One-step enrichment of RNA with NAD cap modification

Abstract

The present invention provides a one-step method for enriching RNA modified with an NAD cap. Specifically, the present invention provides a method for enriching RNA modified with an NAD cap, which is characterized in that a compound represented by formula (I) is contacted with the RNA to be tested in the presence of adenosine diphosphate ribosyl cyclase, so that the compound represented by formula (I) binds to the RNA modified with an NAD cap in the RNA to be tested, and through affinity treatment, RNA modified with an NAD cap is obtained, X-L-B (I), wherein X represents a nucleophilic group, L represents a linking group, B represents biotin or desthiobiotin, and X is linked to B through L. This method is simple to operate and low in cost.

Description

Technical Field

[0001] The present invention relates to the field of biology. Specifically, the present invention relates to a one-step method for enriching RNA modified with NAD caps. Background Art

[0002] Messenger ribonucleic acid (hereinafter referred to as: mRNA) in eukaryotic cells usually has a 5'-end cap modification. For a long time, 7-methylguanylic acid (hereinafter referred to as: m7G) has been considered the only cap modification of eukaryotic cell mRNA. Nicotinamide adenine dinucleotide (hereinafter referred to as: NAD) is an important coenzyme factor in cells. Recent studies have found that NAD can also serve as an RNA 5'-end cap modification (hereinafter referred to as: NAD-RNA). NAD-RNA is widely present in prokaryotic and eukaryotic organisms, indicating that it may be involved in gene regulation. NAD is the core coenzyme of cell oxidation and various metabolic pathways, and NAD significantly decreases during natural aging and aging-related diseases. Systematically characterizing the types and abundances of NAD-RNA provides important clues for understanding cell physiology and pathology. Summary of the Invention

[0003] The inventors found that there are currently two methods in the prior art for enriching RNA with NAD cap modification. One method is the NAD captureSeq technique, that is, under the condition of adenosine diphosphate ribosyl cyclase (hereinafter referred to as: ADPRC), 4-pentyn-1-ol is used to replace the nicotinamide part of NAD with an alkyne-containing molecule, and then the alkynylated product is linked to biotin azide through click chemistry (copper-catalyzed azide-alkyne cycloaddition CuAAC), and the biotin-labeled RNA is enriched with streptavidin resin. Finally, a cDNA library is obtained through next-generation sequencing technology (NGS). The main disadvantages of this technology are mainly two: (1) The participation of metal copper ions will cause serious degradation of RNA, resulting in the loss of full-length RNA information, thus greatly reducing the detection efficiency; (2) Non-specific enrichment occurs for RNA with m7G caps, thus affecting the accuracy of NAD-RNA detection. Another method is the SPAAC-NAD-seq detection technology, which is divided into three steps: (1) Remove RNA with m7G caps through antibody co-immunoprecipitation; (2) Use the ADPRC catalytic reaction to replace the nicotinamide part of NAD with 3-azido-1-propanol to become azide-containing; (3) Connect biotin-PEG4-DBCO through an alkyne cycloaddition reaction, and then enrich the biotin-labeled RNA with streptavidin resin, and then prepare a cDNA library by sequencing through next-generation sequencing technology (NGS). The main disadvantages of this technology are also two: (1) The steps are cumbersome, and multiple reactions reduce the overall detection efficiency; after removing m7G-capped RNA in step 1, it is necessary to add additional transfer RNA (tRNA) to ensure that the total amount of RNA remains unchanged during the reaction process; (2) Multiple reactions cause RNA consumption, and a high starting amount of RNA is required (not less than 400 micrograms of total RNA).

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. Based on the above findings, the present invention proposes a method for enriching RNA with NAD cap modification by a one-step method without metal copper ions. The present invention greatly simplifies the reaction steps, improves the detection efficiency and accuracy, and significantly reduces the RNA usage amount.

