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WO2025113065A1 - Procédé et système de détection de métabolites d'acide nucléique - Google Patents

Procédé et système de détection de métabolites d'acide nucléique Download PDF

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Publication number
WO2025113065A1
WO2025113065A1 PCT/CN2024/128522 CN2024128522W WO2025113065A1 WO 2025113065 A1 WO2025113065 A1 WO 2025113065A1 CN 2024128522 W CN2024128522 W CN 2024128522W WO 2025113065 A1 WO2025113065 A1 WO 2025113065A1
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triphosphate
monophosphate
free
msi
nucleic acid
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Yan Peng
Magdalena Justyna KOZIOL
Houyu ZHANG
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Chinese Institute For Brain Research Beijing
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Chinese Institute For Brain Research Beijing
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present disclosure belongs to the field of biotechnology.
  • the present disclosure relates to a mass spectrometry imaging (MSI) -based method and analysis system for detecting the spatial distribution of the nucleic acid metabolite in the brains.
  • MSI mass spectrometry imaging
  • the present disclosure also relates to a method and system for detecting the modifications on the DNA and/or RNA molecules by detecting free nucleic acid metabolites.
  • RNA modifications are closely related to biological development and diseases. For example, RNA modifications can regulate RNA translation and change RNA stability. Post-transcriptional modifications of RNA are dynamic and critical to cell function. A minor misregulation thereof may have a significant impact on RNA metabolism which is critical to cell function. Therefore, RNA modifications play a vital role in in a variety of biological activities and mediate normal cell function and development.
  • RNA modifications promote mRNA stability and enhance its translation into protein, while others may target mRNAs for degradation and prevent the translation thereof.
  • RNA modifications are also involved in mRNA localization and alternative splicing.
  • N6-methyladenosine (m 6 A) is a modification widely present in mRNA.
  • the formation of m 6 A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (mettl3) as a key factor.
  • mettl3 methyltransferase-like 3
  • Studies have shown that the inactivation of mettl3 in mouse nervous system causes the loss of m 6 A modification, extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression.
  • mettl3 conditional knockout mice are abnormally shrunk and cannot develop into the structure of cerebellums, which would cause balance disorders.
  • mettl3-mediated m 6 A modification plays an important role in the development of mammalian cerebellum.
  • AD Alzheimer's disease
  • RNA modification changes For example, in neurons of patients with AD, the level of m 6 A modifications on mRNA is increased by more than four times with the accumulation of misfolded tau protein. Subsequently, RNA molecules labeled with the m6A modification bind specifically to the misfolded tau protein with the help of an RNA-binding protein HNRNPA2B1. It may be related to the deposition of tau protein in Alzheimer's disease.
  • m6A modification can induce phase separation in cells during stress to isolate some mRNAs involved in cell repair, thereby inhibiting the synthesis of repair proteins to prevent them participating in cell repair.
  • RNA modifications comprise other modifications than m6A modification. It is unclear whether or to what extend other RNA modifications are involved in the progression of AD except the m6A modifications, and how the spatial distribution of RNA modifications is in different regions of the brain.
  • Nucleic acid metabolites comprise of free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides.
  • the spatial distribution of the metabolites in the brain can reflect the metabolic level and modification level before degradation of the nucleic acids in the brain regions.
  • the existing fluorescence-or immune-imaging methods which utilize antibodies for binding modified nucleosides, are prone to false positive results.
  • the corpus callosum and fornix comprise commissural fibers that are difficult to separate and make it even more difficult to localize brain subregions.
  • the spatial distribution of nucleic acid metabolites in the brain plays a crucial role in the study of brain development and brain diseases. However, no related detection methods have so far been reported about the spatial distribution of the free modified nucleosides, free modified (deoxy) nucleotides in the brain.
  • MSI mass-to-charge ratios
  • the present disclosure provides a mass spectrometry imaging (MSI) -based method and system for detecting the DNA and/or RNA modifications through detecting free DNA and RNA metabolites.
  • MSI mass spectrometry imaging
  • the method and system of the present disclosure facilitates the spatial detection of unbound free modified nucleic acid metabolites, shedding light on the dynamic, regional changes in DNA and RNA modifications.
  • the present disclosure provides use of the detection of a free modified nucleic acid metabolite for determining a modification on a nucleic acid molecule.
  • the present disclosure can be used for (e.g. in situ) detecting the free modified nucleic acid metabolites, preferably by using a mass spectrometry imaging (MSI) .
  • MSI mass spectrometry imaging
  • the present disclosure can be used for determining a change in the modification on the nucleic acid molecule by detecting a change in the free modified nucleic acid metabolite.
  • the modification may comprise any chemical modifications affecting DNA and/or RNA metabolites, such as methylation, deoxygenation, acylation and the like.
  • the present disclosure provides a method for detecting a modification on a nucleic acid molecule, which comprises a step of determining a free modified nucleic acid metabolite.
  • the step of determining a free modified nucleic acid metabolite may comprise performing a mass spectrometry imaging (MSI) on a sample to obtain MSI data of a free modified nucleic acid metabolite.
  • MSI mass spectrometry imaging
  • the nucleic acid metabolite may comprise any metabolites of RNA and/or DNA, and comprises one or more selected from a group consisting of free modified (deoxy) nucleoside (s) , free (deoxy) nucleoside (s) , free modified (deoxy) nucleotide (s) , and free (deoxy) nucleotide (s) .
  • the MSI may be performed in a positive-and/or negative-mode scanning.
  • the MSI data may comprise ion intensity of a characteristic adduct ion and/or mass-to-charge ratio (m/z) value thereof for the free nucleic acid metabolite in the sample.
  • the sample may be in a form of cell/tissue homogenate, cells, tissue sections and the like. In some particular embodiments, the sample may be applied on a slide.
  • the method may further comprise a step of comparing the obtained MSI data with a control sample.
  • the control sample may be derived from a healthy subject. With comparing the obtained MSI data of the sample to be detected with a control sample, for example, it may determine if there is a change in the distribution or amount of the modified free nucleic acid metabolite, which mirror the change in the DNA/RNA molecule.
  • the method may comprise the following steps: 1) performing a mass spectrometry imaging (MSI) on a sample to obtain MSI data in positive-and/or negative-mode scanning; 2) extracting the MSI data to obtain ion intensity of a characteristic adduct ion and/or mass-to-charge ratio (m/z) value of the DNA or RNA metabolite in the sample; and, optionally 3) comparing the extracted ion intensity with a control, or obtaining a MSI image.
