CN113355379A

CN113355379A - Economical and practical nucleic acid chain 5' -hydroxyl phosphorylation method

Info

Publication number: CN113355379A
Application number: CN202110154223.3A
Authority: CN
Inventors: 薛丽俊; 张洁; 杨珂新; 胡允金
Original assignee: Kanglong Huacheng Ningbo Technology Development Co ltd; Kanglong Beijing New Drug Technology Ltd By Share Ltd
Current assignee: Kanglong Huacheng Ningbo Technology Development Co ltd; Kanglong Beijing New Drug Technology Ltd By Share Ltd
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2021-09-07

Abstract

The invention discloses an economical method for 5' -hydroxyl phosphorylation on a nucleic acid chain, which comprises the following steps: the method takes a purchased non-modified DNA short fragment as a raw material, the 5 '-end of the non-modified DNA short fragment is hydroxyl, a commercially purchased 10-fold phosphorylation buffer solution component is taken as a phosphate source, and the 5' -hydroxyl of the DNA is phosphorylated under the action of T4 polynucleotide kinase. The method provided by the invention has the advantages of wide application range, mild reaction conditions, simple post-treatment, high recovery rate, low cost and wide application prospect.

Description

Economical and practical nucleic acid chain 5' -hydroxyl phosphorylation method

Technical Field

The invention belongs to the field of biochemistry, and particularly relates to an economical and practical nucleic acid chain 5' -hydroxyl phosphorylation method.

Background

The development of any drug is a lengthy and costly process. According to the research and development of drugsStatistics report that a new drug needs 6-10 years on average from the beginning of research and development to the final approval for market release, and the research cost is more than 10-30 hundred million dollars. The long and expensive development of drugs is due, among other things, to the slow discovery and optimization of lead compounds^[i]. To solve this problem, DNA-encoded library technology (DELT) has been developed. This concept was first followed by Sydney Brenner (Nobel prize winning for physiology and medicine 2002) and Richard Lerner (owned by the Times Scripps institute), both of the American Scripps research institute^[ii]Proposed in 1992. During the development of new drugs, scientists are constantly seeking diverse screening methods to find excellent active compounds in many compounds with binding affinity to biological targets and/or pharmacological potency in a non-differential screening manner. The positive impact of highly automated and deeply optimized multiple high throughput screening in the screening and discovery of active compounds is undisputed. In high-throughput screening, with the improvement of automated technology optimization and screening processes, the improvement of the quality of a chemical molecular library and the increase of the number of compounds, the mainstream drug research and development companies in the world often rely on the method to obtain the lead compound of the target protein. However, the high cost causes limitations in chemical structure and total number of compounds, and is increasingly unable to meet the requirements for new drug development. This traditional approach is futile in the screening practice for many disease proteins. In order to break through the bottleneck of high-throughput screening methods, enable the screened compounds to show geometric leaps in the chemical structure space and quantity, and use a brand-new biological screening mode, DELT just comes into play.

Compared with traditional high-throughput screening, the DNA coding compound library greatly enriches the number and diversity of compounds^[iii]. In reaction tubes of very small volume, e.g. tens of microliters, it is possible to synthesize tens of millions or even hundreds of millions of different compounds after a series of reactions^[iv]. The principle of DELT is to label each small molecule compound in the reaction with a specific DNA sequence, using combinatorial chemistryBy using a split and pool (split and pool) method, with limited cost and time, to synthesize millions to billions of compound libraries linked to specific DNA sequences in large quantities^[v]. The resulting mixture of compounds is then incubated with the protein target, physically separated by washing away compounds that do not bind to the protein target and finding compounds with high binding affinity^[vi]. Since the library of DNA-encoding compounds required for the incubation of the target protein requires only an extremely small dosage scale (micrograms) and can be carried out in a very short time, it is easy to carry out the incubation under different conditions^[vii]Multiple screening assays are performed (e.g., ph of solution, manner of sample protein mixing, protein concentration changes, presence or absence of competitor compounds, presence of different buffers or cofactors). Because the DNA sequence corresponds to the compound one by one, the chemical structural formula of the active compound can be obtained only by reading the DNA sequence of the small molecular compound through PCR amplification and NGS sequencing. The compounds with high affinity are then synthesized separately "off DNA", i.e. without labeling the compounds with DNA sequences, and the binding of the compounds without attached DNA to the target protein is determined to confirm their true biological activity.

