Artificial genes and gene mutation methods

Abstract

To provide an artificial gene which is not subject to gene recombination regulations and which enables the introduction of a local mutation into a target gene and facilitates the quantitation of the mutation during its introduction, and a gene mutation method.SOLUTION: An artificial gene has a 15 N abundance in at least some DNA bases that exceeds the natural abundance. A gene mutation method includes a first step of producing a state in which 15 N is unevenly distributed in a prescribed DNA in a living cell; and a second step of applying a proton beam with an energy at which the 15 N produces a resonant nuclear reaction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an artificial gene and a method for gene mutation. [Background technology]

[0002] The functions of living organisms related to biodiversity, particularly the metabolic production functions of algae that produce natural products such as oils, polysaccharides, and pigments at high densities, are attracting attention in a wide range of fields, including energy, food and beverages, nutritional foods, cosmetics, and pharmaceuticals. Genome editing is used to modify the desired genes (target genes) involved in production in order to create highly productive algal strains. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2018-000129 Summary of the Invention [Problem to be solved by the invention]

[0004] In the process of utilizing wild organisms for various purposes, humans have selected strains with phenotypes that better match their needs, and through crossbreeding and breeding, have utilized organisms with advantageous characteristics. In recent years, it has become relatively easy to analyze the genomic DNA sequences of these organisms. Understanding their genetic information has made it possible to clarify the causes of their beneficial phenotypes. Furthermore, advances in genome editing technology have made it possible to selectively modify specific genes and DNA regions. However, there are many organisms for which the technology for introducing or inserting DNA or genome editing tools into the cell nucleus has not yet been established, and genetic recombination and genome editing technologies have not yet been established for all organisms. Furthermore, because the gene regions whose functions have been elucidated in genomic DNA are only a portion of the whole, not every beneficial organism can be modified as desired. Furthermore, the European Court of Justice ruled that genome editing technology is subject to the same regulations as those for genetically modified organisms under the Cartagena Protocol (the Cartagena Protocol on Biosafety to the Convention on Biological Diversity, adopted at the resumed meeting of the Special Session of the Conference of the Parties to the Convention on Biological Diversity in January 2000), and discussions regarding how to handle the technology are ongoing in various countries.

[0005] Therefore, even today, breeding through mutation introduction using gamma rays, heavy ion beam irradiation, or DNA modification reagents, which are not considered genetic modification, remains essential for industrial applications (see, for example, Patent Document 1). However, conventional mutation breeding methods lack a quantitative method for directly assessing the extent of DNA damage. Therefore, irradiation doses and treatment amounts are determined using easily identifiable phenotypic changes, such as lethality and pigment formation, as indirect indicators of mutation efficiency. Furthermore, random mutations introduced by irradiation require laborious and time-consuming screening of a large number of treated populations to find strains with the desired phenotype. However, until screening results are obtained, it is unclear whether the desired genetic mutations have occurred.

[0006] Therefore, the present invention provides an artificial gene and a gene mutation method that are not subject to genetic recombination regulations, that can introduce localized mutations into target genes, and that can quantify the mutations during the introduction. [Means for solving the problem]

[0007] As will be explained in more detail below with reference to FIG. 15 N( 1 H,α1γ) 12 In the C resonant nuclear reaction, 16 O * emits alpha particles 12 C * In the (p,α1γ) reaction channel, which de-excites the first excited level of 11.6007 MeV, the α( 4 He nuclei) and 12 C and are emitted as secondary particles. 12 C * The first excited state of γ-rays is de-excited to the ground state, emitting γ-rays of 4.43 MeV. After extensive research, the inventors have found that the emission of reactive secondary particles with high ionization activity is 15 If N is located inside or near the DNA, 15 They found that proton beam irradiation exerts a high local ionizing effect on biomolecules near N, increasing the probability of inducing the desired genetic mutation in DNA. Furthermore, the inventors found that the 4.43 MeV gamma rays emitted per nuclear reaction can be easily counted, and that these gamma rays reflect the amount of genetic mutation in DNA caused by proton beam irradiation, making it possible to quantify the amount of genetic mutation in DNA by counting the 4.43 MeV gamma rays during beam irradiation.

[0008] In addition, the present inventors 15 N-labeled DNA samples, or 15 In proton beam irradiation of biological samples such as biological cells containing DNA, the proton beam energy is varied from the resonance energy to a high energy exceeding the resonance energy, for example, by varying the energy step width in a certain range, thereby distributing the protons from the surface to the interior of the target sample. 15 For N, thoroughly 15 N( 1 H,α1γ) 12It has been found that it may be possible to produce C resonant nuclear reactions.

[0009] An artificial gene according to one aspect of the present invention, which is based on at least a part of the above findings of the present inventors, comprises: 15 The abundance of N exceeds the natural abundance. 15 The abundance ratio of N is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more.

[0010] In the above aspect, 15 N unlabeled primer sequence; 15 N-labeled deoxyribonucleotides.

[0011] In the above embodiment, the artificial gene may be formed by ligating the artificial gene according to the above embodiment with another artificial gene. The other artificial gene may be a vector.

[0012] In the above aspect, the biomolecule may have a plurality of biomolecule-binding sites, and the gene sequence of at least some of the plurality of biomolecule-binding sites may be mutated. The biomolecule may be a protein.

[0013] An artificial gene according to one aspect of the present invention comprises: 15 An N-unlabeled artificial gene having a plurality of biomolecule binding sites, at least a portion of which has a mutated gene sequence, and at least one of the biomolecule binding sites has a mutated gene sequence. 15 N-labeled biomolecules can be bound. 15 The biomolecule labeled with N may be a protein. 15 N-labeled biomolecules may be capable of binding.

[0014] The kit according to one aspect of the present invention comprises the above-mentioned 15 N-unlabeled artificial genes and multiple biomolecular binding sites 15and an N-labeled biomolecule.

[0015] A method for mutating a gene according to one embodiment of the present invention comprises: 15 Labeling with N and 15 and irradiating the DNA with a proton beam having an energy such that N undergoes a resonant nuclear reaction.

[0016] In the above embodiment, DNA 15 Labeling with N is the process of replacing the N in DNA with 15 Alternatively, the DNA may be 15 Labeling with N allows the DNA to 15 The method may include labeling the DNA with N. 15 Labeling with N gives DNA 15 The method may include binding a N-labeled biomolecule. 15 The N-labeled biomolecule may be a protein.

