Method for producing a non-specific binding inhibitor of nucleic acids

Abstract

The present invention provides a non-specific binding inhibitor, etc., that exhibits less variability in the non-specific binding inhibitory effect of nucleic acids during hybridization, which is caused by differences between manufacturing lots of nucleic acids consisting of guanine base sequences. [Solution] A nonspecific binding inhibitor is produced by a method characterized by heating a solution containing nucleic acids consisting of a 4-6 guanine base sequence at 70°C to 100°C under conditions where the alkali metal ion concentration is 5 mM or less, and then cooling it to 20°C or below within 5 minutes.

Description

Technical Field

[0001] The present invention relates to a method for producing an inhibitor of non-specific binding of nucleic acids.

Background Art

[0002] A nucleic acid array is one in which nucleic acid probes having complementary base sequences to a large number of target nucleic acids are immobilized on a substrate serving as a support. By previously labeling the target nucleic acid in a sample extracted from a biological sample with a labeling molecule such as a fluorescent substance, applying it to the nucleic acid array, and allowing hybridization between the target nucleic acid and the nucleic acid probe, the expression level of the target nucleic acid can be detected as the fluorescence signal of the formed nucleic acid complex.

[0003] In order to suppress non-specific adsorption to the nucleic acid array substrate and cross-hybridization with nucleic acids having complementary strands of sequences similar to the target nucleic acid, inhibitors (blocking agents) of non-specific binding of these nucleic acids are added to the hybridization reagent. As the inhibitor of non-specific binding of nucleic acids, reagents derived from biological samples such as salmon sperm DNA and chemically synthesized nucleic acid oligos are used.

[0004] Poly-dG, which is a kind of chemically synthesized nucleic acid oligo, is a DNA composed of a single base (guanine). The non-specific binding inhibitor containing poly-dG has an inhibitory effect on non-specific binding equal to or higher than that of conventional inhibitors such as salmon sperm DNA, and since the quality is stable between production lots, the labor for its selection and preparation is unnecessary. In particular, poly-dG has been proposed as an inhibitor of non-specific binding of nucleic acids that can stably achieve an inhibitory effect on cross-hybridization in the detection of target nucleic acids by hybridization (Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] As described in Patent Document 1, in the detection of target nucleic acids by hybridization, nucleic acids consisting of guanine base sequences (hereinafter also referred to as "poly-dG") were actually added to the hybridization reagent as an inhibitor of nonspecific binding of the nucleic acid to be detected. As mentioned above, the document describes the inhibitory effect on nonspecific binding, the stability of quality between manufacturing lots, and the inhibitory effect on cross-hybridization. However, in reality, there was still variability in the inhibitory effect on nonspecific binding and the measurement results of target nucleic acid detection between manufacturing lots of the nucleic acids consisting of guanine base sequences (Comparative Example 1).

[0007] The present invention provides a nonspecific binding inhibitor that exhibits less variability in the nonspecific binding inhibitory effect of nucleic acids during hybridization due to differences between manufacturing lots of nucleic acids consisting of guanine base sequences, a method for producing the same, a hybridization reagent containing the nonspecific binding inhibitor, a hybridization method, and a method for detecting target nucleic acids. [Means for solving the problem]

[0008] The inventors of the present invention considered that the cause of this variation was due to the higher-order structure of nucleic acid sequences consisting of guanine base sequences, and investigated the manufacturing conditions for non-specific binding inhibitors. As a result, they found that the formation of higher-order structures could be suppressed by heating and cooling a solution of nucleic acid consisting of 4-6 guanine base sequences under specific conditions. Furthermore, they found that the formation of these higher-order structures could be suppressed when the alkali metal ions were below a certain concentration during the heating and cooling process. In addition, they found that the non-specific binding inhibitory effect of the non-specific binding inhibitor manufactured under these conditions was uniform regardless of the manufacturing lot of the nucleic acid consisting of guanine base sequences used as the raw material, thus completing the present invention.

[0009] In other words, the present invention consists of the following (1) to (6). (1) A method for producing a nonspecific binding inhibitor that suppresses nonspecific binding of nucleic acids in hybridization between a target nucleic acid and a nucleic acid that can specifically bind to the target nucleic acid, Step A: A solution containing nucleic acids consisting of a 4-6 guanine base sequence is heated at a temperature between 70°C and 100°C under conditions where the alkali metal ion concentration is 5 mM or less, and then cooled to 20°C or below within 5 minutes. Methods that include... (2) The method according to (1), wherein the nucleic acid consisting of the guanine base sequence is DNA. (3) A non-specific binding inhibitor manufactured by the method described in (1) or (2). (4) A hybridization reagent comprising the nonspecific binding inhibitor described in (3). (5) A method for hybridizing nucleic acids, comprising hybridizing a target nucleic acid with a nucleic acid that can specifically bind to the target nucleic acid, the method comprising coexisting with the non-specific binding inhibitor described in (3) during the hybridization. (6) A method for detecting a target nucleic acid, comprising a hybridization step of hybridizing a target nucleic acid with a nucleic acid that can specifically bind to the target nucleic acid, and a detection step of detecting the nucleic acid complex formed in the hybridization step, wherein the nonspecific binding inhibitor described in (3) is present in the hybridization step. [Effects of the Invention]

[0010] The nonspecific binding inhibitor produced by the method of the present invention is characterized by less variability in the nonspecific binding inhibitory effect of nucleic acids during hybridization, which is caused by differences between manufacturing lots of nucleic acids consisting of guanine base sequences used as raw materials. [Brief explanation of the drawing]

