JP5429039B2 - RNA degradation method by acid hydrolysis and RNA sequence analysis method using the same - Google Patents

Description

  The present invention relates to the field of molecular biology, particularly the field of nucleic acid medicine. More specifically, the present invention relates to a ribonucleic acid degradation method and a ribonucleic acid sequence analysis method.

As examples of nucleic acid base sequence determination methods, a sequencer using the Sanger method and a method using a high-speed decoding device collectively referred to as a next-generation type sequencer are known.
As another example of a method for determining the base sequence of a nucleic acid, a method of chemically decomposing a phosphodiester bond with dimethyl sulfate or hydrazine in a base-specific manner is also known.

  Anal. Chem. 2009, 81, 3173-3179 (Non-Patent Document 1), RNA was subjected to acid hydrolysis using a trifluoroacetic acid aqueous solution in a reaction tube, and then 3-hydroxypicolinic acid and ACDB (quenched) were used. A method for determining the base sequence of RNA by mass spectrometry using diammonium acid) is disclosed.

  Japanese Patent Application Laid-Open No. 2008-292422 (Patent Document 1) discloses that 3-hydroxypicolinic acid and ACDB were added to RNA on a stainless steel plate and then subjected to MALDI mass spectrometry.

JP 2008-292422 A

Analytical Chemistry, 2009, Vol. 81, p. 3173-3179

  In the Sanger method, an enzymatic reaction is used for nucleic acid degradation. Since the enzymatic reaction depends on the base sequence, it cannot be decomposed depending on the sequence to be analyzed. In the sequence analysis method using such an enzyme reaction, there is a problem that the sequence analysis cannot be performed depending on the sequence to be analyzed.

  In addition, the above-described chemical decomposition method has problems that a large amount of sample is required and that a very dangerous reagent may be used.

  Furthermore, in the methods described in Patent Document 1 and Non-Patent Document 1, sufficient analysis sensitivity may not be obtained.

  Accordingly, an object of the present invention is to provide a method capable of easily and efficiently degrading RNA, and a method capable of easily analyzing RNA sequences with high analytical sensitivity.

  As a result of intensive studies, the inventor has found that the object of the present invention can be achieved by coexisting hydroxypicolinic acid in an RNA acid hydrolysis reaction system using an aqueous trifluoroacetic acid solution. The invention has been completed.

The present invention includes the following inventions.
The following (1) and (2) are directed to the RNA degradation method, and the following (3) to (6) are directed to the RNA sequence analysis method.

(1) A method for degrading RNA, comprising a step of performing an acid hydrolysis reaction of RNA using trifluoroacetic acid in the presence of hydroxypicolinic acid to obtain a reaction mixture containing the RNA-derived fragment.

(2) The method according to (1), wherein the concentration of the trifluoroacetic acid in the acid hydrolysis reaction system is 1.25 to 3% (v / v).

(3) performing an acid hydrolysis reaction of RNA using trifluoroacetic acid in the presence of hydroxypicolinic acid to obtain a reaction mixture containing the RNA-derived fragment;
Subjecting the reaction mixture to mass spectrometry, and determining the RNA sequence.

(4) The method according to (3), wherein the concentration of the trifluoroacetic acid in the acid hydrolysis reaction system is 1.25 to 3% (v / v).

(5) The method according to (3) or (4), wherein the mass spectrometry is performed by a MALDI mass spectrometer.
In the method (5), a matrix additive can be added to the reaction mixture when subjected to mass spectrometry.

(6) The method according to any one of (3) to (5), wherein the acid hydrolysis reaction and the mass spectrometry are performed on the same plate for mass spectrometry.
(7) The method according to any one of (3) to (6), wherein the RNA has a length of 15 to 25 bases.

  According to the present invention, there are provided a method capable of easily and efficiently degrading RNA and a method capable of analyzing RNA sequences simply and with high analytical sensitivity.