[0017] In the above embodiment, DNA in living cells is 15 It may be labeled with N.

[0018] In the above-mentioned embodiment, in cellular components other than DNA in living cells 15 N in the natural abundance ratio, 15 The abundance ratio of N may be greater than the natural abundance ratio.

[0019] In the above-mentioned embodiment, DNA is extracted from living cells. 15 Labeling with N 15 This may involve providing N-labeled deoxyribonucleotides and a glutamine synthetase inhibitor to living cells.

[0020] In the above-mentioned embodiment, DNA is extracted from living cells. 15 Labeling with N 15 This may involve providing N-labeled deoxyribonucleotides and a ribonucleotide reductase inhibitor to living cells.

[0021] In the above-mentioned embodiment, the DNA is 15 Labeling with N 15 The method may further comprise providing N-unlabeled glutamine to living cells.

[0022] The above-described embodiment may further include detecting a resonant nuclear reaction.

[0023] In the above embodiment, the detection may involve measuring the amount of 4.43 MeV gamma rays.

[0024] The above embodiment may further comprise calculating the number of mutations that have occurred in the DNA based on the measured amount of gamma rays.

[0025] In the above aspect, in the calculation, 15 It is also possible to refer to the amount of gamma rays produced by a resonant nuclear reaction occurring in a standard sample with a known number of N atoms.

[0026] In the above aspect, 15 The energy at which N undergoes a resonant nuclear reaction may be changed. [Effects of the Invention]

[0027] According to the present invention, it is possible to provide an artificial gene and a gene mutation method that are not subject to genetic recombination regulations, that can introduce localized mutations into target genes, and that can quantify the mutations when they are introduced. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows a DNA molecule in which N present in each base pair has been substituted with 15N in a gene mutation method according to an embodiment of the present invention. [Figure 2] FIG. 1 illustrates how resonant nuclear reactions occur in DNA molecules. [Figure 3] FIG. 1 is a diagram showing a case where a gene mutation method using heavy ion beam irradiation, which is a conventional technique, is used. [Figure 4]FIG. 1 shows an artificial DNA standard sample consisting of 84 base pairs. [Figure 5] FIG. 1 shows the 15N(1H,α1γ)12C resonance nuclear reaction. [Figure 6] FIG. 1 is a schematic diagram of a method for determining the presence or absence of DNA breaks. [Figure 7] FIG. 1 is a diagram showing an overview of a method for identifying how much cleavage has occurred in one of the DNA strands. [Figure 8] FIG. 1 shows the synthesis of nitrogen-containing organic compounds in cells. [Figure 9] FIG. 1 is a diagram illustrating a new method for 15N-labeling only nucleic acids in cells when MSX and 15N-unlabeled glutamine are administered. [Figure 10] FIG. 1 is a diagram illustrating a method for labeling only DNA with 15N in a nucleic acid synthesis pathway. [Figure 11] This figure explains a new method for labeling only the target gene or its vicinity with 15N. [Figure 12] FIG. 1 shows the genetic structure of pUC4-KIXX circular plasmid. [Figure 13] 1 is a graph showing the resonance curve of the 15N(p,α1γ)12C resonance nuclear reaction of the 15N_pUC4-KIXX plasmid sample. [Figure 14] This is a graph showing the relationship between the amount of proton irradiation to a plasmid and the damage to the gene. The proton-irradiated plasmid was introduced into E. coli, and the E. coli was cultured in a medium containing ampicillin. [Figure 15] This is a graph showing the relationship between the amount of proton irradiation to a plasmid and the damage to the gene. The proton-irradiated plasmid was introduced into E. coli, and the E. coli was cultured in a medium containing kanamycin. [Figure 16] This is a graph showing the relationship between the amount of proton irradiation to a plasmid and the genetic damage. The proton-irradiated plasmid was introduced into E. coli, and the E. coli was cultured in a medium supplemented with ampicillin and kanamycin. DETAILED DESCRIPTION OF THE INVENTION

[0029] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to the accompanying drawings, in which the same reference numerals denote the same or similar components.

[0030] Nitrogen element N in living organisms is scattered throughout cellular components, including proteins such as enzymes and DNA. In the gene mutation method according to this embodiment, nitrogen, which exists at a natural abundance of 0.364%, is used. 15 Create a situation where N isotopes are concentrated and distributed unevenly in the target gene, 15 N and 1 H 15 N( 1 H,α1γ) 12 By irradiating a target gene with a proton beam with enough energy to cause a C resonant nuclear reaction, large mutations are caused in the target gene.

[0031] In the double-stranded DNA that makes up a gene, adenine (A) has five nitrogen atoms attached to the purine ring and amino group, and thymine (T) has two nitrogen atoms attached to the pyrimidine ring, resulting in seven nitrogen atoms in the adenine-thymine pair (AT pair) of the DNA double strand. Similarly, guanine (G) has five nitrogen atoms attached and cytosine (C) has three nitrogen atoms attached, resulting in eight nitrogen atoms in the guanine-cytosine pair (GC pair) of the DNA double strand.

[0032] Figure 1 shows the N present in each base pair. 15 represents a DNA molecule substituted with N, naturally 15 The isotope ratio of N is enriched by 275.5 times. 15 The figure shows the state of the N-labeled 15 The abundance ratio of N is 0.364%.

[0033] Figure 2 shows how resonant nuclear reactions occur in a DNA molecule. 15 1 in N-labeled DNA 15 N nucleus and proton 15 N( 1 H,α1γ) 12 When a C resonance nuclear reaction occurs, 15 N disappears and the reaction15 Centered on the bonding position of the N atom 4 He and 12 C nuclei are emitted almost isotropically in opposite directions, immediately capture electrons, and pass through the material as ions, generating high-density electronic excitations in nearby atoms along the ion tracks.

[0034] The probability of electron excitation decreases inversely proportional to the square of the distance from the reaction center, and the further an atom is from the reaction center, the smaller the probability. 15 When N undergoes a resonant nuclear reaction, 15 The atoms near the bonding position of the N atom are locally excited with a high probability, 15 Local mutations are more likely to occur in the DNA near the N atom binding site.