[0011] [Figure 1]This figure shows the results of measuring the inter-lot differences in nucleic acids consisting of guanine base sequences in solutions 1X to 1Z obtained after process A, using GPC. [Figure 2] This figure shows the results of measuring the inter-lot differences in nucleic acids consisting of guanine base sequences in solutions 2X to 2Z obtained without going through process A, using GPC. [Figure 3] This figure shows the results of electrophoresis measurements of nucleic acid solutions consisting of guanine base sequences in solutions 3X and 3Y obtained after step A, solutions 4X and 4Y obtained without step A, and solutions 5X and 5Y obtained after cooling at a rate of 1°C per minute for 91 minutes in the presence of 100 mM KCl solution. [Figure 4] This figure shows the results of measuring the lot-to-lot difference in nucleic acids consisting of guanine base sequences in solutions 4X and 4Y, which were obtained without going through step A, using GPC. [Figure 5] This figure shows the results of measuring the lot-to-lot difference in nucleic acids consisting of guanine base sequences in solutions 3X and 3Y obtained after step A, using GPC. [Figure 6] This figure shows the results of measuring lot-to-lot differences in nucleic acids consisting of guanine base sequences in solutions 5X and 5Y, which were obtained by heating in the presence of 100 mM KCl and then cooling at a rate of 1°C per minute for 91 minutes, instead of step A of the present invention, using GPC. [Figure 7] This figure shows the results of comparing solution 4X obtained through process A, solution 3X obtained without going through process A, and solution 6X obtained after heating followed by cooling at a rate of 1°C per minute for 91 minutes, using GPC. [Figure 8] This figure shows the change in GPC in the solution of the nonspecific binding inhibitor obtained when the heating time in step A of the present invention is changed. [Figure 9] This figure shows the change in GPC in the solution of the nonspecific bonding inhibitor obtained when the heating temperature in step A of the present invention is changed. [Figure 10]This is a diagram showing the change in GPC in the solution of the obtained non-specific binding inhibitor when the KCl concentration in Step A of the present invention is changed.

Embodiments for Carrying out the Invention

[0012] The present invention is a method for producing a non-specific binding inhibitor of nucleic acid, which includes a step of heating a solution containing a nucleic acid consisting of a guanine base sequence of 4 to 6 bases under the condition that the concentration of alkali metal ions is 5 mM or less at 70 °C or higher and 100 °C or lower, and then cooling it to 20 °C or lower within 5 minutes to obtain the non-specific binding inhibitor. Hereinafter, the step of cooling to 20 °C or lower within 5 minutes after heating at 70 °C or higher and 100 °C or lower is referred to as "Step A".

[0013] 1. Regarding the nucleic acid consisting of the guanine base sequence as the raw material The nucleic acid serving as the raw material of the non-specific binding inhibitor of the present invention consists of guanine bases. The base length of the nucleic acid consisting of the guanine base sequence of the present invention is 4 to 6 bases, preferably 5 bases. Specifically, it is polyguanine with a base length of 4 bases (that is, the base sequence is GGGG), polyguanine with a base length of 5 bases (that is, the base sequence is GGGGG), or polyguanine with a base length of 6 bases (that is, the base sequence is GGGGGG). The preferred nucleic acid consisting of the guanine base sequence has a base length of 5-base polyguanine (that is, the base sequence is GGGGG).

[0014] The nucleic acid consisting of the guanine base sequence of the present invention can be synthesized by a generally used nucleic acid synthesis method.

[0015] As a method for synthesizing a nucleic acid consisting of a guanine base sequence, either a solid-phase synthesis method or a liquid-phase synthesis method can be used. For example, in the solid-phase synthesis method, a modified nucleotide monomer called an amino-dite is immobilized on beads, and synthesis proceeds by adding nucleotide monomers from the 3'-end side to the 5'-end side of the oligonucleotide sequence. After all chain elongations are completed, the target nucleic acid is obtained by separating it from the solid-phase beads. In the liquid-phase synthesis method, the target nucleic acid is obtained by adding nucleotide monomers to a highly dispersed liquid-phase support or the like.

[0016] The synthesized nucleic acid consisting of a guanine base sequence may be purified by commonly used purification methods such as desalting purification, liquid-phase column purification, HPLC purification, PAGE purification, etc. and then used. Preferably, a nucleic acid purified by liquid-phase column purification, HPLC purification or PAGE purification, more preferably HPLC purification or PAGE purification, is used.

[0017] In addition, in the present invention, a commercially available nucleic acid consisting of a guanine base sequence may be used.

[0018] Examples of the nucleic acid consisting of a guanine base sequence of the present invention include DNA, RNA, artificial nucleic acids, etc. Examples of artificial nucleic acids include LNA, PNA, etc. Also, as the nucleic acid of the present invention, it is possible to use a nucleic acid synthesized by combining multiple types of nucleic acids. As the nucleic acid consisting of a guanine base sequence of the present invention, DNA or RNA is preferable, and DNA having relatively high enzyme stability is more preferable.

[0019] In the present invention, as the guanine base, either an unmodified guanine base or a modified guanine base may be used. As the guanine base of the present invention, a guanine base in which the ribose site is 2'-O-methyl ribonucleoside may be used.

[0020] Furthermore, the nucleic acid comprising the guanine base sequence of the present invention may have either its 5' end, 3' end, or both of its ends modified by any modifying group. Examples of specific modifying groups include phosphate groups, alkyl groups, alkosyl groups, amino groups, adenyl groups, biotin, thiols, halogens, fluorescent dyes, and the like.

[0021] The nucleic acid consisting of a 4-6 guanine base sequence, which is the raw material for the manufacturing method of the present invention, may be one in which the nucleic acids are bonded to each other by bonds other than covalent bonds, resulting in an apparent large molecular weight, that is, one that forms a higher-order structure. This higher-order structure is mostly eliminated by going through step A below.

