CN116973467A

CN116973467A - Modification identification method of phosphorothioate modified nucleic acid sequence

Info

Publication number: CN116973467A
Application number: CN202310485292.1A
Authority: CN
Inventors: 祁明盼; 刘红柳; 刘吉众; 王建鹏; 李竑
Original assignee: Nanjing Jinsirui Science and Technology Biology Corp
Current assignee: Nanjing Jinsirui Science and Technology Biology Corp
Priority date: 2022-04-29
Filing date: 2023-04-28
Publication date: 2023-10-31

Abstract

The present embodiments provide a method for identifying modifications of a phosphorothioate modified nucleic acid sequence, the method comprising: performing enzymolysis on a nucleic acid sequence to be identified by using a nucleic acid mixed enzyme to obtain an enzymolysis product; performing secondary mass spectrometry on the enzymolysis product based on the first mass spectrometry information; and determining whether a target sequence fragment exists in the nucleic acid sequence to be identified based on the first mass spectrum information and the second mass spectrum information. Wherein at least the phosphorothioate modified nucleic acid sequence to be identified is actually obtained based on a preset modification rule; the first mass spectrum information comprises theoretical analysis results generated by analyzing theoretical sequence fragments, wherein the theoretical sequence fragments are determined based on the preset modification rules, the nucleic acid sequences to be identified and the nucleic acid hybrid enzyme. The modification identification method can accurately and efficiently carry out chemical modification identification of the nucleic acid sequence modified by phosphorothioate.

Description

Modification identification method of phosphorothioate modified nucleic acid sequence

Cross reference

The present application claims priority to chinese application 202210467473.7 filed on 4/29 of 2022, which is incorporated herein by reference in its entirety.

Technical Field

The specification relates to the technical field of nucleic acid detection, in particular to a method for identifying a modified nucleic acid sequence modified by phosphorothioate.

Background

The method for identifying the modification of the nucleic acid sequence is mainly used for identifying whether the chemical modification of the nucleic acid sequence accords with the expected design or not, and has wide application in biological, medical and pharmaceutical research and production activities. For example, the CRISPR/Cas9 system widely used for gene editing consists of Cas9 protein and sgRNA (single guide RNA). The sgrnas are designed according to the higher structure formed by crrnas and tracrrnas, bind to Cas9 nuclease protein, guide it to recognize and clip targeting sequences. Since CRISPR/Cas9 comes from the natural acquired immune system of prokaryotes against the defense system of foreign genetic material, cas9 nuclease may inherit the characteristic of low sequence specificity, so that the probability of non-specific cleavage is increased, resulting in increased off-target effect. Thus, chemical modification or editing of Cas9 and sgrnas is often required to reduce off-target effects. The sgRNA is used as a key raw material of CRISPR gene editing technology, and quality research is required when clinical declaration is carried out, namely, the modification of the 5 'end and the 3' end of the sgRNA sequence is required to be identified. For sgrnas with phosphorothioate modifications at the 5 'and 3' ends, if the LC-MS method is directly used to identify the molecular weight of the full-length sequence, the molecular weight identification cannot reveal the nucleotide arrangement sequence of the nucleic acid sequence with chemical modifications, and thus it cannot be determined whether the corresponding chemical modifications are performed based on the correct sequence arrangement, so that the identification result is inaccurate. Therefore, there is a need to provide an accurate and efficient method for identifying modifications of phosphorothioate modified nucleic acid sequences.

Disclosure of Invention

One or more embodiments of the present disclosure provide a method of identifying modifications of a phosphorothioate modified nucleic acid sequence. The method comprises the following steps: performing enzymolysis on a nucleic acid sequence to be identified by using a nucleic acid mixed enzyme to obtain an enzymolysis product; wherein the nucleic acid sequence to be identified is actually obtained based on a preset modification rule, and the modification to be identified of the nucleic acid sequence to be identified at least comprises phosphorothioate modification of internucleotide linkage; performing secondary mass spectrometry on the enzymolysis product based on first mass spectrometry information to obtain second mass spectrometry information actually generated by the enzymolysis product, wherein the first mass spectrometry information comprises theoretical analysis results generated by analyzing theoretical sequence fragments, and the theoretical sequence fragments are determined based on the preset modification rules, the nucleic acid sequences to be identified and the nucleic acid hybrid enzyme; and determining whether a target sequence fragment consistent with the theoretical sequence fragment exists in the nucleic acid sequence to be identified based on the first mass spectrum information and the second mass spectrum information.

In some embodiments, the nucleic acid cocktail is capable of and free of breaking phosphodiester bonds such that the phosphorothioate modified nucleic acid sequence to be identified produces sequence fragments of at least two different molecular weights.

In some embodiments, the nucleic acid cocktail enzyme comprises a snake venom phosphodiesterase and/or bovine spleen phosphodiesterase.

In some embodiments, the nucleic acid-mixed enzyme is enzymatically hydrolyzed for a period of time ranging from 1h to 8h.

In some embodiments, the nucleic acid-mixed enzyme is enzymatically hydrolyzed for 3 hours to 5 hours.

In some embodiments, the modification to be identified of the nucleic acid sequence to be identified may further comprise a modification of pentose sugar and/or a modification of bases.

In some embodiments, the modification of the pentose sugar comprises at least one of the following modifications: 2' -modification, 5' -modification, 3' -modification, locked nucleic acid modification, unlocked nucleic acid modification, and peptide nucleic acid modification.

In some embodiments, the 2' -modification of pentose includes at least one of the following modifications: 2' -O-methyl modification, 2' -fluoro modification, 2' -O-methoxyethyl modification, 2' -O-methylation modification, 2' -O-allyl modification.

In some embodiments, the modification of the base comprises at least one of the following modifications: methylation modifications and methylolation modifications.

In some embodiments, the preset modification rules include: the nucleotide corresponding to the modified pentose is connected with at least one adjacent nucleotide through a phosphorothioate bond; and/or, the preset modification rule comprises: the nucleotides corresponding to the modified bases are linked to at least one adjacent nucleotide by phosphorothioate linkages.

In some embodiments, the preset modification rules include: the 5 'end and/or the 3' end of the nucleic acid sequence to be identified are/is provided with a modified sequence fragment, the length of the modified sequence fragment is 2-5 nucleotides, and adjacent nucleotides in the modified sequence fragment are connected through phosphorothioate bonds.

In some embodiments, adjacent nucleotides of the theoretical sequence fragment are linked by phosphorothioate linkages.

In some embodiments, the first mass spectral information comprises first parent ion information and first child ion information of the theoretical sequence fragment; the first parent ion information at least comprises the mass-to-charge ratio of the first parent ion, and the first child ion information at least comprises the complementary pairing information and the mass-to-charge ratio of the first child ion.

In some embodiments, the second mass spectrometry information comprises second parent ion information and second child ion information; the obtaining of the second mass spectrum information actually generated by the enzymolysis product comprises the following steps: determining the second parent ion information based on the first parent ion information, the second parent ion information including at least a mass-to-charge ratio of a second parent ion; and carrying out secondary mass spectrometry analysis on the enzymolysis product based on the second parent ion information to obtain second child ion information, wherein the second child ion information at least comprises complementary pairing information and mass-to-charge ratio of the second child ion.

In some embodiments, the determining whether a target sequence fragment consistent with the theoretical sequence fragment is present in the nucleic acid sequence to be identified comprises: determining whether the first sub-ion information and the second sub-ion information meet a preset matching condition; if the preset matching condition is met, determining that the target sequence fragment exists in the nucleic acid sequence to be identified.

In some embodiments, the preset matching condition is: there is at least one pair of first sub-ions and at least one pair of second sub-ions, the complementary pairing information and mass-to-charge ratio of the at least one pair of first sub-ions and the at least one pair of second sub-ions being the same.

In some embodiments, the secondary mass spectrometry of the enzymatic hydrolysate is performed by a linear ion well mass spectrometer.