[0035] Figure 3 shows the case of using the conventional gene mutation method using heavy ion beam irradiation. In this conventional gene mutation method using heavy ion beam irradiation, heavy ions are incident from outside the DNA at a constant density over the entire DNA, so atoms near the DNA along the heavy ion trajectory are electronically excited, and the probability of mutation occurring is approximately the same over the entire DNA.

[0036] In contrast, in the method of this embodiment, 15 By increasing the N isotope ratio, 15 It is possible to induce local mutations in the DNA near the N atom binding site.

[0037] In this embodiment, the target gene or its vicinity 15 The technique to increase and concentrate the N isotope ratio is based on the fact that it exists in nature at 0.364% and is also present in living cells at the same ratio. 15 This technology utilizes N as it is. 15 Generated by irradiating N with protons 15 N( 1 H,α1γ) 12 The C resonance nuclear reaction occurs when protons, the most abundant of the hadron components in cosmic rays near the Earth's surface, randomly fall on surface organisms and are present in their DNA.15 This is the same nuclear reaction that occurs when a nuclear bomb collides with a N nucleus, and has similar effects on living cells.

[0038] That is, at or near the target gene 15 Techniques for increasing and enriching N isotope ratios, 15 N-labeled living cells were irradiated with protons. 15 N( 1 H,α1γ) 12 Increasing the mutation probability of a target gene by generating a C resonance nuclear reaction is merely a method for accelerating the process by which life has been exposed to nature since the birth of life on Earth, resulting in mutation of its genetic information, and the basic mechanism is no different from the method of creating mutant strains by conventional radiation (UV, X-rays, gamma rays, heavy ions) irradiation. Therefore, the gene mutation method of this embodiment is fundamentally different in principle from genetic engineering technology, is not subject to regulations on genetic engineering technology, and is a method with the excellent characteristic of enabling the introduction of large mutations into a target gene.

[0039] In or near a target gene in a living cell 15 To quantify DNA mutations caused by proton irradiation of DNA samples with an increased N isotope ratio, 15 The number of N atoms is clearly defined 15 It is necessary to construct N-labeled DNA standards.

[0040] 15 By constructing N-labeled DNA standard samples, 15 N( 1 H,α1γ) 12 The 4.43 MeV gamma ray dose emitted by the C resonant nuclear reaction and the 15 It is possible to obtain calibration data for N concentration. By obtaining the dose calibration value of 4.43 MeV gamma rays, 15 The amount of 4.43 MeV gamma rays emitted by a resonant nuclear reaction simultaneously with proton irradiation of N-labeled living cells is measured, and compared with a calibration value, making it possible to quantify the number of mutations that have occurred in the DNA of living cells.

[0041] In the gene mutation method according to this embodiment, 15 We have constructed a standard sample by artificially synthesizing DNA consisting of N-labeled base pairs using the polymerase chain reaction. It is desirable that the constructed artificial DNA standard sample will consist of base sequences that are important in molecular biology, biochemistry, and biological functions.

[0042] FIG. 4 shows an artificial DNA standard sample consisting of 84 base pairs. The artificial DNA standard sample is prepared, for example, by the following method. 15 N unlabeled, i.e. 14 Primer OR_primer_F has a TACGTTAAATC sequence structure containing N at a natural abundance of 99.636%. 15 N-label, i.e. 15 Deoxynucleoside triphosphates containing 98% or more N 15 Oligonucleotide OR_WT_F is synthesized by polymerase chain reaction using 73 N-labeled dNTPs (deoxyribonucleoside 5'-triphosphates). 15 N unlabeled primer OR_primer_R with the sequence structure TGCAACCATT; 15 N-labeled deoxynucleoside triphosphates 15 Oligonucleotide OR_WT_R is synthesized from 74 N-labeled dNTPs by polymerase chain reaction. Furthermore, OR_WT_F and OR_WT_R form base pairs with each other through hydrogen bonds, 15 N-labeled oligonucleotide double-stranded DNA 15 N-labeled OR_DNA is formed, which serves as the standard sample.

[0043] 15 N-labeled OR_DNA 15 N unlabeled primers OR_primer_F / _R, 15 N-labeled deoxyribonucleotides 15It consists of 84 DNA base pairs (84 bps) including N-labeled dNMPs (Deoxyribonucleotide 5'-monophosphates). The breakdown of the base sequence is as follows: 73 bps of OR_WT_F (excluding the primers): 15 N-labeled dAMP: 19 units, 15 N-labeled dTMP: 22 units, 15 N-labeled dGMP: 17 units, 15 N-labeled dCMP: 15 units. In addition, in the 74 bp of OR_WT_R excluding the primer, 15 N-labeled dAMP: 23 units, 15 N-labeled dTMP: 20 units, 15 N-labeled dGMP: 16 units, 15 N-labeled dCMP: 15. These are summarized in Table 1 below.

[0044] [Table 1]

[0045] 15 N-labeled OR_N contained in the whole DNA ( 14 N+ 15 The total number of N is 623. 15 The standard purity of N-labeling is 98% or higher, and 14 N and 15 The ratio of N isotopes changes. 15 When the N-labeling purity is 98%, the total OR_DNA 15 N is 538.29. Ideally, 15 At 100% N-labeling purity, the total OR_DNA 15 The maximum number of N atoms is 549.27, which accounts for 88.17% of the total number of nitrogen atoms (see Table 1). 15 The molecular weight of N-labeled OR_DNA is also 15 Depending on the N-labeling purity, it is greater than 52,467 g / mol and can reach a maximum of 52,477.95 g / mol.

[0046] Figure 5 shows 15 N( 1 H,α1γ) 12 C resonance nuclear reaction. 15 When proton beams are irradiated onto biological samples labeled with N, 15 The binding energy of the N nucleus and the proton in the center of mass system is 12.1277 MeV, 16 O * When the two nuclei collide at an energy difference of 0.8409 MeV (0.987 MeV in the laboratory proton energy), which is the energy difference between the second excited level of the compound nucleus (12.9686 MeV), the two nuclei resonantly bond together. 16 O * A compound nucleus is formed. 16 O * immediately emits an alpha particle 12 C * The first reaction channel de-excites the nucleon to the first excited level of 11.6007 MeV, and the second reaction channel emits an α particle. 12 A second reaction channel de-excites the C ground state to 7.1616 MeV, and another reaction channel de-excites the C ion by emitting only γ-rays. 16 Three reaction channels occur, including a third reaction channel that de-excites the O ground state at 0 MeV. These are expressed in nuclear reaction equations as (p,αγ), (p,α), and (p,γ).