[0022] 2. Regarding non-specific binding inhibitors The nonspecific binding inhibitor of the present invention refers to a nucleic acid obtained by taking a solution containing a nucleic acid consisting of a guanine base sequence as a raw material, under conditions where the alkali metal ion concentration is 5 mM or less, and proceeding through the following step A. The nonspecific binding inhibitor of the present invention is a nucleic acid consisting of a guanine base sequence as described above, in which most of the higher-order structure has been eliminated.

[0023] The higher-order structure, molecular weight, and binding state of nucleic acids of the non-specific binding inhibitor of the present invention can be analyzed by applying predetermined well-known methods. For example, the higher-order structures of nucleic acids can be compared using NMR or CD. Furthermore, the molecular weight and molecular weight distribution of the non-specific binding inhibitor of the present invention can be evaluated using size exclusion chromatography such as GPC (gel permeation chromatography) or gel electrophoresis.

[0024] 3. Regarding Process A Step A of the present invention is a step in which a solution containing nucleic acid consisting of a 4-6 guanine base sequence is heated at 70°C to 100°C under conditions where the alkali metal ion concentration is 5 mM or less, and then cooled to 20°C or below within 5 minutes.

[0025] 3-1. Heating in Process A The heating in step A of the present invention is performed on a solution containing nucleic acid consisting of a guanine base sequence of 4 to 6 bases.

[0026] The concentration of the solution subjected to the above heating is preferably 0.1 mM or more and 5 mM or less, and more preferably 0.7 mM or more and 1.3 mM or less.

[0027] The solution subjected to heating as described above is preferably an aqueous solution. In addition to the nucleic acid consisting of the guanine base sequence described above, the solution may be heated in the presence of other components such as TE (10-20 mM Tris-HCl, 1 mM EDTA·2Na), MOPS, CaCl2, NaCl, and KCl, as long as the alkali metal concentration conditions described below are met.

[0028] In step A of the present invention, the heating temperature is 70°C or higher and 100°C or lower. More preferably 70°C or higher and 98°C or lower, and even more preferably 80°C or higher and 98°C or lower.

[0029] In step A of the present invention, the heating time range is preferably 1 minute or more and 120 minutes or less, more preferably 2 minutes or more and 60 minutes or less, and even more preferably 5 minutes or more and 15 minutes or less. Here, "heating time" refers to the time during which the temperature of the solution containing the nucleic acid consisting of the guanine base sequence is maintained at the heating temperature. If the volume of the solution is 5 mL or less, the heat transfer medium set to the heating temperature and the container containing the solution may be brought into contact for the "time during which the temperature of the solution containing the nucleic acid consisting of the guanine base sequence is maintained at the heating temperature."

[0030] 3-2. Rapid cooling in process A In step A of the present invention, the cooling temperature is 20°C or lower. The preferred range of the cooling temperature is 0°C to 20°C, more preferably 0°C to 10°C, and most preferably 0°C to 5°C.

[0031] Step A of the present invention is characterized by cooling to 20°C or below within 5 minutes after the above heating. Here, "cooling to 20°C or below within 5 minutes after heating" means that the time required to change the temperature of the solution containing the nucleic acid consisting of the guanine base sequence from the heating temperature to the cooling temperature is 5 minutes or less. If the volume of the solution is 5 mL or less, the container containing the heated solution may be brought into contact with a refrigerant set to the cooling temperature for the "time required to change the temperature of the solution itself from the heating temperature to the cooling temperature". The cooling time range is preferably 1 second to 5 minutes.

[0032] 3-3. Concentration of alkali metal ions in process A Step A of the present invention is carried out under conditions where the concentration of alkali metal ions is 5 mM or less. In carrying out the present invention, it may be necessary to carry out the process under conditions where alkali metal ions originating from components such as buffers are present, but in this case, it is acceptable as long as the concentration of the alkali metal ions is as described above. It is preferable to carry out Step A of the present invention in the absence of alkali metal ions.

[0033] The alkali metal ion concentration referred to here is the sum of the concentrations of alkali metal ions when step A is performed in the presence of multiple alkali metal ions. A preferred alkali metal ion concentration is 1 mM or less.

[0034] In the present invention, alkali metals refer to lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and / or francium (Fr). Alkali metal ions may be included in the solution containing nucleic acids consisting of a guanine base sequence in step A, for example, as salts of sodium, potassium, etc., such as potassium chloride, potassium iodide, potassium acetate, potassium sulfate, sodium chloride, sodium iodide, sodium acetate, and sodium sulfate, as long as they are within the above concentration range.

[0035] The nonspecific binding inhibitor of the present invention can be prepared by performing step A in an aqueous buffer solution having a predetermined salt concentration and optionally containing a surfactant, as long as the above alkali metal concentration conditions are met. For example, within the range of alkali metal concentration conditions, aqueous solutions containing TE (10-20 mM Tris-HCl, 1 mM EDTA·2Na), as well as MOPS and CaCl2, NaCl, KCl, etc., can be used.

[0036] 4. About Hybridization Reagents In the present invention, "hybridization reagent" refers to a reagent containing the above-mentioned nonspecific binding inhibitor, which is used in conjunction with a target nucleic acid and a nucleic acid that can specifically bind to the target nucleic acid.

[0037] The hybridization reagent of the present invention may be a solution or a solid. Preferably, the hybridization reagent of the present invention is a solution. Alternatively, the hybridization reagent of the present invention may be a kit containing a solution, solid, etc., containing the nonspecific binding inhibitor of the present invention, together with other reagents.

[0038] If the hybridization reagent is a solution, the solution containing the nonspecific binding inhibitor obtained through step A above may be used as the hybridization reagent as is, or components other than the nonspecific binding inhibitor may be added to make it the hybridization reagent.

