CN111088252B - Specific labeling method of 5-hydroxymethyl uracil on DNA - Google Patents
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Abstract
The invention discloses a specific labeling method of 5-hydroxymethyl uracil on DNA, belonging to the technical field of molecular biology. The specific labeling method utilizes 5-hmU kinase separated from pseudomonas aeruginosa bacteriophage M6 to realize the transfer of gamma-thiophosphate from 5-O-thiophosphoric acid adenosine triphosphate to 5-hmU and generate 5-thiophosphoric acid methyl uracil; then, the generated 5-thiophosphoric acid methyl uracil is characterized by a liquid chromatography-tandem mass spectrometry method, and the 5-hydroxymethyl uracil on the DNA is specifically marked by utilizing a cross-linking chemical labeling sulfhydryl. The method of the invention effectively overcomes the defects of large demand of the detection sample, harsh chemical reaction conditions, low reaction efficiency and the like.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a specific labeling method of 5-hydroxymethyl uracil on DNA.
Background
The genome contains chemically modified DNA bases in addition to the four classical bases. These modified bases can be produced by endogenous enzymes or exogenous factors, which have the potential to profoundly affect genomic function and cellular processes. A number of modified DNA bases have been discovered, the most notable of which in the mammalian genome are 5-methylcytosine (5-methylcytosine, 5mC) and its oxidized derivative 5-hydroxymethylcytosine (5-hydroxymethythiocine, 5-hmC), 5-fluorocytosine (5-forcylcytosine, 5-fC) and 5-carboxycytosine (5-carboxycytosine, 5-caC). These epigenetic markers have been shown to play an important role in regulating gene expression. Methylation modification of DNA has been a focus of research in the field of epigenetics since its discovery in 1948. Methylation modification of DNA confers epigenetic memory to DNA duplexes in addition to protein coding information, with profound effects on genomic stability, gene expression and development. The result that researchers reported for the first time that TET enzyme can oxidize thymine to 5-hmU in 2014 reveals that oxidation products (5-hmU, 5-fU, 5-caU) of thymine (T) may have certain apparent significance.
5-hmU is a thymine derivative identified in the genome of various organisms. In mammals, 5-hmU is formed by post-replication processing mechanisms including thymine hydroxylation by a 10-11 translocase (TET) or Reactive Oxygen Species (ROS) and 5-hmC deamination. However, the level of modified bases in genomic DNA is affected by many factors, such as the type of cell or tissue and the disease state of the organism. most of 5-hmU in mES cells is produced by TET enzyme oxidase, while the deamination or ROS pathway of 5-hmC is rare. Thus, the matched 5-hmU: a but not mismatched 5-hmU: g is the predominant form of 5-hmU present in the mammalian genome. Studies have been carried out to date which show a composition of 5-hmU with few thymine residues, e.g.5-hmU in mES cells representing about 0.00005% and an abundance of only 0.1% of 5-hmC. This makes analysis of hmU difficult and the structural similarity between 5-hmU and 5-hmC prevents differentiation between these modifications. Therefore, there is a need for an efficient method for labeling 5-hmU on DNA for further identification and joint detection and exploration of its biological functions.
The current method for detecting 5-hmC in DNA samples is to convert 5-hmU to 5-fU by chemical oxidation to detect genome 5-hmU. By forming 5-fU: g base pairing, two induced T to C base changes. However, the efficiency of this induced change was low, the rate of this base change was less than 40% even under optimized conditions, and the functional tag label 5-hmU could not be used.
In addition, there are other detection methods 5-hmU. Research shows that the aldehyde group modified uracil (5fU) may play a certain role in epigenetics, such as base mismatch introduction, DNA structure change and the like. The traditional 5fU-DNA chain is synthesized mainly through Pt catalytic oxidation, osmium oxidation, sodium periodate oxidation and the like, and because catalysts used by the traditional 5fU-DNA chain are extremely toxic and are purchased limitedly, the requirement on equipment is high, so that no significant breakthrough exists in 5fU-DNA synthesis for a long time. To address this issue, researchers have designed and synthesized a biotinylated probe, coupled to Biotin, that can be applied at a later stage to the Biotin-Avidin System (BAS) to achieve selective enrichment for 5 fU. However, this method also has high reactivity to other aldehyde group-bearing components present in genomic DNA.