[0047] The (p,α1γ) reaction channel used in this embodiment is characterized by the α( 4 He nuclei) and 12 C and are emitted as secondary particles in each reaction. 12 C * The first excited level of α0 is de-excited to the ground state, resulting in the emission of a 4.43 MeV gamma ray. In the (p,α0) reaction channel, no gamma ray is emitted, and in the (p,γ0) reaction channel, no highly ionizing secondary particle is emitted. The highly ionizing secondary particle is emitted by 15 If N is located inside or near the DNA, 15This has the effect of exerting a high local ionization effect on biomolecules near N, increasing the probability of causing the desired genetic mutations in DNA. Furthermore, the 4.43 MeV gamma rays emitted per nuclear reaction can be easily counted, and these gamma rays reflect the amount of genetic mutations in DNA caused by proton beam irradiation. Therefore, by counting the 4.43 MeV gamma rays during beam irradiation, it is possible to quantify the amount of genetic mutations in DNA.

[0048] 16 O * The resonance energy with the second excited level is much lower than the Coulomb barrier potential in the collision between the two nuclei. 16 O * When the resonance energy width of 300 eV, which is defined as the width of the second excited level of the compound nucleus, is exceeded and the reaction cross section deviates from the resonance energy, the reaction cross section drops sharply. 15 The proton beam incident on N-labeled OR_DNA at the resonance energy is limited to the range where the fluctuation of the incident energy is within the resonance energy width. 15 N( 1 H,α1γ) 12 C resonance nuclear reaction occurs.

[0049] In actual proton beam irradiation, the resonance energy width is determined by a convolution integral calculation of the resonance energy width and the energy spread of the proton beam, typically about 1 keV, and the proton beam irradiation energy is determined by adding the energy loss when the proton passes through the material to the resonance energy. 15 N-labeled DNA samples, or 15 In proton beam irradiation of biological samples such as biological cells containing DNA, the proton beam energy is varied from the resonance energy to a high energy exceeding the resonance energy, for example, by varying the energy step width in a certain range, thereby distributing the protons from the surface to the interior of the target sample. 15 For N, thoroughly 15 N( 1 H,α1γ) 12It may be possible to generate C resonance nuclear reactions. The energy change range and step size can be selected depending on the target sample. 15 N( 1 H,α1γ) 12 This may be determined by obtaining the resonance curve of the C resonance nuclear reaction. See, for example, FIG. 13.

[0050] 15 N( 1 H,α1γ) 12 Produced in a C resonant nuclear reaction 12 C * and α are emitted in opposite directions, and when they are emitted in the same direction as the incident direction of the proton beam, the kinetic energy reaches its maximum value E(max), 12 C * and α respectively. C (max)=0.6252 MeV, E He (max)=1.2806 MeV. 12 C * When α is emitted in the opposite direction to the incident direction of the proton beam, the kinetic energy reaches a minimum value E(min), and E C (min)=0.1437 MeV, E He (min)=0.7991 MeV. 12 C * and α capture the electron immediately after emission. 12 C. 4 It becomes a He ion and travels through the material.

[0051] 12 C and 4 He ions always have values ​​in these energy ranges, 15 The electrons pass through the N-labeled OR_DNA sample, exciting nearby atoms along the path, especially those with a radius of 5 nm to 10 nm. 15 The samples are not N-labeled OR_DNA samples, but biological samples with cell walls and intracellular organelles, such as eukaryotic unicellular algae. In consideration of the energy loss when protons pass through the cell walls and intracellular organelles, even when proton beam irradiation is performed with an energy higher than the resonance energy, the proton beam and 15 N-labeled DNA or DNA nearby 15N-labeled samples 15 N( 1 H,α1γ) 12 In C resonance nuclear reactions, regardless of the proton beam irradiation energy, 12 C and 4 The He reaction product ions are emitted with a constant energy, and the characteristic of this method is that they excite nearby atoms along their trajectories with a constant intensity.

[0052] moreover, 12 C and 4 The electron excitation of the He reaction product ions is due to the linear energy transfer (LET) (MeVcm 2 / g)), 12 C and 4 The energy range of the He reaction product ions emitted varies, and the LET to carbon, C, one of the constituent elements of the target sample, is 12 C reaction product ions: 2.51 × 10 3 MeVcm 2 / g or more, up to 4.70 × 10 3 MeVcm 2 / g, 4 He reaction product ions: 1.73 × 10 3 MeVcm 2 / g or more, up to 1.99 × 10 3 MeVcm 2 / g. This value is the energy at which the proton reaches its resonance energy. 15 The LET is 17.2 times higher, and up to 27.1 times higher, than that of the N-labeled OR_DNA sample. 15 For biological samples including N-labeled OR_DNA samples, 15 N( 1 H,α1γ) 12 The electron excitation by the ions produced by the C resonance nuclear reaction is overwhelmingly higher than that by proton irradiation. 15 N-labeling makes it possible to selectively introduce large mutations.

[0053] 12 C and 4The LET of the He reaction product ions is 0.58 × 10, which is the LET of C in typical heavy ion irradiation (e.g., 320 MeV C ion irradiation). 3 MeVcm 2 / g), it is 4.3 times higher and up to 8.1 times higher. 12 C and 4 He reaction product ions are released, 15 N-labeled OR_DNA sample and DNA or DNA vicinity 15 In N-labeled living cells, 15 N( 1 H,α1γ) 12 by C resonance nuclear reaction 15 Mutations occur in the N-neighboring genes with a high probability. 12 By measuring the 4.43 MeV gamma ray emitted from the first excited level of C, 15 It is possible to directly quantify the number of gene mutations occurring near N.

[0054] By proton irradiation 15 Mutations that occur in N-labeled OR_DNA samples are not repaired, unlike biological samples that have DNA repair functions. 15 By examining mutations in N-labeled OR_DNA samples, the purely physical mutation status caused by proton irradiation will be revealed. 15 One method for examining mutations that have occurred in N-labeled OR_DNA samples is to determine the presence or absence of DNA breaks using circular plasmid vectors, which are circular DNA present in the cytoplasm of bacteria and other organisms.