[0039] When the hybridization reagent is a solution, the concentration of nucleic acid contained in the solution can be appropriately selected depending on the purpose and method of use. However, assuming that the nucleic acid consisting of guanine base sequences does not form any higher-order structure, a concentration of 0.3 μM to 100 μM is preferred. Since high-concentration nucleic acid solutions are viscous, from the viewpoint of manufacturing control, it is more preferable that the concentration of the nucleic acid be between 0.3 μM and 30 μM.

[0040] If the hybridization reagent is a solid, the solution containing the nonspecific bonding inhibitor obtained through step A above may be used as the hybridization reagent after removing the solvent. Examples of the form after solvent removal include powder, gel, etc. Alternatively, a hybridization solution may be obtained by adding components other than the nonspecific bonding inhibitor to the solution and then removing the solvent. Alternatively, a hybridization solution may be obtained by adding another component to the dried composition obtained by these methods. The hybridization reagent may also be in the form of a semi-solid gel composition.

[0041] The hybridization reagent of the present invention can employ a well-known composition, except that it contains the non-specific binding inhibitor of the present invention. The composition of the hybridization reagent other than the non-specific binding inhibitor of the present invention will be described below.

[0042] The hybridization reagent of the present invention may contain a polysaccharide. A preferred polysaccharide may be sodium dextran sulfate.

[0043] The hybridization reagent of the present invention may contain a denaturing agent. For example, it may contain formamide or dimethyl sulfoxide. The concentration of formamide is preferably 3% to 25% by volume, and the concentration of dimethyl sulfoxide is preferably 3% to 10% by volume.

[0044] The hybridization reagent of the present invention may include an inhibitor of nonspecific adsorption to proteins. Bovine serum albumin (BSA) is preferably used as the inhibitor. BSA is preferably used in a concentration of 5 mg / ml to 20 mg / ml.

[0045] The hybridization reagent of the present invention may contain substances useful for increasing the sensitivity of the hybridization reaction. For example, since bubbles generated during the hybridization reaction may physically inhibit the hybridization reaction, the reagent may contain an antifoaming agent or the like to suppress the generation of bubbles. Examples of antifoaming agents include surfactants such as Tween20, Tween60, Triton X-100, or silicone oil. The antifoaming agent in the hybridization reagent of the present invention can be used in an amount of, for example, 0.01% by mass or more and 1% by mass or less.

[0046] If the hybridization reagent of the present invention is a solution, the hybridization reagent may be prepared by performing step A of the present invention on a solution containing a nucleic acid consisting of the guanine base sequence of the present invention and the above-mentioned components other than the nucleic acid.

[0047] As a specific example of preparation, for instance, the composition described in "Ku WC. et al., Biochemical and Biophysical Research Communications, 2004, Vol. 315, No. 1, pp. 30-37" can be used as a reference, and the preparation can be carried out by performing step A of the present invention on "6xSSPE, 0.1% SDS, 25% formamide, 10 mg / mL BSA, 10% dextran sulfate sodium, 15uM poly-dG5". Here, "poly-dG5" refers to a nucleic acid consisting of a 5-base guanine sequence.

[0048] In the hybridization method of the present invention, the method of coexisting with a nonspecific binding inhibitor includes the method of coexisting with a hybridization reagent containing the nonspecific binding inhibitor of the present invention.

[0049] 5. Effects of non-specific binding inhibitors and hybridization reagents The nonspecific binding inhibitor or hybridization reagent of the present invention exhibits the ability to suppress nonspecific binding when coexisted in the above hybridization process. The nonspecific binding inhibitor and hybridization reagent of the present invention are characterized by small variation in the aforementioned ability due to differences in manufacturing lots of nucleic acids consisting of guanine base sequences.

[0050] In this specification, a nucleic acid probe refers to a nucleic acid that specifically binds to a target nucleic acid and has a base sequence complementary to the target nucleic acid.

[0051] All binding reactions other than hybridization (specific binding) are called non-specific binding of nucleic acids. Examples of non-specific binding of nucleic acids include cross-hybridization that occurs between the target nucleic acid and a nucleic acid having a complementary strand with a similar sequence, when using a support (substrate, etc.) on which a nucleic acid probe used to detect a target nucleic acid is immobilized, such as in a nucleic acid array, or adsorption of the target nucleic acid (physical adsorption by van der Waals forces, chemisorption by hydrophobic interactions) onto the material of the support on which the nucleic acid having a complementary sequence to the target nucleic acid is immobilized.

[0052] 6. Hybridization Methods Aspects of the present invention include a method for hybridizing a nucleic acid, comprising hybridizing a target nucleic acid with a nucleic acid that can specifically bind to the target nucleic acid, the method comprising coexisting with the above-mentioned nonspecific binding inhibitor during the hybridization.

[0053] The hybridization method of the present invention can be carried out by known methods, except for the coexistence of the nonspecific binding inhibitor of the present invention. For example, the method of coexisting the nonspecific binding inhibitor of the present invention can be used in the hybridization step of the method described in "Sambrook, J. et al (1998) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York". Specifically, hybridization can be performed by adding the target nucleic acid and the nonspecific binding inhibitor of the present invention to a support on which a complementary nucleic acid probe has been immobilized in advance, and incubating at a constant temperature. Alternatively, it is also possible to perform hybridization by mixing the target nucleic acid, the nucleic acid probe, and the nonspecific binding inhibitor of the present invention, and incubating at a constant temperature, and then immobilizing the nucleic acid probe on the support.

[0054] In the hybridization method of the present invention, the concentration of the non-specific binding inhibitor of the present invention can be determined according to the detection method. The concentration is preferably 0.3 μM to 30 μM, calculated assuming that the nucleic acid consisting of the guanine base sequence does not form any higher-order structure.