Drawings

The present specification will be further elucidated by way of example embodiments, which will be described in detail by means of the accompanying drawings. The embodiments are not limiting, in which like numerals represent like structures, wherein:

FIG. 1 is an exemplary flow chart of a modification verification method according to some embodiments of the present disclosure;

FIG. 2 is a LC-MS mass spectrum of control sample 1 of example 1 of the present specification;

FIG. 3 is a LC-MS mass spectrum of control sample 2 of example 1 of the present specification;

FIG. 4 is a LC-MS total ion flow diagram of the mixed enzyme reaction buffer of example 1 of the present specification;

FIG. 5 is a mass spectrum of LC-MS of the mixed enzyme reaction buffer of example 1 of the present specification;

FIG. 6 is an LC-MS mass spectrum of an enzymatic hydrolysate for 2min of sample 1 of example 2 of the present specification;

FIG. 7 is a LC-MS mass spectrum of the enzymatic hydrolysis product of sample 1 of example 2 of the present specification for 4 hours;

FIG. 8 is a LC-MS mass spectrum of an enzymatic hydrolysate of sample 1 of example 2 of the present specification for 24 hours;

FIG. 9 is a mass spectrum of LC-MSMS of sample 1 enzymatic hydrolysate corresponding to 5' -end modified fragment mG. Times.mA. Times.rU in example 3 of the present specification;

FIG. 10 is a mass spectrum of LC-MSMS of the 3' -end modified fragment mU rU corresponding to the enzymatic hydrolysate of sample 1 in example 3 of the present specification;

FIG. 11 is a mass spectrum of LC-MSMS of sample 2 enzymatic hydrolysate corresponding to 5' -end modified fragment mA mU mC rA in example 3 of the present specification;

FIG. 12 is a mass spectrum of LC-MSMS of the 3' -end modified fragment mU rU corresponding to the enzymatic hydrolysate of sample 2 in example 3 of the present specification;

wherein, in fig. 2 to 3, and fig. 5 to 8, the abscissa is mass-to-charge ratio m/z, and the ordinate is absolute intensity of ions; in fig. 4, the abscissa is Time and the ordinate is total ion current intensity; in fig. 10 to 12, the abscissa is the mass-to-charge ratio m/z, and the ordinate is the absolute intensity of ions.

Detailed Description

As used in this specification and the claims, the terms "a," "an," "the," and/or "the" are not specific to a singular, but may include a plurality, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.

The following are definitions of some terms used in the present application.

As used in this specification, the term "mass spectrometry" refers to an analytical technique that ionizes a chemical substance and orders it according to its mass to charge ratio. Primary mass spectrometry typically involves, among other things, ionization of chemicals to produce charged ions (ionization process), and measurement of the mass-to-charge ratio of the charged ions. Secondary mass spectrometry typically involves ionization of chemicals to produce charged ions (ionization process), collision-induced dissociation of selected parent ions to produce daughter ions (dissociation process), and measurement of the mass-to-charge ratio of the daughter ions.

The term "parent ion" is an ion of interest generated by the ionization process in mass spectrometry.

The term "daughter ion" is a fragment ion generated by the dissociation process in mass spectrometry. In secondary mass spectrometry, there is a clear relationship between the parent ions generated during ionization and the daughter ions generated during dissociation and their measured ion spectrum peaks.

The present embodiments relate to a method for identifying modifications of phosphorothioate modified nucleic acid sequences. In some embodiments, the modification identification methods can be used to identify chemical modifications of phosphorothioate modified sgRNA sequences. For example, the 3 'and 5' ends of the sgRNA sequence are phosphorothioated and methoxy modified, and the modification identification method can identify both phosphorothioate and methoxy modifications of the sgRNA sequence. In some embodiments, the modification identification methods can be used to identify chemical modifications of phosphorothioate modified antisense oligonucleotides (ASOs). For example, ASO therapeutics have been subjected to a variety of chemical modifications, including Phosphorothioate (PS) framework modifications and pentose and base modifications, to improve pharmacological properties, such that the modified ASO therapeutics exhibit better binding affinity to target RNAs, enhancing binding to proteins. The modification identification method can simultaneously identify Phosphorothioate (PS) framework modification and pentose and base modification of ASO sequence. In some embodiments, the modification identification methods can be used to identify chemical modifications of other nucleic acid sequences that are modified by phosphorothioation, e.g., siRNA. The identification of one or more chemical modifications of the phosphorothioate modified nucleic acid sequence can be accurately and efficiently achieved by the modification identification method.

It should be understood that the application scenarios of the methods for the modified identification of phosphorothioate modified nucleic acid sequences of the present description are merely some examples or embodiments of the present description, and that the present description may also be applied to other similar scenarios according to these figures without the exercise of inventive effort to one of ordinary skill in the art.

The method for identifying a phosphorothioate-modified nucleic acid sequence according to the examples of the present invention will be described in detail with reference to FIG. 1. It is noted that the following examples are only for explanation of the present specification and are not to be construed as limiting the present specification.

FIG. 1 is an exemplary flow chart of a modification verification method according to some embodiments of the present disclosure. In some embodiments, the modification verification process 100 includes at least steps 110 to 130.

In step 110, the nucleic acid sequence to be identified is subjected to enzymolysis by using a nucleic acid hybrid enzyme to obtain an enzymolysis product.

In some embodiments, the nucleic acid sequence to be identified is actually obtained based on a preset modification rule. Specifically, the correctness of one or more chemical modifications of the nucleic acid sequence to be identified is to be identified.

In some embodiments, the modification to be identified of the nucleic acid sequence to be identified comprises at least phosphorothioate modification (PS) of the internucleotide linkage. For example, phosphorothioate linkages are introduced between the 3 'end and the last 3 to 5 nucleotides of the 5' end of the nucleic acid sequence to inhibit the degradation of the nucleic acid sequence by exonucleases.

In some embodiments, the modification to be identified of the nucleic acid sequence to be identified further comprises a modification of pentose, e.g., a modification at pentose position 2 of any one or more nucleotides of the nucleic acid sequence, a modification at the 3 'end of the backbone, or a modification at the 5' end of the backbone. In some embodiments, pentoses include ribose and deoxyribose.

In some embodiments, the modification of pentose includes a 2' -modification, i.e., a modification at position 2 of pentose. In some embodiments, the 2' -modification comprises one or more of the following modifications: 2'-O-methyl modification (2' -OMe), 2 '-fluoro modification (2' -F), 2 '-O-methoxyethyl modification (2' -O-MOE), 2 '-O-methylation modification (2' -O-methyl), 2'-O-Allyl modification (2' -O-Allyl).

In some embodiments, the modification of pentose sugars comprises a 5 '-modification, i.e., a modification of the 5' -end of the backbone. In some embodiments, the 5' -modification comprises one or more of the following modifications: 5'-HEX, 5' -FAM, 5'-CY3, 5' -CY5, 5'-VIC, 5' -ROX, 5'C6 Amino modification (5' -Amino-C6), 5'C12 Amino modification (5' -Amino-C12).

In some embodiments, the modification of pentose sugars comprises a 3 '-modification, i.e., a modification of the 3' end of the backbone. In some embodiments, the 3' -modification comprises one or more of the following modifications: 3'-HEX, 3' -FAM, 3'-CY3, 3' -CY5, 3'-VIC, 3' -ROX, 3'C6 Amino modification (3' -Amino-C6), 3'C12 Amino modification (3' -Amino-C12).

In some embodiments, the modification of pentoses also includes other modifications, such as, for example, locked nucleic acid modifications (LNA), unlocked nucleic acid modifications (UNA), peptide nucleic acid modifications (PNA), and the like.

In some embodiments, the modification to be identified of the nucleic acid sequence to be identified further comprises a modification of a base. In some embodiments, the modification of the base comprises a methylation modification and/or a methylolation modification.