[0055] Figure 6 shows an artificial gene in which the artificial gene shown in Figure 4 is ligated to another artificial gene, and an outline of the method for determining whether or not DNA is broken. 15The circularized plasmid vector, made by ligating N-labeled OR_DNA, is introduced into the E. coli culture medium. The circularized plasmid vector is taken up by the E. coli, and ampicillin-resistant E. coli proliferate. 15 If the N-labeled OR_DNA has been cleaved in advance by proton irradiation, the plasmid vector will not become circular and will not be degraded within the E. coli cells to form a stable gene, resulting in the E. coli cells becoming resistant to ampicillin and dying. 15 In the circular plasmid vector ligated with N-labeled OR_DNA, 15 The more N-labeled OR_DNA there is, the slower the growth of E. coli. 15 N-labeled OR_DNA sample, 15 N unlabeled OR DNA sample, 15 Each of the N-labeled OR_DNA samples that had not been irradiated with proton beams was ligated to T-vector pMD19, and each was placed on ampicillin-containing medium to compare the number of E. coli colonies. 15 N-labeled OR_DNA samples were 15 N( 1 H,α1γ) 12 DNA mutations occur due to C resonant nuclear reactions and proton beams. 15 In the N-unlabeled OR_DNA sample, DNA mutations occur only with the proton beam. In the sample not irradiated with protons, DNA mutations do not occur. 15 The relative frequency of DNA mutations introduced by proton irradiation of N-labeled OR_DNA will be revealed.

[0056] 15 As a more direct method to examine the state of N-labeled OR_DNA cleaved by proton irradiation, 32 One technique involves attaching phosphate groups containing P radioisotopes and separating the fragments by electrophoresis with single-base resolution, which allows the determination of how much breakage has occurred in one strand of DNA.

[0057] Figure 7 shows an overview of how to identify how much cleavage has occurred in one strand of DNA. 15Before synthesizing the N-labeled OR_DNA PCR sample, one of the primers, OR_primer_F or OR_primer_R, is phosphorylated with ATP, and during PCR synthesis, a DNA fragment with a phosphate group attached only to the 5' end of one DNA strand is synthesized. After proton beam irradiation, [γ? 32 P]-ATP is used to bind only the DNA strand that does not have a phosphate group. 32 It is labeled with P. When DNA is electrophoresed at high voltage in a thin gel with a low salt concentration and containing urea, it is heated and separated in a single-stranded state (denatured state), making it possible to distinguish DNA with a single base difference. Because single-base differences can be distinguished by gel electrophoresis, it is possible to determine whether random cleavage is possible or whether only the vicinity of a specific base is susceptible to cleavage.

[0058] DNA in living cells 15 When labeling with N, it is necessary to distinguish between nitrogen atoms that make up DNA and intracellular molecules that contain nitrogen elements other than DNA, such as nitrogen atoms present in proteins such as enzymes that control cell formation and metabolic functions, or nitrogen atoms present in RNAs that have various functions within cells, such as ribosomal RNA that forms ribosomes, transfer RNA that transports amino acids, and messenger RNA that determines the amino acid sequence on ribosomes, and to label only DNA. 15 Label with N.

[0059] Within cells, nitrogen is utilized in a variety of nitrogen-containing organic compounds, including amino acids, nucleotides, proteins, nucleic acids (DNA, RNA), and some phospholipids. All of these nitrogen-containing organic compounds are converted into inorganic nitrogen, the ammonium ion NH4 + The first nitrogen-containing organic compound is produced when glutamine reacts with glutamic acid via glutamine synthetase and is fixed into an organic compound as glutamine (ammonia assimilation). Glutamate synthetase transfers an amino group from glutamine to 2-oxoglutarate, producing two molecules of glutamic acid. The resulting glutamine and glutamic acid are then transformed directly or via transamination reactions to synthesize a variety of nitrogen-containing organic compounds.

[0060] Figure 8 shows the synthesis pathway of nitrogen-containing organic compounds within cells. Nitrogen-containing compounds are broken down into ammonia again within the cell and recycled. Amino acids are used for various purposes in the synthesis of structural proteins, enzymes, nucleic acids, and lipids. On the other hand, DNA synthesis proceeds from deoxyribonucleotides by the hereditary transmission of genetic information. Artificially synthesized 15 When N-labeled deoxyribonucleotides are administered to the culture medium, they are taken up into the cells, some of which are decomposed, ammonium ions are synthesized, and protein synthesis begins. 15 To label N, you need to inhibit that process. 15 To prevent the synthesis of N-labeled proteins, MSX (Methionine sulfoximine), an inhibitor of glutamine synthetase, is administered.

[0061] Figure 9 shows the MSX and 15 When N-unlabeled glutamine was administered, only nucleic acids were released into the cells. 15 This paper presents a new method for N-labeling. Glutamic acid and ammonia as an amino group bind to the active site of glutamine synthetase to synthesize glutamine, which is then released from glutamine synthetase. However, the structural formula of MSX contains a HN=S=O structure, which is similar to the structural formula of glutamine, H2N-CH=O. MSX also has a strong active site, and when glutamic acid and ammonia bind, they cannot leave MSX, and glutamine synthesis stops. In other words, the pathway for synthesizing glutamic acid from glutamine by glutamate synthetase is cut off, 15 N-labeled proteins are no longer synthesized. However, to ensure that synthesis of proteins needed for various purposes is not cut off, 15 N-unlabeled glutamine is administered to the culture medium. 14 Structural proteins, enzymes, and some DNA and RNA with an N abundance ratio of 99.637% are synthesized. 15 The remaining N-labeled deoxyribonucleotides that are not involved in ammonia synthesis 15DNA is synthesized using N-labeled deoxyribonucleotides, 15 N-labeling is limited to DNA only.

