Enzymatic synthesis method of polydeoxyribonucleotide

Abstract

The invention discloses an enzymatic synthesis method of poly (deoxyribonucleotide). The invention provides a synthetic method of polydeoxyribonucleotide. The synthetic method comprises the following steps: 1) synthesizing a single-stranded DNA molecule at the 3'end of a base primer by using TdT enzyme; the base primer is a single-stranded DNA (deoxyribonucleic acid) molecule which is composed of random basic groups and has the length of 5-100nt; 2) amplifying the single-stranded DNA molecule to obtain an amplification product; and 3) breaking the amplification product to obtain the polydeoxyribonucleotide. When the TdT enzymatic ssDNA is synthesized, the length of the synthesized ssDNA can be controlled by accurately adjusting the reaction time. The generated ssDNA is used as a minimum structural unit, and can be broken into PDRN with a specific length after being subjected to single chain cyclization and rolling circle amplification.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and relates to an enzymatic synthesis method of polydeoxyribonucleotides. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is usually extracted from the testicular tissue of fish (such as salmon, trout, or salmon). PDRN is a high molecular weight DNA polymer composed of multiple linearly arranged deoxyribonucleotide units, with a molecular weight of about 50-1500KDa. PDRN has shown significant ability to promote tissue repair and regeneration in the biomedical field. In vitro experiments have shown that PDRN can regulate cell signaling pathways through the adenosine A2A receptor on the cell membrane surface, and play anti-inflammatory, promote cell proliferation and apoptosis, promote angiogenesis and tissue repair. In addition, PDRN can also provide nucleotide raw materials for intracellular DNA synthesis through the salvage pathway, thereby exerting its function. In clinical applications, PDRN has been widely used to treat various skin injuries, ulcers and chronic wounds, showing good efficacy and safety. In addition, PDRN is also used in cosmetic medicine, such as injection to improve skin quality, reduce wrinkles and enhance skin elasticity. With the in-depth study of the mechanism of action of PDRN, its application prospects in tissue engineering, regenerative medicine and other medical fields will be broader.

[0003] Conventional PDRN needs to be extracted from the testicular tissue of fish, and its preparation includes processes such as testicular crushing, DNA extraction, DNA shearing, centrifugation, freeze-drying, quality inspection and packaging. This method has low extraction yield, unstable product quality, high production cost, potential biosafety risks, and difficult to control product molecular weight. PDRN preparation technology that relies on fish extraction may increase fishing pressure on specific fish resources, thereby triggering a series of ecological and ethical issues. With the growth of PDRN market demand, overfishing not only threatens the survival of target fish species, but may also break the balance of marine ecosystems and lead to the loss of biodiversity. In addition, this production method that relies on natural fish resources is questionable in terms of sustainability, as long-term overexploitation may lead to resource depletion. Regulatory restrictions and increased public awareness of marine life protection may also cause instability in the market and supply chain for the PDRN industry. Therefore, developing alternative PDRN production methods and reducing dependence on wild fish is not only responsible for the ecological environment, but also the key to ensuring the long-term stable development of the PDRN industry.

[0004] With the development of bioengineering technology, it has become possible to prepare PDRN by biological methods. The preparation of PDRN by bioengineering technology can ensure the sustainability of the raw material source, no longer be limited to the breeding or fishing of specific fish, and is more eco-friendly. PDRN prepared by biological methods has less protein residue than traditional methods and is not easy to cause allergic reactions. The production efficiency of PDRN prepared by bioengineering technology is high. High yield and greater cost advantage. In addition, the preparation process of bioengineering technology is not suitable for animal-derived materials, which reduces the risk of transmission of pathogenic microorganisms. For example, patent CN117757703A uses sake lactobacillus fermentation to prepare PDRN, and patent CN116396956A extracts PDRN through schizochytrium. Compared with conventional fish testis extraction technology, the above two methods use bacteria and fungi as raw materials, providing a sustainable production route. Patent CN115109775A uses salmon genomic DNA for whole genome amplification (WGA) to produce PDRN, which solves the shortcomings of poor technical sustainability and ecological unfriendliness of existing methods, but it is difficult to control the length of the target product. Patent CN113913420A uses recombinant fermentation technology to construct a salmon genome into a DNA library of a specific length and construct a bacterial vector, and produces a PDRN of a specific length through bacterial fermentation and enzyme digestion. The length of PDRN produced by this method is relatively fixed, but the process flow is complicated. Summary of the invention

