Process for the preparation of polydeoxyribonucleotides and use thereof

Abstract

This invention discloses a method for preparing polydeoxyribonucleic acid (PDRN) and its applications, belonging to the fields of biotechnology, nucleic acid engineering, and daily chemical technology. The invention uses a dual-enzyme composition of a deoxyribonuclease mutant and DNA exonuclease III in a mass ratio of (2-3):(0.1-0.3) to controllably digest genomic DNA derived from salmon testes. Combined with optimized extraction, purification, and fractional ultrafiltration processes, the resulting PDRN is concentrated in the 250-500 bp range. The enzyme mutant is obtained by site-directed mutagenesis of wild-type DNase-1 at two sites (E61K and S144W), achieving an enzyme activity as high as 6.8 × 10⁻⁶. 6 The concentration of PDRN obtained in this invention is 353.3% higher than that of wild-type PDRN. The PDRN synthesized in this invention exhibits excellent cell compatibility and can significantly downregulate UVB-induced DNA damage and response markers such as CPD, 8-OHdG, pATM-S1981, p53-binding protein 1, γH2AX, XPA, and XPC. Its repair effect is superior to existing commercially available products of the same type, and it can be widely used in cosmetics, skincare products, and topical pharmaceutical preparations with DNA damage repair efficacy.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology, nucleic acid engineering and daily chemical technology, specifically relating to a method for preparing polydeoxyribonucleotides and their applications. Background Technology

[0002] Polydeoxyribonucleotide (PDRN) is a low molecular weight deoxyribonucleotide polymer extracted from salmon testes. Its base pair sequence ratio is highly homologous to human DNA. It can promote cell proliferation and migration by activating the cellular purine salvage synthesis pathway, while regulating DNA damage repair pathways, repairing DNA damage induced by external stimuli such as ultraviolet radiation, and improving skin barrier function. It has extremely high application value in the fields of medical aesthetic repair, functional skin care products, and topical dermatological pharmaceutical preparations.

[0003] Currently, PDRN preparation processes are mainly divided into three categories: physical shearing, chemical degradation, and enzymatic digestion. Among them, physical / chemical shearing is a crude process that cannot precisely control the molecular weight distribution of nucleic acid fragments, easily leading to excessive low molecular weight fragmentation. It can also cause irreversible damage to the nucleic acid chain ends, introduce chemical solvent residues, and ultimately result in a low proportion of effective functional fragments, a high risk of cellular stress, and poor biological activity. Although enzymatic digestion can improve fragment uniformity to some extent, it is limited by the enzyme activity and enzyme digestion specificity of deoxyribonuclease, making it impossible to precisely control the nucleic acid fragments within the molecular weight window that skin cells can efficiently utilize. It generally suffers from severe high molecular weight tailing and insufficient enrichment of effective functional fragments, making it difficult for the product's DNA damage repair efficacy to reach the optimal level.

[0004] Deoxyribonuclease-1 (DNase-1) is the most commonly used enzyme in the preparation of PDRN. Wild-type DNase-1 has the drawbacks of low enzyme activity and insufficient digestion efficiency, making it impossible to achieve precise and controllable enzymatic digestion of DNA fragments. Although there are reports of mutating DNase-1 to improve enzyme activity in the existing technology, such as the DNase-1 mutant A69H-S132R-Q215C disclosed in Chinese patent CN118931877B, although its enzyme activity is improved to a certain extent compared with wild type, it still cannot meet the process requirements of efficient and precise enzymatic digestion in the large-scale preparation of PDRN.

[0005] Exonuclease III is a 3'→5' exonuclease specific to double-stranded DNA, capable of progressively removing the 3' end of the DNA strand from blunt ends, 5' overhangs, or nick sites. It has the potential to trim ends and reduce excessively long, high-molecular-weight tails. However, this enzyme primarily acts on DNA ends, making it difficult to achieve rapid and efficient degradation of high-molecular-weight genomic DNA when used alone. Consequently, it is also difficult to obtain the desired PDRN product distribution when used alone. Therefore, current technology still lacks a combined enzyme digestion system that combines highly efficient enzymatic digestion with fine end trimming capabilities to achieve precise control over PDRN fragment distribution and consistent chemical structure at the ends.

[0006] In summary, the lack of high-activity DNase-1 tool enzymes, preparation processes that can precisely control the distribution of PDRN fragments, and PDRN products with high functionality and low stress burden in existing technologies have become the core bottlenecks restricting the in-depth application of PDRN in the field of skin repair. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a high-activity deoxyribonuclease mutant and its matching PDRN dual-enzyme preparation process, thereby achieving precise control of the molecular weight of PDRN nucleic acid fragments and solving the problems of low enzyme activity of existing wild-type deoxyribonuclease, uneven fragment distribution in existing PDRN preparation processes, and insufficient enrichment of effective functional fragments.

[0008] To achieve the above objectives, the present invention discloses the following technical solutions: In a first aspect, the present invention provides a method for preparing polydeoxyribonucleotides, wherein the method includes enzymatic hydrolysis of genomic DNA substrates using a dual-enzyme component.

[0009] Preferably, the genomic DNA substrate is derived from salmon testes.

[0010] Preferably, the dual enzyme components are a mixture of the deoxyribonuclease mutant DNase-1-E61K-S144W and DNA exonuclease III.

[0011] Preferably, the amino acid sequence of the deoxyribonuclease mutant DNase-1-E61K-S144W is shown in SEQ ID NO.2, and the nucleic acid sequence encoding the amino acid is shown in SEQ ID NO.3. The deoxyribonuclease mutant DNase-1-E61K-S144W is obtained by point mutation of wild-type deoxyribonuclease, and the amino acid sequence of the wild-type deoxyribonuclease is shown in SEQ ID NO.1.