The discovery of the lead compound is an important step of new drug development, and the lead compound with a high starting point is one of the key factors for the success of new drug development. Libraries of DNA-encoding compounds are one of the attractive approaches to screening of many lead compounds.^[V]It is unique in terms of number of compounds, structural diversity, and unique binding pattern to biological target proteins. The DNA-encoding compound library technology is a sophisticated complex technology system engineering. Its development needs to span multiple disciplines and areas. The method comprises the following steps: (1) the support of biochemistry is in particular the theory and practice of nucleotide biochemistry. The library of DNA-encoding compounds is a library of small chemical molecules built on DNA. It requires DNA-dependent biosynthetic methods to construct the DNA code. The knowledge and understanding of the DNA biospecificity is one of the keys to the successful application of this technology. (2) Pharmaceutical chemistry, DNA codingThe compound library is a platform established for drug development. The molecular design concept and methodology of medicinal chemistry directly guides the establishment of a DNA coding compound library, and is the key for successfully screening lead compounds. (3) Organic chemistry is the connotation of a library of DNA-encoding compounds. The construction of chemical libraries of small molecule drugs by organic chemistry requires special synthetic chemistry methods for the construction of libraries of DNA-encoding compounds, and the exploration of suitable reaction conditions for organic chemistry and adaptation to DNA is another key point in this technology. (4) Bioinformatics not only provides theoretical support for the design of DNA codes, but also performs result analysis on DNA sequencing, and is an integral important component of DEL technology. (5) Analytical chemistry, DNA is a specific class of macromolecules. Analytical chemistry is required to provide for the analysis and separation of biological macromolecules. Mass spectrum deconvolution functionality is another requirement of this analysis method. The synthesis of a chemical library of DNA can only be performed on the basis of powerful analytical chemistry. (6) The separation, identification and screening of target protein. The field needs complete pathological analysis, and virus protein separation, extraction and identification. The resulting protein is then incubated with a library of DNA-encoding compounds and the substrate is screened for binding. (7) The substrate is amplified by Polymerase Catalyzed Reaction (PCR). Since the DNA obtained is extremely small in number, it must rely on polymerase-catalyzed reactions to amplify the substrate from the screen. (8) And (4) determining the DNA sequence. DNA sequence analysis and determination using NGS (next generation sequencing technology).

The construction of libraries of DNA-encoding compounds requires the labeling of small organic compounds that react in a sequence with a large number of DNA oligonucleotide tags. For the acquisition of large quantities of DNA oligonucleotide tags, the simplest approach is to purchase them on demand by gene companies. The market price of 5' -hydroxyl phosphorylation modified DNA oligonucleotide is high at present, and the label of unphosphorylated DNA oligonucleotide is relatively cheap. It is economically advantageous to select DNA oligonucleotides that are not phosphorylated for self-phosphorylation.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a 5' -hydroxyl phosphorylation method for a nucleic acid chain, which has the advantages of wide application, simple operation, high recovery rate and low cost.

In a first aspect, the present invention relates to a method of phosphorylating the 5' -hydroxyl group of a nucleic acid strand, characterized in that ATP and/or PEG is added to a reaction mixture, wherein the reaction mixture comprises: nucleic acid chain, T4 polynucleotide kinase and phosphorylation buffer.

In one embodiment, the nucleic acid strand is an oligonucleotide strand, wherein the strand length is not limited. In a preferred embodiment, the nucleic acid strand is a DNA oligonucleotide strand. In a more preferred embodiment, the nucleic acid strand is an unphosphorylated DNA oligonucleotide strand.

In one embodiment, the nucleic acid strand is a DNA oligonucleotide strand. In a preferred embodiment, the DNA oligonucleotide strand is single stranded. In a more preferred embodiment, the 5' -end of the DNA oligonucleotide strand is a hydroxyl group. In a more preferred embodiment, the DNA oligonucleotide strand is prepared by polymerization of normal nucleotide monomers, and/or by polymerization of artificially modified nucleotide monomers.

In one embodiment, the phosphorylation reaction is performed in aqueous enucleated or aqueous enucleated enzyme solution.

In one embodiment, the amount of PEG is 0-0.04 microliters of PEG per nanomole of nucleic acid strand. In a preferred embodiment, the amount of PEG is from 0.01 to 0.04 microliters of PEG per nanomole of nucleic acid strand. In a more preferred embodiment, the amount of PEG is 0.025 microliters of PEG per nanomole of nucleic acid strand.

In one embodiment, the PEG is selected from PEG 100 to PEG 8000. In a preferred embodiment, the PEG is PEG 4000.