[0005] The technical problem solved by the present invention is how to produce PDRN with the advantages of high reaction efficiency, high similarity with human genome DNA, high safety, and fixed product length.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a method for synthesizing polydeoxyribonucleotides, comprising the following steps:

[0007] 1) Synthesize single-stranded DNA molecules at the 3' end of the base primer using TdT enzyme;

[0008] The base primer includes a single-stranded DNA molecule with a length of 5-100 nt composed of random bases;

[0009] 2) amplifying the single-stranded DNA molecule to obtain an amplified product;

[0010] 3) The amplified product is interrupted to obtain polydeoxyribonucleotides.

[0011] In the method described above, the specific nucleotide sequence of the base primer in step 1) in the embodiment of the present invention is GAC AAT CCC GGG CAT AA as an example.

[0012] The above-mentioned synthetic system is shown in Table 1, wherein the molar concentration ratios of dATP, dCTP, dGTP, and dTTP in dNTP are 1:1:1:1, respectively.

[0013] The primers required for amplification in step 2) are specifically random primers consisting of 6 Ns in the embodiment of the present invention.

[0014] In the method described above, in step 1), the reaction conditions for the synthesis are 30-40° C., 2-10 min; 98° C., 2-5 min.

[0015] In the method described above, in step 1), the reaction conditions for the synthesis are 30° C. for 5 min; 98° C. for 2 min.

[0016] In the above method, the amplification is PCR amplification, loop-mediated isothermal amplification, recombinant polymerase amplification or rolling circle amplification.

[0017] In the method described above, the amplification method is rolling circle amplification;

[0018] Or, in step 1), the 5' end of the base primer is modified with a phosphate group, and the 3' end is a free hydroxyl group.

[0019] In the method described above, the enzyme used in the rolling circle amplification is Phi29 DNA polymerase.

[0020] In the method described above, in step 2), before the rolling circle amplification, the step of circularizing and digesting the single-stranded DNA is also included.

[0021] The above cyclization system is shown in Table 2. The reaction procedure of cyclization is: 65°C for 1h; 85°C for 10min.

[0022] The digestion system is shown in Table 3. The digestion reaction procedure is: 37°C for 30 min; 80°C for 20 min.

[0023] The rolling circle amplification includes denaturation and amplification before the rolling circle amplification;

[0024] The denaturation system of the rolling circle amplification is shown in Table 4. The reaction procedure of the denaturation before the rolling circle amplification is: 95° C. for 3 min; ice bath for 5 min.

[0025] The amplification system is shown in Table 5; the amplification reaction procedure is: 30°C for 16h; 65°C for 10min, to obtain the RCA product.

[0026] In the method described above, in step 3), the disruption is performed by restriction enzyme disruption or ultrasonic disruption.

[0027] In the present invention, ultrasonic interruption is adopted, and the ultrasonic interruption conditions are shown in Table 6.

[0028] In the above method, the disruption is performed by restriction enzyme cutting;

[0029] The base primer is provided with an enzyme recognition site for the restriction enzyme.

[0030] In the above method, the molecular weight of the polydeoxyribonucleotide is 50-1500 kDa;

[0031] Or the molecular weight of the polydeoxyribonucleotide is 132 kDa.

[0032] The second aspect of the present invention provides a kit for enzymatic synthesis of polydeoxyribonucleotides, comprising the following components:

[0033] 1) TdT enzyme;

[0034] 2) the base primer described in the first aspect;

[0035] 3) Reagents required for amplification;

[0036] 4) Used to interrupt the required reagents or instruments.

[0037] The reagents required for amplification above include Phi29 MAX DNA Polymerase, Phi29 MAX DNA Polymerase Reaction Buffer and Recombinant Albumin.

[0038] The reagents or instruments required for the disruption described above include a sonicator.

[0039] In the above, the kit also includes: 5) reagents required for cyclization, which include ssDNA / RNACircLigase shown in Table 2, etc.

[0040] The third aspect of the present invention is to provide the use of TdT enzyme in the preparation of polydeoxyribonucleotides.

[0041] The fourth aspect of the present invention provides the use of the kit described in the third aspect in the preparation of polydeoxyribonucleotides.