In some embodiments, the predetermined modification rules refer to rules for chemically modifying the original nucleic acid sequence according to a predetermined modification position and modification species. In some embodiments, the preset modification rules include at least: at least one phosphorothioate linkage is present in the nucleic acid sequence to be identified. By utilizing the enzyme cleavage resistance of phosphorothioate bonds, the nucleic acid sequence to be identified can be cracked to generate a target sequence fragment modified by phosphorothioate and a non-target sequence fragment not modified by phosphorothioate after being treated by functional enzyme, and the nucleotide arrangement and chemical modification of the target sequence fragment are easy to identify due to the difference of molecular weight and structure of the target sequence fragment and the non-target sequence fragment.

In some embodiments, the modification to be identified of the nucleic acid sequence to be identified further comprises a modification of pentose; the preset modification rules may also include linkage between the nucleotide corresponding to the modified pentose and at least one adjacent nucleotide via phosphorothioate linkages. In the case where the nucleic acid sequence to be identified is capable of being cleaved to produce a phosphorothioate modified target sequence fragment, modification of pentose at a position in the nucleic acid sequence to be identified corresponding to the target sequence fragment can be performed so that the pentose modification is detected simultaneously with the phosphorothioate modification of the internucleotide linkage. For example, phosphorothioate bonds are introduced between 1 st to 4 th nucleotides from the 5' end of the nucleic acid sequence, 2' -O-methyl modification is introduced at the pentose position 2 of 1 st to 3 rd nucleotides from the 5' end, and the 5' -end modified fragment consisting of the 4 nucleotides is kept intact in the subsequent enzymolysis treatment by utilizing the cleavage resistance property of the phosphorothioate bonds, so that the 2' -O-methyl modification and the phosphorothioate modification can be identified synchronously.

In some embodiments, the modification to be identified of the nucleic acid sequence to be identified further comprises modification of a base; the preset modification rules may also include linkage between the nucleotide corresponding to the modified base and at least one adjacent nucleotide via phosphorothioate linkages. In the case where the nucleic acid sequence to be identified is capable of being cleaved to produce a phosphorothioate modified target sequence fragment, modification of pentose at a position in the nucleic acid sequence to be identified corresponding to the target sequence fragment can be performed so that the pentose modification is detected simultaneously with the phosphorothioate modification of the internucleotide linkage. For example, phosphorothioate bonds are introduced between 1 st to 5 th nucleotides from the 3' end of the nucleic acid sequence, methylation modifications are introduced on bases of 2 nd to 5 th nucleotides from the 3' end, and the 3' end modified fragment consisting of the 5 th nucleotides is kept intact in subsequent enzymolysis treatment by utilizing the cleavage resistance property of the phosphorothioate bonds, so that the phosphorothioate modifications and the methylation modifications of the bases can be identified synchronously.

In some embodiments, the nucleic acid cocktail is capable of and free of breaking phosphodiester bonds such that the phosphorothioate modified nucleic acid sequence to be identified produces sequence fragments of at least two different molecular weights. Specifically, after the nucleic acid sequence to be identified which is modified by phosphorothioate is treated by the nucleic acid mixing enzyme, the phosphodiester bond of the nucleic acid sequence to be identified is broken by the action of the nucleic acid mixing enzyme, so that a plurality of non-target sequence fragments (single nucleotide sequence fragments) are formed; phosphorothioate bonds of the phosphorothioate modified nucleic acid sequences to be identified have cleavage resistance characteristics and are not cleaved by enzymatic hydrolysis by a nucleic acid hybrid enzyme, thereby forming complete target sequence fragments, adjacent nucleotides of which are linked by phosphorothioate bonds. The target sequence fragments and the non-target sequence fragments have obvious differences in length, molecular weight, structure and the like, so that the target sequence fragments can be separated and identified from enzymolysis products through mass spectrometry.

In some embodiments, the nucleic acid-mixing enzyme can include snake venom phosphodiesterase and/or bovine spleen phosphodiesterase. In other embodiments, the nucleic acid-mixing enzyme may further comprise one or more other enzymes capable of disrupting phosphodiester bonds of the nucleic acid sequence to be identified and free of enzymatic cleavage of phosphorothioate bonds, as the present embodiment is not limited.

In some embodiments, the nucleic acid-mixing enzyme has a suitable enzymatic hydrolysis time that enables the nucleic acid-mixing enzyme to substantially enzymatically hydrolyze the nucleic acid sequence to be identified while ensuring enzymatic hydrolysis efficiency. In some embodiments, the enzymatic hydrolysis time of the nucleic acid-mixing enzyme is 1h to 8h. For example, the nucleic acid sequence to be identified may be treated with a nucleic acid cocktail for about 1h, 2h, 3h, 4h, 5h, 6h, 7h, or 8h to allow for sufficient enzymatic cleavage of the nucleic acid sequence to be identified. In some preferred embodiments, the enzymatic hydrolysis time of the nucleic acid-mixing enzyme is 3h to 5h. In some preferred embodiments, the enzymatic hydrolysis time of the nucleic acid-mixing enzyme is about 4 hours.

In step 120, a secondary mass spectrometry is performed on the enzymatic hydrolysate based on the first mass spectrometry information, so as to obtain second mass spectrometry information actually generated by the enzymatic hydrolysate.

In some embodiments, the first mass spectral information comprises theoretical analysis results generated by analysis of theoretical sequence fragments. In some embodiments, the analysis of the theoretical sequence fragments comprises theoretical analysis of primary mass spectrometry. In some embodiments, the analysis of the theoretical sequence fragments comprises theoretical analysis of secondary mass spectrometry. Specifically, for a nucleic acid sequence fragment with a certain molecular weight and structure, the results of the primary mass spectrometry and the secondary mass spectrometry can be obtained by theoretical analysis. For example, for the theoretical sequence fragment a×a ("×representing phosphorothioate modification) determined by the sequence information, information such as charge state (e.g. charge number) and mass-to-charge ratio of a series of charged ions generated by the theoretical sequence fragment a×a after ionization can be obtained by theoretical analysis; after any one charged ion is selected from a series of charged ions as a parent ion, information such as charge state and mass-to-charge ratio of the child ion generated by collision-induced dissociation of the parent ion can also be obtained through theoretical analysis.

In some embodiments, theoretical sequence fragments are determined based on the preset modification rules, the nucleic acid sequence to be identified, and the nucleic acid cocktail. In some embodiments, a theoretical design sequence theoretically available after correct modification of the nucleic acid sequence to be identified with the preset modification rule may be determined based on the preset modification rule and the nucleic acid sequence to be identified. For example, the initial nucleic acid sequence is 5' -AATTCCTT-3', and the preset modification rule is specifically that phosphorothioate bonds are introduced between 1 st and 3 rd nucleotides from the 5' -end of the initial nucleic acid sequence, so that the theoretical design sequence obtained after correct modification is 5' -A-TTCCTT-3 ', wherein ". Times" represents phosphorothioate modification. In some embodiments, a theoretical sequence fragment may be determined based on the theoretical design sequence and the nucleic acid cocktail, the theoretical sequence fragment being present in the theoretical enzymatic product of the theoretical design sequence. For example, the theoretical design sequence is 5 '-A.times.TTCCTT-3', and after nucleic acid mixed enzyme treatment, the theoretical design sequence can be split into modified sequence fragments A.times.T and unmodified mononucleotide sequence fragments, wherein the modified sequence fragments A.times.A.times.T are theoretical sequence fragments.

In some embodiments, adjacent nucleotides of the theoretical sequence fragment are linked by phosphorothioate linkages. The theoretical sequence fragment may be a chemically modified sequence fragment of the theoretical design sequence. Further, the first mass spectrum information (such as molecular weight, charged ion charge state generated after ionization, charged ion mass charge ratio and the like) can be used for representing the nucleotide arrangement sequence, chemical modification and the like of the theoretical sequence fragment, and the second mass spectrum information can be used for representing the nucleotide arrangement sequence, chemical modification and the like of the target sequence fragment subjected to chemical modification in the nucleic acid sequence to be identified, so that the first mass spectrum information and the second mass spectrum information have direct correspondence, and the comparison of subsequent steps is facilitated.