[0055] In the hybridization method described above, the specificity of nucleic acid binding can be further improved by thermally denaturing the target nucleic acid at a high temperature for a certain period of time before adding the non-specific binding inhibitor or hybridization reagent. Alternatively, the target nucleic acid, complementary nucleic acid probe, and the non-specific binding inhibitor or hybridization reagent of the present invention can be mixed, incubated at a constant temperature to perform hybridization, and then the complementary nucleic acid probe can be immobilized on a support.

[0056] Examples of target nucleic acids include DNA and RNA. Examples of DNA include double-stranded DNA (including genomic DNA), single-stranded DNA, cDNA, and synthetic DNA. Examples of RNA include total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, or non-coding RNA, their precursors, or synthetic RNA.

[0057] 7. Methods for detecting target nucleic acids Aspects of the present invention include a method for detecting a target nucleic acid, comprising the steps of performing the above-described hybridization and detecting the nucleic acid complex formed in the hybridization step, wherein the method includes coexisting with the non-specific binding inhibitor in the hybridization step.

[0058] The hybridization step in the method for detecting target nucleic acids of the present invention is a step of hybridizing the target nucleic acid and the nucleic acid probe using the hybridization method described above, which uses the non-specific binding inhibitor of the present invention.

[0059] The nucleic acid complex detection step in the target nucleic acid detection method of the present invention can be carried out by applying well-known methods. Examples include absorbance measurement, fluorescence intensity measurement, luminescence measurement, electrophoresis, PCR, RT-PCR, sequencing analysis, and combinations thereof. Furthermore, by pre-labeling the target nucleic acid with a labeling molecule using hybridization, the labeled molecular weight can be detected as the gene expression level. Specifically, this can be carried out using known methods such as in situ hybridization, colony hybridization, dot blotting, southern blotting, and northern blotting, as well as using nucleic acid arrays (DNA microarrays, DNA chips, etc.) in which nucleic acids that specifically bind to the target nucleic acid and have a base sequence complementary to the target nucleic acid (nucleic acid probes) are immobilized on a support.

[0060] For nucleic acid arrays, commercially available ones can be used. Examples include Affymetric's "GeneChip® Arrays," Agilent Technologies' OligoDNA Chip, and Toray's "3D-Gene®." However, it is preferable to use Toray's "3D-Gene®" because its support is made of resin, resulting in less physical adsorption of nucleic acids. [Examples]

[0061] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0062] (Evaluation of molecular weight distribution of non-specific binding inhibitor solutions) The molecular weight distribution of the non-specific binding inhibitor solution was analyzed by GPC or electrophoresis. The conditions for GPC are shown.

[0063] Equipment configuration: JASCO GPC / SEC system (Pump PU-4185, Column oven CO-4060, UV detector UV-4070, system LC-NetII / ADC) Column: TSKgel UltraSW Aggregate, 3um, 30cm Analysis conditions: Mobile phase PBS, 30°C, flow rate 1 mL / min, detection at 256 nm.

[0064] (Preparation of RNA derived from the subject) Using 300 μL of serum collected from healthy individuals who had given informed consent, RNA derived from the subjects was extracted and purified using a prescribed method.

[0065] (Hybridization reagent) In the examples and comparative examples, the hybridization reagents were prepared as follows: The solutions of the respective non-specific binding inhibitors prepared in each example and comparative example were added to the HB Buffer mix to prepare hybridization reagents such that the final concentration of nucleic acids consisting of guanine base sequences was 15 μM. These reagents were then used for hybridization in the "Measurement of Gene Expression Levels" described later.

[0066] (Measurement of gene expression levels) In the following examples and comparative examples, the expression levels of two types of microRNAs (hsa-miR-4665-5p and hsa-miR-665) were measured using the following method.

[0067] An RNA solution obtained from 300 μL of serum was fluorescently labeled with miRNA using the 3D-Gene® miRNA Labeling kit (Toray Industries, Inc.) according to the protocol (ver2.20) specified by the company. As the oligo DNA chip, the 3D-Gene® Human miRNA Oligo chip (Toray Industries, Inc.), equipped with probes having sequences complementary to 2,632 types of miRNAs registered in miRBase release 22, was used. Hybridization and post-hybridization washing were performed under stringent conditions according to the protocol specified by the company, except that the hybridization reagents mentioned above were used instead of "miRNA Hybridization buffer V3" in the protocol. The DNA chip was scanned using the 3D-Gene® scanner (Toray Industries, Inc.), images were acquired, and the fluorescence intensity was quantified using 3D-Gene® Extraction (Toray Industries, Inc.). The quantified fluorescence intensity was converted to a base-2 logarithmic value to represent the gene expression level, and a blank value was subtracted. As a result, comprehensive miRNA gene expression levels were obtained for 300 μL of serum. Calculations and statistical analyses using the quantified miRNA gene expression levels were performed using R language 4.0.3 (R Development Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, URL https: / / www.r-project.org / ).