In Example 1, a mass spectrum obtained by measuring MALDI-TOF MS of an RNA sample treated by the method of the present invention (ie, using a reagent solution containing trifluoroacetic acid and 3-hydroxypicolinic acid) (a And RNA sequence analysis results (b). In Example 1, the mass spectrum (a) obtained for the RNA sample treated with the reagent solution containing trifluoroacetic acid and 3-hydroxypicolinic acid, and in Comparative Example 1, containing 3-trifluoroacetic acid and 3-hydroxy It is a mass spectrum (b) obtained about the RNA sample processed using the reagent solution which does not contain picolinic acid. In Example 2, it is the mass spectrum obtained in each when the density | concentration of trifluoroacetic acid was changed. In Example 3, it is the mass spectrum obtained in each when the density | concentration of trifluoroacetic acid was changed.

[1. Acid hydrolysis step]
[1-1. RNA]
The RNA (ribonucleic acid) targeted in the present invention is not particularly limited as long as it basically has adenine (A), guanine (G), cytosine (C), and uracil (U) as nucleobases. Absent. Nucleobases are also allowed to be accompanied by changes such as modification. In addition, RNA may be single-stranded or double-stranded, but is preferably single-stranded from the viewpoint of ease of sequence analysis.

  In the present invention, it is preferably a low molecular weight RNA. For example, what has a length of 15-25 bases is mentioned. In particular, since the present invention is useful when performing siRNA sequence analysis, it is preferable that the RNA has a length of 20 to 25 bases or 21 to 23 bases that a general siRNA has. .

  The scale of RNA used in the present invention is not particularly limited. In particular, since the present invention is useful when handling a small amount of RNA, the RNA is preferably at a picomolar level, for example, about 5 to 20 picomolar. If it is a scale of this grade, the process in this invention can be easily implemented on a plate.

[1-2. Hydroxypicolinic acid]
In the present invention, hydroxypicolinic acid is used in the acid hydrolysis of RNA. Hydroxypicolinic acid can include all of the regioisomers that can exist, but 3-hydroxypicolinic acid is particularly preferably used in the present invention. 3-hydroxypicolinic acid is usually used as a matrix for mass spectrometry of nucleic acids.

  The amount of hydroxypicolinic acid used can be, for example, 100 to 10,000 times, preferably 500 to 5000 times (on a molar basis) relative to the amount of RNA. Outside the above range, sufficient acid hydrolysis reaction is unlikely to occur, and ionization tends to be inhibited during mass spectrometry.

[1-3. Trifluoroacetic acid]
The trifluoroacetic acid used in the present invention is used as a reagent for RNA acid hydrolysis. Therefore, it is used at a concentration that allows acid hydrolysis of RNA. The concentration that enables acid hydrolysis may be determined in view of the RNA chain length. For example, when processing shorter RNA lengths, relatively low concentrations are acceptable. Moreover, when processing RNA having a longer chain length, a relatively high concentration tends to be preferable.

  Specifically, the concentration of trifluoroacetic acid in the acid hydrolysis reaction system (that is, in the reaction mixture containing at least RNA, hydroxypicolinic acid and trifluoroacetic acid) is, for example, 1.25 to 3% (v / v ). Below the above range, it tends to be difficult to sufficiently decompose RNA (ie, to obtain a sufficient type of fragment capable of providing sequence information). Exceeding the above range results in excessive degradation beyond what is preferred for sequence analysis (i.e., sequence fragmentation is difficult due to the relative excess of smaller fragments and the difficulty of detecting larger fragments). Tend to be). Furthermore, when performing sequence analysis of RNA having a length of 20 bases or more, from the viewpoint of analysis sensitivity, the lower limit of the above concentration is 1.5% (v / v) or 2.0% (v / v ) May be preferred.

[1-4. Acid hydrolysis reaction]
The acid hydrolysis reaction is performed in a reaction mixture containing at least the above RNA, hydroxypicolinic acid and trifluoroacetic acid. The order in which each component is mixed is not particularly limited. For example, the reaction can be initiated by preparing a sample solution containing RNA and a reagent solution containing hydroxypicolinic acid and trifluoroacetic acid, and mixing the sample solution and the reagent solution. Each solution can be prepared as an aqueous solution.