In one embodiment, the amount of ATP is 0-5 nanomole ATP per nanomole of nucleic acid strand. In a preferred embodiment, the amount of ATP is such that 1-5 nanomoles of ATP are added per nanomole of nucleic acid strand. In a more preferred embodiment, the amount of ATP is such that 2.5 nanomoles of ATP are added per nanomole of nucleic acid strand.

In one embodiment, the amount of T4 polynucleotide kinase is 0-0.50 units of T4 polynucleotide kinase per nanomole of nucleic acid strand. In a preferred embodiment, the amount of T4 polynucleotide kinase is 0.25 units of T4 polynucleotide kinase per nanomole of nucleic acid strand.

In a preferred embodiment, the T4 polynucleotide kinase agent is present at a concentration of 10 units per microliter and is stored under conditions such that: 50 mmol/L potassium chloride, 1 mmol/L dithiothreitol, 0.1. mu. mol/L ATP, 0.1 mmol/L EDTA, 10 mmol/L Tris hydrochloride (pH 7.4), 50% glycerol. In a preferred embodiment, the T4 polynucleotide kinase is NEB product (# M0201S).

In one embodiment, the reaction system is 25 milliliters per 10 nanomolar nucleic acid strand.

In one embodiment, the water of an enucleated enzyme is nuclease-free water, wherein the nuclease is an rnase or dnase. Alternatively, in one embodiment, the aqueous enucleation enzyme solution is a mixed solvent containing water and no nuclease, which contains any one or more of acetonitrile, ethanol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and an inorganic salt buffer. In a preferred embodiment, the total water content of the aqueous solution of the enucleated enzyme is not less than 90% and is nuclease-free. In a preferred embodiment, the enucleated enzyme water is the NEB product (# R0581).

In one embodiment, the phosphorylation buffer component is a 10-fold phosphorylation buffer component comprising 500 mmol/l tris hydrochloride, 100 mmol/l magnesium chloride, 100 mmol/l dithiothreitol, 10 mmol/l ATP. In a preferred embodiment, the phosphorylation buffer has a pH of 7.5 at 25 ℃. In a more preferred embodiment, the 10-fold phosphorylation buffer is NEB product (# B0202S).

In one embodiment, the amount of the phosphorylation buffer is 0-80. mu.l per nanomole of nucleic acid strand. In a preferred embodiment, the amount of the phosphorylation buffer is 1. mu.l per nanomole of nucleic acid strand.

In one embodiment, the reaction temperature is from 0 ℃ to 50 ℃. In a preferred embodiment, the reaction temperature is 37 ℃.

In one embodiment, the reaction time is from 0 hours to 24 hours. In a preferred embodiment, the reaction time is 0.5 hours.

In a second aspect, the invention relates to a kit for phosphorylating a 5' -hydroxyl group of a nucleic acid strand comprising a T4 polynucleotide kinase, a phosphorylation buffer, ATP and/or PEG.

In one embodiment, the phosphorylation buffer comprises 500 mmol/l tris hydrochloride, 100 mmol/l magnesium chloride, 100 mmol/l dithiothreitol, and 10 mmol/l ATP. In a preferred embodiment, the phosphorylation buffer has a pH of 7.5 at 25 ℃.

In one embodiment, the PEG is PEG 4000.

In one embodiment, the kit further comprises instructions for use, wherein the kit components, the amounts and ratios of the reactants of the phosphorylation reaction, and the reaction conditions are as described above.

In a third aspect, the present invention relates to a composition for phosphorylating a 5' -hydroxyl group of a nucleic acid strand, comprising T4 polynucleotide kinase, a phosphorylation buffer, ATP, and/or PEG.

In one embodiment, the composition comprises 0.01 to 0.04 microliters of PEG per nanomolar nucleic acid strand and/or 1 to 5 nanomolar ATP per nanomolar nucleic acid strand, 0.05 to 0.25 units of T4 polynucleotide kinase per nanomolar nucleic acid strand, 0.1 to 80 microliters of phosphorylation buffer per nanomolar nucleic acid strand. In a preferred embodiment, the composition comprises 0.025 microliters PEG per nanomolar nucleic acid strand and/or 2.5 nanomolar ATP per nanomolar nucleic acid strand, 0.25 units T4 polynucleotide kinase per nanomolar nucleic acid strand, 1 microliter phosphorylation buffer per nanomolar nucleic acid strand.

In one embodiment, the phosphorylation buffer comprises 500 mmol/l tris hydrochloride, 100 mmol/l magnesium chloride, 100 mmol/l dithiothreitol, and 10 mmol/l ATP.

