Mass tag DNA adductomics with improved specificity

Abstract

A new type of test for DNA damage, referred to as Enhanced Specificity Mass Tag DNA Adductomics (「ESMD」), which results in advanced DNA adductomics, defines genotoxic exposure accurately and extensively in a practical way, including specific combinations of mass tags targeting amines and techniques selected from strong anion exchange, gentle depurination, and affinity chromatography.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 347,790, filed on June 1, 2022. BACKGROUND OF THE INVENTION

[0002] Cancer prevention screening. There is a heart attack prevention screening (cholesterol), but there is no corresponding cancer prevention screening. More than 50 years ago, cancer epidemiologists proposed that DNA adductomics (covalently damaged nucleotides in DNA) tests would be useful for cancer prevention: people would know the increase in their own adducts and change their environment (defined in the broadest sense) to reduce these adducts, thereby reducing cancer risk. Since then, despite many methods for DNA adducts being reported, there is no effective DNA adductomics test for cancer prevention. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0003] This invention changes the situation by introducing a new type of test for DNA damage called Enhanced Specificity Mass Tag DNA Adductomics (ESMD) that accurately and broadly defines genotoxic exposure in a practical way. This includes using it to optimize treatment for therapeutic drug monitoring of chemotherapeutic agents in terms of maximizing effectiveness while minimizing side effects and secondary cancers. Thereby, ESMD helps prevent both primary and secondary cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0004]

Figure 1

Figure 2

Figure 3

[2014] Nucleoside-Tailored Molecularly Imprinted Polymeric Nanoparticles (MIP NPs), Macromolecules, 47, 6322-6330).

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0005] Urine Urine is a preferred sample of the present invention. It is non-invasive even on a large scale and can contain DNA adducts from the whole body. Although the DNA adducts may be at low levels in urine, their signal intensity in a mass spectrometer is enhanced by labeling them, in particular in the nucleobase form, with an N-targeting quaternary amine mass tag reagent, preferably a CAX (cationic xylyl) mass tag. Other types of mass tags, such as those having a tertiary amine, phosphonium or pyridinium group that provides a positive charge, can also be used in the same way.

[0006] Alternative tests Each genotoxic chemical can give rise to multiple DNA adducts not only by reacting with different monomers of DNA (the general nucleotide, nucleoside or nucleobase components of nucleic acids), but also by reacting with multiple sites on several monomers. To detect or assess a given genotoxic exposure, it is necessary to measure only one of these adducts from a given genotoxic chemical, or an adduct type (as an alternative). Nevertheless, the ESMD test can broaden the genotoxic information obtained by measuring multiple adduct classes in a given procedure or in multiple procedures. It is advantageous to select alternative classes that reflect the different adduct characteristics of each exposure. One class that does this consists of N7-guanine adducts (N7-guanine). This is because many DNA-damaging chemicals attack the highly reactive N7 position of guanine in particular. EDMD can readily measure adducts of this class, as well as other adduct classes.

[0007] Focus on purine Purine nucleobase adducts are preferred for detection. Such adducts are readily formed by genotoxic chemicals; can be readily harvested from DNA as nucleobases (by gentle neutral hydrolysis or gentle acid depurination), and can be efficiently labeled with mass tags for sensitive detection by mass spectrometry. Particularly preferred is the N7-guanine nucleobase adduct (N7-guanine), which is mainly formed by many genotoxic chemicals and is readily released from DNA by gentle neutral hydrolysis because they have a positive charge. Other preferred purine nucleobase adducts for the same reason are N3-guanine, N7-adenine, and N3-adenine. Certain pyrimidine adducts that are hydrolytically labile on DNA are also preferred for measurement by ESMD, O 2 -cytosine and O 2- It contains thymine. The neutral purine containing the adduct can be isolated from DNA by gentle acid hydrolysis. The measurement of the canonical purine in this procedure enables accurate determination of the amount of DNA. The ESMD method can also be applied to the detection of DNA adducts in nucleoside form obtained from DNA by enzymatic digestion.

[0008] Practical cancer prevention tests To stimulate the strong interest of cancer epidemiologists and be introduced into routine clinical diagnosis, DNA damage tests for cancer prevention must be practical. The methods presented herein for the detection of nucleomere adducts are practical, in part, by providing the required resolution in a simple manner using one or more affinity reagents as an alternative to high-performance liquid chromatography (HPLC). HPLC is a common step in the preparation or detection stage in many current valuable methods for DNA adductomics, but this technique tends to be complex, time-consuming, delicate, and expensive, making such methods impractical. Instead, practical and fast short-column chromatography-MS, infusion-MS, or MALDI-MS techniques are used in the detection stage of ESMD, which helps to make this technique practical.

[0009] Impact of cancer prevention tests The environment defined in the broadest sense is the main cause of cancer. The impact of food genotoxicity information is greater, for example, when it is individualized as follows: (1) people know from the ESMD test that the damage to their DNA from their diet is increasing (increase in DNA adducts, "dirty DNA"); (2) they change their diet accordingly to include healthier options; (3) the amount of damaged adducts decreases; (4) this personal feedback of achieving cleaner DNA motivates them to continue the new diet; (5) their cancer risk decreases. By analogy, a person with diabetes, when their HbA1c level decreases and signs that diabetes is better controlled are seen, is motivated to take their medicine properly and practice a low-carb diet.

[0010] Aptamer Aptamers are useful as affinity reagents in ESMD and can assist in the purification of DNA adducts for detection (see below). Aptamers that bind to DNA adducts are known ((McKeague, M.

[2017] Aptamers for DNA Damage and Repair. Int. J. Molecular Sciences, 18, 2212-2228). Aptamers that bind to guanine and xanthine have been prepared (Kiga, D., Futamura, Y., Sakamoto, K., Yokoyama, S.

