Method for analyzing oligonucleotide having phosphoramidate bond or phosphorodiamidate bond

Abstract

Provided is a method for estimating a structure of an oligonucleotide in which a plurality of nucleotides are linked by one or more phosphoramidate bonds or phosphorodiamidate bonds, using a mass spectrometer including an ion source employing a MALDI method, said method comprising: a preparation step for preparing an analysis sample including the oligonucleotide and a matrix; a mass spectrometry step for generating fragment ions by cleaving, in the mass spectrometer, a P-N bond involved in the linkage between the nucleotides in at least one of the phosphoramidate bonds or the phosphorodiamidate bonds of the oligonucleotide contained in the analysis sample, and detecting the fragment ions using the mass spectrometer; and a structure estimation step for estimating at least a portion of the structure of the oligonucleotide by analyzing the result of the mass spectrometry obtained in the mass spectrometry step.

Description

Method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds

[0001] The present invention relates to a method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds.

[0002] Oligonucleotides called morpholino nucleic acids, which have a morpholine ring instead of the sugar moiety found in DNA's deoxyribose and RNA's ribose, are used as nucleic acid medicines. Morpholino nucleic acids are artificially synthesized nucleic acids and belong to the antisense nucleic acid category of nucleic acid medicines. Morpholino nucleic acids are attracting attention as third-generation antisense nucleic acids that overcome the challenges of conventional antisense nucleic acids (specificity, stability, cytotoxicity, etc.).

[0003] Morpholino nucleic acids are synthesized by sequentially linking specific nucleotides to form a designed base sequence. However, if the nucleotide linking reaction does not proceed smoothly or if there is an error in the synthesis procedure, the synthesized morpholino nucleic acid may not have the designed base sequence. Therefore, there is a need to confirm whether the morpholino nucleic acid has been synthesized with the correct base sequence. For common oligonucleotides such as DNA and RNA, the base sequence is confirmed by analyzing fragment ions derived from the obtained sample molecules using LC-MS / MS, which combines a liquid chromatograph (LC) and a tandem mass spectrometer (MS / MS). Specifically, a widely used method involves ionizing sample molecules separated by LC using methods such as electrospray ionization (ESI) and then dissociating the ionized sample molecules by collision-induced dissociation (CID). Then, multiple fragment ions generated by the dissociation of ions of the sample molecule are analyzed, and the fragment ion peaks on the obtained mass spectrum are assigned to fragment ions (theoretical m / z values) corresponding to the partial structure of the sample molecule based on the m / z values ​​(measured values), and the base sequence is deduced based on the assignment results. However, in the case of morpholino nucleic acids, the ion dissociation method using CID does not produce fragment ions useful for deducing the base sequence, so it was not possible to deduce the base sequence of morpholino nucleic acids. This is thought to be due to the fact that morpholino nucleic acids are oligonucleotides having a structure in which methylenemorpholine ring skeletons to which nucleobases are bound are linked by phosphorodiamidate bonds, and their basic structure is different from that of general nucleic acids.

[0004] Recently, one case has been reported in which the base sequence of a morpholino nucleic acid was confirmed by mass spectrometry using LC-MS / MS (Non-Patent Document 1). In the method described in Non-Patent Document 1, ionized morpholino nucleic acid is dissociated by electron capture dissociation (ECD), and the base sequence is estimated from the mass spectrometry results of the generated fragment ions. However, this method generates fragment ions as multiply charged ions, and fragment ions cleaved at various sites are generated as multiple ion species at once, resulting in complex mass spectra and complicated analysis. Therefore, visual analysis is extremely difficult, and the use of analytical software tools is considered essential.

[0005] Kaoru Karasawa, 4 others, "Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry", Analytical Chemistry 2023, 95(44), pp.16352-16358Mikhail S. Shchepinov, 6 others, "Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms", Nucleic Acids Research, 2001, Vo29, No. 18, pp.3864-3872Dejana Jovanovic, and 4 others, "The Enzyme-Free Release of Nucleotides from Phosphoramidates Depends Strongly on the Amino Acid", Angewandte Chemie International Edition, 2020, 59, pp.20154-20160

[0006] The problem to be solved by the present invention is to easily predict the structure of an oligonucleotide having a phosphoramidate or phosphorodiamidate bond, particularly an oligonucleotide in which multiple nucleotides are linked by phosphorodiamidate bonds, such as morpholino nucleic acid.

[0007] The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to the present invention, which has been achieved to solve the above-mentioned problems, is a method for predicting the structure of an oligonucleotide in which multiple nucleotides are linked by one or more phosphoramidate or phosphorodiamidate bonds, using a mass spectrometer equipped with an ion source that utilizes matrix-assisted laser desorption ionization (MALDI), and includes the following steps: a preparation step of preparing an analytical sample containing the oligonucleotide and a matrix; a mass analysis step of generating fragment ions by cleaving the PN bond involved in linking the nucleotides of at least one of the phosphoramidate or phosphorodiamidate bonds of the oligonucleotide contained in the analytical sample in the mass spectrometer, and detecting the fragment ions with the mass spectrometer; and a structure prediction step of predicting at least a part of the structure of the oligonucleotide by analyzing the mass analysis results obtained in the mass analysis step.

[0008] According to the present invention, the structure of an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond can be easily deduced.

[0009] 1 is a schematic diagram showing the configuration of a MALDI-ITMS used in one embodiment of the method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to the present invention.

[0023] FIG. 1 shows the naming rules for fragment ions in morpholine ring-containing oligonucleotides based on the definition in Non-Patent Document 1.

[0024] FIG. 2 shows a proposed mechanism for generating fragment ions.

[0025] FIG. 3 shows a mass spectrum (m / z around 2000 to 7000) of viltolarsen obtained by MALDI-DIT-MS measurement under sequence analysis conditions (pseudo-ISD measurement) in Example 1.

[0026] FIG. 4 shows a mass spectrum (m / z 650 to 5000) of viltolarsen obtained by MALDI-DIT-MS measurement under sequence analysis conditions (pseudo-ISD measurement) in Example 1.

[0027] FIG. 5 shows a mass spectrum (m / z 2000 to 11000) of eteplirsen obtained by MALDI-DIT-MS measurement under sequence analysis conditions (pseudo-ISD measurement) in Example 2. FIG. 1 shows a mass spectrum (m / z 650-5000) of eteplirsen obtained by measurement using MALDI-DIT-MS under conditions for sequence analysis (pseudo-ISD measurement) in Example 2. FIG. 2 shows a mass spectrum (m / z 3600-6500) of viltolarsen obtained by measurement using MALDI-DIT-MS under conditions for sequence analysis (pseudo-ISD measurement) using various matrices in Example 3. FIG. 3 shows a mass spectrum (m / z 650-5000) of viltolarsen obtained by measurement using MALDI-DIT-MS under conditions for sequence analysis (pseudo-ISD measurement) using various matrices in Example 3. FIG. 4 shows another mass spectrum (m / z 3600-6500) of viltolarsen obtained by measurement using MALDI-DIT-MS under conditions for sequence analysis (pseudo-ISD measurement) using various matrices in Example 3. A figure showing another mass spectrum (m / z 650-5000) of viltolarsen when measured using various matrices and sequence analysis conditions (pseudo-ISD measurement) by MALDI-DIT-MS in Example 3.1 shows the mass spectrum (m / z 2000-9000, m / z 4000-7000 in the enlarged view) of casimersen when measurement was performed using MALDI-DIT-MS under sequence analysis conditions (pseudo-ISD measurement) using various matrices in Example 4. 2 shows the mass spectrum (m / z 1000-6800) of viltolarsen when measurement was performed using MALDI-TOFMS under ISD conditions using various matrices in Example 5.