In a fourth aspect, the present invention relates to a method for phosphorylating a 5' -hydroxyl group of a nucleic acid strand, comprising mixing and reacting a composition as described in the third aspect with a nucleic acid strand. In one embodiment, the reaction conditions are as described above.

TABLE-1 DNA Single-stranded 5' -Hydroxyphosphorylation Condition experiments and optimization and results

Standard procedure for zemer feishel scientific phosphorylation: the reaction was completed by 10-50 pmol of non-phosphorylated DNA oligonucleotide, 2. mu.l of 10-fold phosphorylation reaction buffer (500 mmol/l Tris-HCl and pH 7.6 at 25 ℃ C., 100 mmol/l magnesium chloride, 50 mmol/l dithiothreitol, 1 mmol/l spermidine), 2. mu.l of 10 mmol/l ATP, 1. mu.l (10 units) of T4 polynucleotide kinase, and 20. mu.l of enucleated enzyme water (# R0581) added thereto, and reaction at 37 ℃ C. for 20 minutes. New England Biolabs (New England Biolabs, NEB for short) phosphorylation Standard protocol: the reaction was completed by adding up to 300. mu.l of non-phosphorylated DNA oligonucleotide, 5. mu.l of 10-fold phosphorylation buffer (700. mu.l/l of Tris hydrochloride, 100. mu.l/l of magnesium chloride, 50. mu.l/l of dithiothreitol, pH 7.6 as the pH value of the 10-fold phosphorylation buffer component at 25 ℃ C.), 5. mu.l of 10. mu.l/l of ATP, 1. mu.l (10 units) of T4 polynucleotide kinase, adding up to 50. mu.l of enucleate enzyme water, and reacting at 37 ℃ C. for 30 minutes.

The amounts of reagents used in the following experiments of this patent were compared to the international standard amounts of NEB T4 polynucleotide kinase catalyzed reactions. As shown in entry 3 of Table-1, in the small-scale reaction of 5' -hydroxy phosphorylation of single-stranded DNA, we reduced the amount of 10-fold phosphorylation buffer component to 6% of the amount used in NEB standard, and T4 polynucleotide kinase to only 0.75% of the amount used in NEB standard, and the total system volume was reduced to 1.5% of the amount used in NEB standard, and the reaction was complete in 12 hours. Thus, we obtained the phosphorylation effect of the literature method with only 0.75% of T4 polynucleotide kinase. When PEG or ATP is added to the reaction solution, the reaction time can be shortened with a decrease in the amount of T4 polynucleotide kinase. The simultaneous addition of PEG and ATP can further shorten the phosphorylation reaction time. As shown in entries 3-6 of Table-1, the results of comparative experiments on the effect of PEG addition of 0. mu.l, 0.2. mu.l, 0.5. mu.l, 0.8. mu.l on phosphorylation reaction time for 20 nmol of substrate show that the addition of 0.5. mu.l provides the best phosphorylation reaction effect. As shown in entries 7-9 of Table-1, the addition of ATP reduced the phosphorylation reaction time for 20 nanomolar substrate. The results of comparative experiments on the effect of ATP addition at 30, 50 and 70 nanomoles on phosphorylation reaction time show that 50 nanomole addition works best. The phosphorylation reaction was completed after 5 hours and the product yield was high. As shown in entry 10 of Table-1, for the phosphorylation reaction of 20 nmol of substrate, if 0.5. mu.l of PEG and 50 nmol of ATP are added simultaneously, the phosphorylation reaction time can be shortened to 0.5 hours, and the yield of DNA product is high. For 20 nmol of substrate, the reaction was completed in 2.5 hours when the amount of T4 polynucleotide kinase used was 25 units while keeping the concentration of the 10-fold concentration of the phosphorylation buffer, PEG and ATP in 8000. mu.l of the reaction system, as shown in entry 11 of Table-1. However, the DNA recovery rate was low because of the low concentration of the system. This indicates that the method of the present invention using phosphorylase and its small amount is also applicable to conventional phosphorylation reaction.

Further scale-up of the phosphorylation reaction to 2000 nanomolar using the method of entry 10 of Table-1, for example, as shown in entry 12 of Table-1, the reaction can be completed in 2 hours. Based on the reaction conditions of entry 12 in Table-1, the T4 polynucleotide kinase was further reduced to 0.3% of the amount used in the internationally used NEB standard, and the reaction could be completed within 10 hours, as shown in entry 13 in Table-1. The cost of 66667 units of T4 polynucleotide kinase are expensive (5.1 ten thousand) for 2000 nanomolar phosphorylation of a single strand of DNA according to the NEB phosphorylation standard protocol, whereas the cost of the T4 polynucleotide kinase in the present invention is 150 RMB. Therefore, it is sufficient to explain the superiority of the present invention.