[0042] The process flow proposed by the present invention has the following advantages:

[0043] 1) High reaction efficiency: The present invention uses RCA reaction to amplify double-stranded DNA. RCA reaction can be carried out continuously under constant temperature conditions, without the need for complex thermal cycling equipment, which greatly improves the reaction efficiency and can quickly synthesize a large amount of DNA. In addition, the DNA polymerase (such as Phi29) used in the RCA reaction has high activity and stability, which is another guarantee for the high reaction efficiency of this process.

[0044] 2) High similarity with the human genome: The ratio of the four bases ATCG in the human genome is about 60% for A+T and about 40% for C+G. The present invention randomly adds dNTPs to the base primer through TdT enzymatic reaction, and then amplifies and interrupts the random ssDNA sequence obtained by the reaction after single-stranded circularization to produce PDRN of a specific length. Since the addition of the four bases in dNTP during the TdT enzymatic ssDNA synthesis process is a random process, the proportion of the four bases A, T, C, and G can be precisely controlled to make the produced PDRN highly close to the base ratio of the human genome.

[0045] 3) High safety: PDRN produced by conventional fish testicular tissue extraction may have a large amount of protein residues and unknown biosafety risks (such as heavy metals and pathogenic microorganisms contamination during fish growth). PDRN produced by other bacteria, algae, fungi, etc. may also have other residual bioactive ingredients that cause risks such as disease and allergic reactions. The process proposed by the present invention produces PDRN by TdT enzymatic reaction and RCA rolling circle amplification. This method does not require animals or microorganisms as raw materials, and its product has higher biosafety.

[0046] 4) Fixed product length: During TdT-catalyzed ssDNA synthesis, the length of the synthesized ssDNA can be controlled by precisely adjusting the reaction time. The generated ssDNA, as the smallest structural unit, can be broken into PDRNs of a specific length after single-stranded cyclization and rolling circle amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the process of polydeoxyribonucleotide based on biological enzymatic reaction.

[0048] Figure 2 This is the relationship between the TdT enzymatic synthesis amplification time and the product length.

[0049] Figure 3 Obtain PDRN gel map for ultrasonic interruption.

[0050] Figure 4 This is the capillary electrophoresis result of the fragment length distribution of PDRN final product. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0052] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0053] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0054] Figure 1 Schematic diagram of the process of polydeoxyribonucleotide based on biological enzymatic reaction.

[0055] Example 1: Enzymatic synthesis of polydeoxyribonucleotides

[0056] 1. Base primer synthesis

[0057] The synthetic base primer is usually 5-100 nt in length, and the 5' end of the base primer contains a phosphate group modification, and the 3' end must be a free hydroxyl group.

[0058] The specific nucleotide sequence of the base primer is GAC AAT CCC GGG CAT AA (sequence 1) as an example.

[0059] 2. TdT enzyme-catalyzed single-strand DNA (ssDNA) synthesis

[0060] TdT is the abbreviation of Terminal Deoxynucleotidyl transferase. TdT enzyme can add nucleotides to the end of DNA chain, plays an important role in DNA replication and repair process, and participates in important physiological processes such as V(D)J recombination and DNA repair in vivo. In the field of bioengineering, TdT enzyme is often used for enzymatic synthesis of DNA due to its characteristics of template-independent DNA synthesis. The present invention uses TdT enzyme and dNTP to synthesize ssDNA on the base primer. By adjusting the reaction time, the length of ssDNA product can be accurately controlled.

[0061] The TdT enzymatic synthesis reaction was carried out according to the reaction system in Table 1, and the obtained product was recorded as single-stranded ssDNA (with base primer).

[0062] Table 1 is the TdT enzymatic synthesis reaction system

[0063] Reaction system (30uL) Storage concentration Final concentration Input volume(uL) Base Primer 20uM 2uM 3 10X Buffer - 1X 3 dNTP 5mM 0.5mM 3 TdT enzyme 5mg / mL 0.5mg / mL 1 ddH2O - - 13

[0064] The above-mentioned TdT enzyme can also use the Terminal Deoxynucleotidyl Transferase TdT terminal transferase produced by Yisheng Biotechnology, with the product number 10302ES76.

[0065] The molar concentration ratios of dATP, dCTP, dGTP and dTTP in the above dNTPs are 1:1:1:1 respectively.

[0066] The reaction procedure of the above TdT enzymatic synthesis reaction was: 30°C for 5 min; 98°C for 2 min.