[0010] The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to the present invention is a method for predicting the structure of oligonucleotides in which multiple nucleotides are linked by one or more phosphoramidate or phosphorodiamidate bonds, using a mass spectrometer equipped with an ion source that utilizes matrix-assisted laser desorption / ionization (MALDI), and includes the following steps: a preparation step of preparing an analytical sample containing the oligonucleotide and a matrix; a mass analysis step of generating fragment ions by cleaving the PN bond involved in the linkage between the nucleotides of at least one of the phosphoramidate or phosphorodiamidate bonds of the oligonucleotides contained in the analytical sample within the mass spectrometer, and detecting the fragment ions with the mass spectrometer; and a structure prediction step of predicting at least a part of the structure of the oligonucleotide by analyzing the mass analysis results obtained in the mass analysis step.

[0011] The present inventors analyzed the oligonucleotides using a MALDI mass spectrometer (MALDI-MS) under conditions that cause cleavage of sample molecular ions, and found that the PN bond connecting the nucleotides in the phosphoramidate or phosphorodiamidate bond is preferentially cleaved compared to other positions. This finding is utilized in the analytical method of the present invention to estimate the structure of the oligonucleotides.

[0012] <Preparation Step> In the preparation step, an analytical sample is prepared containing an oligonucleotide having a phosphoramidate or phosphorodiamidate bond (hereinafter referred to as PA / PDA oligonucleotide) and a matrix. The PA / PDA oligonucleotide is not particularly limited in terms of the structure or number of nucleotides, as long as it contains a phosphoramidate or phosphorodiamidate bond in the backbone. The term "nucleotide" as used herein includes nucleotide analogs having a backbone other than a sugar. An example of a PA / PDA oligonucleotide is an oligonucleotide in which a methylenemorpholine ring backbone to which a nucleic acid base is bound is linked by a phosphorodiamidate bond (hereinafter referred to as a morpholine ring-containing PDA oligonucleotide), as shown in the following chemical formula (1): Base in chemical formula (1) represents a nucleic acid base, and R1 and R2, which may be the same or different, represent a hydrogen atom, an alkyl group, or a cyclic alkyl group.

[0013]

[0014] Examples of the nucleic acid base of the morpholine ring-containing PDA oligonucleotide include adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), or modified bases thereof. The base length of the oligonucleotide may range from several bases to several tens of bases (e.g., 20-30 bases). Furthermore, R1 and R2 in chemical formula (1) may both be alkyl groups, and preferably both R1 and R2 are methyl groups. The morpholine ring-containing PDA oligonucleotide may be a morpholino nucleic acid used as a nucleic acid drug. Examples of morpholino nucleic acids include viltolarsen, eteplirsen, golodirsen, and casimersen.

[0015] The matrix can be selected appropriately depending on the type of PA / PDA oligonucleotide to be analyzed. For example, when the PA / PDA oligonucleotide is a morpholine ring-containing PDA oligonucleotide, the matrix preferably contains 2',4',6'-trihydroxyacetophenone (THAP) in order to enable sensitive detection of fragment ions in the mass spectrometry step. Alternatively, the matrix may be a mixed matrix containing THAP and 3-hydroxypicolinic acid (3-HPA). In order to enable sensitive detection of fragment ions over a wide range of mass-to-charge ratios from low to high mass, the THAP / (3-HPA + THAP) ratio (the proportion of THAP in the mixed matrix) is preferably 0.75 (corresponding to a 3-HPA:THAP ratio of 1:3) or more and 1 (corresponding to THAP alone). In other words, the ratio of 3-HPA to THAP in the mixed matrix is ​​preferably such that the proportion of THAP is higher than 3-HPA:THAP = 1:3.

[0016] Examples of methods for preparing analytical samples include a method in which a sample solution containing a PA / PDA oligonucleotide is dissolved in a predetermined solvent, and a matrix solution is dissolved in a predetermined solvent, and then the sample solution and the matrix solution are dropped onto wells of a sample plate of a MALDI-Mass Spectrometer (MALDI-MS) and dried (the on-target method), or a method in which the sample solution and the matrix solution are premixed to prepare a sample / matrix mixed solution, and then the sample / matrix mixed solution is dropped onto the wells of a sample plate and dried (the pre-mix method).By these methods, sample / matrix mixed crystals containing the sample and matrix are formed on the wells of the sample plate.

[0017] When the PA / PDA oligonucleotide is a morpholine ring-containing PDA oligonucleotide, the oligonucleotide is water-soluble, so it is preferable to use water as the solvent for the sample solution.In terms of the solubility of the matrix, it is preferable to use water or a mixed solvent of water and an organic solvent (e.g., acetonitrile), and it is particularly preferable to use a mixed solvent of water and an organic solvent.

[0018] The matrix solution may further contain a matrix additive. Examples of matrix additives include diammonium hydrogen citrate (ACD). There are several types of ammonium salts of citric acid, depending on the number of ammonium ions bound to the citrate ion. A preferred matrix additive is a salt in which two ammonium ions are bound to one citrate ion. The concentration of the matrix additive in the matrix solution is preferably 10 mM to 100 mM, more preferably 30 mM to 90 mM.

[0019] In the present invention, a thin film of matrix may be formed on the sample plate before dropping the sample solution and matrix solution onto the sample plate. For example, a thin film may be formed by dissolving the matrix in a predetermined solvent to prepare a thin film-forming solution, and then dropping the thin film-forming solution onto the wells of the sample plate and drying it. When preparing a sample for MALDI-MS analysis, simply dropping the sample solution and matrix solution onto the sample plate may result in the local formation of sample / matrix mixed crystals in a portion of the well of the sample plate. Even if the sample / matrix mixed crystals are formed entirely over the well, a sweet spot (a region where the peaks derived from the sample are particularly intense) may form within the sample / matrix mixed crystals. By preforming a thin film of matrix as described above and then dropping the sample solution and matrix solution onto it, relatively uniform sample / matrix mixed crystals can be prepared. When the PA / PDA oligonucleotide is a morpholine ring-containing PDA oligonucleotide and the matrix solution contains THAP, it is preferable to form a thin film of THAP. Furthermore, since the solvent evaporates easily upon natural drying, it is preferable to use a 100% organic solvent as the solvent for the thin film-forming solution. In particular, from the viewpoints of the solubility of THAP and the ease of evaporation of the solvent, the solvent for the thin film-forming solution is preferably 100% acetonitrile. The concentration of THAP contained in the thin film-forming solution can be 4 mg / mL or more and saturation concentration or less, preferably 40 to 100 mg / mL.

[0020] <Mass spectrometry step> In the mass spectrometry step, a mass spectrometer having a MALDI ion source is used to cleave the PN bond involved in the linkage between nucleotides in at least one phosphoramidate or phosphorodiamidate bond of the PA / PDA oligonucleotide to generate fragment ions, and the fragment ions are detected. Note that in the present invention, both fragments generated by cleavage of the PN bond may be detected as fragment ions, or only one of the fragments may be detected as a fragment ion.

[0021] The mass spectrometer used in the present invention is not particularly limited as long as it has a MALDI ion source. For example, a MALDI-TOFMS, which combines a MALDI ion source with a time-of-flight mass spectrometer (TOFMS), or a MALDI-ITMS, which combines a MALDI ion source with an ion-trap mass spectrometer (ITMS), can be used.