[1998] An RNA aptamer to the xanthine / guanine base with a distinctive mode of purine recognition. Nucleic Acids Res., 26, 1755-1760). Aptamers for use in the present invention can be commercially developed and obtained, such as from Aptagen.

[0011] Chromatographic retention power of multiple hydrogen (H) bonds The high retention of multiple H-bond ligand interactions in chromatography has been demonstrated using the N-[7-(2,4-dimethyl-1,8-naphthyridyl)] ligand, which confers high retention for guanosine derivatives (Feibush, B., Saha, M., Onan, K., Karger, B., Giese, R.

[1987] HPLC Separation of DNA Adducts Based on Hydrogen Bonding. J. Am. Chem. Soc. 109, 7531-7533). This is an example of partner affinity chromatography, where the binding partner for the target substance is similar in size to this substance.

[0012] DNA Nanopore Voltage-Driven Filtration DNA species (DNA nucleotides or DNA oligonucleotides) pass through the narrow channel provided by the channel protein, where the protein is located within the membrane; the solution of DNA species is above the membrane; an appropriate voltage is applied across the membrane; and the DNA species pass through the channel. The channel can be very narrow, for example, 90% of its volume can be occupied by segments of the DNA species passing through. DNA Nanopore Voltage-Driven Filtration provides DNA nanopore sequencing when the current is monitored for the pore. DNA Nanopore Voltage-Driven Filtration is useful in ESMD as part of sample pretreatment (see Figure 1). It is preferably used with 100-mers. Unadducted 100-mers pass quickly through the channel, while 100-mers with bulky adducts get caught there. Reversing the voltage recovers DNA rich in bulky adducts.

[0013] DNA repair enzymes DNA repair enzymes are useful in the present invention. In particular, DNA glycosylases are useful as part of sample pretreatment for releasing DNA adducts as modified nucleobases from DNA. An overview of DNA glycosylases has been provided (Lindahl, T.

[1979] DNA glycosylases, Endonuclease for Apurinc / Apyrmidinic Sites and Base Excision -Repair. Progress in Nucleic Acid Research and Molecular Biology 22, 135-192).

[0014] Quaternary amine Regarding the mass tags of Figure 1, positively charged reagents, particularly quaternary amines, are preferred, and the most preferred are two types of CAX (cationic xylyl) mass tags. The first type is CAX-CHO, which labels exocyclic primary amino groups on damaged nucleomers in the presence of hydride reagents, particularly sodium cyanoborohydride or pyridine borane. The second type is CAX-B (cationic bromo xylyl) for labeling secondary amine sites on modified nucleomers.

[0015] Technical considerations Urine may contain damaged nucleomers from the diet, but the dietary sources for these can be defined or controlled to exclude or define the diet as a source or potential source of modified nucleomers in the samples being tested. For example, this can be done by changing the diet and retesting.

[0016] Damaged nucleomers detected in urine as damaged nucleobases are preferred for detection in the present invention. These can be derived from RNA or DNA. Simultaneous measurement of modified nucleomers from both DNA and RNA is consistent with the goal of defining genotoxic exposure.

[0017] Purine recovery The initial urine can be treated with ribonucleases (e.g., RNase A and RNase T1) to remove RNA, and before initiating the acid depurination reaction, the remaining DNA can be purified from the urine by, for example, ultrafiltration or precipitation. This procedure, in addition to the above techniques (technical considerations), can eliminate diet and RNA as sources of modified nucleomers detected in this method. Positively charged adducts in DNA, such as N7-guanine or N3-adenine, can be recovered from DNA by mild neutral thermal hydrolysis followed by CAX labeling / MS detection. Next, neutral purine nucleomers can be recovered from DNA by mild acid hydrolysis followed by similar detection, which successfully provides a method for quantifying the amount of DNA in a sample. ESMD can be applied to 24-hour urine samples.

[0018] The biosample to be tested Urine is a preferred sample for this detection method, but any type of biosample containing DNA can be tested. Kits are generally available for isolating DNA from biosamples.

[0019] CAX mass tag The labeling reaction with CAX mass tags is important in two general aspects. First, this increases the response of the adduct in the mass spectrometer by up to about 1000-fold, enabling testing of biosamples such as urine with low concentrations of DNA. Second, this helps achieve comprehensive detection of DNA adducts because adducts with lower intrinsic sensitivity in the mass spectrometer would not be detected without this labeling.

[0020] DNA adducts and DNA adductomics Many scientists have contributed numerous publications over the past 50 years regarding the measurement of DNA adducts (damaged nucleotides in DNA) both as single adducts and as groups of adducts (DNA adductomics). DNA adducts can "cause" mutations, and mutations are at the heart of all cancers. The main goals of DNA adduct analysis are to better understand cancer and to help prevent it by learning what causes it in DNA. ESMD is also important for other conditions such as diabetes and aging.

[0021] Many DNA adductomics methods Many methods for measuring DNA adducts differ in their convenience, specificity, range, and cost. Overall, today's DNA adductomics is mostly a failure. There is no place in the world where biosamples can be sent for excellent DNA adduct testing, such as low cost, specificity, robustness, quantitativeness, practicality, extensiveness, etc.

[0022] ESMD enhances prior adductomics Jettison Mass Spectrometry (Wang, P., Shah, G.L., Landau, H., Coulter, M.E., Walsh, C.A., Roider, E., Kramer, C.S., Beuning, P.J., Giese, R.W.

[2020] Jettison-MS of Nucleic Acid Species, J. Am. Soc. Mass Spectrom. 31, 1641-1646) and Prelabeling (Wang, P., Roider, E., Coulter, M.E., Walsh, C.A., Kramer, C.S., Beuning, P.J., Giese, R.W.