Therefore, there is a need for an efficient method for labeling 5-hmU on DNA for further identification and joint detection and exploration of biological functions.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for specifically labeling 5-hydroxymethyl uracil on DNA, which solves the problems of large sample requirement, harsh chemical reaction conditions, low reaction efficiency and the like in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a specific labeling method of 5-hydroxymethyl uracil on DNA, which utilizes 5-hmU kinase separated from pseudomonas aeruginosa bacteriophage M6 to realize the transfer of gamma-thiophosphate from 5-O-thiophosphoric acid adenosine triphosphate to 5-hmU and generate 5-thiophosphoric acid methyl uracil; then, the generated 5-thiophosphoric acid methyl uracil is characterized by a liquid chromatography-tandem mass spectrometry method, and the 5-hydroxymethyl uracil on the DNA is specifically marked by utilizing a cross-linking chemical labeling sulfhydryl.
5-hmU used in the present invention is a kinase isolated from Pseudomonas aeruginosa bacteriophage M6 and purchased directly.
Preferably, the specific procedure for producing 5-thiophosphoryl methyl uracil is as follows:
1) preparing a dsDNA oligonucleotide containing a recognition sequence for restriction endonuclease NcoI as a substrate for 5-hmU kinase;
2) preparing a dsDNA model substrate comprising different base pair sites by in vitro DNA polymerase catalyzed extension of the primers;
3) mixing a mixture containing 5-hmU: incubating the dsDNA product of A with the G template for a strand displacement reaction;
4) phosphorothioate ATP- γ -S with 5-hmU kinase 5-hmU: residues 5-hmU in A to give 5-thiophosphorylmethyl uracil.
Further preferably, in step 1), the recognition sequence for the restriction endonuclease NcoI is 5'-CCAXGG-3', wherein X ═ T, U, hmU and fU.
Further preferably, in step 2), the different base pairs comprise:
5 hmU: a, 5 hmC: g, 5 fC: g, 5 fU: a, U: a or T: A.
further preferably, the step 4) is specifically operated as follows:
10U 5-hmU kinase and 1mM ATP-. gamma. -S were added to the sample, and the reaction was performed in a 1 XCutsmart buffer reaction system at 37 ℃ for 2 hours with shaking at 300rpm, and then the microgel was washed 3 times with 1XPBS to obtain 5-thiophosphoric acid methyluracil.
Preferably, the specific operation of labeling thiol groups with crosslinking chemistry is as follows:
the specific recognition and labeling of the different 5-hmU sites to distinguish 5-hmU in the same genomic DNA sample or single cell was performed by thiol-reacting with phosphorothioate groups by adding different thiol-reactive reagents (iodosyl-alkynyl, haloacetyl, maleimide, etc.).
Further preferably, the thiol reaction is carried out at 37 ℃ for 1h in the absence of light.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes 5-hmU DNA kinase (5-HMUDK) of pseudomonas aeruginosa bacteriophage M6 to realize the transfer of gamma-phosphorothioate from 5-O-adenosine triphosphate (ATP-gamma-S) to 5-hmU, and generate 5-thiophosphate methyl uracil (5 psmU). The resulting 5psmU was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique followed by specific labeling of 5-hmU on DNA by cross-linking chemically labeled thiol groups. The concrete advantages are as follows:
1) the marking method can carry out selective 5-hmU identification and marking, namely, errors caused by chemical modification of other bases can be avoided, and accurate identification and marking can be realized.
2) The ATP-gamma-S of the present invention, which is a substrate and inhibitor of the ATP-dependent enzyme system, is hydrolyzed by phosphatases and most ATPases, but the hydrolysis process is slow. And once the thiophosphatase is phosphorylated, the protein becomes resistant to the protein phosphatase. ATP-gamma-S is a double bond oxygen on the most distant phosphate group in ATP, which is changed into S atom, and gamma-phosphorothioate can be transferred to a target under the action of 5-HMUDK, and then the target on genomic DNA can be identified and labeled, so that the accuracy and sensitivity of analysis are improved.