In some embodiments, there may be one or more theoretical sequence fragments based on the difference of the preset modification rules, and accordingly, there may be one or more target sequence fragments chemically modified in the nucleic acid sequence to be identified, which is not limited in this embodiment. In some embodiments, each theoretical sequence fragment consists of at least two nucleotides.

In some embodiments, the first mass spectral information may include first parent ion information of a theoretical sequence fragment. As used herein, the first parent ion is an ion of interest that the theoretical sequence fragment produces during ionization in mass spectrometry. In some embodiments, charged ions of interest may be screened as the first parent ion from a series of charged ions that are theoretically generated by the theoretical sequence of fragments during ionization. For example, the theoretical sequence of fragments is ionized to produce a series of charged ions including singly, doubly, and tri-charged ions, and any one of the singly, doubly, and tri-charged ions of the theoretical sequence of fragments may be selected as the first parent ion.

In some embodiments, the first parent ion information includes at least a mass-to-charge ratio of the first parent ion. In some embodiments, the first parent ion information further includes a charge state, such as a charge number, of the first parent ion.

In some embodiments, the first parent ion information may be determined by theoretical analysis, such as determining a mass-to-charge ratio, charge state, etc. of the first parent ion by theoretical analysis. In some embodiments, the mass-to-charge ratio of the first parent ion may be determined based on the molecular weight of the theoretical sequence of fragments and the charge state of the first parent ion. For example, the theoretical sequence fragment has a molecular weight of 300, the first parent ion is a singly charged ion, the charge number is 1, and the relationship between mass-to-charge ratio and molecular weight is: molecular weight = mass to charge ratio + charge number, the mass to charge ratio of the first parent ion is determined to be 299.

In some embodiments, the first mass spectral information further comprises first sub-ion information of the theoretical sequence fragment.

As used herein, the first daughter ion is the fragment ion generated by the first parent ion in the dissociation step of mass spectrometry. Specifically, the collision of the first parent ion with the gas causes a specific chemical bond (such as a P-O bond on a phosphodiester bond between adjacent nucleotides) to break, thereby theoretically forming a series of fragment ions, and the fragment ions of the first parent ion may include one or more pairs of first daughter ions capable of complementary pairing. For example, a divalent ion generated by ionization of the theoretical sequence fragment t×a×c×a is selected as the first parent ion. The fragment ions generated by the first parent ion fragmentation may include a pair of complementary paired ions corresponding to fragment T x a and fragment C x a, and may further include a pair of complementary paired ions corresponding to fragment T x a and fragment C x a.

In some embodiments, the first sub-ion information includes at least complementary pairing information and mass-to-charge ratio of the first sub-ion. In some embodiments, the complementary pairing information for the first sub-ion includes a charge state of the first sub-ion and the first sub-ion to which it is complementary paired. In some embodiments, the complementary pairing information for the first sub-ion includes the molecular weight of the fragmentation fragment corresponding to the first sub-ion and the fragmentation fragment complementary paired therewith.

In some embodiments, the first sub-ion information may be determined by theoretical analysis, such as determining a charge state, a mass-to-charge ratio, etc. of the first sub-ion by theoretical analysis. In some embodiments, the mass-to-charge ratio of the first sub-ion may be determined based on the molecular weight of the first sub-ion corresponding to the fragmentation fragment and the charge state of the first sub-ion. For more details on determining the first sub-ion mass-to-charge ratio, reference may be made to the relevant description of determining the first parent ion mass-to-charge ratio.

In some embodiments, the second mass spectrometry information can include second parent ion information. In some embodiments, step 120 may further comprise the step of determining second parent ion information. Specifically, the step of determining the second parent ion information includes determining the second parent ion information based on the first parent ion information.

As used herein, the second parent ion is an ion of interest that is generated during ionization in mass spectrometry following enzymatic hydrolysis of the nucleic acid sequence to be identified. In some embodiments, the charged ion of interest may be selected as the second parent ion from a series of charged ions that are actually generated during ionization of the nucleic acid sequence enzymatic hydrolysate to be identified. In the case of a nucleic acid sequence to be identified which has been subjected to a correct chemical modification based on a preset modification rule, the charged ions which are actually generated during ionization of the enzymatic hydrolysis product of the nucleic acid sequence to be identified may comprise charged ions which are theoretically generated during ionization of the fragment of the theoretical sequence. And determining second parent ion information based on the first parent ion information, so that fragment ions generated by the cleavage of the second parent ion correspond to fragment ions generated by the cleavage of the first parent ion, and the comparison result of the fragment ions and the fragment ions can reflect the modification condition of the nucleic acid sequence to be modified.

In some embodiments, the second parent ion information includes at least a mass to charge ratio of the second parent ion. In some embodiments, the second parent ion information further includes a charge state, such as a charge number, of the second parent ion. For example, in a secondary mass spectrometry analysis of a nucleic acid sequence to be modified, a second parent ion having a mass-to-charge ratio and charge state matching the first parent ion is selected for dissociation and detection. Wherein the mass-to-charge ratio of the second parent ion is the same as the mass-to-charge ratio of the first parent ion, or the difference between the mass-to-charge ratios is within an allowable range (e.g., the difference is less than 0.3, 0.5, 0.8 or 1.3), and the charge state of the second parent ion is the same as the charge state of the first parent ion.

In some embodiments, the second mass spectral information may include second sub-ion information. In some embodiments, step 120 may further comprise the step of determining second sub-ion information. Specifically, the step of determining second sub-ion information includes performing secondary mass spectrometry of the enzymatic hydrolysate based on the second parent ion information to obtain second sub-ion information.

As used herein, the second daughter ion is a fragment ion generated by the second parent ion during dissociation of mass spectrometry. Specifically, the collision of the second parent ion with the gas causes specific chemical bonds to be broken to actually form a series of fragment ions, and one or more pairs of second child ions capable of complementary pairing may be contained in the fragment ions of the second parent ion.

In some embodiments, the second sub-ion information includes at least complementary pairing information and mass-to-charge ratio of the second sub-ion.

In some embodiments, the mass-to-charge ratio of the second sub-ion may be obtained directly from the results of an actual secondary mass spectrometry analysis, such as reading mass-to-charge ratio data of the second sub-ion directly from a secondary mass spectrum.

In some embodiments, the complementary pairing information for the second sub-ion includes a charge state of the second sub-ion and the second sub-ion to which it is complementary paired. In some embodiments, the complementary pairing information for the second sub-ion includes the molecular weight of the fragmentation fragment corresponding to the second sub-ion and the fragmentation fragment complementary paired therewith.

In step 130, it is determined whether a target sequence fragment is present in the nucleic acid sequence to be identified based on the first mass spectrometry information and the second mass spectrometry information. Wherein the target sequence fragment is identical to the theoretical sequence fragment.

In some embodiments, the determining whether a target sequence fragment consistent with the theoretical sequence fragment exists in the nucleic acid sequence to be identified may further include determining whether the first sub-ion information and the second sub-ion information satisfy a preset matching condition; if the preset matching condition is met, determining that the target sequence fragment exists in the nucleic acid sequence to be identified; if the preset matching condition is not met, determining that the target sequence fragment does not exist in the nucleic acid sequence to be identified.

Specifically, determining the existence of the target sequence fragment in the nucleic acid sequence to be identified, namely determining the nucleotide arrangement sequence and the chemical modification type, number and position of the target sequence fragment in the nucleic acid sequence to be identified to meet the requirement of a preset modification rule, wherein the chemical modification of the nucleic acid sequence to be identified is correct. It is understood that the identification of the correct modification means that at least part of the chemical modification of the nucleic acid sequence to be identified in the sample of nucleic acid sequences to be identified is correct. In view of the characteristics of mass spectrometry, if the nucleotide arrangement sequence and/or the chemical modification type, number and position of the nucleic acid sequence to be identified are changed, the mass-charge ratio of the sub-ions generated by the enzymolysis product is obviously changed. Therefore, the comparison result of the actual sub-ion information and the theoretical sub-ion information of the nucleic acid sequence to be identified can accurately reflect whether the nucleotide arrangement sequence and the chemical modification condition in the nucleic acid sequence to be identified are consistent with the expected target sequence fragments.