[2021] DNA Adductomics by mass tag prelabeling, Rapid Commun. Mass Spectrom. 35: e9095) prior adductomics technologies have been brought to new performance levels by ESMD. In both cases, as a first step, the aldehyde mass tag labeling reaction can be applied directly to DNA, followed by the usual steps. The DNA sample can be subjected to enzymatic digestion to generate nucleosides, and at that point, or after partner affinity purification has been applied to the DNA digest, the aldehyde mass tag can also be applied.

[0023] Definition of Terms Canonical Nucleomer: Adenine, adenosine, 2'-deoxyadenosine, guanine, guanosine, 2'-deoxyguanosine, cytosine, cytidine, 2'-deoxycytidine, 5-methylcytosine, 5-methylcytidine, 5-methyl-2'-deoxycytidine, thymine, thymidine, uracil, uridine, or one of these naturally modified forms that contribute to the native structure or function of a nucleic acid molecule.

[0024] Noncanonical Nucleomer: A standard nucleomer that has been chemically modified in an unnatural way (causing loss of natural function or structure) by, for example, alkylation, acylation, amination, halogenation, hydrolysis, radical attack, electromagnetic radiation, or oxidation, and includes products resulting from combinations of these events, as well as products from molecular rearrangements due to such events. Other terms for noncanonical nucleomers here are "adduct", "nucleomer adduct", "damaged nucleomer", "modified nucleomer", "DNA adduct", or "RNA adduct".

[0025] Targeted Nucleomer: A nucleomer to be detected by a given procedure.

[0026] Nontargeted Nucleomer: A nucleomer that should not be detected in a given procedure.

[0027] Nucleomer: A standard or noncanonical nucleomer.

[0028] Nucleomer Component: A standard or noncanonical nucleomer that is part of DNA, part of RNA, or part of a nucleotide of the DNA or RNA type.

[0029] Guanine: Nonstandard guanine such as N7-guanine.

[0030] Adenine: Nonstandard adenine such as N3-adenine.

[0031] Canonical Nucleobase: A nucleomer in the form of an unmodified nucleobase such as adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil.

[0032] DNA: Genomic DNA, fragmented DNA, or DNA oligomer

[0033] Noncanonical Nucleobase: A nucleobase that has been chemically modified by the above-described non-natural methods (see nonstandard nucleomers).

[0034] Nonstandard DNA: DNA that has been chemically modified by non-natural methods; also called "adducted DNA".

[0035] Nonstandard RNA: RNA that has been chemically modified by non-natural methods (methods that are not natural in nature).

[0036] Nucleobase: A nucleobase that is either standard or nonstandard.

[0037] Nucleoside: Standard or nonstandard nucleosides are in deoxy form (characteristic of DNA) or non-deoxy form (characteristic of RNA).

[0038] Nucleobase: A purine or pyrimidine that is either standard (e.g., adenine, guanine, thymine, cytosine, uracil, 5-methylcytosine, or a naturally modified form of one of these) or nonstandard.

[0039] Nucleoside: A sugar-purine or sugar-pyrimidine compound in which the sugar is either ribose or deoxyribose and the compound is either standard or nonstandard.

[0040] Purine: Guanine and adenine.

[0041] Biosample: A biological or physiological fluid, cell, aerosol, or tissue including urine, blood, saliva, cerebrospinal fluid, white blood cells, plasma, skin, hair, placenta, semen, feces, liver, sweat, tears, breath, feces, lung, or liver. Urine is preferred.

[0042] Bio - sample extract: A bio - sample, as a material of the first phase, after being treated with a material of the second phase, is recovered in, on, or through a second material that is either a liquid phase or a solid phase. The treatment process includes purification steps such as solid - phase extraction, liquid - phase extraction, partitioning, centrifugation, precipitation, redissolution, or liquid chromatography.

[0043] Affinity Reagent: A lysed or solid-phase material that provides two or more attractive interaction sites for its target molecule (the non-standard nucleomer to be detected or the interfering nucleomer to be removed), such as via an affinity ligand, thereby enabling facile purification of the non-standard nucleomer of interest prior to detection. Examples of types of affinity ligands or reagents useful in the present invention include bidentate diacylphenyl templates (one option is shown in FIGS. 3 and 4); (a part of FIG. 5); β-cyclodextrin (FIG. 6 uses, for example, DEXSORB from CycloPure); molecularly imprinted polymers (e.g., MIPs prepared using imidazole or guanine as ligands, and the ligands can be linked to a solid support for MIP preparation); nucleic acid aptamers; peptide aptamers (peptimers); nanobodies, antibodies, aptabodies, vacancy G-quadruplex scaffolds, imidazolium-based cyclophanes, or riboswitches. Affinity reagents for guanine have been reviewed (Li, Y., Liu, J.,

[2020] Sensing guanine and its derivatives: From molecular recognition to applications. Sensors and Actuators Reports 2, 100020). Examples of non-standard nucleomer affinity ligands are 8-oxoguanine (which recognizes adenine) and hypoxanthine (which recognizes cytosine). Affinity ligands in polymeric form, such as polycytidylic acid bound to a strong anion exchange resin, can be used. Affinity reagents tend to be more retentive for their target molecules when used at lower temperatures, e.g., 5°C. By adding an organic solvent such as acetonitrile or methanol, the temperature can even be lowered below the freezing point of water.

[0044] Affinity Ligand Template: A molecule or molecular group that is extended by synthesis to yield an affinity ligand. In the case of the affinity ligands shown in FIGS. 3 and 4, the template is 2-nitrophthaloyl chloride, a diacylphenyl-type affinity ligand template. Other examples of affinity ligand templates for use in the present invention are 2-nitro-p-phenylenediamine, and 2-fluoro-1,3-diaminobenzene derived from 2-fluoro-1,3-dinitrobenzene.

[0045] Partner Affinity: An affinity step in which the reagent binding partner for the nucleomer of interest has a size similar to that of the nucleomer, e.g., less than about twice as large.

[0046] Biomarker: A substance derived from an animal, plant, or human whose measurement helps answer questions about health or disease.

