法で行った。
また、主鎖が2’-O-メチル化RNAである異種核酸プローブの代わりに、主鎖がDNAである同種核酸プローブ(配列番号7:5’-TGCAGGACCACTCGAGGCTGCCAC-3’)を用いた以外は、同様の方法で標的核酸を測定した。
標的核酸(DNA)のヌクレオチド配列は5’-T[C]GTGGTGGACTTCTCTCAATTTTCTAGGGGG-3’(配列番号8:[C]は5-メチルシトシンを示す)、標的核酸を捕捉するための異種核酸プローブのヌクレオチド配列は5’-CCCCCUAGAAAAUUGAGAGAAGUCCACCACAAAAAAAAAA-3’(配列番号9、核酸の主鎖は2’-O-メチル化RNA、5’末端はビオチン標識)であり、北海道システムサイエンス社により人工合成したものをそれぞれ使用した。異種核酸プローブは、標的核酸との異種核酸ハイブリッドが形成されたとき、異種核酸ハイブリッドの一本鎖構造部分において修飾核酸塩基[C]が存在するように設計した。
異種核酸プローブを用いた修飾核酸塩基の測定は、上述のとおり異なる標的核酸および異種核酸プローブを用いた以外は、実施例1と同様の方法で行った。
また、主鎖が2’-O-メチル化RNAである異種核酸プローブの代わりに、主鎖がDNAである同種核酸プローブ(配列番号10:5’-CCCCCTAGAAAATTGAGAGAAGTCCACCACAAAAAAAAAA-3’)を用いた以外は、同様の方法で標的核酸を測定した。
標的核酸(DNA)のヌクレオチド配列は5’-GGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTT[C]GT-3’(配列番号11:[C]は5-メチルシトシンを示す)、標的核酸を捕捉するための異種核酸プローブのヌクレオチド配列は5’-CACUGAACAAAUGGCACUAGUAAACUGAGCCAAAAAAAAAA-3’(配列番号12、核酸の主鎖は2’-O-メチル化RNA、5’末端はビオチン標識)であり、北海道システムサイエンス社により人工合成したものをそれぞれ使用した。異種核酸プローブは、標的核酸との異種核酸ハイブリッドが形成されたとき、異種核酸ハイブリッドの一本鎖構造部分において修飾核酸塩基[C]が存在するように設計した。
異種核酸プローブを用いた修飾核酸塩基の測定は、上述のとおり異なる標的核酸および異種核酸プローブを用いた以外は、実施例1と同様の方法で行った。
また、主鎖が2’-O-メチル化RNAである異種核酸プローブの代わりに、主鎖がDNAである同種核酸プローブ(配列番号13:5’-CACTGAACAAATGGCACTAGTAAACTGAGCCAAAAAAAAAA-3’)を用いた以外は、同様の方法で標的核酸を測定した。
以下に記載された条件で標的核酸を測定した。その他の実験条件(例、反応系の容量)は、実施例1と同様であった。
標的核酸:配列番号1のヌクレオチド配列からなる、修飾核酸塩基を含むDNA(10pmol);
核酸プローブ:配列番号3のヌクレオチド配列からなる同種核酸(DNA)プローブ(5pmol);
修飾核酸塩基に対する抗体:100ng/mLの抗メチルシトシン抗体(Millipore社製、Clone33D3)。
その結果、標的核酸の非存在下(標的核酸(-))において、抗メチルシトシン抗体または同種核酸プローブの双方の存在下では、検出シグナル値は高かったが、抗メチルシトシン抗体または同種核酸プローブのいずれかの非存在下では、検出シグナルのバックグラウンド値の低下が認められた(表6)。このことは、抗メチルシトシン抗体または同種核酸プローブの双方の存在下で計測された高い検出シグナル値がそれらの非特異的結合に起因することを示唆する。
Claims (12)
- 以下を含む、修飾核酸塩基の測定方法:
(1)核酸サンプルおよび異種核酸プローブを溶液中でインキュベートすること;ならびに
(2)(1)で得られた溶液において、修飾核酸塩基に対する抗体を用いて修飾核酸塩基を測定すること。 - 核酸サンプルが、修飾核酸塩基を含む標的核酸を含有し、かつ工程(1)および(2)がそれぞれ(1’)および(2’)により行われる、請求項1記載の方法:
(1’)修飾核酸塩基を含む標的核酸を含有する核酸サンプル、および異種核酸プローブを溶液中でインキュベートにより反応させて、当該標的核酸および異種核酸プローブから構成される異種核酸ハイブリッドを形成すること;ならびに
(2’)当該異種核酸ハイブリッドを含む溶液において、修飾核酸塩基に対する抗体を用いて修飾核酸塩基を測定すること。 - 標的核酸が、2以上の修飾核酸塩基を含む可能性がある標的核酸である、請求項1または2記載の方法。
- 前記核酸サンプルを含有する溶液に異種核酸プローブを添加して、核酸サンプルおよび異種核酸プローブの双方を含有する溶液を調製することをさらに含む、請求項1~3のいずれか一項記載の方法。
- 前記核酸サンプルが、修飾核酸塩基を含む標的DNAを含有するサンプルである、請求項1~4のいずれか一項記載の方法。
- 異種核酸プローブがRNAプローブである、請求項1~5のいずれか一項記載の方法。
- 修飾核酸塩基を構成する核酸塩基がシトシンである、請求項1~6のいずれか一項記載の方法。
- 修飾核酸塩基がメチルシトシンである、請求項1~7のいずれか一項記載の方法。
- 異種核酸ハイブリッドの二本鎖構造部分において修飾核酸塩基の不対部分が形成されるように、異種核酸プローブが設計される、請求項2~8のいずれか一項記載の方法。
- 異種核酸ハイブリッドの一本鎖構造部分において修飾核酸塩基が存在するように、異種核酸プローブが設計される、請求項2~9のいずれか一項記載の方法。
- 修飾核酸塩基に対する抗体を用いる修飾核酸塩基の測定が、ELISAにより行われる、請求項1~10のいずれか一項記載の方法。
- 以下を含む、修飾核酸塩基の測定用キット:
(I)異種核酸プローブ;および
(II)修飾核酸塩基に対する抗体。
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WO2020054782A1 (ja) | 2018-09-13 | 2020-03-19 | 国立研究開発法人国立がん研究センター | 乳癌細胞存在率の推定方法 |
Families Citing this family (1)
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004011644A1 (ja) | 2002-07-30 | 2004-02-05 | Riken | 体細胞相同組換えの促進方法及び特異的抗体の作製方法 |