[0068] (Example 1) As nucleic acids consisting of guanine base sequences, DNA consisting of a 5-base guanine sequence (commissioned synthesis by Fasmac, HPLC purified grade, synthetic sequence: GGGGG, hereinafter the same) was dissolved in TE buffer (Nacalai Tesque) to a final concentration of 1 mM for each manufacturing lot (lot X, lot Y, lot Z). 100 μL of each nucleic acid solution consisting of a guanine base sequence, dispensed into 1.5 ml tubes, was placed on a heat block (Shibata Chemical) heated to 95°C and heated for 5 minutes. After heating, the 1.5 ml tubes were transferred to an aluminum block on ice and cooled for 5 minutes to cool the nucleic acid solutions consisting of the guanine base sequence to 4°C. In this way, solutions of the non-specific binding inhibitor were obtained. The manufacturing conditions for the above non-specific binding inhibitor solutions are shown in Table 1. Hereinafter, the solutions obtained here will be referred to as Solution 1X from lot X, Solution 1Y from lot Y, and Solution 1Z from lot Z. GPC analysis of all solutions 1X to 1Z showed that the peak shifted towards the lower molecular weight side (longer elution time) compared to the unheated sample (Comparative Example 1), and no difference in molecular weight distribution was observed between manufacturing lots. In other words, it was considered that the higher-order structure of nucleic acids consisting of guanine base sequences was eliminated by step A of the present invention, and molecules that eluted in about 9 minutes became the main components. The results are shown in Figure 1. Hybridization reagents were prepared for each of solutions 1X to 1Z using the method described above, applied to the 3D-Gene chip, and the hybridization and detection methods described above were performed. When the gene expression levels were measured, the difference in the maximum signal intensity of microRNA between manufacturing lots in solutions 1X to 1Z obtained after heating and rapid cooling was 0.03. Table 2 shows the difference in signal intensity between manufacturing lots of miR-665 and miR-4665-5p among the measured microRNAs.

[0069] [Table 1]

[0070] [Table 2] (Comparative Example 1) Solutions of the nonspecific binding inhibitor were prepared for each of the lots X to Z under the same conditions as in Example 1, except that heating with a heat block and cooling for 5 minutes on an aluminum block on ice were not performed. The manufacturing conditions for the above nonspecific binding inhibitor solutions are shown in Table 1. Hereinafter, the solutions obtained here will be referred to as Solution 2X from Lot X, Solution 2Y from Lot Y, and Solution 2Z from Lot Z. GPC analysis revealed high molecular weight components, and differences in molecular weight distribution were observed for each manufacturing lot. The results are shown in Figure 2.

[0071] Hybridization reagents were prepared for each of the solutions 2X to 2Z using the method described above, and applied to the 3D-Gene chip to perform the hybridization and detection methods described above. The results of the gene expression measurements showed that the difference in maximum microRNA signal intensity between manufacturing lots for solutions 2X to 2Z was 0.16 (logarithmic scale), confirming that the amount of each microRNA differed slightly from lot to lot. Table 3 shows the difference in signal intensity between manufacturing lots for miR-665 and miR-4665-5p among the measured microRNAs.

[0072] [Table 3] (Comparison between Example 1 and Comparative Example 1) Based on Example 1 and Comparative Example 1 described above, we compared the differences between manufacturing lots of nucleic acids consisting of guanine base sequences.

[0073] In the solution of the non-specific binding inhibitor produced by the method following step A of the present invention, almost no higher-order structures were observed to elute within 7 minutes, and most of the solution consisted of nucleic acids consisting of guanine base sequences that did not form higher-order structures and eluted around 9 minutes. On the other hand, in the solution of the non-specific binding inhibitor produced without following step A, it was found that the fraction mainly consisted of higher-order structures that eluted within 7 minutes.

[0074] Furthermore, it was found that the solution of the nonspecific binding inhibitor produced by the method following step A of the present invention showed smaller spectral differences between production lots of nucleic acids consisting of guanine base sequences, as observed by GPC analysis, compared to the solution of the nonspecific binding inhibitor produced without following step A.

[0075] Furthermore, the hybridization reagent produced by the method following step A of the present invention showed a smaller difference in fluorescence intensity signal intensity in the measurement results of RNA by hybridization compared to the hybridization reagent produced without following step A.

[0076] (Example 2) As nucleic acids consisting of guanine base sequences, solutions of a non-specific binding inhibitor were prepared using the same procedure as in Example 1 for Lot X and Lot Y of DNA consisting of a 5-base guanine sequence (commissioned synthesis by Fasmac). The manufacturing conditions for the above non-specific binding inhibitor solutions are shown in Table 1. Hereinafter, the solutions obtained here will be referred to as Solution 3X from Lot X and Solution 3Y from Lot Y. Electrophoresis revealed that over 80% were low-molecular-weight molecules of 20mer or less. The results are shown in Figure 3. Furthermore, GPC analysis showed that the peak shifted to the low molecular weight side (longer elution time), and no difference in molecular weight distribution was observed between manufacturing lots. That is, it was considered that the higher-order structure of the nucleic acids consisting of guanine base sequences was eliminated by step A of the present invention, and molecules that eluted in about 9 minutes became the main components. The results are shown in Figure 5. Hybridization reagents were prepared using Solution 3X and Solution 3Y, applied to a 3D-Gene chip, and the above hybridization method and detection method were performed. As a result of measuring gene expression levels, the difference in maximum signal intensity of microRNA between manufacturing lots in heated and rapidly cooled solutions 3X and 3Y was 0.04 (logarithmic value). Table 4 shows the difference in signal intensity (absolute value) between manufacturing lots of miR-665 and miR-4665-5p among the measured microRNAs.

[0077] [Table 4] (Comparative Example 2) A solution of the non-specific binding inhibitor was prepared under the same conditions as in Example 2, except that heating with a heat block and cooling for 5 minutes on an aluminum block on ice were not performed. The manufacturing conditions for the above non-specific binding inhibitor solution are shown in Table 1. Hereinafter, the solutions obtained here will be referred to as Solution 4X from Lot X and Solution 4Y from Lot Y. Electrophoresis revealed high molecular weight components with a molecular weight of 100mer or more. The results are shown in Figure 3. Furthermore, GPC analysis showed a main peak on the high molecular weight side (elution time of 7 minutes or less), and differences in molecular weight distribution were observed between manufacturing lots. That is, it was considered that nucleic acids consisting of guanine base sequences remained in their higher-order structures. The results are shown in Figure 4. Hybridization reagents were prepared using each solution and applied to a 3D-Gene chip to perform the above hybridization and detection methods. When gene expression levels were measured, the difference in the maximum signal intensity of microRNA between manufacturing lots for Solution 4X and Solution 4Y was 0.1 (logarithmic value), confirming that the amount of each microRNA differed for each manufacturing lot. Table 5 shows the difference in signal intensity (absolute value) between manufacturing lots of miR-665 and miR-4665-5p among the measured microRNAs.