3) The 5-thiophosphoryl methyl uracil (5psmU) is generated by transferring gamma thiophosphoric acid to 5-hmU under the action of HMUDK by ATP-gamma-S. The specific labeling of 5-hmU on the DNA was then achieved by cross-linking chemically labeled thiol groups. The reaction is a chemical reaction with high yield, simple reaction conditions, high reaction speed and easily obtained raw materials and reaction reagents.
4) The LC-MS/MS can only separate volatile and non-decomposed substances by gas chromatography, the separation range of liquid chromatography is greatly widened, LC is combined with high-selectivity and high-sensitivity MS/MS, a complex sample can be analyzed in real time, and even under the condition that LC is difficult to separate, only neutral fragment scanning is carried out on a target compound by MS1 and MS2, the target compound in a mixture can be found and highlighted, and the signal-to-noise ratio is obviously improved.
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FIG. 1 is a schematic diagram of the present invention;
FIG. 2 is a melting curve of DNA;
FIG. 3 is a nucleoside chromatogram;
FIG. 4 is a mass spectrum of labeled 5-hmU.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
referring to FIG. 1, a schematic diagram of the method for specifically labeling DNA 5-hmU of the present invention, which achieves the transfer of gamma-phosphorothioate from ATP-gamma-S to 5-hmU using 5-HMUDK, yielding 5 psmU; specific labeling of 5-hmU was then achieved using a thiol-reactive iodoacyl-alkynyl reagent to react with the thiol group of the phosphorothioate group. Specifically, the method comprises the following steps:
1) preparing dsDNA oligonucleotides containing recognition sequences for the restriction endonuclease NcoI (5'-CCAXGG-3', X ═ T, U, hmU and fU) as substrates for 5-HMUDK;
2) extension of the primer by in vitro DNA polymerase catalysis, a primer containing a different base pair was prepared (5 hmU: a, 5 hmC: g, 5 fC: g, 5 fU: a, U: a or T: A) dsDNA model substrates for the sites;
3) to form a gel containing 5 hmU: g dsDNA, will contain 5-hmU: incubating the dsDNA product of A with a G template for a strand displacement reaction;
4) phosphorothioate ATP- γ -S5-hmU using the 5-hmU DNA kinase of pseudomonas aeruginosa bacteriophage M6: residues 5-hmU in A, making 5 psmU;
5) adding a sulfhydryl-reactive iodoacyl-alkynyl reagent to react with sulfhydryl of a phosphorothioate group, then degrading the dsDNA sample into nucleosides through nuclease and phosphatase, and detecting the nucleosides of each sample obtained through LC-MS/MS to complete the discrimination of different bases.
Example 1: the specific labeling method of 5-hmU is used for realizing the differentiation of different bases.
dsDNA oligonucleotide sequences containing different bases (containing restriction endonuclease NcoI sites 5'-CCAXGG-3', X ═ T, U, hmU and fU) were prepared, and dsDNA model substrates containing different base pair (5 hmU: a, 5 hmC: G, 5 fC: G, 5 fU: a, U: a or T: a) sites were prepared by in vitro DNA polymerase catalyzed extension of the primers.
Wherein, to form a composition comprising 5 hmU: g dsDNA, will contain 5-hmU: the dsDNA product of A was incubated with the G template for strand displacement reactions. The products were characterized in a model DNA labeling reaction by 5-HMUDK and SH reagents. As shown in FIG. 2, which is a melting curve of a DNA sample cleaved by restriction endonuclease NcoI, it can be seen that 5psmU of the reaction product can block the cleavage of DNA by NcoI.