[0005] In the first aspect of the present invention, a method for enriching NAD-capped RNA is proposed. According to an embodiment of the present invention, the method includes contacting a compound represented by formula (I) with the RNA to be tested in the presence of adenosine diphosphate ribosyl cyclase, so that the compound represented by formula (I) binds to the RNA containing NAD cap modification in the RNA to be tested, and through affinity treatment, NAD-capped RNA is obtained, X-L-B (I), where X represents a nucleophilic group, L represents a linking group, B represents biotin or desthiobiotin, and X is connected to B through L. It should be noted that the affinity treatment here is a conventional operation method, which can be the specific reaction of biotin or desthiobiotin group with streptavidin resin, so as to achieve the effect of affinity enrichment. The inventors found that the X of the compound represented by formula (I) is a nucleophilic group, which can undergo nucleophilic substitution with the nicotinamide group on the NAD cap in the RNA, replacing the nicotinamide group. The B group is an affinity group, which is convenient for affinity purification after the nucleophilic substitution reaction, and there is no need to go through cumbersome steps (such as click chemistry), simplifying the operation process and improving the efficiency.

[0006] According to an embodiment of the present invention, the above method may further include at least one of the following technical features:

[0007] According to an embodiment of the present invention, X is selected from -OH, -SH, -NH2, -NHR, -NRR', and N-containing heteroaryl, and R and R' are independently selected from optionally substituted alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl. The inventors found that the above groups can all undergo nucleophilic substitution reactions with the nicotinamide group on the NAD cap, and when X is OH, the efficiency of nucleophilic substitution can reach 76%.

[0008] According to an embodiment of the present invention, L is selected from n is selected from 1, 2, or 3. The inventors found that since biotin or desthiobiotin is highly hydrophobic, a hydrophilic linking fragment (L) needs to be added between biotin and the nucleophilic group to enable the final tool molecule to stably exist in the water-soluble sample at a relatively high concentration. By comparing PEG (with oxygen atoms) and fully carbonated alkane chains, the tool molecule with PEG has better solubility in the water-soluble sample, so PEG is used as the L part of the substrate molecule.

[0009] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0010]

[0011] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0012]

[0013] In another aspect of the present invention, the present invention also provides a method for enriching RNA. According to an embodiment of the present invention, the method includes enriching NAD-cap modified RNA according to the method described above. The inventors found that the method of the embodiment of the present invention is simple to operate and highly efficient.

[0014] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0015] According to an embodiment of the present invention, the above method further includes performing a decapping treatment on the enriched NAD-cap modified RNA.

[0016] According to an embodiment of the present invention, the above decapping treatment is catalyzed by NudC pyrophosphatase. The inventors found that treatment with NudC pyrophosphatase can specifically decap RNA containing NAD-cap modification, and the released RNA can be used for subsequent operations (such as constructing an RNA sequencing library). RNA containing an m7G cap, because its cap structure contains three phosphate groups, will not be catalyzed by NudC pyrophosphatase to undergo a decapping reaction. The application of NudC pyrophosphatase is equivalent to "secondary purification" in the process, without the need for pretreatment with m7G antibody, simplifying the operation and reducing the amount of RNA used. The method according to the embodiment of the present invention can achieve the enrichment of RNA from a small number of cells.

[0017] In another aspect of the present invention, the present invention also provides the use of the compound shown in formula (I) in enriching NAD-cap modified RNA, X-L-B (I), wherein X represents a nucleophilic group, L represents a linking group, and B represents biotin or desthiobiotin, and X is linked to B through L. The inventors first used the compound shown in formula (I) to enrich NAD-cap modified RNA, and only one-step reaction is required to achieve enrichment using this compound, with simple operation and low cost.

[0018] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0019] According to an embodiment of the present invention, X is selected from -OH, -SH, -NH2, -NHR, -NRR', and N-containing heteroaryl, and R and R' are each independently selected from optionally substituted alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl. The inventors found that the above groups can all undergo nucleophilic substitution reactions with the NAD group, and when X is OH, the efficiency of nucleophilic substitution can reach 76%.