In some embodiments, the preset matching condition is set based on the complementary pairing information and the mass-to-charge ratio of the first and second sub-ions. In some embodiments, the preset matching condition is: there is at least one pair of first sub-ions and at least one pair of second sub-ions, the complementary pairing information and mass-to-charge ratio of the at least one pair of first sub-ions and the at least one pair of second sub-ions being the same.

For example, the series of fragment ions formed by cleavage of the divalent parent ion of the theoretical sequence fragment A.times.C.times.G comprises the complementarily paired first daughter ion A-1 and first daughter ion A-2, and the complementarily paired first daughter ion B-1 and first daughter ion B-2. Wherein the first sub-ion A-1 and the first sub-ion A-2 correspond to the fragmentation fragment A.times.C and the fragmentation fragment A.times.G, respectively, and the first sub-ion B-1 and the first sub-ion B-2 correspond to the fragmentation fragment A.times.C and the fragmentation fragment A.times.G, respectively. If the second sub-ion A '-1 and the second sub-ion A' -2 appear in fragment ions generated by the enzymolysis product of the nucleic acid sequence to be identified, wherein the mass-to-charge ratio of the second sub-ion A '-1 and the first sub-ion A-1 is the same, the mass-to-charge ratio of the second sub-ion A' -2 and the first sub-ion A-2 is the same, and the second sub-ion A '-1 and the second sub-ion A' -2 are in a complementary pairing relation, the first sub-ion information and the second sub-ion information can be determined to meet the preset matching condition. If only the second sub-ion A '-1 and the second sub-ion B' -1 with the same mass-to-charge ratio as the first sub-ion B-1 are present in the second sub-ion obtained from the enzymatic hydrolysis product of the nucleic acid sequence to be identified, the mass-to-charge ratio of the second sub-ion A '-1 and the first sub-ion A-1 are the same, the mass-to-charge ratio of the second sub-ion B' -1 and the first sub-ion B-1 are the same, and the second sub-ion A '-1 and the second sub-ion B' -1 have no complementary pairing relation, the first sub-ion information and the second sub-ion information can be determined to not meet the preset matching condition.

In some embodiments, secondary mass spectrometry of the enzymatic hydrolysis product can be performed by a linear ion well mass spectrometer. In other embodiments, the secondary mass spectrometry of the enzymatic hydrolysis products can be performed by other instruments having the same or similar functions, which is not limited in this embodiment.

The methods of identifying modifications of phosphorothioate modified nucleic acid sequences disclosed herein may provide benefits including, but not limited to: (1) The modification and identification method of the embodiment of the specification is based on the comparison of the information of the actually generated sub-ions of the nucleic acid sequence to be identified and the information of the theoretically generated sub-ions through enzymolysis and secondary mass spectrometry, and the method determines whether the chemical modification of the nucleic acid sequence to be identified is correct or not, and the identification result is accurate and visual; (2) In addition to the identification of phosphorothioate modifications, the modification identification methods of the embodiments of the present disclosure may also be synchronized to the identification of other chemical modifications of pentoses and/or bases, with high efficiency; (3) The modification and identification method of the embodiment of the specification directly characterizes the nucleic acid sequence to be identified through secondary mass spectrometry after enzymolysis, omits complicated steps such as purification of enzymolysis products, and saves time and samples. It should be noted that, the advantages that may be generated by different embodiments may be different, and in different embodiments, the advantages that may be generated may be any one or a combination of several of the above, or any other possible advantages that may be obtained.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

The materials and instruments used in the following examples include：

LC-MS mobile phase matching：

LC-MS-buffer A:2mL of HFIPA+250. Mu.L of TEA+10mL of EDTA was added to 1L of water, and the mixture was sonicated to give mobile phase A;

LC-MS-buffer B: 2mL of HFIPA+250. Mu.L of TEA+10mL of EDTA+190mL of water was added to 800mL of acetonitrile, and the mixture was sonicated to give mobile phase B.

LC-MSMS mobile phase configuration:

LC-MSMS-buffer A:2mL of HFIPA+800. Mu.L of DIEA+8mL of LC-MS EDTA was added to 792mL of water, and the mixture was sonicated to prepare mobile phase A;

LC-MSMS-buffer B:600 μl HFIPA+300 μl DIEA+8mL LC-MS EDTA+160mL water was added to 640mL acetonitrile, and the mixture was sonicated to give mobile phase B.

Example 1 background interference of nucleic acid Mixed enzymes

1.1 LC-MS analysis of control sample 1

1.1.1 enzymolysis treatment

Control sample 1 is an enterprise control of single-stranded RNA fragment 5'-sgRNA-4nt, and relevant information of RNA fragment 5' -sgRNA-4nt is shown in Table 1.1.

TABLE 1.1-5' -sgRNA-4nt sequence information

Name of the name	Sequence(s)	Molecular weight
			5’-sgRNA-4nt	5’mGmAmG*rU 3’	1353.84

Note that: m represents a 2' -OMe modification, r represents a phosphorothioate modification, and r represents RNA.

The control sample 1 is taken as a substrate, enzymolysis reaction liquid of the control sample 1 is prepared according to the table 1.2, and after uniform mixing, the mixture is centrifuged and placed in a digital display temperature control metal bath at 37 ℃ for incubation, so that an enzymolysis product is obtained.

TABLE 1.2 Components of the enzymatic hydrolysis reaction solution

1.1.2 LC-MS sample analysis

5. Mu.L of the enzymatic hydrolysate of step 1.1.1 was diluted with 45. Mu.L of water, and the diluted enzymatic hydrolysate was subjected to LC-MS analysis using a time-of-flight mass spectrometer using the aforementioned LC-MS mobile phase configuration.

1.1.3 LC-MS Mass Spectrometry results and analysis

As shown in Table 1.1, the theoretical molecular weight of the RNA fragment 5'-sgRNA-4nt was 1353.84, and it was found by theoretical analysis that the charged ion of the RNA fragment 5' -sgRNA-4nt included M/z= 1352.84 ([ M-H)] ^- )、m/z＝675.92([M-2H] ^2- ) And M/z= 450.28 ([ M-3H)] ^3- )。

FIG. 2 is a LC-MS mass spectrum of control sample 1. As shown in FIG. 2, the characteristic peaks of M/z= 1352.1973, M/z= 675.5928 and M/z= 450.0583 can correspond to [ M-H ] of the RNA fragment 5' -sgRNA-4nt, respectively] ^- 、[M-2H] ^2- And [ M-3H] ^3- . From this, it is clear that the actual parent ion information (type and mass-to-charge ratio) of the control sample 1 matches the theoretical parent ion information. The nucleic acid mixing enzyme does not cleave phosphorothioate bonds between adjacent nucleotides in the RNA fragment of the control sample 1, and the enzymolysis effect is in line with expectations.

1.2 LC-MS analysis of control sample 2

1.2.1 enzymolysis treatment

Control sample 2 is an enterprise control of single-stranded RNA fragment 3'-sgRNA-4nt, and relevant information of RNA fragment 3' -sgRNA-4nt is shown in Table 1.3. The control sample 2 is taken as a substrate, enzymolysis reaction liquid of the control sample 2 is prepared according to the table 1.2, and after uniform mixing, the mixture is centrifuged and placed in a digital display temperature control metal bath at 37 ℃ for incubation, so that an enzymolysis product is obtained.