  • MSI mass spectrometry imaging
  • the ion intensity of the characteristic adduct ion and/or m/z value thereof for the free nucleic acid metabolite may be determined by using a standard sample including the metabolite.
  • the standard sample may comprise the metabolite in a matrix, which is derived from the same origin as the sample to be detected.
  • the matrix may be brain homogenate or a brain section if the sample to be detected is a brain sample (e.g. a brain section) , and may be cell suspension or cells if the sample to be detected is the cell.
  • the characteristic adduct ion and/or m/z value of the free nucleic acid metabolite may be determined by a process including the following steps: a) performing mass spectrometry imaging (MSI) on the standard sample to obtain MSI data in positive-and/or negative-mode scanning; b) extracting the MSI data; and c) selecting the adduct ion and/or m/z value which has the highest peak intensity as the characteristic ion intensity and/or m/z value thereof for the free nucleic acid metabolite.
  • MSI mass spectrometry imaging
  • the adduct ion may comprise, but is not limited to, one or more selected from a group consisting of [M+H] + , [M+Na] + , [M+K] + , [M+NH 4 ] + , [M+H-H 2 O] + , [M] + , [M-H] - , [M+Cl] - and [M-H-H 2 O] - .
  • the adduct ion which is generated in the positive mode scanning may comprise one or more selected from a group consisting of [M+H] + , [M+Na] + , [M+K] + , [M+NH 4 ] + , [M+H-H 2 O] + and [M] + .
  • the adduct ion which is generated in the negative mode scanning may comprise one or more selected from a group consisting of [M-H] - , [M+Cl] - and [M-H-H 2 O] - .
  • the m/z value of the positive-and/or negative-mode scanning may range from 0 to 1000. In some embodiments, the m/z value of the positive-and/or negative-mode scanning may range 10 to 1000, preferably 20 to 900, and more preferably 30 to 800.
  • the m/z values of the characteristic adduct ions may be selected from those listed in Table 1.
  • the MSI may comprise a step of applying a spray solution to the sample.
  • the spray solution may comprise an acetonitrile-water mixture having a volume ratio of acetonitrile : water of (1 : 10) to (10 : 1) or any value therebetween.
  • the spray solution may comprise an acetonitrile-water mixture having a volume ratio of acetonitrile : water of 1: 10, 1: 9.5, 1: 9, 1: 8.5, 1: 8, 1: 7.5, 1: 7, 1: 6.5, 1: 6, 1: 5.5, 1: 5, 1: 4.5, 1: 4, 1: 3.5, 1: 3, 1: 2.5, 1: 2, 1: 1.5, 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 9: 1, or 10: 1.
  • the spray solution is sprayed by means of a needle with a diameter of 0.1-100 ⁇ m or any value therebetween, preferably 1-90 ⁇ m or any value therebetween, more preferably 10-30 ⁇ m or any value therebetween.
  • the spray solution may be sprayed by means of a needle with a diameter of 0.1 ⁇ m, 0.5 ⁇ m, 1 ⁇ m, 5 ⁇ m, 10 ⁇ m, 15 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, 75 ⁇ m, 80 ⁇ m, 85 ⁇ m, 90 ⁇ m, 95 ⁇ m, or 100 ⁇ m.
  • the above method may further comprise a step of a MSI image through a ion channel of a certain m/z value, for example any m/z value in a range of 10 to 1000 or any value therebetween, preferably 20 to 900 or any value therebetween, and more preferably 30 to 800 or any value therebetween.
  • a MSI image through a ion channel of a certain m/z value, for example any m/z value in a range of 10 to 1000 or any value therebetween, preferably 20 to 900 or any value therebetween, and more preferably 30 to 800 or any value therebetween.
  • the free nucleic acid metabolite may comprise one or more selected from a group consisting of free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides.
  • the free nucleic acid metabolite may comprise one or more selected from a group consisting of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, uridine, pseudouridine, adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate
  • the method of the present disclosure can detect one or more free nucleic acid metabolite (s) at one time.
  • the MSI may be ionization MSI.
  • the MSI may be desorption electrospray ionization (DESI) MSI.
  • DESI desorption electrospray ionization
  • the sample may be biological sample.
  • the sample may be animal or human tissue, body fluids (e.g. saliva, blood, urine, lymphatic fluids and the like) or cells, and may be healthy or diseased.
  • body fluids e.g. saliva, blood, urine, lymphatic fluids and the like
  • tissue sample may include, but is not limited to, testicular, prostate, liver, breast, colon, kidney, lymph, and brain tissues.
  • the method of the present disclosure can make an in-situ and/or spatial detection of the free modified nucleic acid metabolite (s) in the samples.
  • the method of the present disclosure can detect the changes in the modifications of free nucleic acid metabolite (s) , which could mirror the modification changes in the DNA or RNA molecules, e.g. in different regions of one sample.
  • control is not specifically limited and may be selected depending on actual practice.
  • the control may be the sample which is not subjected to treatment, and the sample may have been subjected to the treatment.
  • control may be derived from a healthy subject and the sample may be derived from a diseased subject.
  • the spray flow rate is controlled so that a spraying spot of the spray solution has an elliptical shape on the sample.
  • the present disclosure provides a system for performing the method of the present disclosure.
  • the system may comprise: a mass spectrometry imaging (MSI) unit for performing MSI on a sample to obtain MSI data in positive-and/or negative-mode scanning; a MSI data extracting unit for extracting the MSI data to obtain ion intensity of a characteristic adduct ion and/or mass-to-charge ratio (m/z) value of the DNA or RNA metabolite in the sample; and, optionally, a data analysis unit for comparing the extracted ion intensity with a control, or obtaining a MSI image.
  • MSI mass spectrometry imaging
  • the system can implement the above method.