[0067] The reaction time at 30°C in the above TdT enzymatic synthesis was set to different times. Figure 2 As shown, it can be seen that the size of single-stranded ssDNA is different at different reaction times. It has reached 500nt after 5 minutes of reaction and can be used for subsequent shearing.

[0068] Therefore, a shorter reaction time of 30°C for 5 min was selected.

[0069] 3. Single-chain cyclization

[0070] The single-stranded ssDNA synthesized by the above TdT enzyme is cyclized by ligase. Since the 5' end of the base primer has a phosphate group, it can form a phosphodiester bond with the 3' end hydroxyl of ssDNA to make the single strand into a ring. After the single strand is cyclized, a digestion step can be added to degrade the uncyclized ssDNA, as follows:

[0071] 1) Purification

[0072] The single-stranded ssDNA obtained in step 2 (single-stranded ssDNA obtained at 30°C for 5 min; 98°C for 2 min) was purified using Thermo Fisher purified magnetic beads (purified magnetic beads, Thermo Fisher, A58523). 30uL of purified magnetic beads (1X) were added to each reaction, incubated for 10 min, washed twice with 70% ethanol, and eluted with 20uL ddH2O to obtain purified single-stranded ssDNA (with base primers).

[0073] 2) Synthetic product quality inspection

[0074] The purified single-stranded ssDNA obtained in 1) above was subjected to electrophoresis using 8% denaturing polyacrylamide gel. The electrophoresis conditions were set as follows: 260 V, 2 h. The ssDNA ladder used was BioBio, catalog number B1003.

[0075] The results showed that the purified single-stranded ssDNA was a 500nt sequence with a single bright band, and the quality inspection was qualified.

[0076] 3) Single-stranded DNA circularization

[0077] The purified single-stranded ssDNA that has passed the quality inspection in 2) is cyclized using a single-stranded cyclase. The specific reaction system is as shown in Table 2 to obtain a cyclized product.

[0078] Table 2 shows the cyclization reaction system

[0079] Reaction system (30uL) Input volume(uL) ssDNA / RNA CircLigase (Sinogene, D2602) 1 Purified single-stranded ssDNA (5' phosphorylation modification) (10 pmol) 18 10×CircLigase Buffer (Sinogene, D2602) 3 50mM MnCl2 1.5 ddH20 6.5

[0080] The reaction procedure of the above cyclization was: 65°C for 1 h; 85°C for 10 min.

[0081] 4) Digestion

[0082] The above cyclization product is then digested according to the system in Table 3 to obtain a digested cyclization product.

[0083] Table 3 shows the digestion reaction system

[0084] Reaction system (32uL) Input volume(uL) Cyclization product 30 Exonuclease I (Sinogene, C5014) 2

[0085] The reaction program of the above digestion was: 37°C for 30 min; 80°C for 20 min.

[0086] 5) Quality inspection

[0087] Step 4) The digested cyclized product obtained was subjected to electrophoresis using 8% denaturing polyacrylamide gel, and the electrophoresis conditions were: 260V, 2h. Qubit quantification: ssDNA Qubit quantification reagent was used to quantify the digested cyclized product, and the operation was strictly carried out in accordance with the Qubit operating procedures.

[0088] The results showed that the cyclization product after digestion had a single bright band and passed the quality inspection.

[0089] 4. Rolling circle amplification (RCA)

[0090] The circularized product is amplified using random primers and a DNA polymerase (such as Phi29 DNA polymerase) to generate a large amount of double-stranded DNA sequences.

[0091] 1) Denaturation before rolling circle amplification

[0092] The digested cyclized product obtained in step 3 is denatured before rolling circle amplification according to the system in Table 4 to obtain a rolling circle amplification denatured product, maintaining the single-stranded state of the circular DNA.

[0093] Table 4 is the rolling circle amplification denaturation reaction system

[0094]

[0095] In the above table, the random primers are 6 Ns.

[0096] The reaction procedure of the above rolling circle amplification denaturation was: 95°C for 3 min; ice bath for 5 min.

[0097] 2) Amplification

[0098] Table 5 shows the amplification system

[0099] Reaction system (50uL) Input volume(uL) Rolling circle amplification denatured product 48 Recombinant Albumin (NEB, M0269S) 0.25 Phi29MAX DNA Polymerase (NEB, M0269S) 2

[0100] The above amplification reaction procedure was: 30°C for 16h; 65°C for 10min, to obtain the RCA product.