TABLE 1 sequence information of 3-3' -sgRNA-4nt

Name of the name	Sequence(s)	Molecular weight
			3’-sgRNA-4nt	5’mUmUmU*rU 3’	1252.72

1.2.2 LC-MS sample analysis

5. Mu.L of the enzymatic hydrolysate of step 1.2.1 was diluted with 45. Mu.L of water, and the diluted enzymatic hydrolysate was subjected to LC-MS analysis using a time-of-flight mass spectrometer using the aforementioned LC-MS mobile phase configuration.

1.2.3 LC-MS Mass Spectrometry results and analysis

As shown in Table 1.3, the molecular weight of the RNA fragment 3'-sgRNA-4nt was 1252.72, and it was found by theoretical analysis that the charged ion of the RNA fragment 3' -sgRNA-4nt included M/z= 1251.72 ([ M-H)] ^- ) And M/z= 625.36 ([ M-2H)] ^2- )。

FIG. 3 is a LC-MS mass spectrum of control sample 2. As shown in FIG. 3, the characteristic peaks of M/z= 1251.1244 and M/z= 625.0575 can correspond to [ M-H ] of the RNA fragment 3' -sgRNA-4nt, respectively] ^- And [ M-2H] ^2- . From this, it can be seen that the actual charged ion information of the control sample 2 was separated from the theoretical charge The sub-information is consistent. The nucleic acid mixing enzyme does not cleave phosphorothioate bonds between adjacent nucleotides in the RNA fragment of the control sample 2, and the enzymolysis effect is expected.

1.3 LC-MS analysis of Mixed enzyme reaction buffers

1.3.1, preparing a mixed enzyme reaction buffer solution: a substrate-free mixed enzyme reaction buffer was prepared for use as per Table 1.2.

1.3.2 LC-MS sample analysis

5. Mu.L of the mixed enzyme reaction buffer of step 1.3.1 was diluted with 45. Mu.L of water, and LC-MS analysis was performed on the diluted mixed enzyme reaction buffer using a time-of-flight mass spectrometer using the aforementioned LC-MS mobile phase configuration.

1.3.3, LC-MS Mass Spectrometry results and analysis

FIG. 4 is a LC-MS total ion flow diagram of the mixed enzyme reaction buffer, and FIG. 5 is a mass spectrum of the mixed enzyme reaction buffer LC-MS data at 5.925 min. As shown in FIGS. 4 and 5, no characteristic peaks of the RNA fragment 5'-sgRNA-4nt and the RNA fragment 3' -sgRNA-4nt appeared within 3 to 10min of the gradient, except for the background peak at the retention time of 0.5 min. It can be seen that the mixed enzyme reaction buffer does not interfere with the mass spectrum results of the enzymatic hydrolysis products.

Example 2 Mass spectrometry of enzymatic products of sample 1 at different enzymatic hydrolysis times

2.1 enzymolysis reaction

Sample 1 is a sample to be identified of single-stranded RNA fragment ET-02sgRNA, and the relevant information of the RNA fragment ET-02sgRNA is shown in Table 2.1. Wherein the sequence of the 5 '-end modified fragment of the RNA fragment ET-02sgRNA is 5' -mG mA mG rU-3', and is consistent with the sequence of the RNA fragment 5' -sgRNA-4 nt; the sequence of the 3 '-end modified fragment of the RNA fragment ET-02sgRNA is 5' -mU rU-3', which is consistent with the sequence of the 3' -sgRNA-4nt of the RNA fragment. Sample 1 is taken as a substrate, an enzymolysis reaction liquid of sample 1 is prepared according to the table 1.2 of the embodiment 1, and after uniform mixing, the mixture is centrifuged and placed in a 37 ℃ digital display temperature control metal bath for incubation, so that an enzymolysis product is obtained.

TABLE 2 sequence information of 1-ET-02sgRNA

Note that: n represents A, G, C, T or U.

2.2 LC-MS sample analysis

After 2min, 4h and 24h of enzymolysis, 5 μl of the enzymolysis product of step 2.1 is diluted with 45 μl of water, the LC-MS mobile phase configuration is adopted, and the diluted enzymolysis product is subjected to LC-MS analysis by using a time-of-flight mass spectrometer.

2.3 LC-MS Mass Spectrometry results and analysis

FIG. 6 is a LC-MS mass spectrum of the enzymatic hydrolysate of sample 1 for 2 min. Wherein the characteristic peak of M/z= 1251.1328 shown in FIG. 6A may correspond to [ M-H ] of the 3' -end modified fragment of ET-02sgRNA] ^- . The characteristic peaks shown in FIG. 6B for M/z= 1352.2057 and M/z= 675.6022 can correspond to [ M-H ] of the 5' -end modified fragment of ET-02sgRNA, respectively] ^- ，[M-H] ^2- . The corresponding parent ion signals of the 5 'end modified fragment and the 3' end modified fragment of the ET-02sgRNA appear in the mass spectrum result of the enzymolysis product, which indicates that the nucleic acid mixed enzyme can generate partial enzymolysis product after carrying out enzymolysis treatment on the sample 1 for a short time.

FIG. 7 is a LC-MS mass spectrum of the enzymatic hydrolysate of sample 1 for 4 h. Wherein, the characteristic peaks of M/z= 1251.1318 and M/z= 625.0617 shown in FIG. 7A can correspond to [ M-H ] of the 3' -end modified fragment, respectively] ^- ，[M-H] ^2- . The characteristic peaks shown in FIG. 7B at M/z= 1352.2027 and M/z= 675.5978 can correspond to [ M-H ] of the 5' -terminal modified fragment, respectively ] ^- ，[M-H] ^2- . No corresponding parent ion signal for the complete sequence fragment of ET-02sgRNA was seen in the LC-MS mass spectrum results. Compared with the enzymolysis product obtained by enzymolysis of the sample 1 for 2min, in the LC-MS mass spectrum result of the enzymolysis product obtained by enzymolysis of the sample 1 for 4h, the corresponding parent ion signal response (such as absolute intensity) of the 5 '-end modified fragment and the 3' -end modified fragment of the ET-02sgRNA is obviously increased.

FIG. 8 is a LC-MS mass spectrum of the enzymatic hydrolysate of sample 1 for 24 h. Wherein, the characteristic peaks of M/z= 1251.1223 and M/z= 625.0563 shown in FIG. 8A can correspond to [ M-H ] of the 3' -end modified fragment, respectively] ^- ，[M-H] ^2- . The characteristic peaks shown in fig. 8B for m/z= 1352.1922 and m/z= 675.5920 can be separated[ M-H ] of modified fragment corresponding to 5' end] ^- ，[M-H] ^2- . Compared with the enzymolysis product obtained by enzymolysis of sample 1 for 4 hours, in the LC-MS mass spectrum result of the enzymolysis product obtained by enzymolysis of sample 1 for 24 hours, the corresponding parent ion signal response of the 5 '-end modified fragment and the 3' -end modified fragment of the ET-02sgRNA is not obviously increased.

In conclusion, after the nucleic acid mixed enzyme is subjected to enzymolysis for 4 hours, the enzymolysis of the substrate is more sufficient, so that the 5 '-end modified fragment and the 3' -end modified fragment of the ET-02sgRNA are accurately obtained.

Example 3, identification of modifications of sample 1

3.1 enzymolysis treatment

Sample 1 is taken as a substrate, an enzymolysis reaction liquid of sample 1 is prepared according to the table 1.2 of the embodiment 1, and after uniform mixing, the mixture is centrifuged and placed in a 37 ℃ digital display temperature control metal bath for incubation for 4 hours, so that an enzymolysis product is obtained. The relevant information for sample 1 is shown in table 2.1 of example 2.

3.2 LC-MSMS sample injection analysis

Taking 5 mu L of the enzymolysis product obtained in the step 3.1, supplementing water to 10 mu L, adopting the LC-MSMS analysis mobile phase configuration, and carrying out LC-MSMS analysis on the diluted enzymolysis product by using a linear ion well mass spectrometer, wherein the LC-MSMS is injected with 10 mu L at one time.