[0078] [Table 5] (Comparative Example 3) As nucleic acids consisting of guanine base sequences, DNA consisting of a 5-base guanine sequence (commissioned synthesis by Fasmac Corporation) was dissolved in TE buffer (Nacalai Tesque) to a final concentration of 1 mM for each manufacturing lot (Lot X, Lot Y), and KCl was added to bring the concentration to 100 mM, which is a condition that facilitates the formation of guanine quadruplexes. 100 μL of each nucleic acid solution consisting of guanine base sequences was placed in a heat block (Shibata Chemical) heated to 95°C and heated for 5 minutes. After heating, it was slowly cooled to 4°C at a rate of 1°C per minute for 91 minutes to obtain a solution of the non-specific binding inhibitor. The manufacturing conditions for the above non-specific binding inhibitor solution are shown in Table 1. Hereafter, the solutions obtained here will be referred to as Solution 5X from Lot X and Solution 5Y from Lot Y. Electrophoresis revealed high molecular weight components with a molecular weight of 100mer or more. The results are shown in Figure 3. Furthermore, GPC analysis showed that the main peak was on the high molecular weight side (elution time of 7 minutes or less), and differences in molecular weight distribution were observed for each manufacturing lot. In other words, it was considered that the nucleic acids consisting of guanine base sequences remained in their higher-order structure. The results are shown in Figure 6. Hybridization reagents were prepared using solution 5X and solution 5Y, applied to the 3D-Gene chip, and the hybridization and detection methods described above were performed. When the gene expression levels were measured, the difference in the maximum signal intensity of microRNA between manufacturing lots for heated and rapidly cooled solution 5X and solution 5Y was 0.16. Table 6 shows the difference in signal intensity (absolute value) between manufacturing lots of miR-665 and miR-4665-5p among the measured microRNAs.

[0079] [Table 6] (Comparison of Example 2, Comparative Example 2, and Comparative Example 3) For Example 2, Comparative Example 2, and Comparative Example 3 described above, the apparent molecular weight distribution of the non-specific binding inhibitor solutions obtained in each example and comparative example was confirmed by GPC and electrophoresis. In the non-specific binding inhibitor solution produced by the method following step A of the present invention, almost no higher-order structures eluted within 7 minutes were observed, and most of the solution consisted of nucleic acids made of guanine base sequences that did not form higher-order structures and eluted around 9 minutes. In contrast, in the non-specific binding inhibitor solution produced without following step A, and the non-specific binding inhibitor solution produced by heating in the presence of 100 mM KCl and then slowly cooling at a rate of 1°C per minute for 91 minutes, it was found that the main component was a fraction of higher-order structures that eluted within 7 minutes.

[0080] Furthermore, it was found that the non-specific binding inhibitor solution produced by the method following step A of the present invention showed smaller spectral differences between production lots of nucleic acids consisting of guanine base sequences, as observed by GPC analysis, compared to the non-specific binding inhibitor solution produced without following step A and the non-specific binding inhibitor solution produced by heating in the presence of 100 mM KCl and then slowly cooling at a rate of 1°C per minute for 91 minutes.

[0081] Furthermore, the hybridization reagent produced by the method following step A of the present invention showed a smaller difference in fluorescence intensity signal intensity in the RNA measurement results by hybridization compared to a hybridization reagent produced without following step A and a hybridization reagent produced by heating in the presence of 100 mM KCl and then slowly cooling at a rate of 1°C per minute for 91 minutes.

[0082] (Comparative Example 4) A solution of the nonspecific binding inhibitor was prepared under the same conditions as in Example 2, except that instead of cooling on an aluminum block on ice for 5 minutes, it was slowly cooled to 4°C at a rate of 1°C per minute for 91 minutes. The production conditions for the above nonspecific binding inhibitor solution are shown in Table 7. Hereinafter, the solutions obtained here will be referred to as Solution 6X if derived from Lot X, and Solution 6Y if derived from Lot Y.

[0083] [Table 7] (Comparison of Example 2, Comparative Example 2, and Comparative Example 4) GPC analysis of solution 4X (Comparative Example 2), solution 3X (Example 2), and solution 6X (Comparative Example 4) revealed that solution 4X, which did not undergo step A, and solution 6X, which was heated and then slowly cooled at a rate of 1°C per minute for 91 minutes, shifted towards the high molecular weight side (shorter elution time), meaning that nucleic acids consisting of guanine base sequences forming higher-order structures were the main components. In contrast, solution 3X, which underwent step A, shifted towards the low molecular weight side (longer elution time), eluting in approximately 9 minutes, and was observed to mainly consist of nucleic acids consisting of guanine base sequences that do not form higher-order structures. The results are shown in Figure 7. The GPC analysis spectrum of solution 6X obtained in Comparative Example 4 is shifted towards the high molecular weight side, suggesting that it cannot exhibit a stable inhibitory effect on nonspecific binding.

[0084] (Comparative Example 5, Example 3, Example 4, Example 5, Example 6) In Comparative Example 5, Example 3, Example 4, Example 5, and Example 6, the effect of heating time on the apparent molecular weight distribution of the nonspecific bonding inhibitor solution was investigated.