[0101] 3) Ethanol precipitation to obtain the product

[0102] A. Add 1 / 10 volume of sodium acetate aqueous solution (3M, pH=5.2) to the RCA product obtained in step 2) and mix thoroughly to a concentration of 0.3M;

[0103] B. Add 2 times the volume of pre-cooled anhydrous ethanol to the reaction tube in the previous step, mix thoroughly, and place the reaction tube in a -80℃ freezer for 20-30 minutes;

[0104] C. Place the reaction tube in a high-speed centrifuge at 12,000 rpm for 15 minutes, and remove the supernatant.

[0105] D. Add 70% alcohol to the reaction tube in the previous step, incubate at 12000 rpm for 5 min, and remove the supernatant.

[0106] E. Repeat step D.

[0107] F. Dry at room temperature until the alcohol evaporates;

[0108] G. Add ddH2O to the reaction tube for elution to obtain the RCA product.

[0109] 5. Product interruption to obtain the final product PDRN

[0110] By ultrasonic means, the RCA product is broken into double-stranded DNA fragments of fixed length, which is a PDRN solution. The RCA product obtained in 4 above is ultrasonically broken to obtain an ultrasonic product, which is the final product PDRN.

[0111] The ultrasonic conditions are set as shown in Table 6:

[0112] Table 6 shows the ultrasonic interruption conditions.

[0113] Target fragment peak Target 200bp accounts for >50% Maximum incident power(W) 240 Working coefficient 20％ Number of cycles per pulse 200 Duration(S) 740

[0114] 6. Identification

[0115] The ultrasonic product obtained in 5 above was subjected to agarose electrophoresis and capillary electrophoresis, and the electrophoresis conditions were: 1.5%, 140V, 30min.

[0116] Agarose electrophoresis results Figure 3 As shown, it can be seen that the fragment of the product after shearing is about 200 bp.

[0117] The above results show that the product obtained by the method of the present invention has a fixed length and contains only DNA without any protein residue, and has a high degree of safety.

[0118] A product with a molecular weight of 132 kDa was obtained.

[0119] Capillary electrophoresis results are as follows Figure 4 As shown, it can be seen that the target product fragment distribution is confirmed to be less than 1kb, proving that PDRN is obtained.

[0120] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for synthesizing polydeoxyribonucleotides, comprising the following steps: 1) Synthesize single-stranded DNA molecules at the 3' end of the base primer using TdT enzyme; The base primer includes a single-stranded DNA molecule with a length of 5-100 nt composed of random bases; 2) amplifying the single-stranded DNA molecule to obtain an amplified product; 3) The amplified product is interrupted to obtain polydeoxyribonucleotides.

2. The method according to claim 1, characterized in that: In step 1), the reaction conditions for the synthesis are 30-40° C., 2-10 min; 98° C., 2-5 min.

3. The method according to claim 1 or 2, characterized in that: In step 1), the reaction conditions for the synthesis are 30° C. for 5 min; 98° C. for 2 min.

4. The method according to any one of claims 1 to 3, characterized in that: The amplification is PCR amplification, loop-mediated isothermal amplification, recombinant polymerase amplification or rolling circle amplification.

5. The method according to any one of claims 1 to 4, characterized in that: The amplification method is rolling circle amplification; Or, in step 1), the 5' end of the base primer is modified with a phosphate group, and the 3' end is a free hydroxyl group.

6. The method according to claim 5, characterized in that: The enzyme used in the rolling circle amplification is Phi29 DNA polymerase.

7. The method according to any one of claims 1 to 6, characterized in that: In step 3), the fragmentation is performed by restriction enzyme cutting or ultrasonic fragmentation.

8. The method according to claim 7, characterized in that: The disruption is achieved by restriction enzyme cutting; The base primer is provided with an enzyme recognition site for the restriction enzyme.

9. The method according to any one of claims 1 to 8, characterized in that: The molecular weight of the polydeoxyribonucleotide is 50-1500 kDa; Or the molecular weight of the polydeoxyribonucleotide is 132 kDa.

10. A kit for enzymatic synthesis of polydeoxyribonucleotides, comprising the following components: 1) TdT enzyme; 2) The base primer according to any one of claims 1 to 9; 3) Reagents required for amplification; 4) Reagents or instruments required for interruption; or, use of TdT enzyme in the preparation of polydeoxyribonucleotides; Or use of the kit in preparing polydeoxyribonucleotides.

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