[0062] Furthermore, in the intracellular nucleic acid synthesis pathway, only DNA is synthesized. 15 The process of labeling with N is shown in more detail in Figure 10. 15 To synthesize N-labeled DNA, (1) each of the four bases is 15 N-labeled deoxyribonucleotides are added to the medium and the cells are cultured. Next, since deoxyribonucleotides, which synthesize DNA, are synthesized only from ribonucleotides by ribonucleotide reductase (RNR) in the cells, (2) an RNR inhibitor (i-RNR) is administered. 15 This creates conditions in which deoxyribonucleotides not labeled with N cannot be synthesized within the cell. Furthermore, within the cell, (3) base transfer occurs between deoxyribonucleotides and ribonucleotides, and the deoxyribonucleotides introduced from outside the cell are converted to ribonucleotides. 15 The N-labeled base is transferred to the base of ribonucleotide in the cell and then 15 N_RNA can be synthesized, and conversely, ribonucleotides 15 The bases not labeled with N may also be transposed to the bases of deoxyribonucleotides. 14 Metabolized from N-amino acids, etc. 14 The amount of N_ribonucleotides increases due to the inhibition of RNR in (2), 15 The amount of base transition from N-deoxyribonucleotide to ribonucleotide can be suppressed. 15 Excessive amounts exceed those required for DNA synthesis 15 When N-deoxyribonucleotides are added to the medium, (3) 15 There is a possibility that the N base may be transferred from a deoxyribonucleotide to a ribonucleotide. 15 High efficiency of N_DNA synthesis, 15 To suppress the synthesis of N_RNA, 15The amount of N-deoxyribonucleotides input can be adjusted appropriately.

[0063] Figure 11 shows the results of analyzing only the target gene or its vicinity. 15 A new method for N labeling is shown in Figure 4. 15 The base sequence of N-labeled OR_DNA is part of the gene of lambda (λ) phage, a virus that parasitizes Escherichia coli as a host. It is a regulatory gene that inhibits the λ phage's own proliferation so that it can parasitize the host Escherichia coli without killing it. 15 N-labeled OR_DNA has three Cro protein binding sites, and when Cro protein binds to one of these sites, the regulatory gene is activated. By introducing a mutation in the gene sequence among the three binding sites, the frequency at which the regulatory gene is activated can be controlled. Figure 4 explains how the polymerase chain reaction (PCR) 15 A method for preparing N-labeled OR_DNA was described. 15 After synthesizing it without N labeling, it is added to the culture medium, and the E. coli that has incorporated it into the control gene 15 When N-labeled Cro protein is administered, 15 The N-labeled Cro protein binds to the regulatory gene of E. coli. When irradiated with protons, 15 Near specific genes where N-labeled Cro protein is bound 15 N( 1 H,α1γ) 12 A C resonance nuclear reaction occurs. This method makes it possible to induce mutations in specific genes with a high probability.

[0064] Example 1 15 Proton irradiation results for N-labeled OR_DNA samples] (1) 15 Preparation of N-labeled OR_DNA samples [OR_primer_R phosphorylation] The phosphorylation process for assessing DNA breakage after proton irradiation is described below. 16 μL of ATP (10 mM = mmol / L), 10 μL of OR_primer_R (100 μM), 16 μL of 10x diluted buffer, 8 μL of T4PNK, and 110 μL of sterile water were reacted at 37°C for 35 minutes, then the reaction was stopped by raising the temperature to 72°C. Proteins and lipids were removed by PCI extraction (Phenol Chloroform Isoamylalcohol (25:24:1) extraction), followed by ethanol fixation and extraction.

[0065] [By PCR method 15 Synthesis of N-labeled OR DNA] Two types of primers were synthesized: with and without phosphorylated primers. Heat-stable DNA polymerase (PrimeSTAR): 0.5 μL, 5x diluted buffer solution: 10 μL, 15 N sign ( 15 (N purity 98% or higher) dNTP: 6 μL, OR_primer_F (10 μM): 2.5 μL, OR_primer_RP (phosphorylated primer 10 μM): 7.5 μL, template: 1 μL, sterile water: 22.5 μL, thermostable DNA synthase (Prime STAR): 0.5 μL, 5x dilution buffer: 10 μL, 15 N sign ( 15 6 μL of dNTP (purity 98% or higher), 2.5 μL of OR_primer_F (10 μM), 2.5 μL of OR_primer_R (non-phosphorylated primer 10 μM), 1 μL of template, and 27.5 μL of sterile water were used for 35 PCR cycles (98°C for 10 minutes, 31°C for 20 minutes, 72°C for 30 minutes). Synthesis of an 84-base pair DNA was confirmed by electrophoresis.

[0066] (2) Proton beam irradiation 15 N-labeled OR_DNA sample, 15 The irradiated sample was prepared by dropping N-unlabeled OR_DNA onto a gold substrate. The OR_DNA solution concentration was 31.2 ng / μL, the drop volume was 13 μL, and the drop area was 0.23 cm. 2The sample dropped contains 4.7 × 10 12 Each DNA contains more than 538 15 N atoms are bonded. Converted to a sample area, it is 1.1 x 10 16 cm -2 The proton beam irradiation was performed using a 4 MV Pelletron electrostatic accelerator at the Tsukuba Center of the National Institute of Advanced Industrial Science and Technology (AIST) and a 1 MV tandem electrostatic accelerator at the Research Infrastructure Center of the University of Tsukuba. The beam current was 0.1 nA to 5 nA, and the beam irradiation area was 0.071 cm. 2 It was. 15 N( 1 H,α1γ) 12 The 4.43 MeV gamma ray emitted by the C resonant nuclear reaction is the most efficiently detected high-energy gamma ray containing Bi (atomic number 83) in BGO (Bi4Ge3O 12 Crystalline scintillator, specific gravity 7.3 gcm -3 The detector (76.2 mm diameter x 79.2 mm length) was placed outside the vacuum at a distance of 24 mm from the irradiated sample, and detection was performed. The solid angle that the detector can detect is 10% of the total azimuthal angle. If the detection sensitivity for 4.43 MeV gamma rays is a maximum of 0.1, the detection efficiency for 4.43 MeV gamma rays is a maximum of about 1%. 15 When N-labeled OR_DNA samples were irradiated with protons at a constant energy, the count of 4.43 MeV gamma rays was 4,000 counts, and the total count when irradiating eight points with an energy of 8 keV was 25,000 counts. Considering the detection efficiency, at least 2.5 × 10 6 of 15 N-labeled OR_DNA 15 This means that a mutation occurred due to an N resonance nuclear reaction.