The phosphorylation reaction was further scaled up to 10000 nanomolar using the method of entry 13 of table-1, and the reaction could be completed within 16 hours as shown in entry 14 of table-1. Based on the reaction conditions of entry 14 in Table-1, the T4 polynucleotide kinase was further reduced to 0.15% of the amount used in the internationally used NEB standard, and the reaction could be completed within 24 hours, as shown in entry 15 in Table-1. The cost of the T4 polynucleotide kinase of the present invention is 375 yuan. 10000 nanomolar DNA single strand phosphorylation requires 333333 units of T4 polynucleotide kinase, which is expensive (25.67 ten thousand) according to the NEB phosphorylation standard.

The T4 polynucleotide kinase is the most material-costly reagent used in the experiment. According to the invention, through a series of condition experiments, the use amount of expensive T4 polynucleotide kinase is reduced to 0.15% of the standard use amount of the international new England biological laboratory T4 polynucleotide kinase; 0.25% of the standard amount of the Saimer Feishell technology T4 polynucleotide kinase. The phosphorylation method can greatly reduce the material cost.

The total substrate concentration in the present invention was 67-fold higher than the internationally common NEB conventional phosphorylation concentration, and 160-fold higher than the concentration used in the sermer feishel scientific phosphorylation standard protocol. At high substrate concentrations, phosphorylation was performed using a very small amount of enucleated enzyme water (1/100 in the amount of nuclease used for routine phosphorylation of NEB) and 10-fold amount of phosphorylation buffer component (6/100 in the amount of 10-fold amount of phosphorylation buffer component used for routine NEB). Therefore, a high product yield can be obtained in the process of DNA single strand precipitation. The method of the invention is carried out at high concentration, is convenient for the precipitation of DNA, and has higher recovery rate than other methods. Of course, it is also applicable to DNA substrates at normal concentrations. In addition, because of the high concentration of the DNA substrate in the present invention, a relatively small container can be used for the precipitation operation, which facilitates the standing at a low temperature. The operation is more convenient and faster. It is expected that the method of the invention will have excellent economic benefit and wide application prospect in industrial large-scale application!

Drawings

FIG. 1 shows a DNA single strand 1: the length of the DNA is 12 bases, the base sequence is CGCCGAAATAGG, the relative molecular mass before phosphorylation is 3679, and the result is shown in FIG. 1 after liquid chromatography mass spectrometry detection.

FIG. 2 shows the DNA single strand 2 of the DNA fragment before phosphorylation: the length of 19 bases, the base sequence of ACTGGTAGCGTCGACGTCC, the relative molecular mass before phosphorylation of 5805, through liquid chromatography mass spectrometry, the results are shown in figure 2.

FIG. 3 shows the DNA fragment DNA single strand 1: length of 12 bases, base sequence of CGCCGAAATAGG, relative molecular mass of 3759 after phosphorylation, DNA single strand 2: the length is 19 bases, the base sequence is ACTGGTAGCGTCGACGTCC, the relative molecular mass after phosphorylation is 5885, and the result is shown in figure 3 after liquid chromatography mass spectrometry detection.

FIG. 4 shows the DNA fragment DNA single strand before phosphorylation 3: the length of 17 bases, the base sequence of CAGTGATTCGGTCGGAG, the relative molecular mass before phosphorylation of 5266, through liquid chromatography mass spectrometry detection, the results are shown in figure 4.

FIG. 5 shows the DNA fragment DNA single strand after phosphorylation 3: the length is 17 bases, the base sequence is CAGTGATGAGTCTCGGG, the relative molecular mass after phosphorylation is 5346, and the result is shown in FIG. 5 after liquid chromatography mass spectrometry detection.

FIG. 6 shows the DNA fragment DNA single strand before phosphorylation 4: the length of 10 bases, the base sequence of ATGCGAGCGT, the relative molecular mass before phosphorylation of 3069, and the results of liquid chromatography mass spectrometry are shown in FIG. 6.

Detailed Description

The technical solution of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely a subset of the embodiments of the invention and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. All DNA starting materials in the case of the practice of the present invention are substrates which are not phosphorylated on the 5' -hydroxyl group.

Example 1, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component and 5 units of T4 polynucleotide kinase were added, and enucleating enzyme water was added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 12 hours. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 17.5 nanomole with a recovery of 88%.