JP2012230019A (ja) | 2011-04-27 | 2012-11-22 | National Institute Of Advanced Industrial & Technology | メチル化核酸検出法 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2558172B1 (fr) * | 1984-01-16 | 1986-06-13 | Inst Nat Sante Rech Med | Sonde contenant un acide nucleique modifie et reconnaissance par des anticorps specifiques et utilisation de cette sonde et de ces anticorps specifiques pour detecter et caracteriser une sequence d'adn homologue |
FR2722799B1 (fr) * | 1994-07-21 | 1996-10-04 | Parteurop | Procede d'amplification d'acide nucleique a l'aide d'un nucleoside modifie, et detection du produit d'amplification a l'aide d'anticorps |
WO2001096604A2 (en) * | 2000-06-12 | 2001-12-20 | Genicon Sciences Corporation | Assay for genetic polymorphisms using scattered light detectable labels |
JP4233807B2 (ja) * | 2002-05-31 | 2009-03-04 | 住友精密工業株式会社 | 生化学反応体の検出方法とバイオチップ |
JP3854943B2 (ja) * | 2003-05-23 | 2006-12-06 | 独立行政法人科学技術振興機構 | Dnaメチル化率の測定方法 |
EP1914320A3 (en) * | 2006-10-16 | 2009-02-11 | Epigenomics AG | A method for detection of one or more CpG positions |
JP2009247260A (ja) * | 2008-04-04 | 2009-10-29 | Sumitomo Chemical Co Ltd | Dnaメチル化測定方法 |
US20120107808A1 (en) * | 2010-10-27 | 2012-05-03 | Weiwei Li | High throughput detection of gene-specific hydroxymethylation |
-
2014
- 2014-08-20 JP JP2015532868A patent/JP6451632B2/ja not_active Expired - Fee Related
- 2014-08-20 EP EP14837506.6A patent/EP3037531A4/en not_active Withdrawn
- 2014-08-20 US US14/912,348 patent/US20160187325A1/en not_active Abandoned
- 2014-08-20 WO PCT/JP2014/071703 patent/WO2015025862A1/ja active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004011644A1 (ja) | 2002-07-30 | 2004-02-05 | Riken | 体細胞相同組換えの促進方法及び特異的抗体の作製方法 |
JP2012230019A (ja) | 2011-04-27 | 2012-11-22 | National Institute Of Advanced Industrial & Technology | メチル化核酸検出法 |
Non-Patent Citations (7)
Title |
---|
ANNEX 2, TABLE 2: MODIFIED BASE TABLE, December 2009 (2009-12-01) |
GUIDELINES FOR PREPARATION OF SPECIFICATIONS CONTAINING NUCLEOTIDE SEQUENCES OR AMINO ACID SEQUENCES, July 2002 (2002-07-01) |
KURITA ET AL., ANAL. CHEM., vol. 84, 2012, pages 7533 - 7538 |
KURITA R. ET AL.: "DNA methylation analysis triggered by bulge specific immuno-recognition.", ANAL. CHEM., vol. 84, no. 17, 2012, pages 7533 - 7538, XP055213627 * |
PROLL ET AL., DNA RESEARCH, vol. 13, 2006, pages 37 - 42 |
RYOJI KURITA: "DNA Methylation Analysis by Electrogenerated Chemiluminescence and Bulge- Specific Immuno-Recognition", KAGAKU TO MICRO NANO SYSTEM, vol. 12, no. 1, March 2013 (2013-03-01), pages 8 - 15, XP008182408 * |
ULMAN ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 1184 - 89 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016052368A1 (ja) * | 2014-09-29 | 2016-04-07 | 富士レビオ株式会社 | 修飾核酸塩基を含む標的核酸の測定方法およびキット |
WO2017217530A1 (ja) * | 2016-06-17 | 2017-12-21 | 富士レビオ株式会社 | 修飾核酸塩基を含む標的核酸の測定方法 |
WO2020054782A1 (ja) | 2018-09-13 | 2020-03-19 | 国立研究開発法人国立がん研究センター | 乳癌細胞存在率の推定方法 |
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JP6451632B2 (ja) | 2019-01-16 |
JPWO2015025862A1 (ja) | 2017-03-02 |
EP3037531A1 (en) | 2016-06-29 |
US20160187325A1 (en) | 2016-06-30 |
EP3037531A4 (en) | 2017-04-26 |
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