  The acid hydrolysis reaction can be performed at room temperature (25 ± 5 ° C.). The reaction time is not particularly limited. If it is a micro scale, specifically about 5-20 pmol mentioned above, it can be set to 5-10 minutes, for example, about 10 minutes. For example, when performing mass spectrometry described later after the acid hydrolysis reaction, a mixed solution of the sample solution and the reagent solution is dropped on the plate for mass spectrometry, and during the reaction, for example, until the mixed solution is naturally dried, Can be left unattended.

  The acid hydrolysis reaction breaks down the phosphodiester bond in the RNA, resulting in multiple types of RNA fragments with different lengths. Therefore, a reaction mixture (regardless of liquid and solid) containing plural kinds of RNA fragments having different lengths can be obtained by the acid hydrolysis reaction of the present invention.

[2. Mass spectrometry process]
By subjecting the reaction mixture obtained by acid hydrolysis of the above RNA to mass spectrometry, the sequence of RNA can be determined.
When mass spectrometry is performed by the MALDI method, the hydroxypicolinic acid used in the acid hydrolysis reaction present in the reaction mixture functions as a matrix for mass spectrometry. Thus, in mass spectrometry, it is not necessary to add a matrix to the reaction mixture. However, the addition of a matrix to the reaction mixture is not specifically excluded.

[2-1. Matrix additive]
In mass spectrometry using the MALDI method, a matrix additive is usually used. As the matrix additive, an ammonium salt of an organic acid or an inorganic acid can be used. Specifically, diammonium citrate (Ammonium citrate dibase (ACDB)), ammonium acetate (Ammonium Acetate (AA)), ammonium chloride (Ammonium Chloride (ACl)), triammonium citrate (Ammonium citrate tribase (ACTB)) , Ammonium fluoride (AF), ammonium tartarate (AT), and the like. In the present invention, it may be preferable to use ACDB among the above-mentioned additives.

[2-2. Preparation of sample for mass spectrometry]
The reaction mixture obtained by acid hydrolysis can be directly contacted with the matrix additive. When the reaction mixture is obtained in a dry spot state on the plate, a sample for mass spectrometry can be prepared by dropping a matrix additive solution dropwise on the dry spot and drying it. Thus, according to the method of the present invention, both the acid hydrolysis step and the mass spectrometry step, which are pretreatment steps of RNA to be analyzed, can be easily performed on the same plate. Therefore, the target plate that is usually used in mass spectrometry can be used without any particular limitation.

[2-3. Mass spectrometry]
The mass analysis is preferably performed by a MALDI (Matrix Assisted Laser Desorption / Ionization) method, and is further performed by a combination (MALDI-TOF MS) of a MALDI method and a TOF type mass spectrometer (time-of-flight mass spectrometer). Are preferred.
In the RNA sequence analysis method of the present invention, a plurality of types of RNA fragments sufficient to give sequence information can be obtained in a balanced manner by acid hydrolysis, which is RNA pretreatment. For this reason, the mass spectrum obtained by mass spectrometry can be obtained as the mass difference between peaks derived from each RNA fragment indicates the mass of nucleotides constituting RNA. Therefore, RNA sequence analysis can be performed very easily by reading the mass difference between the peaks.