Example 2, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.2. mu.l of PEG were added, and enucleating enzyme water was added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 2.5 hours. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 17.4 nanomole with a recovery of 87%.

Example 3, 20 nmol of the DNA single strand 3 before phosphorylation, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, 5266 in relative molecular mass before phosphorylation, dissolved in enucleation enzyme water, added with 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.5. mu.l of PEG, and added with enucleation enzyme water until the reaction system was 50. mu.l, reacted at 37 ℃ for 2 hours completely. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 17.2 nanomole with a recovery of 86%.

Example 4, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleated enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.8. mu.l of PEG were added, and enucleated enzyme water was added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 3 hours. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 16.8 nanomole with a recovery of 84%.

Example 5, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 30 nmol of ATP were added, and enucleating enzyme water was added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 8 hours. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 16.6 nanomole with a recovery of 83%.

Example 6, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 50 nmol of ATP were added, and enucleating enzyme water was added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 5 hours. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine that the phosphorylated product amount was 17 nanomole and the recovery rate was 85%.

Example 7, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 70 nmol of ATP were added, and enucleating enzyme water was added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 6 hours. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 16.8 nanomole with a recovery of 84%.

Example 8, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.5. mu.l of PEG, 50 nmol of ATP, and additional enucleating enzyme water were added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 0.5 hour. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 17.4 nanomole with a recovery of 87%.

Example 9, 20 nmol of the DNA single strand 1 before phosphorylation, 12 bases in length, CGCCGAAATAGG in base sequence, 3679 in relative molecular mass before phosphorylation, was dissolved in enucleation enzyme water, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.5. mu.l of PEG, 50 nmol of ATP, and enucleation enzyme water were added to the reaction system to 50. mu.l, and the reaction was completed at 37 ℃ for 0.5 hour. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry, the product amount was found to be phosphorylated 17.4 nanomole with a recovery of 87%.

Example 10, 20 nmol of the pre-phosphorylation DNA single strand 2, 19 bases in length, ACTGGTAGCGTCGACGTCC in base sequence, relative molecular mass before phosphorylation 5805, was dissolved in water containing an enucleating enzyme, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.5. mu.l of PEG, 50 nmol of ATP, and additional enucleating enzyme water were added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 0.5 hour. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 to 25 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 17.4 nanomole with a recovery of 87%.

Example 11, 20 nmol of the Pre-phosphorylation DNA Single strand 4, 10 bases in length, ATGGCGTCGA in base sequence, 3069 in relative molecular mass before phosphorylation, was dissolved in enucleation enzyme water, 20. mu.l of 10-fold phosphorylation buffer component, 5 units of T4 polynucleotide kinase, 0.5. mu.l of PEG, 50 nmol of ATP, and enucleation enzyme water were added until the reaction system was 50. mu.l, and the reaction was completed at 37 ℃ for 0.5 hour. 5 microliter of 5 mol/L sodium chloride aqueous solution and 125 microliter of absolute ethyl alcohol are added into the reaction solution, uniformly mixed by shaking, placed at minus 80 ℃ for freezing for 10 to 30 minutes in a refrigerator, and subjected to high-speed freezing and centrifugation (4 ℃, 12000 rpm, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product as 17.4 nanomole with a recovery of 87%.

Example 12, 20 nmol of the pre-phosphorylation DNA single strand 3, 17 bases in length, CAGTGATGAGTCGGTCG in base sequence, and 5266 in relative molecular mass before phosphorylation were dissolved in water containing enucleating enzyme, 1600. mu.l of 10-fold phosphorylation buffer component, 25 units of T4 polynucleotide kinase, 40. mu.l of PEG, 4000 nmol of ATP were added, and enucleating enzyme water was added until the reaction system was 8000. mu.l, and the reaction was completed at 37 ℃ for 2.5 hours. Adding 800 microliters of 5 mol/liter sodium chloride aqueous solution and 20 nanoliters of absolute ethyl alcohol into the reaction solution, shaking and uniformly mixing, standing at-80 ℃ for freezing for 10-30 minutes in a refrigerator, and performing high-speed freezing and centrifugation (4 ℃, 4000 revolutions/minute and 5 minutes) to obtain a precipitate. The precipitate was dissolved in 20 μ l of water and quantitatively determined by DNA ultraviolet spectrophotometry to determine the amount of phosphorylated product to be 14.2 nanomole with a recovery of 71%.