3.3 LC-MSMS Mass Spectrometry results and analysis

3.3.1 identification of the 5' -terminal modification of the RNA sequence of sample 1

From the study of example 1, it was confirmed that the phosphorothioate bonds of the 5' -end modified fragment mG rU of ET-02sgRNA were not broken and remained as intact fragments after cleavage of ET-02sgRNA by nucleic acid mixture enzyme. In the theoretical analysis result of LC-MSMS mass spectrometry, 5' end modified fragments mgma rU are ionized to form a series of charged ions. One of the charged ions is selected as a parent ion (e.g., a divalent parent ion of m/z= 675.92), and a gas is used to generate collision-induced dissociation of a certain energy to break specific chemical bonds of the parent ion (typically P-O bonds on phosphodiester bonds between adjacent nucleotides) and form a plurality of daughter ions. The plurality of sub-ions include a plurality of pairs of sub-ions capable of complementary pairing, for example, monovalent sub-ions (m/z=733.6) corresponding to mgma of the fragmentation fragment and monovalent sub-ions (m/z= 618.5) corresponding to mgru of the fragmentation fragment are a pair of complementary paired sub-ions.

Divalent parent ions with m/z= 675.92 were screened in negative mode for LC-MSMS analysis of sample 1 enzymatic hydrolysis products, and the mass spectrum results are shown in fig. 9. Fig. 9 shows fragmentation information corresponding to characteristic peaks and the number of sub-ion charges (Z represents the number of charges). As shown in fig. 9, sample 1 enzymatic hydrolysate was cleaved in LC-MSMS analysis to generate several daughter ions. Wherein, the characteristic peaks of m/z= 618.12 and m/z= 733.08 can correspond to a pair of complementary paired ion ions, namely, a pair of valence ion ions corresponding to the fragment mgxu and fragment mgxu; the characteristic peaks of m/z= 655.13 and m/z= 733.08 may correspond to a pair of complementary paired ion ions, i.e. a pair of valence ion ions corresponding to fragmentation fragment mgma and fragmentation fragment mgru.

Thus, the information of the sub-ions actually generated by the enzymatic hydrolysate of sample 1 can be matched with the information of the sub-ions theoretically generated by mGlu of the 5' -end modified fragment. The RNA sequence of sample 1 has a target sequence fragment consistent with the 5 'end modified fragment mgma mgru, and the nucleotide arrangement sequence and chemical modification condition of the target sequence fragment and the 5' end modified fragment mgma mgru are the same, so that the 5 'end of the RNA sequence of sample 1 is verified to have correct 2' -O-methylation modification and phosphorothioate modification.

3.3.2 identification of the 3' -terminal modification of the RNA sequence of sample 1

From the study of example 1, it was confirmed that the phosphorothioate bonds of the 3' -end modified fragment mU rU of ET-02sgRNA remained intact after cleavage of ET-02sgRNA by nucleic acid mixing enzyme due to the anti-cleavage effect of phosphorothioate bonds. In the theoretical analysis result of LC-MSMS mass spectrometry, after screening a suitable parent ion (such as a divalent parent ion with m/z= 625.36), the 3' -end modified fragment mu×mu×ru×ru may form several child ions. The plurality of sub-ions include a plurality of pairs of sub-ions capable of complementary pairing, for example, a monovalent sub-ion (m/z= 671.5) corresponding to the fragmentation fragment mU and a monovalent sub-ion (m/z= 579.5) corresponding to the fragmentation fragment mU are a pair of complementary paired sub-ions.

Divalent parent ions with m/z= 625.36 were screened in negative mode for LC-MSMS analysis of sample 1 enzymatic hydrolysis products, and mass spectrometry results are shown in fig. 10. The fragmentation information and the number of sub-ion charges corresponding to the characteristic peaks are labeled in fig. 10. As shown in fig. 10, sample 1 enzymatic hydrolysate was cleaved in LC-MSMS analysis to generate several daughter ions. Wherein, the characteristic peaks of m/z= 579.09 and m/z= 671.01 can correspond to a pair of complementary paired ion ions, i.e. fragmentation fragment mu×ru and fragmentation fragment mu×mu corresponding to a pair of valence ion ions; the characteristic peaks of m/z= 593.10 and m/z= 656.99 may correspond to a pair of complementary paired ion ions, i.e., fragmentation fragment mu×mu and fragmentation fragment mu×ru correspond to a pair of valence ion ions; the characteristic peaks of m/z= 321.00 and m/z= 929.08 may correspond to a pair of complementary paired ion ions, i.e., fragmentation fragment xu and fragmentation fragment mU.

Thus, the information of the sub-ions actually generated by the enzymatic hydrolysate of sample 1 can be matched with the information of the sub-ions theoretically generated by the 3' -end modified fragment mU rU. The RNA sequence of the sample 1 has a target sequence fragment consistent with a 3 '-end modified fragment mU rU, and the target sequence fragment is identical with the nucleotide arrangement sequence and chemical modification condition of the 3' -end modified fragment mU rU, so that the 3 '-end of the RNA sequence of the sample 1 is verified to have correct 2' -O-methylation modification and phosphorothioate modification.

Example 4, identification of modifications of sample 2

4.1 enzymolysis treatment

Sample 2 is the sample to be identified of single-stranded RNA fragment ET-01sgRNA, and the relevant information of the RNA fragment ET-01sgRNA is shown in Table 4.1. The sequence of the 5' -end modified fragment of the RNA fragment ET-01sgRNA is 5' -mA mU mC rA-3'; the sequence of the 3 '-end modified fragment of the RNA fragment ET-01sgRNA is 5' -mU rU-3', which is consistent with the sequence of the 3' -sgRNA-4nt of the RNA fragment. Sample 2 is taken as a substrate, an enzymolysis reaction solution of sample 2 is prepared according to the table 1.2 of the embodiment 1, and after uniform mixing, the mixture is centrifuged and placed in a 37 ℃ digital display temperature control metal bath for incubation for 4 hours, so that an enzymolysis product is obtained.

TABLE 4 sequence information of 1-ET-01sgRNA

4.2 LC-MSMS sample analysis

Taking 5 mu L of the enzymolysis product obtained in the step 4.1, supplementing water to 10 mu L, adopting the LC-MSMS mobile phase configuration, and carrying out LC-MSMS analysis on the diluted enzymolysis product by using a linear ion well mass spectrometer, wherein the LC-MSMS is injected with 10 mu L at one time.

4.3 LC-MSMS Mass Spectrometry results and analysis

4.3.1 identification of the 5' -end modification of the RNA sequence of sample 2

Due to the anti-cleavage effect of phosphorothioate bonds, the 5' -end modified fragment mA, mU, mC, rA of ET-01sgRNA is not cleaved by the enzyme and remains as an intact fragment. The theoretical molecular weight of the 5' -end modified fragment mA mU mC rA is 1298.1. In the theoretical analysis result of LC-MSMS mass spectrometry, after screening a suitable parent ion (such as a divalent parent ion with m/z=648), a 5' modified fragment ma×mu×mc×ra may form several child ions. The plurality of sub-ions include a plurality of pairs of sub-ions capable of complementary pairing, for example, monovalent sub-ions (m/z= 694.5) corresponding to fragmentation fragment ma×mu and monovalent sub-ions (m/z=601.5) corresponding to fragmentation fragment mc×ra are a pair of complementary paired sub-ions.

Divalent parent ions with m/z=648 were screened in negative mode for LC-MSMS analysis of sample 2 enzymatic hydrolysis products, and the mass spectrum results are shown in fig. 11. The fragmentation information and the number of sub-ion charges corresponding to the characteristic peaks are labeled in fig. 11. As shown in fig. 11, sample 2 enzymatic hydrolysate was cleaved in LC-MSMS analysis to generate several daughter ions. Wherein, the characteristic peaks of m/z= 616.00 and m/z= 678.95 can correspond to a pair of complementary paired ions, namely a pair of valence ions corresponding to fragmentation fragment ma×mu and fragmentation fragment mc×ra; the characteristic peaks of m/z= 601.02 and m/z= 693.93 may correspond to a pair of complementary paired ion ions, i.e. a pair of valence ion ions corresponding to fragmentation fragment mc×ra and fragmentation fragment ma×mu×mu; the characteristic peaks of m/z= 343.98 and m/z= 950.99 may correspond to a pair of complementary paired ion ions, i.e. a pair of valence ion ions corresponding to fragmentation fragment x rA and fragmentation fragment mA x mU x mC.