[0047] Semipolar Solid Phase Extraction: Using a semipolar chromatography bed (e.g., bonded silica, OASIS) to retain nonpolar analytes while polar interferences are washed away, or to retain nonpolar interferences while polar analytes are washed away. Semipolar solid phase extraction can be useful in the present invention as an affinity reagent based on providing simultaneous nonpolar and polar attractive contact with the target molecule of interest. Even methylated purines that are only slightly nonpolar can be retained by semipolar extraction (Still, W.G., Xu, H.-X., Adkins, J.A., Wishnok, J.S., Tannenbaum, S.R.

[1989] Analysis of Methylated and Oxidized Purine in Urine by Capillary Gas Chromatography-Mass Spectrometry. Chem Res. Toxicol. 2, 94-99), which can be enhanced at low temperatures such as 5°C.

[0048] Mixed-mode Solid Phase Extraction: Using a mixed-mode solid phase extraction chromatography bed to retain non-polar analytes while polar interferences are washed away, or to retain non-polar interferences while polar analytes are washed away. Semi-polar solid phase extraction can be useful as an affinity reagent based on providing simultaneous divergent attractive contacts (e.g., non-polar and ionic) with the target molecule of interest. An example is provided by Hu et al. ((Hu, K., Zhao, G., Liu, J., Xie, F., Zhang, S., Liu, H., Liu, M.,

[2018] Simultaneous quantification of three alkylated purine adducts in human urine using sulfonic acid poly(glycidyl methacrylate-divinlbenzene)-based microsphere as sorbent combined with LC-MS / MS. J. Chromatogr. B, 1082, 15-24). This technique can be enhanced by operating at low temperatures such as 5°C.

[0049] Mass spectrometry: Forms of mass spectrometry for organic substances such as liquid chromatography / electrospray / Orbitrap mass spectrometry; liquid chromatography / electrospray / triple quadrupole mass spectrometry; liquid chromatography / electrospray / ion trap mass spectrometry; or MALDI-TOF mass spectrometry (including MALDI-TOF / TOF and TIMS-TOF-MS).

[0050] Exemplary nucleoside procedure: DNA or RNA that has been enzymatically digested to yield nucleosides is subjected to chromatography such as affinity chromatography, and the target nucleosides having primary amino groups are labeled with a positively charged reagent having a reactive aldehyde group in the presence of a hydride reagent, and the labeled nucleosides are detected by mass spectrometry.

[0051] Method Nine exemplary measurement procedures (designated as Methods A - L) are presented below, some of which utilize one or more affinity reagents to detect non - standard nucleomers. In some cases, specific types of damage are considered as examples. Conventional extraction steps such as chromatography, partitioning, centrifugation, or filtration may be part of each measurement procedure, particularly in the pretreatment stage (see Figure 1), to help purify a given type of non - standard nucleomer analyte from background substances in the biological sample. Since the final detection is by mass spectrometry, each procedure can detect one or more nucleomers.

[0052] Method A: Detection of N7 - guanine adducts (N7 - guanine) and application to cancer prevention Step 1. Urine is collected into a standard screw - top urine collection cup on one or more occasions, and the cup containing the urine is stored in a freezer or by another method for storage at room temperature such as adding rubbing alcohol. The urine may be collected in a jar or bottle. Step 2. Warm the urine to enable spontaneous depurination of N7 - guanine, such as under conditions of 3 days at 37°C, pH 7.0 (gentle neutral thermal hydrolysis), and maintain it to obtain N7 - guanine. Step 3. Affinity extract guanine that is not substituted on the I - side (see Figure 2) by base - pairing using a cytosine - substitution chromatography material such as the cytosine affinity material option in Figure 5 that recognizes the I - side of guanine (see Figure 2). Since guanine is less likely to form under neutral thermal hydrolysis, it may not be seen very often. A β - cyclodextrin ligand can also be used for the affinity extraction of guanine (Figure 6 shows a guanosine / β - cyclodextrin complex). Step 4. Optionally, treat the sample with adenosine deaminase to render potentially interfering adenine(s) non - reactive with the aldehyde - functionalized mass tag in subsequent steps. Step 5. Optionally, remove non-target guanine by affinity chromatography using a ligand-substituted chromatography material, where the ligand recognizes the II side of guanine (see Figure 2). Step 6. As shown in Figure 7, label the recovered N7-guanine with a CAX-CHO mass tag. CAX-CHO-2 can also be used. Step 7. In the presence of a hydride reagent such as sodium cyanoborohydride or pyridine borane, remove the residual CAX-CHO mass tag by reaction with an amine chromatography material as shown in Figure 5, or a chromatography material having a primary alkylamine or arylamine or hydrazide group such as ethylenediamine-polyacrylamide. Step 8. Detect CAX-labeled N7-guanine by mass spectrometry. Step 9. By informing the urine donor about any increased adducts, the urine donor can potentially reduce their cancer risk by reducing their exposure to the chemicals or conditions that cause these adducts. (This is instead a follow-up [second] test, and if they have already modified their exposure and brought about a reduction in adducts, this feedback can encourage them to continue their improved behavior, thereby sustaining their lower cancer risk.)

[0053] It is recognized that the order of some steps can be changed; note that similar reagents and conditions can be used.