[0022] The MALDI method is generally known as a soft ionization method capable of ionizing large molecular weight polymers without significant fragmentation. Furthermore, by appropriately setting the ionization conditions (e.g., the type of matrix contained in the analytical sample and / or the intensity of the laser beam irradiated onto the analytical sample), the MALDI ion source can dissociate ions of sample molecules simultaneously with or immediately after ionization to generate fragment ions. The above-described ion dissociation in MALDI-TOFMS is known to occur when an appropriate matrix is ​​used and the laser beam intensity is set high, and is generally referred to as in-source decay (ISD). In MALDI-ITMS, fragment ions can be generated by appropriately adjusting the instrument settings, but the mechanism by which fragment ions are generated has not yet been elucidated. For convenience, in this specification, the dissociation that occurs simultaneously with or immediately after ionization in the MALDI-ITMS ion source, as well as subsequent ion dissociation within the instrument, will be referred to as pseudo-ISD.

[0023] To detect fragment ions generated by ISD in MALDI-TOFMS, a matrix appropriate for the PA / PDA oligonucleotide to be analyzed is used, and the laser intensity is adjusted to the ion (protonated molecule [M+H]) that gives molecular weight information of the sample molecule. + or the deprotonated molecule [MH] -The conditions can be set higher than those used for measurements to detect ions that provide molecular weight information for the sample molecule (e.g., M is a molecule, H is a hydrogen atom) (i.e., measurements that generate ions that provide molecular weight information for the sample molecule while minimizing the generation of fragment ions). On the other hand, to detect fragment ions generated by pseudo-ISD using MALDI-ITMS, a matrix appropriate for the PA / PDA oligonucleotide to be analyzed must be used, and the MALDI-ITMS instrument conditions must be set to specific conditions. The details of how to set the MALDI-ITMS instrument conditions are described below.

[0024] 1 is a schematic diagram showing the configuration of a MALDI-ITMS used in one embodiment of the method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to the present invention. The MALDI-ITMS used in this embodiment includes an ion source 1 that ionizes a sample, an ion trap 2 that temporarily traps ions of a predetermined mass-to-charge ratio (m / z) from among the ions generated by the ion source 1 using a radio-frequency electric field and separates the trapped ions according to their mass-to-charge ratios, and a detector 3 that detects the separated ions.

[0025] The ion source 1 is an ion source that utilizes the MALDI method and includes a laser irradiation unit 11 that irradiates a sample with laser light and a sample stage 12 on which a sample plate S with a sample placed thereon is placed. The ion trap 2 is a quadrupole ion trap that includes a circular ring electrode 21 and a pair of end cap electrodes 22, 23 that are arranged opposite each other across the ring electrode 21. An ion entrance aperture 22a is formed in the entrance end cap electrode 22, and an ion exit aperture 23a is formed in the exit end cap electrode 23. The detection unit 3 includes a conversion dynode 31 that converts ions into electrons, and a detector (secondary electron multiplier) 32 that amplifies and detects the electrons arriving from the conversion dynode 31.

[0026] A predetermined radio-frequency voltage is applied to the ring electrode 21 and the endcap electrodes 22, 23 of the ion trap 2. The radio-frequency electric field formed by this radio-frequency voltage can trap ions in the internal space enclosed by the ring electrode 21 and the endcap electrodes 22, 23, or eject ions from the internal space through the ion exit hole 23a. The MALDI-ITMS in this embodiment may be a mass spectrometer that utilizes the mass separation function of the ion trap 2 itself to eject ions trapped in the ion trap 2 in ascending order of mass-to-charge ratio, and then detects the ions with a detector 3 located outside the ion trap 2. Alternatively, the MALDI-ITMS may be a mass spectrometer that separates ions simultaneously ejected from the ion trap 2 according to their mass-to-charge ratio with a mass separator located outside the ion trap 2, such as a time-of-flight mass separator, and then detects the ions with a detector 3 also located outside the ion trap 2.

[0027] The radio frequency voltage applied to the ring electrode 21 and the end cap electrodes 22, 23 may be a sinusoidal voltage or a square wave voltage. An ITMS that utilizes the electric field generated by a square wave voltage is called a digital ion trap mass spectrometer (DIT-MS). When the radio frequency voltage is a sinusoidal voltage, a resonator is required for the voltage generator that generates the sinusoidal voltage, making it difficult to change the frequency of the voltage. Therefore, in an ITMS that applies a sinusoidal voltage, the mass-to-charge ratio range of ions trapped in the ion trap 2 is controlled by changing the amplitude of the voltage. On the other hand, when the radio frequency voltage is a square wave voltage, a square wave voltage can be generated by rapidly switching between two different voltages without using a resonator for the voltage generator. Therefore, in a DIT-MS, the mass-to-charge ratio range of ions trapped in the ion trap 2 is controlled by changing the frequency while keeping the voltage amplitude constant. Specifically, the low mass cut-off (LMCO) is set by adjusting the frequency of the radio frequency voltage, and the higher the frequency, the smaller the LMCO is set. Because there is an upper limit to the amount of ions that can be trapped in the ion trap 2, the mass-to-charge ratio range of ions trapped in the ion trap 2 (i.e., the mass-to-charge ratio range measured by DIT-MS) is set to the low mass-to-charge ratio side when the frequency is increased and the LMCO is set to a small value, and vice versa.

[0028] By changing the time (hereinafter referred to as the delay time) between when the sample is irradiated with laser light from the ion source 1 and when a radio frequency voltage for trapping ions is applied to the ring electrode 21 of the ion trap 2, it is possible to control the mass-to-charge ratio range of ions that are preferentially trapped in the ion trap 2. By increasing the delay time, ions on the higher mass-to-charge ratio side are preferentially trapped, whereas by decreasing the delay time, ions on the lower mass-to-charge ratio side are preferentially trapped compared to other ions.

[0029] The following describes how to set the MALDI-ITMS instrument conditions. First, the ions ([M+H] + or [MH] - Standard analysis conditions are acquired that can detect ions such as ions generated by the ion source 1 (ion amount setting item), ions trapped in the ion trap 2 (mass-to-charge ratio range setting item), and ions in the detection unit 3 (signal intensity setting item).

[0030] The standard analytical conditions are [M+H] of PA / PDA oligonucleotides. + or [MH] - These are typical conditions under which sample molecular ions such as [M+H] of PA / PDA oligonucleotides can be detected. For example, as a method for obtaining standard analytical conditions, mass spectrometry was performed using the default values ​​of various setting items pre-set in ITMS. + or [MH] - If the [M+H] of the PA / PDA oligonucleotide is detected as an appropriate ion peak with sufficient sensitivity, the default value may be used as the standard analysis condition. In this case, reading the default value from a memory unit or the like in which the default value is stored corresponds to obtaining the standard analysis condition. Alternatively, the setting values ​​of various setting items may be changed relative to the default value to obtain the [M+H] of the PA / PDA oligonucleotide. + or [MH] - Alternatively, a value (threshold value, etc.) that can be detected with sufficient sensitivity and resolution may be obtained.

[0031] The ion quantity setting item includes the intensity (laser power) of the laser light irradiated by the laser irradiation unit 11. The mass-to-charge ratio range setting item includes an RF delay value corresponding to the delay time. The signal intensity setting item includes the voltage applied to the conversion dynode 31 (hereinafter referred to as the dynode voltage) and the voltage applied to the detector 32 (hereinafter referred to as the detector voltage).