Example 13, 1000 nanomoles of Pre-phosphorylated DNA Single Strand 1 and 1000 nanomoles of Pre-phosphorylated DNA Single Strand 2, having base sequences of CGCCGAAATAGG and ACTGGTAGCGTCGACGTCC, were dissolved in enucleated enzyme water, 2000. mu.l of 10-fold phosphorylation buffer composition, 500 units of T4 polynucleotide kinase, 50. mu.l of PEG, 5000 nanomoles of ATP were added, and enucleated enzyme water was added until the reaction system was 5000. mu.l, and the reaction was completed at 37 ℃ for 2 hours. Adding 500 microliters of 5 mol/liter sodium chloride aqueous solution and 12500 microliters of anhydrous ethanol into the reaction solution, shaking and uniformly mixing, standing at-80 ℃ for freezing for 10-60 minutes in a refrigerator, and performing high-speed freezing and centrifugation (4 ℃, 4000 rpm/minute, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 2000 microliters of water and subjected to quantitative detection by DNA ultraviolet spectrophotometry to determine that the amounts of the two phosphorylated products were 1800 nanomoles and the recovery rate was 90%.

Example 14, 1000 nanomoles of Pre-phosphorylated DNA Single Strand 1 and 1000 nanomoles of Pre-phosphorylated DNA Single Strand 2, having base sequences of CGCCGAAATAGG and ACTGGTAGCGTCGACGTCC, were dissolved in enucleated enzyme water, 2000. mu.l of 10-fold phosphorylated buffer components, 200 units of T4 polynucleotide kinase, 50. mu.l of PEG, 5000 nanomoles of ATP were added, and enucleated enzyme water was added until the reaction system was 5000. mu.l, and the reaction was completed at 37 ℃ for 10 hours. Adding 500 microliters of 5 mol/liter sodium chloride aqueous solution and 12500 microliters of anhydrous ethanol into the reaction solution, shaking and uniformly mixing, standing at-80 ℃ for freezing for 10-60 minutes in a refrigerator, and performing high-speed freezing and centrifugation (4 ℃, 4000 rpm/minute, 5 minutes) to obtain a precipitate. The precipitate was dissolved in 2000 microliters of water and subjected to quantitative detection by DNA ultraviolet spectrophotometry to determine that the amounts of the two phosphorylated products were 1800 nanomoles and the recovery rate was 90%.

Example 15, 5000. mu.l of Pre-phosphorylation DNA Single Strand 1 and 5000. mu.l of Pre-phosphorylation DNA Single Strand 2, which have base sequences of CGCCGAAATAGG and ACTGGTAGCGTCGACGTCC, were dissolved in enucleated enzyme-containing water, 10000. mu.l of 10-fold phosphorylation buffer component, 1000 units of T4 polynucleotide kinase, 250. mu.l of PEG, 25000. mu.l of ATP were added, and enucleated enzyme-containing water was added thereto until the reaction system became 25000. mu.l, and the reaction was completed at 37 ℃ for 16 hours. Adding 2500 μ l of 5 mol/l sodium chloride aqueous solution and 62500 μ l of anhydrous ethanol into the reaction solution, shaking, mixing, standing at-80 deg.C, freezing for 10-100 min, and high-speed freezing and centrifuging (4 deg.C, 4000 rpm/min, 5 min) to obtain precipitate. The precipitate was dissolved in 10000. mu.l of water and quantified by DNA UV spectrophotometry to determine the amount of the two phosphorylated products to be 9200 nanomoles and the recovery rate to be 92%.

Example 16, 5000. mu.l of Pre-phosphorylation DNA Single strand 1 and 5000. mu.l of Pre-phosphorylation DNA Single strand 2, which had base sequences of CGCCGAAATAGG and ACTGGTAGCGTCGACGTCC, were dissolved in enucleated enzyme water, 10000. mu.l of 10-fold phosphorylation buffer component, 500 units of T4 polynucleotide kinase, 250. mu.l of PEG, 25000. mu.l of ATP were added, and enucleated enzyme water was added until the reaction system was 25000. mu.l, and the reaction was completed at 37 ℃ for 24 hours. Adding 2500 μ l of 5 mol/l sodium chloride aqueous solution and 62500 μ l of anhydrous ethanol into the reaction solution, shaking, mixing, standing at-80 deg.C, freezing for 10-100 min, and high-speed freezing and centrifuging (4 deg.C, 4000 rpm/min, 5 min) to obtain precipitate. The precipitate was dissolved in 10000. mu.l of water and quantified by DNA ultraviolet spectrophotometry to determine the amount of the two phosphorylated products to be 9500 nanomoles and the recovery rate to be 95%.