  • the present disclosure provides a mass spectrometry imaging (MSI) -based method for detecting spatial distribution of nucleic acid metabolite in a brain, wherein the method comprises the following steps:
  • sample preparation mounting a frozen brain section to be tested on a positively charged glass slide to obtain a sample slide
  • MSI spraying a spray in the needle evenly onto a surface of the brain section by using a needle capillary with a diameter of 10-30 microns, adjusting an angle between the needle and the sample slide to 55°-57° and height therebetween to 35-36.5 mm; performing in-situ desorption and ionization of metabolites on the brain section; and imaging by selecting a corresponding ion channel in a negative ion acquisition mode depending on whether the brain section is sagittal or coronal, so as to obtain MSI data; and
  • step (3) spatial quantitative analysis of nucleic acid metabolite: based on the MSI data obtained in step (2) , extracting ion intensity corresponding to a target in different brain regions according to a monitored mass-to-charge ratio of ions of the nucleic acid metabolite, so as to obtain spatial distribution of the nucleic acid metabolite in different brain regions of the brain;
  • nucleic acid metabolite comprises metabolite of RNA and/or DNA, and comprises one or more selected from a group consisting of free modified (deoxy) nucleoside (s) , free (deoxy) nucleoside (s) , free modified (deoxy) nucleotide (s) , and free (deoxy) nucleotide (s) .
  • the spray comprises an acetonitrile-water mixture having a volume ratio of acetonitrile : water of (5 ⁇ 8) : (2 ⁇ 5) , preferably of 8: 2.
  • the step of spraying comprises using a needle capillary with a diameter of 20 microns, adjusting the angle between the needle and the sample slide to 57° and the height therebetween to 36.5 mm.
  • the spray flow rate is controlled so that a spraying spot has an elliptical shape, and the spray is evenly sprayed onto the surface of the brain section in the direction of mass spectrometry.
  • the brain section comprises a sagittal plane and is imaged by selecting an m/z 303.2330 ion channel; and the brain section comprises one or more brain region (s) selected from a group consisting of fornix, corpus callosum, as well as olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.
  • s brain region
  • the brain section comprises a coronal plane and is imaged by selecting an m/z 309.2794 ion channel; and the brain section comprises a dentate gyrus in a hippocampal subregion.
  • the nucleic acid metabolite comprise metabolite of RNA and/or DNA, and comprise free modified (deoxy) nucleoside (s) , free (deoxy) nucleoside (s) , free modified (deoxy) nucleotide (s) and free (deoxy) nucleotide (s) .
  • the free modified (deoxy) nucleoside (s) may comprise one or more selected from a group consisting of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, and pseudouridine.
  • methyladenosine N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-
  • the free (deoxy) nucleoside (s) may comprise one or more selected from a group consisting of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine.
  • the free modified (deoxy) nucleotide (s) may comprise one or more selected from a group consisting of methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, and pseudouridine triphosphates.
  • the free (deoxy) nucleotide (s) may comprise one or more selected from a group consisting of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate.
  • the present disclosure further provides a MSI data-based system for analyzing spatial distribution of nucleic acid metabolite in a brain, which comprises:
  • a brain mass spectrometry image-acquisition module for obtaining groups of mass spectrometry images of a brain sagittal or coronal plane, and submitting to a data extraction module;
  • a data extraction module for, depending on the obtained brain mass spectrometry images, extracting ion intensity corresponding to a target which is nucleic acid metabolite, in different brain regions according to brain structure, and transmitting the ion intensity and the extracted information of the brain regions to a data analysis module;
  • the nucleic acid metabolite comprises metabolite of RNA and/or DNA, and comprises one or more selected from a group consisting of free modified (deoxy) nucleoside (s) , free (deoxy) nucleoside (s) , free modified (deoxy) nucleotide (s) and free (deoxy) nucleotide (s) ; and
  • a data analysis module for performing differentiation analysis of content of the target between the different brain regions and a control based on the ion intensity and extracted information of the brain region corresponding to the target, and outputting analysis result.
  • the brain mass spectrometry images are obtained in accordance with the method as described in the present disclosure.
  • the brain may comprise a sagittal plane and be imaged by selecting an m/z 303.2330 ion channel, and the brain may comprise one or more brain regions selected from a group consisting of fornix, corpus callosum, as well as olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.
  • the brain may comprise a coronal plane and be imaged by selecting an m/z 309.2794 ion channel, and the brain may comprise a dentate gyrus in a hippocampal subregion.
  • the above technical solutions of the present disclosure can obtain MSI ion diagrams of the brain sections which can clearly identify more different brain regions by improving the needle and sprays. Further, the present disclosure has the following beneficial effects:
  • the MSI-based method for detecting the spatial distribution of the nucleic acid metabolite (also called as metabolite) in the brain utilizes the needle capillary with a diameter of 10-30 microns, and adjusts the angle between the needle and the sample slide to 55°-57° and the height therebetween to 35-36.5 mm.
  • the mass spectrometry image obtained in the present disclosure can clearly identify the structures of different brain regions such as the fornix and corpus callosum on the sagittal plane and the dentate gyrus in the hippocampal subregion on the coronal plane, and thus can be used for accurately detecting and analyzing the spatial distribution of the nucleic acid metabolite in different brain regions.
  • the method of the present disclosure has higher sensitivity and accuracy for the detection of the nucleic acid metabolite in the brain by improving the sprays and constructing the MSI databases of the nucleic acid metabolite.
  • the present disclosure provides a MSI data-based system for analyzing the spatial distribution of the nucleic acid metabolite in the brain extracts the ion intensity corresponding to the targets in different brain regions according to the monitored mass-to-charge ratios of ions of the nucleic acid metabolite, and obtains the spatial distribution of the nucleic acid metabolite in different brain regions.
  • the system of the present disclosure can be used for screening and evaluating the therapeutic effects of different drugs on the nucleic acid modification-related diseases, by analyzing the differences in the contents and distributions of various nucleic acid metabolite in different brain regions between the experimental and control groups.
  • the present disclosure can promote the research and development of drugs for related diseases.
  • the present disclosure can accurately extract the ion intensity corresponding to the targets in 14 brain regions, including olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix, and corpus callosum. It facilitates rapid detection of the spatial distribution of various nucleic acid metabolite in the brain.
  • the present disclosure can obtain MSI ion diagrams of various samples with high resolution.
  • the method and system of the present disclosure can be used for screening and evaluating the therapeutic effects of drugs, environmental conditions and treatments on nucleic acid modifications and diseases, by analyzing the differences in the contents and distributions of various nucleic acid metabolite in different brain regions between the experimental and control groups.
  • Figure 1 shows a schematic diagram of the experimental process of Example 1.
  • Figure 2 shows a comparison of the MSI ion diagrams obtained from m/z 309.2794 ion channel by using needle capillaries with different diameters.