[0032] Next, modified analysis conditions are set by changing at least one of the ion quantity setting item, mass-to-charge ratio range setting item, and signal intensity setting item from the acquired standard analysis conditions. At this time, the ion quantity setting item is changed relative to the standard analysis conditions so that the quantity of ions generated in the ion source 1 is increased. The mass-to-charge ratio range setting item is changed relative to the standard analysis conditions so that ions on the lower mass side are preferentially trapped in the ion trap 2. The signal intensity setting item is changed relative to the standard analysis conditions so that the signal intensity of ions in the detection unit 3 is increased.

[0033] Specifically, when changing the laser light intensity, which is an ion quantity setting item, it is preferable to set it higher than the standard analysis conditions. For example, it is preferable to set it about 1 to 40% higher than the standard analysis conditions, and more preferably about 1 to 30% higher. When changing the RF delay value, which is a mass-to-charge ratio range setting item, it is preferable to set it lower than the standard analysis conditions. For example, it is preferable to set it about 10 to 40% lower than the standard analysis conditions, and more preferably about 20 to 30% lower (so that the delay time is 5 to 8 μs shorter). When changing the dynode voltage, which is a signal intensity setting item, it is preferable to set it higher than the standard analysis conditions. For example, it is preferable to set it about 5 to 30% higher, and more preferably about 10 to 30% higher. When changing the detector voltage, which is also a signal intensity setting item, it is preferable to set it higher than the standard analysis conditions. For example, it is preferable to set it about 5 to 50% higher, and more preferably about 10 to 50% higher.

[0034] The MALDI-ITMS used in this embodiment further includes a control processor 4 that acquires data relating to the mass-to-charge ratio and / or detection intensity of ions detected by the detector 3, and analyzes and processes the acquired data. The control processor 4 includes a controller 41, a memory 42, an analysis processor 43, an operation input unit 44, and a display unit 45. The control processor 4 is generally a computer such as a personal computer (PC), and in this case, the operation input unit 44 is a pointing device such as a keyboard or mouse attached to the PC, and the display unit 45 is a monitor display attached to the PC.

[0035] The above-mentioned MALDI-ITMS instrument conditions (ion amount setting item, mass-to-charge ratio range setting item, and / or signal intensity setting item) are set, for example, by an operator inputting specific numerical values ​​into the operation input unit 44. The control unit 41 inputs each set instrument condition into the MALDI-ITMS. The control unit (not shown) of the MALDI-ITMS controls the ion source 1, ion trap 2, and detection unit 3 in accordance with the instrument conditions input from the control unit 41.

[0036] The memory unit 42 acquires and stores data relating to the mass-to-charge ratio and / or detection intensity of the ions detected by the detection unit 3. The analysis processing unit 43 reads the data stored in the memory unit 42 and creates mass spectrum data based on the data. The analysis processing unit 43 also creates a peak list including the mass spectrum and / or the mass-to-charge ratio and / or detection intensity of the detected ions based on the created mass spectrum data, and displays it on the display unit 45 or outputs it as a file.

[0037] As mentioned above, depending on the characteristics of the matrix used, MALDI-MS samples can form mixed crystals of the sample and matrix locally on the well, or sweet spots can form on the sample / matrix mixed crystal. In such cases, ionization efficiency varies depending on the location of the laser beam irradiated on the sample / matrix mixed crystal. In such cases, the operator must select the location where the mixed crystals are formed or the sweet spot on the sample / matrix mixed crystal where the sample is most likely to be ionized, and then irradiate the laser beam onto those locations for measurement. This can lead to variations in the quality of the acquired data depending on the operator's skill level. Therefore, to obtain data with as high reproducibility as possible, it is desirable to form relatively uniform mixed crystals of the sample / matrix and to detect sample molecular ion peaks relatively uniformly on the mixed crystals. Alternatively, MALDI-MS systems may be equipped with a function called raster scanning. In raster scanning, multiple measurement points are set in a predetermined array (e.g., a grid) on the well, and mass spectral data is acquired by irradiating each measurement point with the laser a predetermined number of times. The final mass spectrum data is then obtained by integrating all of the acquired mass spectrum data. Therefore, measurements using the raster scan function allow for the acquisition of objective (fair) data that is not affected by the skill level of the operator. Furthermore, if the sample / matrix mixed crystal is formed relatively uniformly and the sample ions are detected relatively uniformly on the sample / matrix mixed crystal, the raster scan function can be used to acquire mass spectrum data with higher reproducibility.

[0038] In the structure estimation step, the mass spectrometry results obtained in the mass spectrometry step are analyzed to estimate at least a portion of the structure of the PA / PDA oligonucleotide. Specifically, fragment ion peaks generated by PN bond cleavage in the phosphoramidate or phosphorodiamidate bond are extracted from the mass spectrum data obtained in the mass spectrometry step, and the peaks are assigned to fragment ions corresponding to partial structures of the PA / PDA oligonucleotide based on their mass-to-charge ratios.

[0039] When the PA / PDA oligonucleotide is a morpholine ring-containing PDA oligonucleotide having an N,N-dimethyl-phosphorodiamidate bond (when R1 and R2 in the chemical formula (1) are both methyl groups), the fragment ions include b-ions and (x-10)-ions (ions with a mass-to-charge ratio 10 lower than that of the x-ions). Here, the b-ions and x-ions are named according to the naming rules for fragment ions in morpholine ring-containing oligonucleotides proposed in Non-Patent Document 1. Figure 2 shows the naming rules proposed in Non-Patent Document 1. The fragment ions containing the 5'-end are b-ions and (x-10)-ions (ions with a mass-to-charge ratio 10 lower than that of the x-ions). n -ion, c n -ion, d n -ions, and fragment ions containing the 3' end in the opposite direction are denoted x m -ion, y m -ion, z m -ions. The subscripts n and m indicate the number of constituent units from the corresponding end to the dissociation site. In this specification, fragment ions are represented according to the naming rules proposed in Non-Patent Document 1.

[0040] Although the details of the generation mechanism of the b-ion and (x-10)-ion are unknown, a hypothetical generation mechanism (generation scheme) such as that shown in Figure 3 can be hypothesized based on prior literature. First, Non-Patent Document 2, a prior literature, describes, as a conventional technique, that when a compound containing one phosphoramidate bond is reacted under acidic conditions in an aqueous solution, hydrolysis-like cleavage occurs at the PN bond of the phosphoramidate bond, and that sequence information can be confirmed as mass values ​​by analyzing the fragmented sample using MALDI-TOFMS or the like. Furthermore, Non-Patent Document 2 describes that by mixing an acidic MALDI matrix (3-hydroxypicolinic acid: 3-HPA) dissolved in a solvent containing trifluoroacetic acid (TFA) with DNA containing one phosphoramidate bond in an aqueous solution, the reaction conditions (acidic and in the presence of water) are the same as those of conventional techniques, the resulting sample / matrix mixture is dropped onto a sample plate, dried, and measured by MALDI-TOFMS. Similar to conventional solution reactions, hydrolysis-like cleavage occurs at the PN bond of the phosphoramidate bond, and the resulting sample fragments can be analyzed to confirm sequence information as mass values. That is, Non-Patent Document 2 shows that using an acidic matrix solution shortens the reaction process that causes cleavage of the PN bond portion of the phosphoramidate bond, resulting in easier and more rapid fragmentation and the acquisition of sequence information. On the other hand, Non-Patent Document 3 describes that when an amino acid and a nucleotide are linked by a phosphoramidate bond, and particularly when the amino acid is proline, cleavage of the PN bond portion of the phosphoramidate bond by hydrolysis proceeds rapidly under neutral conditions of 37°C and pH 7.5.