[0085] A solution of the nonspecific binding inhibitor was prepared under the same conditions as in Example 1, except for the presence or absence of heating by a heat block and the heating time. In Comparative Example 5, no heating was performed, while in Examples 3, 4, 5, and 6, heating was performed for 1 minute, 3 minutes, 5 minutes, and 10 minutes, respectively. The production conditions for the above nonspecific binding inhibitor solutions are shown in Table 7. The molecular weight distribution of each obtained solution was compared by GPC analysis. As a result, in the case without heating (Comparative Example 5), high molecular weight substances that eluted within 7 minutes, i.e., nucleic acids consisting of guanine base sequences that form higher-order structures, remained. However, when heated at 90°C for 1 minute (Example 3), 3 minutes (Example 4), 5 minutes (Example 5), or 10 minutes (Example 6), the high molecular weight substances almost disappeared in all cases, and nucleic acids consisting of guanine base sequences that do not form higher-order structures, which eluted around 9 minutes, were the main component. The results are shown in Figure 8. From this, it was considered that the nonspecific binding inhibitor solution obtained by heating at a temperature of 70°C or higher has a stable nonspecific binding inhibitory effect, similar to Examples 1 and 2.

[0086] (Comparative Example 6, Comparative Example 7, Example 7, Example 8, Example 9, Example 10) In Comparative Examples 6, 7, 7, 8, 9, and 10, the effect of heating temperature on the apparent molecular weight distribution of the nonspecific bonding inhibitor solution was investigated.

[0087] Solutions of nonspecific binding inhibitors were prepared under the same conditions as in Example 1, except for the presence or absence of heating by a heat block and the heating time. The production conditions for the above nonspecific binding inhibitor solutions are shown in Table 7. In each case, no heating was performed (Comparative Example 6), heating at 60°C for 5 minutes (Comparative Example 7), heating at 70°C for 5 minutes (Example 7), heating at 80°C for 5 minutes (Example 8), heating at 90°C for 5 minutes (Example 9), and heating at 98°C for 5 minutes (Example 10), followed by cooling on an aluminum block on ice. The obtained solutions of each nonspecific binding inhibitor were subjected to GPC analysis, and the molecular weight distribution was compared. As a result, in the case of no heating and heating temperatures below 60°C, high molecular weight substances, i.e., nucleic acids consisting of guanine base sequences that form higher-order structures, remained, but in all cases at heating temperatures above 70°C, the high molecular weight substances disappeared, and nucleic acids consisting of guanine base sequences that do not form higher-order structures, which eluted in about 9 minutes, were the main component. The results are shown in Figure 9. From this, it is considered that the solution of the nonspecific bonding inhibitor obtained by heating to a temperature of 70°C or higher has a stable nonspecific bonding inhibitory effect, similar to that of Examples 1 and 2.

[0088] (Comparative Example 9, Comparative Example 10, Example 11, Example 12) In Comparative Examples 9, 10, 11, and 12, the effect of alkali metal ion concentration in step A on the apparent molecular weight distribution in the nonspecific bonding inhibitor solution was investigated.

[0089] Nucleic acid solutions consisting of guanine base sequences were prepared under the same conditions as in Example 1, except that KCl was added at the following concentrations before heating. The production conditions for the above guanine base sequence nucleic acid solutions are shown in Table 7. KCl was added to each solution to a concentration of 0 mM (Example 1), 1 mM (Example 11), 10 mM (Comparative Example 9), and 100 mM (Comparative Example 10), and then the heating and subsequent steps were carried out. The molecular weight distribution of each obtained guanine base sequence nucleic acid solution was compared by GPC analysis. As a result, in the cases without heating and with KCl concentrations of 10 mM and 100 mM, high molecular weight molecules remained, i.e., nucleic acids consisting of guanine base sequences that form higher-order structures remained. However, in the cases of 0 mM and 1 mM, the high molecular weight molecules disappeared, and the main component was nucleic acid consisting of guanine base sequences that do not form higher-order structures, which eluted in about 9 minutes. The results are shown in Figure 10. From these results, it was concluded that if the alkali metal ion concentration is 5 mM or less, it does not hinder the disappearance of high molecular weight molecules, and that the nucleic acid solution consisting of guanine base sequences obtained under these conditions has a stable non-specific binding inhibitory effect, similar to that of Examples 1 and 2.

Claims

1. A method for producing a nonspecific binding inhibitor that suppresses nonspecific binding of nucleic acids in hybridization between a target nucleic acid and a nucleic acid that can specifically bind to the target nucleic acid, Step A: A step in which a solution containing nucleic acids consisting of a 4-6 guanine base sequence is heated at a temperature between 70°C and 100°C under conditions where the alkali metal ion concentration is 5 mM or less, and then cooled to 20°C or below within 5 minutes. Methods that include...

2. The method according to claim 1, wherein the nucleic acid consisting of the guanine base sequence is DNA.

3. A non-specific binding inhibitor manufactured by the method described in claim 1 or 2.

4. A hybridization reagent comprising the nonspecific binding inhibitor described in claim 3.

5. A method for hybridizing nucleic acids, comprising hybridizing a target nucleic acid with a nucleic acid that can specifically bind to the target nucleic acid, the method comprising coexisting with the non-specific binding inhibitor described in claim 3 during the hybridization.

6. A method for detecting a target nucleic acid, comprising a hybridization step of hybridizing a target nucleic acid with a nucleic acid that can specifically bind to the target nucleic acid, and a detection step of detecting the nucleic acid complex formed in the hybridization step, wherein the hybridization step includes the coexistence of the nonspecific binding inhibitor described in claim 3.

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