  • Figure 2A shows the MSI ion diagram obtained by using a needle capillary of 100 micron; and
  • Figure 2B shows the MSI ion diagram obtained by using a needle capillary of 20 micron.
  • Figure 3 shows a comparison of the MSI ion diagrams obtained from m/z 309.2794 ion channel in a negative ion mode with different heights and angles between the needle and the slide.
  • Figure 3A shows the MSI ion diagram obtained with the angle between the needle and the slide being 60° and the height therebetween being 37 mm; and
  • Figure 3B shows the MSI ion diagram obtained with the angle between the needle and the slide being 57° and the height therebetween being 36.5 mm.
  • Figure 4 shows a schematic diagram of the experimental process of Example 2.
  • Figure 5 shows the ion intensity corresponding to the targets detected by different spray solutions in a positive ion mode.
  • Figure 6 shows the ion intensity corresponding to the targets detected by different spray solutions in a negative ion mode.
  • Figure 7 shows a schematic diagram of the experimental process of Example 3.
  • Figure 8 shows a schematic diagram of the mount of the mouse brain sections.
  • Figure 9 shows a schematic diagram of the spatial quantitative analysis process of the nucleic acid metabolite.
  • Figure 9A shows a schematic diagram of the brain structure of the sagittal plane of the brain;
  • Figure 9B shows the MSI ion diagram obtained from m/z 303.2330 ion channel in a negative ion mode;
  • Figure 9C shows the 14 brain regions selected.
  • Figure 10 shows the mass spectrometry images of 14 brain regions.
  • Figure 10A shows the mass spectrometry image of the brain regions including olfactory bulb, cerebral cortex, anterior olfactory nucleus and corpus callosum;
  • Figure 10B shows the mass spectrometry image of the brain regions including caudate putamen, ventral striatum, fornix, and basal forebrain;
  • Figure 10C shows the mass spectrometry image of the brain regions including hippocampus, thalamus, hypothalamus, and midbrain;
  • Figure 10D shows the mass spectrometry image of the brain regions including cerebellum, pons and medulla.
  • Figure 11 shows box plots of the differential analysis results of the target methyluridine in different brain regions.
  • Figure 12 shows box plots of the differential analysis results of the nucleic acid metabolites determined by MSI according to one embodiment of the present disclosure.
  • Figure 13 shows the differential analysis results of the nucleic acid metabolites determined by LC-MS/MS according to one embodiment of the present disclosure.
  • DNA a fundamental biological molecule encoding genetic instructions, is composed of nucleic acids present in both DNA and RNA. These nucleic acids undergo chemical modifications catalyzed by specialized cellular enzymes, directly influencing genetic instructions and crucial cellular processes like transcription and degradation. While research has predominantly focused on DNA modifications affecting deoxycytidine (dC) , the exploration of other nucleic acid modifications is emerging. High-throughput sequencing methods, although valuable, only indirectly infer modifications through sequencing patterns and are therefore error prone, unlike Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) , which provides direct identification and quantification of modifications.
  • LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry
  • LC-MS/MS enables the precise study of DNA and RNA modification changes and can reliably quantify and even identify less abundant and even novel DNA and RNA modifications, after the isolation, digestion and processing of DNA and RNA into individual nucleosides. Yet material requirements and the labor-intensive nature, including the need to process DNA and RNA into individual nucleosides, hinder its application for comprehensive spatial analysis of DNA and RNA modifications within tissues or organs.
  • the present disclosure pinpoints local alterations in DNA and RNA modifications through identification of spatial changes in free modified nucleic acid metabolites. Since the free modified nucleic acid metabolites present in cells stem from DNA and RNA degradation processes, the local alterations in DNA and RNA modifications can mirror the DNA and RNA modification changes in the cells or tissues. Exploring free modified nucleic acid metabolites as indicators of local modification alterations could present a promising screening approach for identifying areas warranting further investigation in epigenetic and epitranscriptomic research.
  • the MSI technology can be combined with professional image processing software, to directly analyze the biological tissue sections, generate two-dimensional ion intensity diagrams of any compounds with specified mass-to-charge ratios (m/z) , and realize the high-throughput, comprehensive and rapid analysis of the composition, relative abundance and distribution of compounds in tissues.
  • Potential biomarkers can be discovered and monitored by obtaining the spatial distribution of the biomarkers.
  • DREAMS mass spectrometry imaging
  • MSI Desorption Electrospray Ionization
  • DESI Desorption Electrospray Ionization
  • aqueous droplets acting as projectiles are directed towards a moving biological sample, continuously freeing molecules while mass spectrometry (MS) proceeds, revealing the relative spatial distribution of biomolecules.
  • MS mass spectrometry
  • DREAMS has been refined to specifically target the release, detection and analysis of free unmodified and modified nucleic acid metabolites, while retaining the capability to detect a wide range of other metabolites.
  • the method and system of the present disclosure have emerged as a powerful tool for spatially detecting free modified nucleic acid metabolites, which can provide unprecedented insights into DNA and RNA modifications.
  • the method and system of the present disclosure can offer valuable insights into epigenetic and epitranscriptomic regulation in both health and disease contexts, serving as a crucial spatial screening approach for identifying regions of interest and potential hotspots of changes in tissues, cells and the like.
  • it can be easily applied to screen cells or tissues to determine effects of drugs, treatments or environmental stimuli on these cells or tissues to evaluate changes in their free and potentially DNA and RNA modifications.
  • the present disclosure utilizes MSI technology by using mass spectrometry as a detector, determines the monitoring ions of the nucleic acid metabolite (e.g. modified (deoxy) nucleosides, (deoxy) nucleosides, modified (deoxy) nucleotides and (deoxy) nucleotide) through experiments, and qualitatively determines the target nucleic acid metabolite in brain by detecting the monitored ionic mass-to-charge ratios of each target. It is found in the experiments that the outer contour of the brain sections and the general structures of different brain regions can be clearly seen in the MSI ion diagram obtained by using a needle capillary with a diameter of 10-30 microns to spray the brain sections.
  • the nucleic acid metabolite e.g. modified (deoxy) nucleosides, (deoxy) nucleosides, modified (deoxy) nucleotides and (deoxy) nucleotide
  • the different brain structures such as the dentate gyrus in the hippocampal subregion on the coronal plane and the corpus callosum and fornix on the sagittal plane, can be clearly identified.