[0041] Generally, in MALDI mass spectrometry, b-ions and (x-10)-ions are not detected unless the measurement is performed under conditions that cause fragmentation. Therefore, it is highly likely that the fragmentation that produces b-ions and (x-10)-ions occurs not in the sample / matrix mixed solution but in the gas phase. Furthermore, in MALDI, protons (H ＋ It is known that the [M+H] is easily detected in the positive mode. + It has also been reported that protons may also originate from acidic matrices.

[0042] Based on these findings, one hypothesis is that a hydrolysis-like reaction, as shown in Figure 3, may be occurring in the gas phase. Figure 3 shows an example in which the PA / PDA oligonucleotide is a morpholine ring-containing PDA oligonucleotide with an N,N-dimethyl-phosphorodiamidate bond. However, if the oligonucleotide has a phosphoramidate or phosphorodiamidate bond, the PN bond of the phosphoramidate or phosphorodiamidate bond is cleaved, generating fragment ions, by a mechanism similar to that shown in Figure 3.

[0043] In the present invention, the PN bond connecting nucleotides in phosphoramidate or phosphorodiamidate bonds is preferentially cleaved compared to other sites, and the resulting fragment ions are detected. Therefore, in the mass spectrum obtained by the mass analysis process, peaks of fragment ions generated by PN bond cleavage of phosphoramidate or phosphorodiamidate bonds appear regularly (in a ladder-like pattern) with particularly high sensitivity. Similarly, in the peak list obtained by the mass analysis process, including the mass-to-charge ratio and peak intensity values ​​of the detected ions, peaks of fragment ions generated by PN bond cleavage of phosphoramidate or phosphorodiamidate bonds are shown with sufficiently high peak intensity values. Therefore, in the structure estimation process of the present invention, the peaks of fragment ions generated by PN bond cleavage can be easily confirmed, for example, visually, from the mass spectrum and / or peak list displayed on the display unit of the computer equipped with the MALDI-MS (or output as a file). Alternatively, the analysis processing unit of the computer equipped with the MALDI-MS may automatically extract peaks with a predetermined peak intensity or higher. Alternatively, the mass-to-charge ratio (theoretical value) of the fragment ion generated by PN bond cleavage corresponding to the PA / PDA oligonucleotide to be analyzed may be stored in advance in the memory of a computer equipped with a MALDI-MS, and the peak may be extracted by comparing the stored mass-to-charge ratio with the mass-to-charge ratio (actual measured value) of the peak in the obtained mass spectrum by the analysis processing unit of the computer equipped with the MALDI-MS. The data processing by the analysis processing unit described above may be realized by executing an analysis processing program pre-stored in the memory of the computer equipped with the MALDI-MS by the control unit of the computer, or by using a separate analysis software tool. The assignment of the extracted peak may also be performed by the analysis processing program or by a separate analysis software tool.

[0044] The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to the present invention will be described below with reference to examples, but these are merely illustrative examples and the present invention is not limited thereto.

[0045] [1. Preparation of sample solution] A 20 pmol / μL aqueous solution of viltolarsen (MedChemExpress, 5'-CCTCCGGTTCTGAAGGTGTTC-3': SEQ ID NO: 1, 21 bases long), a morpholino nucleic acid, was prepared as a sample solution. The chemical structural formula of viltolarsen is shown in chemical formula (2). In the formula, B(n) represents the nth base from the 5' end (B(21) represents the 21st base).

[0046]

[0047] [2. Preparation of Matrix Solution] Disodium hydrogen citrate (ACD) was dissolved in 50% acetonitrile (ACN) aqueous solution to a concentration of 70 mM to prepare an ACD solution. 2',4',6'-trihydroxyacetophenone (THAP) monohydrate was dissolved in the ACD solution to a concentration of 40 mg / mL to prepare a matrix solution.

[0048] [3. Preparation of analytical sample] The sample solution prepared in 1. and the matrix solution prepared in 2. were mixed at a 1:1 (v / v) ratio to prepare a sample / matrix mixed solution. 1 μL of this sample / matrix mixed solution was dropped onto a well of a sample plate (SUS plate) and dried to prepare an analytical sample (sample / matrix mixed crystal).

[0049] [4. Mass spectrometry] The sample plate carrying the analytical sample was placed on a MALDI-DIT-MS (Shimadzu Corporation, MALDImini TM -1) and measure the [M+H] of the sample molecule in positive mode. +Measurements were performed to detect fragment ions derived from sample molecules (MS measurements). The instrument's default settings were: detector voltage (DV-1): 1400, dynode voltage (DV-2): 7000, and RF delay (RF): 25. The raster scan settings were 4 shots / point and 25 points. The detector voltage, dynode voltage, and RF delay were changed to DV-1: 1900, DV-2: 8000, and RF: 17, respectively, and measurements were performed using the pseudo-ISD sequence analysis conditions to detect fragment ions derived from sample molecules (pseudo-ISD measurements). Each measurement was performed using the raster scan function to ensure the most objective evaluation of the data obtained.

[0050] Figures 4 and 5 show the mass spectra of viltolarsen obtained by pseudo-ISD measurement. The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z), and the vertical axis represents the relative intensity (int%). The main peaks in the mass spectra in Figures 4 and 5 are labeled with the names of the fragment ion species of viltolarsen corresponding to the peaks (based on the naming convention proposed in Non-Patent Document 1).

[0051] In Figures 4 and 5, many fragment ions were detected as singly charged ions, with b- and (x-10)-ions being detected with high intensity and in a regular pattern (ladder-like pattern). These results allowed the complete base sequence of viltolarsen to be determined. Because the fragment ion detection pattern in the mass spectrum in Figures 4 and 5 is simple, the structure of viltolarsen could be easily deduced. Furthermore, there have been no reports of the detection of (x-10)-ions during mass spectrometry of morpholino nucleic acids, and the detection of (x-10)-ions was confirmed for the first time by the present invention.

[0052] Analysis of eteplirsen was carried out in the same manner as in Example 1, except that in [1. Preparation of sample solution], the morpholino nucleic acid eteplirsen (MedChemExpress, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3': SEQ ID NO: 2, 30 bases long) was used instead of viltolarsen. The chemical structural formula of eteplirsen is shown in chemical formula (3). In the formula, B(n) represents the nth base from the 5' end (B(30) represents the 30th base).

[0053]

[0054] Figures 6 and 7 show the mass spectra of eteplirsen obtained by pseudo-ISD measurement. As with viltolarsen in Example 1, many fragment ions were detected as singly charged ions, with b-ions and (x-10)-ions being particularly intense and detected regularly (in a ladder pattern). From these results, the entire base sequence of eteplirsen was determined.

[0055] [1. Preparation of sample solution] A sample solution containing viltolarsen was prepared in the same manner as in Example 1.

[0056] 2. Preparation of Matrix Solutions: Disodium hydrogen citrate (ACD) was dissolved in 50% acetonitrile (ACN) aqueous solution to a concentration of 70 mM to prepare an ACD solution. 3-Hydroxypicolinic acid (3-HPA), 2',4',6'-trihydroxyacetophenone (THAP) monohydrate, 2,4-dihydroxyacetophenone (DHAP), or 6-aza-2-thiothymine (ATT) was dissolved in the ACD solution to a concentration of 40 mg / mL to prepare four matrix solutions (3-HPA solution, THAP solution, DHAP solution, and ATT solution). The 3-HPA solution and the THAP solution were then mixed at 1:1, 1:3, 3:1, 1:5, and 1:10 (v / v) ratios to prepare 3-HPA / THAP mixed matrix solutions. In addition, a mixed matrix DHAP / THAP solution was prepared by mixing the DHAP solution and the THAP solution at a ratio of 1:1 (v / v).