5-HMUDK phosphorothioates ATP- γ -S5-hmU: specifically, 5-HMUDK phosphorothioates ATP- γ -S5-hmU: residues 5-hmU in A; adding 10U 5-HMUDK and 1mM ATP-gamma-S into a sample, carrying out a shaking reaction for 2 hours at 37 ℃ and 300rpm in a 1 XCutsmart buffer reaction system, and then washing the microgel with 1XPBS for 3 times to obtain 5 psmU; after a DNA sample is cut by restriction endonuclease NcoI, the sample is subjected to chromatographic detection, the result is shown in figure 3, and as can be seen from figure 3, different retention times of liquid phase mass spectrometry prove that the method has good specificity and selectivity on base pairs.
Then adding a thiol-reactive iodoacyl-alkynyl reagent to react with the thiol group of the phosphorothioate group, adding 100 mu M of the iodoacyl-alkynyl reagent to the sample, and further reacting for 1 hour at 37 ℃ in the dark to complete the thiol reaction on the phosphorothioate group.
Degrading the dsDNA sample into nucleosides by nuclease and phosphatase after the reaction is finished, and degrading all purified dsDNA samples into nucleosides by nuclease and phosphatase before analysis so as to ensure the accuracy of LC-MS/MS detection; and detecting the obtained nucleoside of each sample by LC-MS/MS, and analyzing the result to complete the discrimination of different bases. The results are shown in FIG. 4, and it can be seen from FIG. 4 that the mass spectrum and data after labeling demonstrate that the labeling method successfully achieves the specific labeling of 5-hmU sites on dsDNA samples.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (7)
1. A method for specifically labeling 5-hydroxymethyl uracil on DNA is characterized in that 5-hmU kinase is used for realizing the transfer of gamma-phosphorothioate from 5-O-thiophosphoric acid adenosine triphosphate to 5-hmU to generate 5-thiophosphoric acid methyl uracil; then, the generated 5-thiophosphoric acid methyl uracil is characterized by a liquid chromatography-tandem mass spectrometry method, and the 5-hydroxymethyl uracil on the DNA is specifically marked by utilizing a cross-linking chemical labeling sulfhydryl.
2. The method for labeling 5-hydroxymethyluracil on DNA according to claim 1, wherein the specific procedures for producing 5-phosphorothioate methylglycine are as follows:
1) preparing a dsDNA oligonucleotide containing a recognition sequence for restriction endonuclease NcoI as a substrate for 5-hmU kinase;
2) preparing a dsDNA model substrate comprising different base pair sites by in vitro DNA polymerase catalyzed extension of the primers;
3) mixing a mixture containing 5-hmU: incubating the dsDNA product of A with the G template for a strand displacement reaction;
4) phosphorothioate ATP- γ -S with 5-hmU kinase 5-hmU: residues 5-hmU in A to give 5-thiophosphorylmethyl uracil.
3. The method for labeling 5-hydroxymethyluracil on DNA as set forth in claim 2, wherein the recognition sequence for restriction endonuclease NcoI in step 1) is 5'-CCAXGG-3', wherein X is T, U, hmU and fU.
4. The method for labeling 5-hydroxymethyluracil on DNA according to claim 2, wherein the different base pairs in step 2) include:
5 hmU: a, 5 hmC: g, 5 fC: g, 5 fU: a, U: a or T: A.
5. the method for labeling 5-hydroxymethyluracil on DNA according to claim 2, wherein the step 4) is specifically performed as follows:
10U 5-hmU kinase and 1mM ATP-. gamma. -S were added to the sample, and the reaction was performed in a 1 XCutsmart buffer reaction system at 37 ℃ for 2 hours with shaking at 300rpm, and then the microgel was washed 3 times with 1xPBS to obtain 5-thiophosphoric acid methyluracil.
6. The method for labeling 5-hydroxymethyluracil on DNA according to claim 1, wherein the thiol group is labeled with a crosslinking chemistry by the following procedure:
the specific recognition and labeling of the different 5-hmU sites by thiol reaction with phosphorothioate groups by the addition of different thiol-reactive reagents to distinguish 5-hmU in the same genomic DNA sample or single cell.
7. The method for labeling 5-hydroxymethyluracil on DNA according to claim 6, wherein the thiol group is reacted at 37 ℃ for 1 hour under a dark condition.
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