[0067] (3) 15 Ligation of N-labeled OR_DNA into a linearized plasmid vector: [ 15 Creation of A-overhangs in N-labeled OR DNA Proton beam irradiation 15Four types of mixed solutions were prepared: N-labeled OR_DNA (9 ng / μL): 6.9 μL, buffer solution (Ex_taq) 1 μL, Ex_taq 0.1 μL, dATP (10 mM) 2 μL; unirradiated OR_DNA (8 ng / μL): 6.9 μL, 10-fold diluted buffer solution (Ex_taq) 1 μL, Ex_taq 0.1 μL, dATP (10 mM) 2 μL; negative insert DNA sample: 6.9 μL, 10-fold diluted buffer solution (Ex_taq) 1 μL, Ex_taq 0.1 μL, dATP (10 mM) 2 μL; control insert CI sample (10 ng / μL): 6.9 μL, 10-fold diluted buffer solution (Ex_taq) 1 μL, Ex_taq 0.1 μL, dATP (10 mM) 2 μL; and the mixtures were incubated at 72 °C for 1 hour.

[0068] [Ligation with a linear plasmid vector] A linear plasmid vector (T-Vector pMD19, 2,692 base pairs) was mixed with the above four mixed solutions on ice and left to stand for 30 minutes, after which it was subjected to a heat shock at 42°C for 30 seconds to increase the transformation efficiency.

[0069] [Ampicillin-resistant E. coli culture] E. coli (JM109) cultured in M9 medium at 37°C with shaking at 180 rpm was inoculated into ampicillin-containing medium containing four types of circular plasmid vectors, and colony formation of JM109 was compared. In the medium containing the circular plasmid vector ligated from the non-irradiated sample, the number of colonies was 46 / 11 ng DNA, while in the medium containing the circular plasmid vector ligated from the non-irradiated sample, the number of colonies was 46 / 11 ng DNA. 15 In the medium administered with the circular plasmid vector ligated with N-labeled OR_DNA, the number of colonies decreased to 13 / 11 ng DNA, or less than one-third.

[0070] [Example 2: Results of proton irradiation on Escherichia coli biological samples] Because proton beam irradiation is generally performed in a vacuum, we first freeze-dried E. coli (JM109) and examined its resistance. 10 μL of JM109 E. coli solution was placed in an Eppendorf tube and divided into two samples: one that was frozen at liquid nitrogen temperature and one that was not frozen in liquid nitrogen. Each was placed in a vacuum dryer at -20°C for 4 hours, then removed and left at 4°C for an additional 24 hours to create freeze-dried samples. The survival rate of JM109 E. coli at this point was 26.8% for the sample that was frozen in liquid nitrogen and 39.1% for the sample that was not frozen in liquid nitrogen.

[0071] JM109 E. coli has 514 million base pairs of DNA, which can be split in two ways. 15 N labeling was performed. 15 In JM109 Escherichia coli cultured in a medium containing N-labeled ammonium ions without administering MSX, an inhibitor of glutamine synthetase, all nitrogen in the cells, including proteins and nucleic acids, was 15 N-labeled (sample F). 15 In the medium containing N-labeled deoxynucleotides, MSX and 15 In JM109 E. coli cells cultured with N-unlabeled glutamine, only DNA and RNA were detected. 15 N-labeled biological sample (sample D).

[0072] Two types 15 N-labeled JM109 E. coli and 15 Three types of solutions of N-unlabeled JM109 E. coli (sample E) were prepared to create proton beam irradiated samples. The E. coli concentration, solution drip volume, drip area, number of dripped cells, area density of the cell count, and the number of cells were measured for each irradiated sample. 15 The N area density is as follows:

[0073] [Sample D] E. coli concentration: 3.17×10 8 cells / mL, solution dropping volume: 10 μL, dropping area: 1.26 cm 2 , Number of cells dropped: 3.17 × 10 6 cells, area density of cell number: 2.52 × 10 6cells / cm 2 , 15 N area density: 9.50×10 13 cm -2 .

[0074] [Sample E] E. coli concentration: 3.17×10 8 cells / mL, solution dropping volume: 10 μL, dropping area: 1.20 cm 2 , Number of cells dropped: 3.17 × 10 6 cells, area density of cell number: 2.64 × 10 6 cells / cm 2 , 15 N area density: 3.66×10 11 cm -2 .

[0075] [Sample F] E. coli concentration: 8.62×10 7 cells / mL, solution dropping volume: 20 μL, dropping area: 1.56 cm 2 , Number of cells dropped: 1.72 × 10 6 cells, area density of cell number: 1.11 × 10 6 cells / cm 2 , 15 N area density: 4.18×10 13 cm -2 .

[0076] The 4.43 MeV gamma ray dose measured simultaneously with proton irradiation revealed that only DNA and RNA in sample D were 15 N-labeled 15 The amount of N was calculated by dividing the total amount of nitrogen in the F sample by the total amount of nitrogen in the F sample. 15 It was found that the ratio was 44.6 times lower than that of N-labeled.

[0077] Example 3 15 Proton irradiation results for N-labeled drug resistance gene plasmid samples] (1) pUC4-KIXX Plasmid and Its Construction Method Figure 12 shows the genetic structure of the pUC4-KIXX plasmid (plasmid DNA). This plasmid is a circular plasmid (3853 bps) in which the kanamycin resistance gene (Kanamycin-R, 1248 bps) has been inserted into the gene pUC4 (2605 bps (base pairs)) which has the ampicillin resistance gene (Ampicillin-R) and Ori. The gene sizes occupied by the promoters and transcription regions of the two drug resistance genes are 934 bps for the ampicillin resistance gene, 953 bps for the kanamycin resistance gene, and 621 bps for the Ori region required for plasmid replication. The types of base pairs in each gene region and 15 Table 2 shows the N number (100% substitution rate). [Table 2]