[0054] Method B: O 6 -guanine adduct (O 6 -guanine) detection and application Step 1. The same as Step 1 in Method A. Step 2. Thaw the urine, treat it with an acid to lower its pH to 5.0, and heat it at 45 °C for 8 hours to effect depurination of non-standard DNA, DNA-oligos, deoxynucleotides, and the corresponding RNA species to obtain O 6 -guanine. Unmodified guanine on the I side (see Figure 2) is affinity-removed by base pairing using a cytosine-substituted chromatography material, where the cytosine ligand, which is one of the options in Figure 5, is linked via its exocyclic amino group. Step 4. O lacking substitution on the II side (see Figure 2 shown for guanine) 6 -Guanine and adenine are affinity-extracted by an affinity reagent directed to region II, such as those using the affinity ligands of Figures 3 and 4. Step 5. Optionally, the sample is treated with adenosine deaminase to render potentially interfering adenine having a primary amine group non-reactive with a mass tag reagent having an aldehyde in subsequent steps. Step 6. As shown in Figure 7 for N7-guanine, O 6 -Guanine is labeled with a CAX-CHO mass tag. Step 7. Residual CAX-CHO mass tag is removed by reaction with a chromatography material having a primary amine group, such as the primary amine chromatography material (Poma et al.) shown in Figure 5. Step 8. CAX-labeled N7-guanine is detected by mass spectrometry. Step 9. It is the same as Step 9 of Method A.

[0055] Method C: N 2 -Detection and application of guanine Step 1. The same as Step 1 in Method A. Step 2. Thaw the urine, treat it with an acid to lower its pH to 5.0, heat it at 45 °C for 8 hours to effect depurination of non-standard DNA, DNA-oligos, deoxynucleotides, and corresponding RNA species, and obtain purines containing N 2 -Guanine. Step 3. Unmodified guanine in region I (see Figure 2) is affinity-removed by base pairing using a cytosine-substituted chromatography material, where the cytosine ligand, which is one of the options in Figure 5, is linked via its exocyclic amino group. Step 4. Using the conditions described by Wang et al. (Wang, P., Zhang, Q., Yao, Y., Giese, R.W. (2015) Cationic Xylene Tag for Increasing Sensitivity in Mass Spectrometry, J. Am. Soc. Mass Spectrom. 26, 1713 - 1721), label N 6 -guanosine with the CAX - B mass tag in the presence of triethylamine as shown in Figure 8 for 1,N 2 -guanosine. Step 5. Remove residual CAX - B by reacting overnight with a chromatographic material having a nucleophilic group such as guanine (see Figure 5), phenol, sulfhydryl, or imidazole in the presence of triethylamine. Step 6. Detect the CAX - labeled N 2 -guanosine (N 2 -guanosine adduct) by mass spectrometry. Step 7. The same as Step 9 in Method A.

[0056] Method D: Detection and Application of C8 - Guanosine Step 1. The same as Step 1 in Method A. Step 2. Thaw the urine, treat it with acid to lower its pH to 5.0, and heat it at 45 °C for 8 hours to depurinate non - standard DNA, DNA - oligos, deoxynucleotides, and the corresponding RNA species to obtain purines containing C8 - guanosine. Step 3. Extract guanosine not substituted in the I region using the cytosine - substituted chromatographic material of Figure 5. Step 4. Optionally, treat the sample with adenosine deaminase to render potentially interfering adenine having a primary amine group non - reactive towards the mass tag having an aldehyde in subsequent steps. Step 5. Label C8 - guanosine with the CAX - CHO mass tag as shown in Figure 7 for N7 - guanosine. Step 6. Remove the residual CAX-CHO mass tag by reaction with a chromatography material having a primary amine group, such as the amine chromatography material shown in FIG. 5, in the presence of a hydride reagent. Step 7. Detect CAX-labeled C8-guanine by mass spectrometry. Step 8. It is the same as step 9 of Method A.

[0057] Method E: Detection and application of N3-adenine Step 1. It is the same as step 1 of Method A. Step 2. Warm the urine and maintain it at 37 °C and pH 7.0 for 3 days (gentle neutral thermal hydrolysis) to allow spontaneous depurination of N3-adenine to obtain N3-adenine. Step 3. Label N3-adenine with CAX-CHO. (Labeling of standard guanine and adenine may be tolerated when these species are present in very low amounts.) Step 4. Remove the residual CAX-CHO by reaction with a chromatography material having a primary amine group, such as the primary amine chromatography material shown in FIG. 5, in the presence of a hydride reagent. Step 5. Detect CAX-labeled N3-adenine by mass spectrometry. Step 6. It is the same as step 9 in Method A. Note: As in Method C, N3-adenine may be labeled with CAX-B for detection and then the residual CAX-B removed.

[0058] Method F: 1,N 6 -adenine detection and application Step 1. It is the same as step 1 of Method A. Step 2. Thaw the urine, treat it with acid to lower its pH to 5.0, and heat it at 45 °C for 8 hours to effect depurination of DNA, DNA-oligos, deoxynucleotides, and the corresponding RNA species, to obtain purines containing 1,N 6 -adenine. Extract the guanine that has not been replaced in the step 3.I region (see Figure 2) using the cytosine replacement chromatography material of Figure 5. Step 4. Deaminate adenine with adenosine deaminase. Step 5. As shown in Figure 8, in the presence of triethylamine, 1,N 6 -ethenoadenine is labeled with CAX-B. Step 6. Remove the remaining CAX-B by reaction with a chromatography material having a nucleophilic group such as imidazole, phenol, sulfhydryl, or guanine (see Figure 5 for guanine) overnight in the presence of triethylamine. Step 7. Detect CAX-labeled 1,N 6 -ethenoadenine by mass spectrometry. Step 8. It is the same as step 9 in method A.

[0059] Method G. Detection and application steps of N7-guanine from non-standard DNA in urine Step 1. Treat urine with ribonuclease to convert RNA species to nucleotides. Step 2. Treat urine with a strong anion exchanger such as Bio-Rad's Nuvia Q Anion Exchange Resin to bind non-standard DNA. Step 3. Use a low-salt buffer to wash away nucleomers, nucleotides, and background substances from the resin, leaving the non-standard DNA bound. Step 4. As in the corresponding method above, for example, if performing neutral thermal hydrolysis, as in method A (steps 3-9), or if performing acid depurination, as in method B (steps 3-9), subject the resin to gentle neutral thermal hydrolysis or gentle acid depurination to release the nucleomers to be detected. Step 5. It is the same as step 9 in method A.