In summary, the above embodiments and drawings are merely illustrative of the broad applicability of the present invention, and can be applied to DNA fragments with different lengths, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

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Claims

1. A method of phosphorylating a 5' -hydroxyl group of a nucleic acid strand, characterized by adding ATP and/or PEG to a reaction mixture, wherein said reaction mixture comprises: nucleic acid chain, T4 polynucleotide kinase, phosphorylation buffer solution;

preferably, the nucleic acid strand is an oligonucleotide strand; more preferably, the nucleic acid strand is a DNA oligonucleotide strand; more preferably, the nucleic acid strand is an unphosphorylated DNA oligonucleotide strand;

preferably, the reaction is carried out in aqueous enucleated or aqueous enucleated enzyme solution.

2. The method according to claim 1, wherein the amount of PEG is 0-0.04 microliters PEG, preferably 0.01-0.04 microliters PEG, preferably 0.025 microliters PEG, per nanomole of nucleic acid strand;

preferably, the PEG is selected from PEG 100 to PEG 8000; more preferably, the PEG is PEG 4000.

3. The method according to claim 1 or 2, wherein the amount of ATP is 0-5 nanomole ATP, preferably 1-5 nanomole ATP, preferably 2.5 nanomole ATP per nanomole of nucleic acid strand.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid strand is a DNA oligonucleotide strand; more preferably, the DNA oligonucleotide strand is single-stranded; more preferably, the 5' -end of the DNA oligonucleotide chain is a hydroxyl group; more preferably, the DNA oligonucleotide chain is prepared by polymerizing normal nucleotide monomers, and/or is prepared by polymerizing artificially modified nucleotide monomers.

5. The method according to any one of claims 1 to 4, wherein the amount of T4 polynucleotide kinase is 0-0.50 units of T4 polynucleotide kinase, preferably 0.25 units of T4 polynucleotide kinase, per nanomole of nucleic acid strand.

6. The method according to any one of claims 1 to 5, wherein the reaction system is 25 ml per 10 nmol of nucleic acid strands.

7. The method according to any one of the preceding claims, wherein the enucleated enzyme water is nuclease-free water; or

The denuclease enzyme aqueous solution is a mixed solvent which contains water and does not contain nuclease and contains any one or more of acetonitrile, ethanol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide and inorganic salt buffer solution.

8. The method according to any one of the preceding claims, wherein the phosphorylation buffer component is a 10-fold phosphorylation buffer component comprising 500 mmol/l tris hydrochloride, 100 mmol/l magnesium chloride, 100 mmol/l dithiothreitol, 10 mmol/l ATP;

preferably, the amount of the phosphorylation buffer is 0-80. mu.l, preferably 1. mu.l, per nanomole of nucleic acid strand.

9. The process according to any of the preceding claims, wherein the reaction temperature is from 0 ℃ to 50 ℃, preferably 37 ℃;

preferably, the reaction time is from 0 hour to 24 hours; more preferably, the reaction time is 0.5 hour.

10. A kit for phosphorylating a 5' -hydroxyl group of a nucleic acid strand comprising T4 polynucleotide kinase, a phosphorylation buffer, ATP and/or PEG;

preferably, the phosphorylation buffer comprises 500 mmol/l tris hydrochloride, 100 mmol/l magnesium chloride, 100 mmol/l dithiothreitol, 10 mmol/l ATP, pH 7.5 at 25 ℃;

preferably, the PEG is PEG 4000.

11. A composition for phosphorylating a 5' -hydroxyl group of a nucleic acid strand comprising T4 polynucleotide kinase, a phosphorylation buffer, ATP and/or PEG;

preferably, the composition comprises 0.01-0.04 microliters of PEG per nanomolar nucleic acid strand and/or 1-5 nanomolar ATP per nanomolar nucleic acid strand, 0.05-0.25 units of T4 polynucleotide kinase per nanomolar nucleic acid strand, 0.1-80 microliters of phosphorylation buffer per nanomolar nucleic acid strand;

preferably, the composition comprises 0.025 microliters PEG per nanomolar nucleic acid strand and/or 2.5 nanomolar ATP per nanomolar nucleic acid strand, 0.25 units T4 polynucleotide kinase per nanomolar nucleic acid strand, 1 microliter of phosphorylation buffer per nanomolar nucleic acid strand;

preferably, the phosphorylation buffer comprises 500 mmol/l tris hydrochloride, 100 mmol/l magnesium chloride, 100 mmol/l dithiothreitol, 10 mmol/l ATP.

12. A method for phosphorylating a 5' -hydroxyl group of a nucleic acid strand comprising mixing and reacting a composition according to claim 11 with a nucleic acid strand.