[0057] [3. Preparation of analytical samples] The sample solution prepared in 1. was mixed 1:1 (v / v) with either one of the matrix solutions or the mixed matrix solution prepared in 2. to prepare a sample / matrix mixed solution. 1 μL of this sample / matrix mixed solution was dropped onto a well of a sample plate (SUS plate) and dried to prepare an analytical sample (sample / matrix mixed crystal).

[0058] [4. Mass spectrometry] The sample plate carrying the analytical sample was placed on a MALDI-DIT-MS (Shimadzu Corporation, MALDImini TM -1) and measure the [M+H] of the sample molecule using the raster scan function in positive mode. + Measurements were performed to detect fragment ions derived from sample molecules (MS measurements). The default detector voltage, dynode voltage, and RF delay for the instrument were DV-1: 1300, DV-2: 7000, and RF: 25, respectively. The raster scan settings were 4 shots / point and 25 points. The detector voltage, dynode voltage, and RF delay were changed to DV-1: 1800, DV-2: 8000, and RF: 17, respectively, and measurements were performed using sequence analysis conditions (pseudo-ISD measurements) to detect fragment ions derived from sample molecules using pseudo-ISD.

[0059] Figures 8 and 9 show mass spectra of viltolarsen obtained by pseudo-ISD measurements using various matrices. Figure 8 shows the mass spectrum from m / z 3600 to 6500, and Figure 9 shows the mass spectrum from m / z 650 to 5000. Figure 8 shows the peak intensity (mV) of the most intense fragment ion on the mass spectrum. In the mass range from m / z 3600 to 6500 (Figure 8), fragment ions were generally easily detected. Fragment ions were particularly sensitively detected in DHAP, THAP, 3-HPA / DHAP (1:1), 3-HPA / THAP (1:1), 3-HPA / THAP (1:3), and 3-HPA / THAP (3:1). In the mass range from m / z 650 to 5000 (Figure 9), many fragment ions were detected with sufficient sensitivity, especially in THAP and 3-HPA / THAP (1:3). From the above, it was confirmed that many fragment ions could be detected with high sensitivity over a wide range of mass-to-charge ratios, from low to high mass ranges, especially when using THAP or 3-HPA / THAP (1:3) matrices.

[0060] Figures 10 and 11 show mass spectra of viltolarsen obtained by pseudo-ISD measurement using various matrices, measured on different days from the data shown in Figures 8 and 9. Figure 10 shows the mass spectrum from m / z 2000 to 7500, and Figure 11 shows the mass spectrum from m / z 650 to 5000. In the mass range from m / z 2000 to 7500 (Figure 10), fragments were generally easily obtained, and many fragment ions were detected with particularly high sensitivity in THAP, 3-HPA / DHAP (1:1), 3-HPA / THAP (1:1), 3-HPA / THAP (1:3), 3-HPA / THAP (1:5), and 3-HPA / THAP (1:10). On the other hand, in the mass range of m / z 650 to 5000 (Figure 11), many fragment ions were detected with sufficient sensitivity for THAP, 3-HPA / THAP (1:3), 3-HPA / THAP (1:5), and 3-HPA / THAP (1:10). These results confirm that many fragment ions were detected over a wide range of mass-to-charge ratios, from low to high, when THAP, 3-HPA / THAP (1:3), 3-HPA / THAP (1:5), and 3-HPA / THAP (1:10) were used, particularly when the THAP ratio was higher than that of 3-HPA / THAP (1:3).

[0061] [1. Preparation of sample solution] A 20 pmol / μL aqueous solution of the morpholino nucleic acid casimersen (MedChemExpress, 5'-CAATGCCATCCTGGAGTTCCTG-3': SEQ ID NO: 3, 22 bases in length) was prepared as a sample solution. The chemical structural formula of casimersen is shown in chemical formula (4). Note that B(n) in the formula indicates the nth base from the 5' end (B(22) indicates the 22nd base).

[0062]

[0063] [2. Preparation of Matrix Solutions] Two types of matrix solutions (3-HPA solution and THAP solution) were prepared in the same manner as in Example 3. Next, the 3-HPA solution and the THAP solution were mixed at a ratio of 1:3 (v / v) to prepare a mixed matrix solution (3-HPA / THAP solution). THAP was also dissolved in 100% ACN to a concentration of 40 mg / mL to prepare a matrix solution for thin film formation (THAP-2 solution).

[0064] [3. Preparation of Analytical Samples] The sample solution prepared in 1. and the matrix solution (THAP solution) or mixed matrix solution (3-HPA / THAP solution) prepared in 2. were mixed at a 1:1 (v / v) ratio to prepare a sample / matrix mixed solution. 1 μL of this sample / matrix mixed solution was dropped onto a well of a sample plate (SUS plate) and dried to prepare an analytical sample (sample / matrix mixed crystals). Alternatively, 0.5 μL of the thin-film-forming matrix solution (THAP-2 solution) prepared in 2. was dropped onto a well of a sample plate (SUS plate) and dried to form a thin film of matrix (THAP). Then, 1 μL of a sample / matrix mixed solution, prepared by mixing the sample solution and the 3-HPA / THAP solution, was dropped onto the thin film and dried to prepare an analytical sample (sample / matrix mixed crystals).

[0065] [4. Mass spectrometry] The sample plate carrying the analytical sample was placed on a MALDI-DIT-MS (Shimadzu Corporation, MALDImini TM -1) and measure the [M+H] of the sample molecule using the raster scan function in positive mode. +Measurements were performed to detect fragment ions derived from sample molecules (MS measurements). The default detector voltage, dynode voltage, and RF delay for the instrument were DV-1: 1300, DV-2: 7000, and RF: 25, respectively. The raster scan settings were 4 shots / point and 25 points. For sequence analysis, the detector voltage, dynode voltage, and RF delay were changed to DV-1: 1800, DV-2: 8000, and RF: 17, respectively, and measurements were performed to detect fragment ions derived from sample molecules using a pseudo-ISD (pseudo-ISD measurements).

[0066] Figure 12 shows the mass spectra (m / z 2000-9000, enlarged view: m / z 4000-7000) of casimersen when pseudo-ISD measurements were performed using various matrices. When THAP and 3-HPA / THAP (1:3) were used as matrices, numerous fragment ions due to PN bond cleavage were detected. Furthermore, when a sample / matrix mixed crystal with a 3-HPA / THAP (1:3) matrix was prepared on a thin THAP film, numerous fragment ions were also detected. In this case, preparing a sample / matrix mixed crystal on a thin THAP film enabled more uniform peak detection. The enlarged view of m / z 4000-7000 also confirmed improved sensitivity for fragment ions.

[0067] In the sample / matrix mixed crystal formed on the thin film of THAP, the [M+H] of the morpholine ring-containing PDA oligonucleotide was measured by the above MS. + It has been confirmed that the detection of morpholine ring-containing PDA oligonucleotides is highly sensitive, highly uniform, and with suppressed fragment ions. Therefore, by using the sample / matrix mixed crystals formed on a thin film of THAP as the analytical sample, both molecular weight analysis and sequence analysis can be performed using the same analytical sample, enabling efficient and rapid analysis of morpholine ring-containing PDA oligonucleotides.