[0060] Method H. Detection and application of DNA-protein crosslinks in DNA in urine Steps 1-3. The same as method G. Using the conditions reported by Barker et al. (Barker, S., Murray, D., Zhene, J., Li, L., Weinfeld, M.

[2005] A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis. Anal. Biochem. 344, 204-215), the resin is subjected to an aqueous chaotrope in the presence of proteinase K and ribonuclease, followed by washing, and the non-standard DNA remaining on the resin is subjected to gentle acid depurination, and then steps 3-9 of Method B are followed.

[0061] Method I. Detection and Application of 1,N 6 -Adenine in Urinary DNA Step 1. Treat urine with ribonuclease and proteinase K. Step 2. Isolate DNA from urine as described by Yun (Yun, B.H., Belliamri, M., Rosenquist, T.A., Turesky, R.J.

[2018] A Method for Biomonitoring of DNA Adducts in Exfoliated Urinary Cell by Mass Spectrometry. Anal. Chem. 90, 9943-9950), or by ultrafiltration. Step 3. Treat urine with acid to lower its pH to 5.0 and heat at 45 °C for 8 hours for depurination. Step 4. 1,N 6 -Adenine is recovered in the filtrate when urine is subjected to ultrafiltration to remove residual non-standard DNA. Step 5. Extract guanine that is not substituted in the I region (see Figure 2) using the cytosine substitution chromatography material shown in Figure 5. Step 6. Deaminate adenine with adenosine deaminase. Step 7. As shown in Figure 8, in the presence of triethylamine, 1,N 6 -ethenoadenine is labeled with CAX-B. Step 8. In the presence of triethylamine, residual CAX-B is removed by reacting overnight with a chromatography material having a covalently linked nucleophilic group such as, for example, guanine (see Figure 5), imidazole, phenol, or sulfhydryl. Step 9. Detect CAX-labeled 1,N 6 -ethenoadenine by mass spectrometry. Step 10. It is the same as Step 9 in Method A.

[0062] Method J. Alternative treatment of urine for the detection of N7-guanine Urine is injected, either as is or after treatment with a cell lysis reagent such as rubbing alcohol, into a strong anion exchange chromatography column or filter, followed by rinsing with water and then rubbing alcohol. The column or filter is then warmed in buffer to release N7-guanine for further steps (Steps 3 - 8) that result in detection as described in Method A.

[0063] Method K. Testing of DNA samples A DNA sample in physiological saline is incubated with CAX-CHO in the presence of a hydride reagent. The DNA is precipitated with isopropyl alcohol, washed with an aqueous isopropyl alcohol solution, and subjected to Jettison MS as described (Wang, P., Shah, G.L., Landau, H., Coulter, M.E., Walsh, C.A., Roider, E., Kramer, C.S., Beuning, P.J., Giese, R.W.

[2020] Jettison-MS of Nucleic Acid Species, J. Am. Soc. Mass Spectrom. 31, 1641 - 1646).

[0064] Method L. Mass spectrometry using CAX-CHO In the described procedure (Wang, P., Roider, E., Coulter, M.E., Walsh, C.A., Kramer, C.S., Beuning, P.J., Giese, R.W.

[2021] DNA Adductomics by mass tag prelabeling, Rapid Commun. Mass Spectrom. 35: e9095), CAX-CHO is used instead of CAX-B in the presence of a hydride reagent, resulting in mass spectrometry of CAX-CHO-labeled nucleotides. The CAX-CHO-labeled nucleotides can also be digested to the corresponding CAX-CHO-labeled nucleosides prior to detection by mass spectrometry.

[0065] Synthesis of CAX-CHO-2. 3-Aminobenzaldehyde ethylene acetal (AlfaEasar) is reacted with CAX-B in an aqueous acetonitrile solution in the presence of triethylamine (Wang, P., Zhang, Q. Yao, Y., Giese, R.W. (2015) Cationic Xylene Tag for Increasing Sensitivity in Mass Spectrometry, J. Am. Soc. Mass Spectrom. 26, 1713-1721), followed by acidification to remove the ethylene acetal protecting group.

[0066] Synthesis of CAX-CHO. 4-Hydroxybenzaldehyde (Sigma Aldrich) is refluxed with 1.1 equivalents of ethylene glycol and 0.1 equivalent of tosic acid in toluene using a Dean Stark Trap. Workup is performed by minimally diluting with water, extracting with toluene, and subsequently evaporating. 4-Hydroxybenzaldehyde ethylene acetal is reacted with CAX-B in an aqueous acetonitrile solution in the presence of triethylamine, followed by acidification to remove the ethylene acetal protecting group to obtain the product.

Example

[0067] Example 1. Labeling of target purines having exocyclic primary amine groups with CAX-CHO and removal of residual CAX-CHO using amines linked to a solid phase In a capped Eppendorf tube, damaged purines dissolved in 5 μL of LCMS-grade acetonitrile are mixed with 2 μL of acetonitrile containing 5 μg of CAX-CHO and 2 μL of LCMS-grade methanol containing 60 μg of NaCNBH3. The resulting sample is vortexed for 10 seconds and then left to stand overnight at room temperature. Residual CAX-CHO is removed by adding a primary amine reagent linked to the beads described in Figure 5 and 30 μg of NaCNBH3 and continuing the incubation.