  • the spray of acetonitrile-water mixture especially in a volume ratio of acetonitrile to water of 8: 2, has superior detection results, generates higher ion intensities of most nucleic acid metabolites (e.g. standards of modified nucleosides, nucleosides, modified (deoxy) nucleotides and (deoxy) nucleotides) , and thus has higher detection sensitivity with respect to that of other sprays.
  • nucleic acid metabolites e.g. standards of modified nucleosides, nucleosides, modified (deoxy) nucleotides and (deoxy) nucleotides
  • the present disclosure provides a MSI-based method for detecting spatial distribution of nucleic acid metabolite in a brain, which specifically comprises the following steps:
  • sample preparation mounting a frozen brain section to be tested on a slide, such as a positively charged glass slide, to obtain a sample slide;
  • MSI spraying a spray in the needle evenly onto a surface of the brain section by using a needle capillary with a diameter of 10-30 microns; adjusting an angle between the needle and the sample slide to 55°-57° and height therebetween to 35-36.5 mm; performing in-situ desorption and ionization of metabolites on the brain section; and transmitting to a mass spectrometer for separation and detection according to an ionic mass-to-charge ratio;
  • the nucleic acid metabolite comprises the metabolite of RNA and/or DNA, and comprises one or more selected from a group consisting of free modified (deoxy) nucleoside, free (deoxy) nucleoside, free modified (deoxy) nucleotide, and free (deoxy) nucleotide.
  • the brain sections are detected by using the spraying conditions as defined in step (2) for spraying.
  • the different brain region structures such as the corpus callosum and cerebral fornix on the sagittal plane and the dentate gyrus in the hippocampal subregion on the coronal plane, in the obtained MSI ion diagram can be clearly identified.
  • the slide to be tested is placed on an automatic moving stage; the spray is sprayed with very small droplets onto the surface of the sample line by line with the assistance of high-speed air flow; the metabolites on the brain section are desorbed and ionized in situ; the whole sample is scanned line by line; and the obtained mass spectrometry data is used for ion imaging to obtain a MSI ion diagram.
  • a needle capillary with a diameter of 20 microns is used, the angle between the needle and the slide to be tested is adjusted to 57° and the height therebetween is adjusted to 36.5 mm.
  • the spray flow rate is controlled so that the spraying spot is elliptical and oriented to the mass spectrometry. The spray can thus be evenly sprayed onto the surface of the brain section, and the desorption of ions on the current sampling point will not affect that of the next sampling point, in favor of further improving the spatial resolution of MSI.
  • the experimental results showed that the spray of acetonitrile-water mixture with the volume ratio of acetonitrile : water of (5 ⁇ 8) : (2 ⁇ 5) , is helpful to improve the sensitivity and coverage of MSI for the detection of the nucleic acid metabolite such as modified (deoxy) nucleosides, (deoxy) nucleosides, modified (deoxy) nucleotides and (deoxy) nucleotides.
  • the spray with a volume ratio of acetonitrile : water of 8 : 2 has significantly higher detection sensitivity than that of the other sprays.
  • the brain is sliced along the sagittal plane.
  • the mass spectrometry images corresponding to different brain regions which include olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix, and corpus callosum, are extracted according to the brain structure of sagittal plane by selecting the m/z 303.2330 ion channel in the negative ion acquisition mode.
  • the brain is sliced along the coronal plane.
  • the mass spectrometry image corresponding to different brain regions, which include the dentate gyrus in the hippocampal subregion, are extracted according to the brain structure of coronal plane by selecting the m/z 309.2794 ion channel in the negative ion acquisition mode is selected.
  • the nucleic acid metabolite comprises the metabolite of RNA and/or DNA, including free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides and free (deoxy) nucleotides.
  • the free modified nucleoside comprises one or more selected from a group consisting of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, and pseudouridine.
  • the free nucleoside comprises one or more selected from a group consisting of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine.
  • the free modified (deoxy) nucleotide comprises one or more selected from a group consisting of methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, and pseudouridine triphosphates.
  • the free (deoxy) nucleotide comprises one or more selected from a group consisting of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate.
  • the preferred spray comprises a acetonitrile-water mixture, which has a volume ratio of acetonitrile : water of (5 ⁇ 8) : (2 ⁇ 5) , preferably of 8: 2.
  • the positive and negative full scannings are performed under the condition that the MSI system is set to have the nitrogen pressure of 0.63-0.67 MPa and the spray flow rate of 5 ⁇ L/min, the spray is acetonitrile-water mixture, and the scanning mass-to-charge ratio range of 78-780, to obtain mass spectrometry data.
  • the quantitative analysis of the spatial distribution of nucleic acid metabolites in the brain can be realized by selecting the m/z 303.2330 ion channel from the MSI data collected in a negative ion mode, extracting the corresponding mass spectrometry image of different brain regions according to the brain structure of sagittal plane, and obtaining the ion intensities of free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides and free (deoxy) nucleotides in different brain regions.
  • the present disclosure also provides a MSI data-based system for analyzing the spatial distribution of nucleic acid metabolites in a brain.
  • the system comprises a brain mass spectrometry image acquisition module, a data extraction module and a data analysis module.
  • the brain mass spectrometry image acquisition module selects a corresponding ion channel in a negative ion acquisition mode for imaging depending on whether the brain section comprises sagittal plane or coronal plane, obtains mass spectrometry images, and submits them to the data extraction module.
  • the brain section comprises a sagittal plane, and is imaged by selecting an m/z 303.2330 ion channel in a negative ion acquisition mode.
  • the negative ion-mass spectrometry image corresponding to different brain regions are extracted according to the brain structure of the sagittal plane.
  • the brain section comprises a coronal plane, and is imaged by selecting an m/z 309.2794 ion channel in a negative ion acquisition mode.
  • the negative ion mass spectrometry image corresponding to different brain regions are extracted according to the brain structure of the coronal plane.
  • the data extraction module extracts the mass spectrometry images of different brain regions according to the brain structures of the sagittal or coronal plane, extracts mass spectrometry data according to the monitored ionic mass-to-charge ratio of the nucleic acid metabolite, obtains the ion intensities of the metabolite in different brain regions, and transmits the extracted ion intensities of the target and the extracted region information corresponding to the brain regions to the data analysis module.