[0020] According to an embodiment of the present invention, L is selected from n is selected from 1, 2 or 3. The inventors found that since biotin or desthiobiotin is highly hydrophobic, it is necessary to introduce a hydrophilic linker fragment (L) between biotin and the nucleophilic group so that the final tool molecule can stably exist in the water-soluble sample at a relatively high concentration. By comparing PEG (with oxygen atoms) and fully carbon alkane chains, the tool molecule with PEG has better solubility in the water-soluble sample. Therefore, PEG is used as the L part of the substrate molecule.

[0021] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0022]

[0023] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0024]

[0025] Definitions and General Terms

[0026] The present invention will list in detail the documents corresponding to the specific embodiments. The examples are accompanied by diagrams of structural formulas and chemical formulas. The present invention is expected to cover all options, variants and equivalents, which may be included within the scope of the present invention as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances to those described herein, which can be applied to the practice of the present invention. The present invention is not limited to the description of methods and substances. There are many documents and similar substances that are different from or conflict with the present application, including but not limited to the definitions of terms, the usage of terms, the technologies described, or the scope controlled by the present application.

[0027] The present invention will apply the following definitions unless otherwise indicated. For the purposes of the present invention, chemical elements are defined according to the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75 th Ed., 1994. Additionally, general principles of organic chemistry are found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito, 1999, and "March's Advanced Organic Chemistry", Michael B. Smith and Jerry March, John Wiley & Sons, New York, 2007. All of the above references are incorporated herein by reference.

[0028] As described in the present invention, the compounds of the present invention may optionally be substituted by one or more substituents, such as the compounds of the general formula above, or as in the specific examples, subclasses, and classes of compounds encompassed by the present invention. It should be understood that the term "optionally substituted" may be used interchangeably with the term "substituted or unsubstituted". Generally, the term "optionally", whether or not preceding the term "substituted", means that one or more hydrogen atoms in the given structure are replaced by specific substituents. Unless otherwise indicated, an optional substituent group may have a substituent at each substitutable position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be the same or different at each position. The substituents may be, but are not limited to, deuterium, hydroxy, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyl, alkenyl, alkynyl, heterocyclic, mercapto, nitro, aryloxy, heteroaryloxy, oxo(=O), carboxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O)-, alkyl-C(=O)-, alkyl-S(=O)-, alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O)-, hydroxy-substituted alkyl-S(=O)2-, carboxy-substituted alkoxy, and the like.

[0029] Unless otherwise specified, the term "alkyl" refers to a saturated straight-chain or branched-chain monovalent hydrocarbon group having 1 to 20 carbon atoms, or 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms, wherein the alkyl group can be independently and optionally substituted by one or more substituents described in the present invention, and the substituents include, but are not limited to, deuterium, amino, hydroxy, cyano, F, Cl, Br, I, mercapto, nitro, oxo (=O), and the like. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, and the like. The term "alkyl" and its prefix "alk" as used herein include both straight-chain and branched-chain saturated carbon chains. The term "alkylene" as used herein refers to a saturated divalent hydrocarbon group obtained by eliminating two hydrogen atoms from a straight-chain or branched-chain saturated hydrocarbon, and examples of such include, but are not limited to, methylene, ethylene, isopropylidene, and the like.

[0030] The term "heteroalkyl" means that one or more heteroatoms can be inserted into an alkyl chain, where the alkyl group and the heteroatom have the meanings as described in the present invention. Unless otherwise specified in detail, the heteroalkyl group contains 1-10 carbon atoms. In some other embodiments, the heteroalkyl group contains 1-8 carbon atoms. In some other embodiments, the heteroalkyl group contains 1-6 carbon atoms. In some other embodiments, the heteroalkyl group contains 1-4 carbon atoms. In some other embodiments, the heteroalkyl group contains 1-3 carbon atoms. Such examples include, but are not limited to, CH3OCH2-, CH3CH2OCH2-, CH3SCH2-, (CH3)2NCH2-, (CH3)2CH2OCH2-, CH3OCH2CH2-, CH3CH2OCH2CH2-, etc.