[0068] Example 2. Labeling of N3-adenine with CAX-Br and removal of residual CAX-B using guanine linked to beads (one of the options in Figure 5) The evaporated N3-adenine in the vial was redissolved in 6 μL of 50% acetonitrile containing 1 mg / mL of CAX-B (prepared as described in Wang et al.,

[2015] Cationic Xylene Tag for Increasing Sensitivity in Mass Spectrometry, J. Am Soc. Mass Spectrometry, 26, 173 - 1721) and 10 μL / mL of triethylamine. The vial was capped, placed in the dark, and maintained at 38 °C for 16 h. 10 mg of silica-linked guanine (prepared as described in Poma et al.,

[2014] Nucleoside-Tailored Molecularly Imprinted Polymeric Nanoparticles (MIP NPs), Macromolecules, 47, 6322 - 6330) was added. The vial was placed on a rotating plate and subsequently maintained at 38 °C for an additional 16 h. By adding the bead-leashed guanine reagent shown in Figure 5 and continuing the incubation, the residual CAX-B was removed. The CAX-labeled N3-adenine was recovered in the filtrate after ultrafiltration.

[0069] Example 3. Preparation of bidentate diacylphenyl-type affinity substances and analogs shown in Figures 3 and 4 by multi-step or combinatorial chemistry Dissolve 2-nitrophthaloyl chloride (1 g) in acetonitrile (30 mL), and slowly add 30 mL of acetonitrile containing one or more reagents (or analogs of these reagents) such as ethanolamine, 3-amino-1-propanol, 4-amino-1-butanol, and 5-amino-1-pentanol (100 mg each) together with 10 mL of triethylamine at ice-bath temperature. A second round can use a second such reagent. Stir the mixture overnight at room temperature in the dark. Dissolve the mixture in water or an aqueous methanol solution and apply it to the bead-immobilized guanine reagent of Figure 5. Wash away the unbound compounds, then wash away the bound compounds and identify them by NMR and mass spectrometry. Dissolve the most strongly bound compound in 30 mL of 50% methanol and reduce the nitro group to an amino group with NaBH4 as described (Pina et al., Reduction of nitrobenzene derivatives using sodium borohydride and transition metal sulfides

[2014] Tetrahedron Letters, 55, 5408 - 5470). Then immobilize it by reductive amination as shown in Figure 5 for use as an affinity reagent to bind to the II side of the purine that is not modified on that side (see Figure 2).

[0070] Example 4. Preparation of a bidentate diaminophenyl affinity substance for binding to the II side of guanine that is not modified on that side (see Figure 2) by multi-step or combinatorial chemistry React 2-nitro-para-phenylenediamine in acetonitrile or acetonitrile / methanol with a mixture of R-N-hydroxysuccinimide esters; where R contains an alkyl, alkenyl, or aryl moiety, R contains 10 or fewer carbon atoms, and R may contain one or more functional groups such as OH, methoxy, ether, ketone, amide, secondary amine, tertiary amine, or thioether. These NHS esters are prepared by reacting RCO2H with N-hydroxysuccinimide in the presence of N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride in water. The reaction of the R-N-hydroxysuccinimide ester with 2-nitro-para-phenylenediamine is carried out in an aqueous buffer at pH 8.3 - 8.5 or in an organic solvent such as DMF or DMSO in the presence of 1 equivalent of a tertiary amine base such as triethylamine. After evaporation, the mixture is dissolved in water or an aqueous methanol solution and applied to the immobilized guanine reagent of Figure 5. Unbound compounds are washed away, followed by washing away the bound compounds, which are then identified by NMR and mass spectrometry. The most strongly bound compound is dissolved in 30 mL of 50% methanol, and the nitro group is reduced to an amino group with NaBH4 as described (Pina et al., Reduction of nitrobenzene derivatives using sodium borohydride and transition metal sulfides

[2014] Tetrahedron Letters, 55, 5408 - 5470). This product is then immobilized on the beads by reductive amination as shown in Figure 5 for use as an affinity reagent to bind to the II side of the purine that is not modified on that side (see Figure 2).

[0071] Example 5. Affinity Chromatography Affinity chromatography is performed in methanol:water, 1:10, v / v, or a similar solvent by preparing a cartridge solid-phase extraction column with the target affinity substance, applying the target sample in this solvent, washing away unbound substances with this solvent or another solvent, and eluting the bound substances with this solvent or another solvent.

[0072] Example 6. Preparation of an affinity reagent that recognizes the III side of guanine (see Figure 2) Ligands such as 4-aminophthalimide, 1-carboxymethyluracil, 6-carboxymethyluracil, 1-carboxymethylthymine, or N7-carboxymethylxanthine are used and then coupled to a solid support having a primary amine group by activation to an NHS ester. The thymine affinity reagent of Van Breemen et al. can be used (Van Breemen, R.B., Tan, Y., Lai, J., Huang, C.-R., Zhao, X.

[1998] Immobilized thymine chromatography-mass-spectrometry of oligonucleotides. J. Chromatogr. A 806, 67 - 76).

[0073] Example 7. Preparation and use of an antibody or nanobody affinity reagent that recognizes PhIP, a genotoxic chemical derived from grilled red meat Prepare N-(2’-deoxyguanosin-8-yl)-PhIP as described (Guo, J., Koopmeiners, S., Walmsley, S.J., Villalta, P.W., Yao, L., Murugan, P., Tejpaul, R., Weight, C.J., Turesky, R.J. The Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazole[4,5-b]pyridine Hair Dosimeter, DNA Adductomics Discover, and Associations with Prostate Cancer Pathology Biomarkers, Chemical Research in Toxicology, https: / / doi.org / 10.1021 / acs.chemrestox.2c00012). Then conjugate this compound to 4-carboxybenzaldehyde in the presence of sodium cyanoborohydride; as described in Example 4, convert the resulting product to an NHS ester and subsequently react it with albumin to obtain an antigen PhIP-albumin having PhIP as a hapten for the production of an anti-PhIP antibody or nanobody. Then use the resulting column as an affinity reagent to isolate PhIP-guanine from urine, which is then detected by CAX-CHO labeling and subsequently detected by mass spectrometry after removing CAX-CHO as in Method A. The antibody can be immobilized on a chromatographic streptavidin column by reacting the antibody with extended biotin-NHS.