  • the nucleic acid metabolite comprises metabolite of RNA and/or DNA, including one or more selected from free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides and free (deoxy) nucleotides.
  • the data extraction module is used for, based on the obtained positive-and negative-ion-mass spectrometry image in different brain regions of the brain, extracting the ion intensities of a corresponding target in each brain region according to the monitored ionic mass-to-charge ratio of the target and taking the nucleic acid metabolite as the target; and transmitting the extracted ion intensities of the target and the extracted region information of the corresponding brain regions to the data analysis module.
  • the negative ion-mass spectrometry image corresponding to different brain regions are extracted according to the brain structure of the sagittal plane, in which the brain regions comprise one or more selected from a group consisting of fornix, corpus callosum, olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.
  • the negative ion-mass spectrometry image corresponding to different brain regions are extracted according to the brain structure of the coronal plane, in which the brain regions comprise the dentate gyrus in the hippocampal subregion.
  • the data analysis module is used for performing differentiation analysis of the content of each target between the different brain regions and the control based on the received ion intensity data and the corresponding brain region of the target, and outputting the analysis result, such as drawing box plots.
  • the nucleic acid metabolite comprises the metabolite of RNA and/or DNA, including free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides and free (deoxy) nucleotides.
  • the free modified (deoxy) nucleoside comprises one or more selected from a group consisting of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, and pseudouridine.
  • the free (deoxy) nucleoside comprises one or more selected from a group consisting of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine.
  • the free modified (deoxy) nucleotide comprises one or more selected from a group consisting of methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, and pseudouridine triphosphates.
  • the free (deoxy) nucleotide comprises one or more selected from a group consisting of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate.
  • brain section MSI data is obtained according to the MSI detection method of the present disclosure:
  • the monitored ionic mass-to-charge ratio of the target to be tested is critical for the accuracy of this MSI method.
  • the present disclosure constructs a MSI database through experiments to identify free modified (deoxy) nucleosides, free (deoxy) nucleosides, free modified (deoxy) nucleotides and free (deoxy) nucleotides in the mouse brains. It is conducive to plotting the spatial distribution and content thereof.
  • the analysis system can be used for screening and evaluating the therapeutic effects of different drugs or treatments on RNA and/or DNA modification-and metabolism-disorders, for screening for effects of different treatments on cells, homogenate or tissues, or for diagnosing RNA and/or DNA modification-related diseases or effects on cells or tissue regions.
  • the RNA modification-related diseases comprise, such as Alzheimer's disease.
  • the term “about” or “approximately” refers to an acceptable degree of error for the quantity measured, given the nature or precision of the measurements. Typical exemplary degrees of error may be within 20%, 10%, or 5%of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” refer to values within an order of magnitude, potentially within 5-fold or 2-fold of a given value.
  • mice are obtained from the Experimental Animal Center of Chinese Institute for Brain Research, Beijing. The animal experiments in the following examples have been approved by the Animal Protection and Welfare Committee.
  • Example 1 Determination of the parameters of mass spectrometry imaging (MSI) for the spatial resolution
  • MSI provides non-targeted analysis in tissue sections or on differentially cultured cells.
  • An array of different ion distribution patterns can be obtained in a single MSI experiment, and can be used to reveal the in situ spatial distribution of endogenous metabolites.
  • This example compared the effects of the changes in various parameters such as the capillary diameters of the needle, the heights and angles between the needle and the slide on the spatial resolution of MSI, so as to screen out the optimized parameters of the needle.
  • the experimental process was shown in Figure 1. The specific experiments were performed as follows:
  • mice anesthetize the mice by intraperitoneal injection of 2.5%avertin at a dose of 0.35 g/kg; after sacrifice, take the brains of the mice, including olfactory bulb to medulla regions; place on ice and wash with pre-cooled phosphate-buffered saline; carefully suck the remaining phosphate-buffered saline on the surface of the brain samples with dust-free paper; place the mouse brains in cell culture dishes; and store in a -80°C freezer for later use.
  • Preparing brain sections take out the mouse brains obtained in step (1) ; place in the chamber of the freezing microtome at -22 °C for 20 minutes; fix it on the sample holder; cut sections with a thickness of 20 microns per section along the coronal direction in this example, because the dentate gyrus in the hippocampal subregions locates on the coronal plane; and mount the sections on positively charged glass slides.
  • MSI the experiment was carried out by using an aerodynamically assisted ionization MSI device and an Orbitrap Exploris 480 mass spectrometer. In particular: place the slides to be tested on an automatic moving stage and move line by line; spraying the spray-solution with very small droplets onto the surfaces of the samples line by line with the assistance of high-speed air flow; in situ desorb and ionize the metabolites on the brain sections; then transfer to the mass spectrometer for separation and detection depending on the ionic mass-to-charge ratios.
  • the spray- solution was sprayed under the conditions in which the needle capillaries had diameters of about 100 microns and about 20 microns respectively, the angle between the needles and the sample slides was about 55°, and the height between the needles and the sample slides was about 35 mm.
  • the MSI system was set to have nitrogen pressure of 0.63-0.67 MPa, spray flow rate of 5 ⁇ L/min, horizontal x-direction scanning rate of 0.2 mm/s, y-direction vertical interval of 0.1 mm, ion transfer tube temperature of 350°C, resolution ratio of 1.2 million, full negative ion scanning, scanning mass-to-charge ratio range of 78 to 780, target of automatic gain control (AGC) of 1E7, and maximum injection time of 100 ms.
  • Imaging the mouse brain sections convert the data to CDF format using Xcalibur software; process using MassImager software to subtract the background, with the mass tolerance being set to 10 ppm.
  • the MSI data from the m/z 309.2794 ion channel were selected from the MSI data acquired in a negative ion mode.
  • the above-mentioned ion channel corresponded to an endogenous metabolite with characteristic distribution in the mouse brains.
  • the spatial resolution of the MSI process was examined by its MSI ion diagrams. Then the MSI ion diagrams obtained by scanning with different parameters were compared to determine the optimal conditions for the needles, in order to improve the spatial resolution of the MSI process.
  • the MSI ion diagrams obtained from the m/z 309.2794 ion channel in a negative ion mode were shown in Figure 2.
  • Figure 2A shows a MSI ion diagram obtained from the m/z 309.2794 ion channel in a negative ion mode by using the needle capillary with a diameter of 100 ⁇ m; and Figure 2B shows a MSI ion diagram obtained from the m/z 309.2794 ion channel in a negative ion mode by using the needle capillary with a diameter of 20 ⁇ m.