[0031] The terms "heterocyclic", "heterocyclic group", "heteroalicyclic" or "heterocyclic" are used interchangeably herein and all refer to a monocyclic, bicyclic or tricyclic system, where one or more carbon atoms in the ring are independently and optionally replaced by heteroatoms, and the heteroatoms have the meanings as described in the present invention. The ring can be completely saturated or contain one or more degrees of unsaturation, but is never aromatic, and has one or more connection points connecting to other parts of the molecule. The hydrogen atoms on one or more rings are independently and optionally replaced by one or more substituents described in the present invention. In some of these embodiments, the "heterocyclic", "heterocyclic group", "heteroalicyclic" or "heterocyclic" group is a monocyclic ring of 3-7 members (1-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, S, where S or P is optionally replaced by one or more oxygen atoms to give groups such as SO, SO2, PO, PO2, and when the ring is a three-membered ring, there is only one heteroatom), or a bicyclic ring of 7-10 members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, S, where S or P is optionally replaced by one or more oxygen atoms to give groups such as SO, SO2, PO, PO2).

[0032] The heterocyclic group can be carbon-based or heteroatom-based. The "heterocyclic group" also includes groups formed by the fusion of a heterocyclic group with a saturated or partially unsaturated ring or heterocycle. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, thiazolyl, oxazolidinyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, epoxypropyl, azepanyl, oxepanyl, thiepanyl, 4-methoxypiperidin-1-yl, 1,2,3,6-tetrahydropyridin-1-yl, oxaaza yl, diaza yl, thiaza groups, pyrrolin-1-yl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,6-thiadiazinan-1,1-dioxo-2-yl, 4-hydroxy-1,4-azaphospholane 4-oxide-1-yl, 2-hydroxy-1-(piperazin-1-yl)ethan-4-yl, 2-hydroxy-1-(5,6-dihydro-1,2,4-triazin-1(4H)-yl)ethan-4-yl, 5,6-dihydro-4H-1,2,4-oxadiazin-4-yl, 2-hydroxy-1-(5,6-dihydropyridin-1(2H)-yl)ethan-4-yl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 2-methyl-5,6,7,8-tetrahydro-[1.2.4]triazolo[1,5-c]pyrimidin-6-yl, 4,5,6,7-tetrahydroisoxazolo[4,3-c]pyridin-5-yl, 3H-indol-2-yl-5-azabicyclo[2.2.1]heptan-5-yl, 2-oxo-5-azabicyclo[2.2.2]octan-5-yl, quinolizinyl and N-pyridylurea. Examples of the heterocyclic groups also include 1,1-dioxothiomorpholinyl and those in which two carbon atoms on the ring are replaced by oxygen atoms such as pyrimidinedione group. And the heterocyclic group can be substituted or unsubstituted, and the substituents can be, but are not limited to, deuterium, oxo(=O), hydroxy, amino, halogen, cyano, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclic group, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O)-, alkyl-C(=O)-, alkyl-S(=O)-, alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O)-, hydroxy-substituted alkyl-S(=O)2-, carboxy-substituted alkoxy, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0034] Figure 1 is a test chart of the reaction efficiency of different nucleophilic groups according to an embodiment of the present invention;

[0035] Figure 2 is a test chart of the reaction efficiency of different PEG lengths according to an embodiment of the present invention;

[0036] Figure 3 is an 8% polyacrylamide urea TBE gel electrophoresis chart according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0038] Example 1 Substrate molecule design and testing:

[0039] 1) Selection of nucleophilic groups:

[0040] By comparing the reactivity of nucleophilic groups -OH, -SH, -NH2, -NHCH3, -N(CH3)2, or pyridine, the reaction efficiency of -OH is the best (see Figure 1 ), so -OH is used as the nucleophilic group of the substrate molecule.