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In the following, the amount expressed in% is based on volume unless otherwise specified. In the mass spectra obtained in Examples, the horizontal axis represents mass / charge (m / z), and the vertical axis represents relative intensity.
[Example 1]
In this example, siRNA was used as a sample. The sequence of this siRNA is 5′-UAUCACUUGAUCUCGUACAdTdT-3 ′ (UAUCACUUGAUCUCGUACA: SEQ ID NO: 1).
siRNA samples were prepared at 10 pmol / μl. Furthermore, a reagent solution containing 3-hydroxypicolinic acid (3-HPA) at a concentration of 50 mg / ml in a 5% aqueous trifluoroacetic acid solution was prepared.
After the same volume of the sample solution and the reagent solution were mixed to prepare a reaction mixture (ie, the final concentration of trifluoroacetic acid was 2.5%), 1 μl of the reaction mixture was immediately applied to a MALDI stainless steel plate and air-dried. I let you. 0.5 μl of 10 mg / ml diammonium citrate (ACDB) aqueous solution was applied to the obtained dried spot, and further air-dried. After drying, measurement was performed by MALDI-TOF MS.

  A mass spectrum obtained by MALDI-TOF MS measurement is shown in FIG. Moreover, the sequence analysis results are shown in FIG. As shown in FIG. 1 (b), all sequences could be analyzed.

[Comparative Example 1]
The same operation as in Example 1 above, except that the reagent solution does not contain 3-hydroxypicolinic acid and that 50 mg / ml of 3-hydroxypicolinic acid is further contained in a diammonium citrate (ACDB) aqueous solution. Went. The mass spectrum thus obtained is shown in FIG. FIG. 2A is a mass spectrum obtained in Example 1. FIG. As shown in FIG. 2, when the reagent solution did not contain 3-hydroxypicolinic acid, the entire sequence of the sample could not be decoded. This is presumably because the acid hydrolysis reaction did not occur sufficiently.

[Example 2]
In this example, the same operation as in Example 1 was performed except that the concentration of trifluoroacetic acid in the reaction mixture was changed to 0.25%, 0.5%, 1.25%, or 2.5%. An example in which the trifluoroacetic acid concentration is 0% is shown for comparison and corresponds to Comparative Example 1.
Each mass spectrum obtained by this is shown in FIGS. As shown in FIG. 3, sequence analysis could be performed at a final concentration of trifluoroacetic acid of 1.25% to 2.5%. In particular, when the final concentration of trifluoroacetic acid was 2.5%, good analysis results could be obtained.

[Example 3]
In this example, the sample RNA was changed to one having the sequence 5′-UCGAAGUAUUCCGCGUACGdTdT-3 ′ (UCGAAGUAUUCCGCGUACG: SEQ ID NO: 2), and the trifluoroacetic acid concentration in the reaction mixture was 1.5%, 2.0%, 2.5% Alternatively, the same operation as in Example 1 was performed except that the content was changed to 3.0%.
Each mass spectrum obtained by this is shown in FIGS. As shown in FIG. 4, good analysis results could be obtained when the final concentration of trifluoroacetic acid was 1.5% to 3.0%. Among them, particularly good analysis results could be stably obtained when the final concentration of trifluoroacetic acid was 2.0% to 3.0%.

SEQ ID NO: 1 is a synthetic oligonucleotide.
SEQ ID NO: 2 is a synthetic oligonucleotide.

Claims (7)

  A method for degrading RNA, comprising a step of performing an acid hydrolysis reaction of RNA using trifluoroacetic acid in the presence of hydroxypicolinic acid to obtain a reaction mixture containing the RNA-derived fragment.   The method according to claim 1, wherein the concentration of the trifluoroacetic acid in the acid hydrolysis reaction system is 1.25 to 3% (v / v). Performing an acid hydrolysis reaction of RNA using trifluoroacetic acid in the presence of hydroxypicolinic acid to obtain a reaction mixture containing the RNA-derived fragment;
Subjecting the reaction mixture to mass spectrometry, and determining the RNA sequence.   The method according to claim 3, wherein the concentration of the trifluoroacetic acid in the acid hydrolysis reaction system is 1.25 to 3% (v / v).   The method according to claim 3 or 4, wherein the mass spectrometry is performed by a MALDI mass spectrometer.   The method according to any one of claims 3 to 5, wherein the acid hydrolysis reaction and the mass spectrometry are performed on the same mass spectrometry plate.   The method according to any one of claims 3 to 6, wherein the RNA has a length of 15 to 25 bases.