[0074] The present invention can also be carried out using a fluorescent dye having an aldehyde group, such as fluorescein aldehyde (Ettenauer, J., Semak, V., Brandl, M. (2018) Synthesis of Fluorescein Aldehydes for the Sensitive Detection of L-Cysteine, Proceedings 2, 895), fluorescein PEG aldehyde, or 3-(2-furoyl)quinoline-2-carboxaldehyde, and the fluorescence detection is carried out by capillary electrophoresis with fluorescence detection, high performance liquid chromatography with fluorescence detection, or array fluorescence detection, etc.

Claims

1. A method for detecting nucleic acid bases, a) A step of subjecting a biosample or biosample extract containing non-standard DNA to strong anion exchange extraction; b) Incubating the biosample or biosample extract from an anion exchanger to elute nucleic acid bases from DNA; c) The step of covalently labeling the nucleic acid base with a positively charged reagent having a reactive aldehyde or alkylating group to obtain a tagged nucleic acid base; and d) Step of detecting the tagged nucleic acid base by mass spectrometry. The method, including the method described above.

2. The method according to claim 1, wherein the nucleic acid base contains a primary amino group and is labeled with a positively charged aldehyde-containing mass tag, the labeling being carried out in the presence of sodium cyanoborohydride or pyridineborane.

3. The method according to claim 2, wherein the aldehyde-containing mass tag is added to a nucleic acid base using a reagent containing a group selected from the group consisting of quaternary amines, tertiary amines, phosphonium, and pyridinium.

4. The method according to any one of claims 1 to 3, further comprising performing an affinity step immediately after incubation step b), wherein the target or non-target nucleomer undergoes non-covalent polyvalent interactions with complementary groups on the dissolved or undissolved material, and the target nucleomer is recovered.

5. The method according to claim 4, wherein the affinity step is a partner affinity step.

6. The method according to any one of claims 1 to 3, wherein the incubation step b) comprises mild neutral thermal hydrolysis or mild acid depurination.

7. The method according to any one of claims 1 to 3, wherein the positively charged reagent is CAX-CHO or CAX-CHO-2.

8. A method for detecting nucleomers having a primary amine group, a) A step of exposing a biosample or biosample extract to an affinity reagent, wherein the biosample or biosample extract contains a target nucleomer that is a primary amine and is non-standard; b) The step of recovering the target nucleomer and covalently reacting the primary amine group on the nucleomer with a positively charged reagent having an aldehyde group in the presence of a hydride reagent; c) Step of obtaining a conjugate having an amino-alkyl bond detected by mass spectrometry. The method, including the method described above.

9. The method according to claim 8, wherein the positively charged reagent comprises a group selected from the group consisting of quaternary amines, tertiary amines, pyridinium, and phosphonium.

10. The method according to claim 8, wherein the nucleomer is a nucleic acid base or a nucleoside.

11. The method according to claim 8, wherein the biosample is urine.

12. The method according to any one of claims 8 to 11, wherein the hydride reagent is sodium borocyanohydride or pyridineborane.

13. The method according to any one of claims 8 to 11, wherein the affinity reagent is selected from the group consisting of a standard nucleomer, a non-standard nucleomer, a diacylphenyl moiety, a diaminophenyl moiety, a β-cyclodextrin moiety, a molecular imprinted polymer, a nucleic acid aptamer, a peptide aptamer, a nanobody, an antibody, and a riboswitch.

14. The method according to claim 13, wherein the affinity reagent is a mixed-mode or semi-polar chromatography material.

15. The method according to any one of claims 8 to 11, wherein the affinity step a) is performed at a temperature of less than 10°C.

16. The method according to any one of claims 8 to 11, wherein a non-target nucleic acid base is extracted in the affinity step a).

17. A method for detecting nucleomers having a secondary amine, a) A step of exposing a biosample or biosample extract to an affinity reagent, wherein the biosample or biosample extract contains a target nucleomer having a secondary amine and being non-standard; b) The step of recovering the target nucleomer and covalently reacting the secondary amine group on the nucleomer with a positively charged reagent having an alkylating group; and c) Step of obtaining a conjugate having an amino-alkyl bond detected by mass spectrometry. The method, including the method described above.

18. The method according to claim 17, wherein the nucleomer is a nucleic acid base or a nucleoside.

19. The method according to claim 17, wherein the biosample is urine.

20. The method according to any one of claims 17 to 19, wherein the positively charged reagent has a quaternary amine group.

21. The method according to any one of claims 17 to 19, wherein the alkylating group is benzyl bromide, xylyl bromide, xylyl acetate, xylyl tosylate, alkyl chloride, alkyl bromide, alkyl iodide, or alkyl tosylate.

22. The method according to claims 17 to 19, wherein the affinity reagent is selected from the group consisting of standard nucleomers, non-standard nucleomers, diacylphenyl moieties, diaminophenyl moieties, β-cyclodextrins, molecular imprinted polymers, nucleic acid aptamers, peptide aptamers, nanobodies, antibodies, aptabodies, and riboswitches.

23. The method according to claims 17 to 19, wherein the alkylation reaction is carried out in the presence of a tertiary amine.

24. The method according to any one of claims 17 to 19, wherein the affinity reagent is a mixed-mode or semipolar chromatography material.

25. The method according to any one of claims 17 to 19, wherein the affinity step a) is performed at a temperature below 10°C.

26. The method according to any one of claims 17 to 19, wherein a non-target nucleomer is extracted in the affinity step a).

27. A method for detecting compounds having a primary amino group, a) A step of reacting a primary amino group with a positively charged reagent having a reactive aldehyde group in the presence of a hydride reagent to obtain a conjugate having an aminoalkyl bond; and b) Step of detecting the conjugate by mass spectrometry. The method, including the method described above.

28. The method according to claim 27, wherein the positively charged reagent is CAX-CHO or CAX-CHO-2.

29. The method according to claim 27 or 28, wherein the compound is a nucleomer.

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