[0041] The compounds shown in formula I) were reacted with NAD-RNA at 37 °C for 1 hour under the catalysis of ADPRC enzyme, and then the reacted RNA was purified with ZymoColumn. The purified RNA was verified by 8% polyacrylamide urea TBE gel electrophoresis and the reaction efficiency was calculated quantitatively. As Figure 1 shown, the nucleophilic group -OH achieved the highest reaction efficiency of 76%.

[0042] 2) Selection of PEG groups:

[0043] The compounds shown in formula (I) with different PEG lengths (n = 1, 2, 3) were reacted with NAD-RNA at 37 °C for 1 hour under the catalysis of ADPRC enzyme, and the reacted RNA was purified with Zymo Column, and then verified by 8% polyacrylamide urea TBE gel electrophoresis. As Figure 2 shown, the product of PEG (n = 2) biotinylated the most RNA, indicating that PEG (n = 2) has the highest reactivity.

[0044] Example 2

[0045] The substrate molecule and NAD-RNA were reacted at 37 °C for 1 hour under the catalysis of ADPRC enzyme. The reacted RNA was purified with ZymoColumn and then incubated with streptavidin resin magnetic beads at 25 °C for 30 minutes. The RNA bound to the magnetic beads was extracted by Trizol and verified by 8% polyacrylamide urea gel electrophoresis (as Figure 2 shown) and the enrichment efficiency was calculated. Under the condition of 60 mM substrate molecule, starting from 1 μg of NAD-RNA, finally 0.16 μg of RNA could be enriched, and the enrichment rate was 16%.

[0046] Example 3

[0047] Incubate the NAD-RNA standard with the ADPRC enzyme at 37 °C for 1 hour. Purify the RNA after the reaction using a Zymo Column and incubate it with streptavidin resin magnetic beads at 25 °C for 30 minutes. Catalyze the reaction with NudC pyrophosphatase at 37 °C for 1 hour and collect the supernatant. As shown by 8% polyacrylamide urea TBE gel electrophoresis (such as Figure 3 shown), NudC pyrophosphatase releases NAD-RNA through a decapping reaction.

[0048] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0049] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for enriching NAD-cap modified RNA, characterized in that, Contact the compound shown in formula (I) with the RNA to be tested in the presence of adenosine diphosphate ribosyl cyclase so that the compound shown in formula (I) binds to the RNA containing NAD cap modification in the RNA to be tested, and through affinity treatment, to obtain the RNA with NAD cap modification. X-L-B (I), wherein X represents a nucleophilic group, L represents a linking group, B represents biotin or desthiobiotin, and X is linked to B through L; X is selected from -OH, -SH, -NH2, -NHCH3, -N(CH3)2 or pyridine; L is selected from n is selected from 1, 2 or 3.

2. The method according to claim 1, characterized in that, The compound shown in formula (I) is selected from the following structures:

3. The method according to claim 1, characterized in that, The compound shown in formula (I) is selected from the following structures:

4. A method for enriching RNA, characterized in that, Enrich the RNA with NAD cap modification according to the method described in any one of claims 1-3.

5. The method according to claim 4, characterized in that, Further include performing decapping treatment on the enriched RNA with NAD cap modification.

6. The method according to claim 5, characterized in that, The decapping treatment is catalyzed by NudC pyrophosphatase.

7. Use of the compound shown in formula (I) in enriching NAD-cap modified RNA, X-L-B (I), wherein, X represents a nucleophilic group, L represents a linking group, B represents biotin or desthiobiotin, and X is linked to B through L; X is selected from -OH, -SH, -NH2, -NHCH3, -N(CH3)2 or pyridine; L is selected from n is selected from 1, 2 or 3.

8. The use according to claim 7, characterized in that, The compound shown in formula (I) is selected from the following structures:

9. The use according to claim 7, characterized in that, The compound shown in formula (I) is selected from the following structures:

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