  • the shapes and orientations of the spraying spots were fine-adjusted by continuously adjusting the angles (50° to 60°) and heights (35 to 37 mm) between the needles and the sample slides.
  • the spraying spots should be small and stable, but not be blurry or moved, which is essential for obtaining stable ion signals.
  • the angle of 55°-57° and height of 35-36.5 mm which could clearly identify the structures of different brain regions on the coronal plane, were determined, by comparing the MSI ion diagrams of m/z 309.2794 obtained under different parameters (heights and angles between the needle and the slide) .
  • the optimal angle is 57° and the optimal height is 36.5 mm. With these parameters, the structures of different brain regions, even the dentate gyrus in the hippocampal subregions, on the coronal plane can be clearly identified. It is shown in Figure 3.
  • Figure 3A shows the MSI ion diagrams obtained from the m/z 309.2794 ion channel by using needle capillary with a diameter of 20 ⁇ m, an angle between the needle and the slide of 60° and a height therebetween of 37 mm. Under such a condition, the structure of the dentate gyrus in the hippocampal subregion on the coronal plane cannot be clearly identified.
  • Figure 3B shows the MSI ion diagram obtained from the m/z 309.2794 ion channel by using needle capillary with a diameter of 20 ⁇ m, an angle between the needle and the slide of 57° and a height therebetween of 36.5 mm.
  • the structures of different brain regions on the coronal plane, even of the dentate gyrus in the hippocampal subregion, can be clearly identified.
  • it can improve the spatial resolution of MSI.
  • the spraying spot having an elliptical shape and oriented to the mass spectrometry, the desorption of ions on the current sampling point did not affect that of the next sampling point, thereby further improving the spatial resolution of MSI.
  • the optimal detection conditions determined in this example comprise the followings: the diameter of the needle capillary is 20 microns, the angle between the needle and the glass slide is 57° and the height is 36.5 mm.
  • the structures of different brain regions, such as the structure of the dentate gyrus in the hippocampal subregion, on the coronal plane can be clearly distinguished.
  • sample to be tested which contained the standard samples of modified nucleosides and nucleosides: grind the whole mouse brain mentioned above; add the same volume of water to the obtained brain homogenate; add 1 ⁇ L of 400 ⁇ M standard samples of the modified nucleosides and nucleosides to the brain homogenate to make the final concentrations of the samples to be 200 ⁇ M; and mount to positively charged glass slides adhered with PVC adhesive tapes having 2 ⁇ 5 mm rectangular wells.
  • the standard samples of modified nucleosides and nucleosides comprised cytidine, guanosine, methylcytidine, inosine, adenosine, uridine, pseudouridine, methylguanosine, and methyladenosine.
  • 2 ⁇ L of the mixtures were pipetted into each rectangular well, and the blank brain matrix was added to the same slide as a negative control. The slides were vacuum-dried for subsequent MSI scanning.
  • MSI the experiment was carried out by using an aerodynamically assisted ionization MSI device and an Orbitrap Exploris 480 mass spectrometer.
  • MSI system it was set to have nitrogen pressure of 0.63-0.67 MPa, spray flow rate of 5 ⁇ L/min.
  • the above-mentioned 8 kinds of spray solutions were used for spraying the samples, respectively.
  • the angle between the needle and the slide was 57° and the height therebetween was 36.5 mm.
  • the horizontal x-direction scanning rate was set to be 0.2 mm/s.
  • the y-direction vertical interval was set to be 0.1 mm.
  • the duration of the detection process was equal to the movement time of the MSI sample platform.
  • the temperature of the ion transfer tube was set to be 350°C, and the resolution ratio was set to be 1.2 million. Full positive or negative scanning was performed, with the scanning mass-to-charge ratio range of 78-780.
  • the target of AGC was set to be 1E7.
  • the maximum injection time was set to be 100 ms, to obtain the mass spectrometry data.
  • nucleic acid metabolites convert the data to CDF format using Xcalibur software; process using MassImager software to subtract the background, with the mass tolerance being set to 10 ppm.
  • the ion intensities of the targets were extracted by using the 8 kinds of spray solutions, and analyzing the MSI data obtained from positive-and negative-mode scanning.
  • the nucleic acid metabolites comprised free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides.
  • the detection results of partial nucleic acid metabolite were intercept and shown in Figures 5 and 6.
  • Figure 5 shows the ion intensities corresponding to the targets detected by different spray solutions in a positive ion mode.
  • Figure 6 shows the ion intensities corresponding to the targets detected by different spray solutions in a negative ion mode.
  • the ion intensities of each target were high, especially in the positive-and negative-ion acquisition modes, when the spray solution was acetonitrile-water mixture with the volume ratio of acetonitrile to water of (5 ⁇ 8) : (2 ⁇ 5) .
  • the obtained ion intensities were significantly higher for the acetonitrile-water mixture with the volume ratio of acetonitrile to water of 8 : 2 than that of other spray solutions, and thus had the highest detection sensitivity.
  • the standard samples comprised: modified nucleosides (methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, and pseudouridine) ; nucleosides (adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'- deoxycytidine, and thymidine) ; modified nucleotides (methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-
  • the horizontal angle between the slide and the needle was 57° and the height therebetween was 36.5 mm.
  • the horizontal x-direction scanning rate was set to be 0.2 mm/s.
  • the y-direction vertical interval was set to be 0.1 mm.
  • the temperature of the ion transfer tube temperature was 350°C, and the resolution ratio was set to be 1.2 million. Full positive or negative scannings was performed, with the scanning mass-to-charge ratio range of 78-780.
  • the target of AGC was set to be 1E7.
  • the maximum injection time was set to be 100 ms.
  • the m/z values of 9 adduct ions for specific DNA/RNA metabolites were screened and calculated based on the molecular weights of the target modified nucleosides, nucleosides, modified nucleotides and nucleotides.
  • the adduct ions generated in the positive ion mode comprised [M+H] + , [M+Na] + , [M+K] + , [M+NH 4 ] + , [M+H-H 2 O] + and [M] + .
  • the adduct ions generated in the negative ion mode comprised [M-H] - , [M+Cl] - and [M-H-H 2 O] - .