Thus, the information of the sub-ions actually generated by the enzymatic hydrolysate of the sample 2 can be matched with the information of the sub-ions theoretically generated by the modified fragment mA, mU, mC, rA at the 5' end. The RNA sequence of the sample 2 has a target sequence fragment consistent with the 5 '-end modified fragment mA [ mU ] mA, and the target sequence fragment has the same nucleotide arrangement sequence and chemical modification condition as the 5' -end modified fragment mA [ mU ] mC ] rA, so that the 5 '-end of the RNA sequence of the sample 2 is verified to have correct 2' -O-methylation modification and phosphorothioate modification.

4.3.2 identification of the 3' -end modification of the RNA sequence of sample 2

Referring to example 3.3.2, divalent parent ions with m/z= 625.36 were screened in negative mode for LC-MSMS analysis of sample 2 enzymatic hydrolysis products, and mass spectrometry results are shown in fig. 12. The fragmentation information and the number of sub-ion charges corresponding to the characteristic peaks are labeled in fig. 12. As shown in fig. 12, sample 2 enzymatic hydrolysate was cleaved in LC-MSMS analysis to generate several daughter ions. Wherein, the characteristic peaks of m/z= 578.98 and m/z= 670.89 can correspond to a pair of complementary paired ion ions, i.e. fragmentation fragment mu×ru and fragmentation fragment mu×mu corresponding to a pair of valence ion ions; the characteristic peaks of m/z= 592.98 and m/z= 656.88 may correspond to a pair of complementary paired ion ions, i.e., fragmentation fragment mu×mu and fragmentation fragment mu×ru correspond to a pair of valence ion ions; the characteristic peaks of m/z= 320.94 and m/z= 928.93 may correspond to a pair of complementary paired ion ions, i.e., fragmentation fragment xu and fragmentation fragment mU.

Thus, the information of the sub-ions actually generated by the enzymatic hydrolysate of sample 2 can be matched with the information of the sub-ions theoretically generated by the 3' -end modified fragment mU rU. The RNA sequence of sample 2 has a target sequence fragment consistent with the 3 'end modified fragment mU rU, and the target sequence fragment is identical to the nucleotide arrangement sequence and chemical modification condition of the 3' end modified fragment mU rU, so that the 3 'end of the RNA sequence of sample 2 is verified to have correct 2' -O-methylation modification and phosphorothioate modification.

It will be appreciated by those skilled in the art that the above examples are illustrative of the invention and are not to be construed as limiting the invention. Any modifications, equivalent substitutions and variations, etc., which are within the spirit and principles of the present invention, are intended to be included within the scope of the present invention.

Claims

1. A method for identifying modifications of a phosphorothioate modified nucleic acid sequence, said method comprising:

performing enzymolysis on a nucleic acid sequence to be identified by using a nucleic acid mixed enzyme to obtain an enzymolysis product; wherein the nucleic acid sequence to be identified is actually obtained based on a preset modification rule, and the modification to be identified of the nucleic acid sequence to be identified at least comprises phosphorothioate modification of internucleotide linkage;

Performing secondary mass spectrometry on the enzymolysis product based on first mass spectrometry information to obtain second mass spectrometry information actually generated by the enzymolysis product, wherein the first mass spectrometry information comprises theoretical analysis results generated by analyzing theoretical sequence fragments, and the theoretical sequence fragments are determined based on the preset modification rules, the nucleic acid sequences to be identified and the nucleic acid hybrid enzyme; and

determining whether a target sequence fragment consistent with the theoretical sequence fragment exists in the nucleic acid sequence to be identified based on the first mass spectrum information and the second mass spectrum information.

2. The method of claim 1, wherein the nucleic acid cocktail is capable of and free of breaking phosphodiester bonds such that the phosphorothioate modified nucleic acid sequence to be identified produces at least two sequence fragments of different molecular weights.

3. The method of claim 2, wherein the nucleic acid cocktail enzyme comprises snake venom phosphodiesterase and/or bovine spleen phosphodiesterase.

4. The method of claim 1, wherein the nucleic acid-mixing enzyme is enzymatically hydrolyzed for a period of time ranging from 1h to 8h.

5. The method of claim 1, wherein the nucleic acid-mixing enzyme has an enzymatic hydrolysis time of 3 hours to 5 hours.

6. The method according to any one of claims 1 to 5, wherein the modification to be identified of the nucleic acid sequence to be identified further comprises a modification of pentose sugar and/or a modification of bases.

7. The method of claim 6, wherein the modification of pentose sugar comprises at least one of the following modifications: 2' -modification, 5' -modification, 3' -modification, locked nucleic acid modification, unlocked nucleic acid modification, and peptide nucleic acid modification.

8. The method of claim 7, wherein the 2 '-modification of pentose is selected from the group consisting of a 2' -O-methyl modification, a 2 '-fluoro modification, a 2' -O-methoxyethyl modification, a 2 '-O-methylation modification, and a 2' -O-allyl modification.

9. The method of claim 6, wherein the modification of the base comprises at least one of the following modifications: methylation modifications and methylolation modifications.

10. The method of claim 6, wherein the preset modification rules comprise: the nucleotide corresponding to the modified pentose is connected with at least one adjacent nucleotide through a phosphorothioate bond; and/or, the preset modification rule comprises: the nucleotides corresponding to the modified bases are linked to at least one adjacent nucleotide by phosphorothioate linkages.

11. The method of any one of claims 1-5, wherein the preset modification rules comprise: the 5 'end and/or the 3' end of the nucleic acid sequence to be identified are/is provided with a modified sequence fragment, the length of the modified sequence fragment is 2-5 nucleotides, and adjacent nucleotides in the modified sequence fragment are connected through phosphorothioate bonds.

12. The method of any one of claims 1-5, wherein adjacent nucleotides of the theoretical sequence of fragments are linked by phosphorothioate linkages.

13. The method of any one of claims 1-5, wherein the first mass spectral information comprises first parent ion information and first child ion information of the theoretical sequence fragment; the first parent ion information at least comprises the mass-to-charge ratio of the first parent ion, and the first child ion information at least comprises the complementary pairing information and the mass-to-charge ratio of the first child ion.

14. The method of claim 13, wherein the second mass spectrometry information comprises second parent ion information and second child ion information; the obtaining of the second mass spectrum information actually generated by the enzymolysis product comprises the following steps:

Determining the second parent ion information based on the first parent ion information, the second parent ion information including at least a mass-to-charge ratio of a second parent ion;

and carrying out secondary mass spectrometry analysis on the enzymolysis product based on the second parent ion information to obtain second child ion information, wherein the second child ion information at least comprises complementary pairing information and mass-to-charge ratio of the second child ion.

15. The method of claim 14, wherein said determining whether a fragment of the target sequence is present in the nucleic acid sequence to be identified that corresponds to the fragment of the theoretical sequence comprises:

determining whether the first sub-ion information and the second sub-ion information meet a preset matching condition;

if the preset matching condition is met, determining that the target sequence fragment exists in the nucleic acid sequence to be identified.

16. The method of claim 15, wherein the predetermined matching condition is: there is at least one pair of first sub-ions and at least one pair of second sub-ions, the complementary pairing information and mass-to-charge ratio of the at least one pair of first sub-ions and the at least one pair of second sub-ions being the same.

17. The method of any one of claims 1-5, wherein the secondary mass spectrometry of the enzymatic hydrolysate is performed by a linear ion well mass spectrometer.