[0068] [1. Preparation of sample solution] A sample solution containing viltolarsen was prepared in the same manner as in Example 1.

[0069] [2. Preparation of Matrix Solutions] Two types of matrix solutions (3-HPA solution and THAP solution) were prepared in the same manner as in Example 3. Next, the 3-HPA solution and the THAP solution were mixed at 3:1, 1:1, and 1:3 (v / v) ratios to prepare mixed matrix solutions (3-HPA / THAP solutions) with various mixing ratios. Furthermore, a matrix solution for thin film formation (THAP-2 solution) was prepared in the same manner as in Example 4.

[0070] [3. Preparation of Analytical Samples] The sample solution prepared in 1. and the matrix solution (THAP solution) prepared in 2. or mixed matrix solutions (3-HPA / THAP solutions) at various mixing ratios were mixed at a 1:1 (v / v) ratio to prepare a sample / matrix mixture. 1 μL of this sample / matrix mixture was dropped onto a well of a sample plate (SUS plate) and dried to prepare an analytical sample (sample / matrix mixed crystals). Alternatively, 0.3 μL of the thin-film-forming matrix solution (THAP-2 solution) prepared in 2. was dropped onto a well of a sample plate (SUS plate) and dried to form a thin matrix film. Then, 1 μL of a sample / matrix mixture, which was a mixture of the sample solution and various 3-HPA / THAP solutions, was dropped onto the thin film and dried to prepare an analytical sample (sample / matrix mixed crystals).

[0071] [4. Mass spectrometry] The sample plate carrying the analytical sample was inserted into a MALDI-TOFMS (Shimadzu Corporation, MALDI-8030), and a measurement (ISD measurement) was performed to detect fragment ions derived from the sample molecules using the ISD in positive mode using the raster scan function. Specifically, the [M+H] of the sample molecule, which is usually performed, was measured. + In contrast to measurements to detect ions (MS measurements) (specifically, measurements in which the laser power is adjusted to a threshold value so that sample molecules can be measured while minimizing the generation of fragment ions), ISD measurements were performed by increasing the laser power above normal to appropriately generate fragment ions.

[0072] Figure 13 shows the mass spectra (m / z 1000-6750) of viltolarsen measured by MALDI-TOFMS using various matrices and ISD. Fragment ions due to PN bond cleavage were detected under all conditions, regardless of whether a thin film was formed or not. The detected fragment ions roughly matched those obtained by MALDI-DIT-MS pseudo-ISD analysis. These results confirm that THAP and the mixed matrix 3-HPA / THAP (3:1, 1:1, 1:3), which produced highly sensitive fragment ion detection by MALDI-DIT-MS, are also effective for MALDI-TOFMS ISD analysis. Furthermore, the fragmentation patterns obtained by MALDI-DIT-MS pseudo-ISD analysis and MALDI-TOFMS ISD analysis were roughly consistent.

[0073] According to the method of the present invention for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds, the structure of the oligonucleotides having phosphoramidate or phosphorodiamidate bonds (such as the structure of the nucleotides and / or the order of the linked nucleotides) can be easily analyzed from the mass spectrometry results of fragment ions generated by cleavage of the PN bond involved in the nucleotide bond of the phosphoramidate or phosphorodiamidate bond.

[0074] Aspects It will be apparent to those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0075] (Item 1) A method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to one embodiment of the present invention is a method for predicting the structure of an oligonucleotide in which multiple nucleotides are linked by one or more phosphoramidate or phosphorodiamidate bonds, using a mass spectrometer equipped with an ion source that utilizes matrix-assisted laser desorption ionization (MALDI), and includes the following steps: a preparation step of preparing an analytical sample containing the oligonucleotide and a matrix; a mass analysis step of generating fragment ions by cleaving, in the mass spectrometer, the P-N bond involved in the linkage between the nucleotides of at least one of the phosphoramidate or phosphorodiamidate bonds of the oligonucleotide contained in the analytical sample, and detecting the fragment ions with the mass spectrometer; and a structure prediction step of predicting at least a part of the structure of the oligonucleotide by analyzing the mass analysis results obtained in the mass analysis step.

[0076] According to the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond of the first aspect, fragment ions generated by cleavage of the PN bond connecting the nucleotides in the oligonucleotide are detected, and therefore the structure of the oligonucleotide can be easily estimated.

[0077] (Item 2) The analytical method according to Item 2 is the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to Item 1, wherein the mass spectrometer is a MALDI-ion trap mass spectrometer comprising an ion trap for separating and trapping ions generated in the ion source, and a detection unit for detecting ions trapped in the ion trap; In the mass analysis step, at least one of the analysis conditions of the MALDI-ion trap mass spectrometer, namely, an ion amount setting item relating to the amount of ions generated in the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped in the ion trap, and a signal intensity setting item relating to the signal intensity of ions in the detection unit, can be changed from standard analysis conditions set during measurement to detect protonated or deprotonated molecules of the oligonucleotide, so that the mass-to-charge ratio range setting item increases the amount of ions generated in the ion source, the mass-to-charge ratio range setting item increases the amount of ions generated in the ion source, the mass-to-charge ratio range setting item increases the amount of ions lower in mass than the mass-to-charge ratio of the protonated or deprotonated molecule of the oligonucleotide is preferentially trapped in the ion trap, and the signal intensity setting item increases the signal intensity of ions in the detection unit, and the fragment ions can be detected under these conditions.

[0078] According to the method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to the second aspect, a large number of fragment ions, particularly those ranging from low mass to high mass regions, can be detected with high sensitivity.

[0079] (Item 3) The analytical method according to item 3 may be the analytical method according to item 2 for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds, wherein the mass spectrometer is a MALDI-digital ion trap mass spectrometer.

[0080] Digital ion trap MALDI-MS (MALDI-DIT-MS), which is one type of ion trap MALDI-MS (MALDI-IT-MS), is available in a compact instrument, so that the method for analyzing oligonucleotides having (phosphoramidate or) phosphorodiamidate bonds according to paragraph 3 can estimate the structure of the oligonucleotide without being restricted by the location of the mass spectrometer. Furthermore, it can more effectively detect a large number of fragment ions ranging from low to high mass regions with high sensitivity.

[0081] (4) The analytical method according to 4 is the method according to 1 for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond, wherein the mass spectrometer is a MALDI-time-of-flight mass spectrometer equipped with a time-of-flight separation unit, and in the mass analysis step, the fragment ions can be detected by increasing the intensity of the laser light in the ion source of the MALDI-time-of-flight mass spectrometer to a value set during measurement for detecting protonated or deprotonated molecules of the oligonucleotide.

[0082] According to the method for analyzing oligonucleotides with phosphoramidate or phosphorodiamidate bonds according to item 4, the structure of the oligonucleotides can be estimated using MALDI-TOFMS.

[0083] (Item 5) The analytical method according to item 5 is the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to any one of items 1 to 4, wherein the oligonucleotide is an oligonucleotide in which a plurality of nucleotides having a morpholine ring skeleton are linked by one or more phosphorodiamidate bonds.

[0084] (Item 6) The analytical method according to Item 6 may be the analytical method according to Item 5 for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond, wherein the oligonucleotide is a morpholino nucleic acid.

[0085] According to the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to item 5 or 6, it is possible to estimate the base sequence of an oligonucleotide having a morpholine ring and a morpholino nucleic acid used as a nucleic acid drug.