[0078] By the following method, 15 a plasmid containing N at natural abundance; 15 Plasmids containing more than 98% N were generated. 15 250 mL of M9 minimal medium containing N at its natural abundance (0.364%) and using NH4Cl as the nitrogen source was prepared. 15 Contains more than 98% N 15M9 minimal medium containing NH4Cl as a nitrogen source was prepared in 250 mL volumes. Specifically, each M9 minimal medium was prepared by dissolving Na2HPO4 (15 g), KH2PO4 (7.5 g), NaCl (1.25 g), NH4Cl (2.5 g), 1 M (mol / L) MgSO4 (250 μL), 20% (w / v) glucose (2.5 mL), and 1 M CaCl2 (25 μL) in 250 mL of ultrapure water, autoclaving, cooling to room temperature, and then adding 250 μL of 1% thiamine-HCl. E. coli (JM109) harboring the pUC4-KIXX plasmid was cultured in each medium. Specifically, 25 μL of E. coli was added to the M9 minimal medium and cultured for 2 days at 37 °C with shaking at 200 rpm. The E. coli was then collected and the plasmid was purified using a plasmid extraction kit, Plasmid Midi kit (Qiagen). The plasmid was dissolved in sterile water, and the DNA concentration was quantified using a fluorometer NanoDrop (Thermo Fisher Science) and adjusted to 115 ng / μL. 15 It is a plasmid containing N at natural abundance. 14 N_pUC4-KIXX and 15 It is a plasmid containing more than 98% N 15 N_pUC4-KIXX was generated.

[0079] (2) Proton irradiation of pUC4-KIXX plasmid 1 μL of each of the two types of plasmid samples was dropped onto a Si wafer substrate and dried, after which the substrate was placed in a vacuum chamber and irradiated with a proton beam. The proton beam irradiation was carried out using a 1 MV tandem electrostatic accelerator at the Research Infrastructure Center of the University of Tsukuba, a national university corporation. The proton beam irradiation conditions were the same as in Example 2 (2). Figure 13 shows the results. 15 N_pUC4-KIXX plasmid sample 15 N( 1 H,α1γ) 12 The resonance curve of the C resonance nuclear reaction is shown below. 15 N-labeled deoxyribonucleotides 15The resonance curve obtained for N-labeled dGTP is also shown. Proton beam irradiation was performed at three different doses, varying the proton energy from 890 keV to 906 keV in 4 keV increments. The three different doses are shown in Table 3. [Table 3]

[0080] (3) Transformation of proton-irradiated plasmids 1×10 10 8.6 ng of proton-irradiated plasmid was added to 50 μL of E. coli cells / mL, and the plasmid was introduced into the E. coli cells by electroporation. During the electroporation process, a direct current (DC) field was applied at a voltage of 1500 V, a resistance of 200 Ω, and a capacitance of 25 μF for 3.5–3.7 ms. 1 mL of LB medium was added, and the cells were cultured for one hour at 37°C and 200 rpm. During this culture process, the plasmid was damaged by proton irradiation, resulting in linearization of the circular form. This prevented replication within the E. coli cells; therefore, only the proton-irradiated plasmids that maintained their circular form were amplified and transformed.

[0081] (4) Evaluation of the extent of damage to drug resistance genes Three types of culture plates containing two drugs were prepared. 100 μL of E. coli transformed with the proton-irradiated plasmid was plated onto each culture plate containing (a) 100 μg / mL ampicillin, (b) 50 μg / mL kanamycin, and (c) 100 μg / mL ampicillin + 50 μg / mL kanamycin, and the number of colonies formed was measured. As a control sample, 100 μL of unirradiated plasmid was plated onto each of the three culture plates containing drugs. Figures 14, 15, and 16 show the number of colonies formed on the three culture plates containing drugs. The vertical axis represents the amount normalized by the number of colonies formed in the control sample (=1.0) and shows the average of three measurements. The error indicates the upper and lower limits of the three measurements. Although the number of colonies formed at 10 μC was zero in all figures, the detection limit value was plotted in the figure. The vertical axis represents the percentage of the two drug resistance genes in the plasmid that function normally without being damaged by proton irradiation. At a total dose of 2.5 μC, 15 This result indicates that the N_pUC4-KIXX plasmid was more damaged by radiation than the normal plasmid. 15 N( 1 H,α1γ) 12 This shows that C resonance nuclear reactions cause greater damage to genes than the ionization effect of proton beams. In addition, in Figure 14, when the total irradiation dose is approximately 5.0 μC or more, genes are thought to suffer nonspecific damage.

[0082] The above-described embodiments are intended to facilitate understanding of the present invention and are not intended to limit the present invention. The elements of the embodiments, as well as their arrangement, materials, conditions, shapes, sizes, etc., are not limited to those illustrated and can be modified as appropriate. Furthermore, configurations shown in different embodiments can be partially substituted or combined with each other.

Claims

1. DNA 15 labeling with N; 15 Irradiating the DNA with a proton beam having an energy that causes a resonant nuclear reaction of N; Any method of mutating genes, including, but not limited to, methods of mutating genes in the human body.

2. The DNA 15 Labeling with N is 15 The method of claim 1 , comprising substituting with N.

3. The DNA 15 Labeling with N allows the vicinity of the DNA to be 15 The method of claim 1, comprising labeling with N.

4. The DNA in living cells 15 The method according to any one of claims 1 to 3, wherein the compound is labeled with N.

5. In the cellular components other than DNA in the living cells 15 N is maintained at its natural abundance, and 15 The method according to claim 4, wherein the abundance ratio of N is increased above its natural abundance ratio.

6. The DNA in the living cell 15 Labeling with N 15 The method of claim 4 or 5, comprising administering to said living cells N-labeled deoxyribonucleotides and an inhibitor of glutamine synthetase.

7. The DNA in the living cell 15 Labeling with N 15 The method of claim 4 or 5, comprising administering to said living cells N-labeled deoxyribonucleotides and an inhibitor of ribonucleotide reductase.

8. The DNA in the living cell 15 Labeling with N 15 The method of claim 6 or 7, further comprising providing the living cells with N-unlabeled glutamine.

9. The method of claim 8, further comprising detecting the resonant nuclear reaction. In the detecting step, a gamma ray dose of 4.43 MeV is measured; The method according to any one of claims 1 to 8, further comprising calculating the number of mutations that have occurred in the DNA based on the measured amount of gamma rays.

10. In the calculation, 15 The method according to claim 9, wherein the amount of gamma rays generated by a resonant nuclear reaction occurring in a standard sample with a known number of N atoms is referenced.

11. The method of claim 1 , further comprising varying the energy of the proton beam.

Images