  • the adduct ions which had the m/z value corresponding to the highest peak intensity, were selected among the 9 adduct ions as the monitoring ions of the specific DNA/RNA metabolite.
  • the monitoring ions of different target i.e., modified nucleosides, nucleosides, modified nucleotides and nucleotides were shown in the following table.
  • Adduct ions are formed by the interaction of a precursor ion with one or more atom (s) or molecule (s) , resulting in ions which contain all the atoms of the precursor ion and the additional atoms of the associated atom (s) or molecule (s) .
  • Other compounds in the sample mixture which are called matrix, can also form adduct ions with precursor ions, and the resulted adduct ions having no analyte (s) belong to background ions.
  • the MSI data obtained by scanning in positive and negative modes were analyzed by MassImager software.
  • the mass spectrometry data from the sample regions were extracted, and the peak intensities corresponding to the ions were obtained based on the calculated m/z values of the adduct ions. Further, the adduct ions with the highest ion intensities were selected as the monitoring ions to establish a database.
  • mice 5-FAD Alzheimer's disease mice and C57BL/6J wild-type mice were used as experimental objects, which are obtained from the Experimental Animal Center of Chinese Institute for Brain Research, Beijing.
  • mice the brains of 6 mice were detected, with 3 of Alzheimer's disease mice as the experimental group and 3 of wild-type mice as the control group.
  • the experimental group may also use knockout mice.
  • the experiments were specifically performed as follows:
  • Preparing brain sections take out the mouse brains obtained above; place in the chamber of the freezing microtome at -22 °C for 20 minutes; cut the left and right sides of the brains with a knife and fix them on the sample holder; cut sections with a thickness of 20 microns per section, along the sagittal direction in this example (because both the sagittal and coronal brain regions can be identified by this method, and the sagittal plane can expose 14 brain regions at a time which is more than the brain regions exposed by the coronal plane) ; and mount the sections on positively charged glass slides.
  • the brain sections of the mice of each group were applied on the positively charged slides.
  • One glass slide was applied with 6 brain sections from 6 mice (1-3 from the experimental group and 4-6 from the control group) .
  • Two more slides were applied in the same order, marked and stored in a -80°C freezer.
  • MSI take out the slides from the -80 °C freezer before the MSI experiment; dry at room temperature for 20 minutes; and perform the MSI in the same manner as that in Example 2 to obtain mass spectrometry data.
  • nucleic acid metabolites convert the data to CDF format using Xcalibur software; process using MassImager software to subtract the background, with the mass tolerance being set to 10 ppm.
  • the nucleic acid metabolites comprised the metabolites of RNA and/or DNA, such as free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides.
  • FIG. 9A The schematic diagram of the brain structure of the sagittal plane is as shown in Figure 9A.
  • the image obtained through m/z 303.2330 ion channel (which corresponds to arachidonic acid) selected from the MSI data acquired in a negative ion mode is as shown in Figure 9B.
  • 14 brain regions were selected using MassImager software, as shown in Figure 9C, and comprised olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix and corpus callosum.
  • Figure 10A shows the mass spectrometry image of the brain regions including olfactory bulb, cerebral cortex, anterior olfactory nucleus, and corpus callosum
  • Figure 10B shows the mass spectrometry image of the brain regions including caudate putamen, ventral striatum, fornix, and basal forebrain
  • Figure 10C shows the mass spectrometry image of the brain regions including hippocampus, thalamus, hypothalamus, and midbrain
  • Figure 10D shows the mass spectrometry image of brain regions including cerebellum, pons and medulla.
  • MSI data analysis procedures were as follows:
  • Step 0 automatically create Excel files for storing the mass spectrometry data of all brain regions of interest;
  • Step 1 select the ion intensities according to the monitoring ions of the targets
  • Step 2 integrate the data in all Excel files
  • Step 3 normalize the data with at least three technological or biological replicates
  • Step 4 compare the data of the mice from the experimental and control groups, and perform differential analysis of the content of each target free modified nucleoside, free nucleoside, free modified nucleotide or free nucleotide in these 14 brain regions one by one, to generate box plots.
  • the analysis results of the target methyluridine were exemplarily shown in Figure 11. Other targets can also be detected in these 14 brain regions to generate corresponding box plots.
  • Example 5 The changes of free modified nucleosides detected by MSI can reflect the changes in the modifications of RNA molecules.
  • This example used the brains from 6 mice, which included 3 of conditional knocked-out Pus7 mice (KO) as the experimental group and 3 of Pus7 loxP mice as the control group.
  • the experiments were specifically performed as follows:
  • Adeno-associated plasmids pAAV-EF1 ⁇ -Cre-P2A-EGFP (AAV-Cre-EGFP) and pAAV-EF1 ⁇ -EGFP (AAV-EGFP) were constructed.
  • AAV-Cre-EGFP the coding sequence of CRE recombinase (Uniprot: P06956) was cloned into an AAV vector (Addgene) carrying the Human elongation factor-1 alpha (EF-1 ⁇ ) promoter.
  • the enhanced green fluorescent protein (EGFP, Uniprot: A0A6G6D467) was cloned into the C-terminal of Cre for co-expression, linked through the P2A peptide.
  • AAV-EGFP no P2A and no Cre were included in the vector.
  • each plasmid was co-transfected into HEK293T cells with a rep/cap containing plasmid pUCmini-iCAP-PHP.
  • eB Additional plasmid pAdDeltaF6
  • polyethylenimine polyethylenimine
  • the mass spectrometer was set in a positive ion mode and operated in selective reaction monitoring. Precursor ion was m/z 245.1 and product ion was m/z 113.1 for uridine and m/z 125.1 for pseudouridine.
  • the EIC of the base fragment was used for quantification. Accurate mass of the corresponding base-fragment was extracted using the XCalibur Qual Browser and Xcalibur Quan Browser software (Thermo Fisher Scientific) and used for quantification. Quantification was performed by comparison with the corresponding standard curve obtained from the pure nucleoside standards running with the same batch of samples.
  • the results are show in Figure 13.

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Abstract

Un procédé de détection d'un métabolite d'acide nucléique modifié libre est utilisé pour déterminer une modification sur une molécule d'acide nucléique. Un système est utilisé pour mettre en œuvre le procédé.
PCT/CN2024/128522 2023-11-28 2024-10-30 Procédé et système de détection de métabolites d'acide nucléique Pending WO2025113065A1 (fr)

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