[0086] (Item 7) The analytical method according to Item 7 is the analytical method according to Item 5 or 6 for an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond, wherein the fragment ions may include (x-10)-ions generated by cleavage of the PN bond.

[0087] (Item 8) The analytical method according to item 8 is the analytical method for an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to any one of items 5 to 7, wherein the fragment ions may include b-ions generated by cleavage of the PN bond.

[0088] According to the method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to item 7 or 8, analysis can be performed based on preferentially detected fragment ions, thereby enabling more rapid sequence analysis.

[0089] (Item 9) The analytical method according to Item 9 is a method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to any one of Items 5 to 8, wherein the matrix may contain 2',4',6'-trihydroxyacetophenone (THAP).

[0090] According to the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to Item 9, a large number of fragment ions resulting from cleavage of the PN bond of the morpholine ring-containing oligonucleotide can be detected with high sensitivity.

[0091] (Item 10) The analytical method according to Item 10 is the method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to any one of Items 5 to 9, wherein the matrix is ​​2',4',6'-trihydroxyacetophenone (THAP) or a mixed matrix containing 2',4',6'-trihydroxyacetophenone (THAP) and 3-hydroxypicolinic acid (3-HPA), and the ratio of THAP / (3-HPA + THAP) can be 0.75 or more and 1 or less.

[0092] According to the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to Item 10, fragment ions resulting from cleavage of the P-N bond of a morpholine ring-containing oligonucleotide can be detected with high sensitivity over a wide range of mass-to-charge ratios.

[0093] (Item 11) The analytical method according to Item 11 may be the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to Item 9 or 10, wherein in the preparation step, a solution of THAP dissolved in a solvent is dropped onto a sample plate of the mass spectrometer and dried to form a thin film of THAP, and then a matrix solution containing the matrix and a sample solution containing the oligonucleotide are dropped onto the thin film, or a mixed solution of the matrix solution and the sample solution is dropped onto the thin film and dried to form a sample / matrix mixed crystal on the thin film, thereby preparing the analytical sample.

[0094] According to the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond of item 11, fragment ions resulting from cleavage of the PN bond of a morpholine ring-containing oligonucleotide can be detected more uniformly, more reproducibly, and more reliably with high sensitivity.

[0095] (Item 12) The analytical method according to Item 12 is a method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to any one of Items 1 to 11, further comprising a display step of displaying the mass analysis results obtained in the mass analysis step as a mass spectrum and / or a peak list on a display unit, and the structure estimation step may involve extracting peaks of the fragment ions from the mass spectrum and / or peak list displayed in the display step, and estimating at least a part of the structure of the oligonucleotide based on the mass-to-charge ratio of the peak.

[0096] According to the method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond of item 12, the peak of the fragment ion preferentially detected in the detection unit can be confirmed visually (or by using an analytical software tool) from the mass spectrum and / or peak list, thereby making it possible to more easily, quickly and reliably estimate the structure of the oligonucleotide.

[0097] REFERENCE SIGNS LIST 1 ion source 11 laser irradiation unit 12 sample stage 2 ion trap 21 ring electrode 22 entrance end cap electrode 22a ion entrance hole 23 exit end cap electrode 23a ion exit hole 3 detection unit 31 conversion dynode 32 detector

Claims

1. A method for predicting the structure of an oligonucleotide in which multiple nucleotides are linked by one or more phosphoramidate or phosphorodiamidate bonds, using a mass spectrometer equipped with an ion source that utilizes matrix-assisted laser desorption ionization (MALDI), comprising: a preparation step of preparing an analytical sample containing the oligonucleotide and a matrix; a mass analysis step of generating fragment ions by cleaving the PN bond involved in the linkage between the nucleotides of at least one of the phosphoramidate or phosphorodiamidate bonds of the oligonucleotide contained in the analytical sample within the mass spectrometer, and detecting the fragment ions with the mass spectrometer; and a structure prediction step of predicting at least a part of the structure of the oligonucleotide by analyzing the mass analysis results obtained in the mass analysis step.

2. The mass spectrometer is a MALDI-ion trap type mass spectrometer comprising an ion trap for separating and trapping ions generated in the ion source, and a detection unit for detecting ions trapped in the ion trap, 2. The method for analyzing oligonucleotides having phosphoroamidate or phosphorodiamidate bonds according to claim 1, wherein in the mass analysis step, at least one of the analysis conditions of the MALDI-ion trap mass spectrometer, i.e., an ion amount setting item relating to the amount of ions generated in the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped in the ion trap, and a signal intensity setting item relating to the signal intensity of ions in the detection unit, is changed from standard analysis conditions set during measurement for detecting protonated or deprotonated molecules of the oligonucleotide, so that the mass-to-charge ratio range setting item increases the amount of ions generated in the ion source, the mass-to-charge ratio range setting item is changed so that ions lower in mass than the mass-to-charge ratio of the protonated or deprotonated molecule of the oligonucleotide are preferentially trapped in the ion trap, and the signal intensity setting item is changed so that the signal intensity of ions in the detection unit increases.

3. The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to claim 2, wherein the mass spectrometer is a MALDI-digital ion trap mass spectrometer.

4. The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to claim 1, wherein the mass spectrometer is a MALDI-time-of-flight mass spectrometer equipped with a time-of-flight separation section, and in the mass analysis step, the fragment ions are detected by increasing the intensity of the laser light in the ion source of the MALDI-time-of-flight mass spectrometer above a value set during measurement to detect protonated or deprotonated molecules of the oligonucleotide.

5. A method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to claim 1, wherein the oligonucleotide is an oligonucleotide in which a plurality of nucleotides having a morpholine ring skeleton are linked by one or more phosphorodiamidate bonds.

6. The method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to claim 5, wherein the oligonucleotide is a morpholino nucleic acid.

7. The method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to claim 5, wherein the fragment ions include (x-10)-ions generated by cleavage of the PN bond.

8. The method for analyzing an oligonucleotide having a phosphoramidate bond or a phosphorodiamidate bond according to claim 5, wherein the fragment ions include b-ions generated by cleavage of the PN bond.

9. The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to claim 5, wherein the matrix contains 2',4',6'-trihydroxyacetophenone (THAP).

10. A method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to claim 5, wherein the matrix is ​​2',4',6'-trihydroxyacetophenone (THAP) or a mixed matrix containing 2',4',6'-trihydroxyacetophenone (THAP) and 3-hydroxypicolinic acid (3-HPA), and the ratio of THAP / (3-HPA + THAP) is 0.75 or more and 1 or less.

11. A method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds as described in claim 9, wherein in the preparation step, a solution of THAP dissolved in a solvent is dropped onto a sample plate of the mass spectrometer and dried to form a thin film of THAP, and then a matrix solution containing the matrix and a sample solution containing the oligonucleotide are dropped onto the thin film, or a mixed solution of the matrix solution and the sample solution is dropped onto the thin film and dried to form a sample / matrix mixed crystal on the thin film, thereby preparing a sample for analysis.

12. The method for analyzing oligonucleotides having phosphoramidate or phosphorodiamidate bonds according to claim 1, further comprising a display step of displaying the mass analysis results obtained in the mass analysis step as a mass spectrum and / or a peak list on a display unit, wherein in the structure estimation step, peaks of the fragment ions are extracted from the mass spectrum and / or peak list displayed in the display step, and at least a part of the structure of the oligonucleotide is estimated based on the mass-to-charge ratio of the peaks.

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