[0012] The exonuclease III is used to trim the ends of DNA fragments formed after endonuclease digestion, reduce excessively long high molecular weight tails, improve the concentration of fragment distribution, and enhance the enrichment of target functional fragments.

[0013] Preferably, in the dual-enzyme component, the mass ratio of the deoxyribonuclease mutant DNase-1-E61K-S144W to DNA exonuclease III is (2-3):(0.1-0.3).

[0014] More preferably, the preparation method specifically includes the following steps: Step 1: Take frozen salmon testes, thaw at 4℃, cut into 1-2 mm tissue blocks, rinse 3 times with sterile physiological saline at 4℃, drain, add pre-cooled lysis buffer at a material-to-liquid ratio of 1g:5mL, homogenize at 4℃ and 10000rpm for 30s each time with 1min interval, repeat 3 times, let the homogenate stand at 4℃ for 2h to fully release genomic nucleic acids, and obtain the solution to be processed; Step 2: Add an equal volume of chloroform-isoamyl alcohol mixture to the solution to be treated, gently invert and mix for 10 min, centrifuge at 8000 rpm for 15 min at 4 °C, collect the upper aqueous phase, and repeat the extraction twice until no protein precipitation occurs in the middle; add RNase A to the upper aqueous phase to a final concentration of 50 μg / mL, incubate at 37 °C for 30 min to completely degrade RNA, and extract once more with chloroform-isoamyl alcohol to remove enzymes and residual proteins, obtaining a purified aqueous phase; Step 3: Add 1 / 10 volume of 3 mol / L sodium acetate solution to the purified aqueous phase, mix well, add 2.5 volumes of pre-cooled anhydrous ethanol at -20℃, let stand at -20℃ for 2 hours to precipitate DNA, centrifuge at 12000 rpm at 4℃ for 20 minutes and discard the supernatant, wash the precipitate twice with 70% pre-cooled ethanol, vacuum dry at low temperature for 5 minutes, prepare a 4 mg / mL DNA substrate solution with enzyme digestion buffer, and filter through a 0.22 μm filter membrane for sterilization; Step 4: Add the dual enzyme components to the DNA substrate solution at a dosage of 0.1 wt% of the substrate solution, and incubate at 37°C for 1 hour. After inactivation, inactivate each enzyme by bathing at 75°C for 12 minutes. Centrifuge the inactivated enzyme solution at 4°C and 12,000 rpm for 10 minutes. Collect the supernatant and filter it through a 0.45 μm filter membrane. First, ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and collect the permeate. Then, concentrate the permeate through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and discard the permeate. Collect the retentate and freeze-dry it under vacuum to obtain polydeoxyribonucleotides.

[0015] More preferably, the lysis buffer in step 1 is a buffer solution of 10 mmol / L Tris-HCl, 1 mmol / L EDTA, and pH 8.0.

[0016] More preferably, the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixture in step 2 is 24:1.

[0017] More preferably, the enzymatic digestion buffer in step 3 is a buffer solution containing 10 mmol / L Tris-HCl, 5 mmol / L MgCl2, 1 mmol / L CaCl2, and pH 7.5 calibrated at 37°C.

[0018] In a second aspect, the present invention provides a polydeoxyribonucleotide, which is prepared by the preparation method described in the first aspect.

[0019] Preferably, the molecular weight of the polydeoxyribonucleotide fragments is concentrated in the range of 250-500 bp.

[0020] It should be noted that the molecular weight cutoff (MWCO) of ultrafiltration membranes is often defined using globular proteins. However, since the polydeoxyribonucleotides (PDRNs) in this invention have a linear double-stranded structure, their hydrodynamic radius differs from that of globular proteins. During ultrafiltration, linear DNA can pass through the membrane pores along its long axis. Therefore, a 100kDa ultrafiltration membrane primarily retains ultra-long fragments >1000bp, while a 50kDa ultrafiltration membrane can effectively retain fragments of 250bp and above. This invention achieves efficient and precise enrichment of PDRNs in the 250-500bp range through a combination of 100kDa permeation and 50kDa retention.

[0021] Thirdly, the present invention provides the application of the polydeoxyribonucleotides described in the second aspect in the preparation of products with DNA damage repair functions.

[0022] Preferably, the product is a cosmetic, skin care product, or topical pharmaceutical preparation.

[0023] The beneficial effects of this invention are: 1. The deoxyribonuclease mutant DNase-1-E61K-S144W provided by this invention achieves a significant increase in enzyme activity by performing site-directed mutations at two sites (E61K and S144W) on wild-type DNase-1, with an enzyme activity reaching 6.8 × 10⁻⁶. 6 U / mL, compared with wild-type DNase-1, the enzyme activity is increased by 353.3%, which greatly improves the DNA digestion efficiency, shortens the digestion reaction time, and reduces the production cost of PDRN large-scale preparation, providing a high-performance tool enzyme for achieving precise and controllable DNA fragment digestion; 2. The PDRN dual-enzyme preparation process provided by this invention uses the above-mentioned high-enzyme-activity mutant and DNA exonuclease III in a mass ratio of (2-3):(0.1-0.3) dual-enzyme components, combined with optimized nucleic acid extraction, purification, enzymatic digestion and fractional ultrafiltration processes, which can precisely control the nucleic acid fragments of PDRN products within the optimal functional window for skin cells of 250-500bp, while ensuring the chemical structure consistency of the nucleic acid fragment end sequences, thus solving the problems of severe polymer tailing and insufficient enrichment of effective fragments in the single enzyme digestion method; 3. The PDRN provided by this invention, at a concentration of 0.03% (m / m), showed an inhibition rate of 56.75% against the DNA damage marker CPD and 42.70% against 8-OHdG in UVB irradiation-induced keratinocyte and 3D epidermal skin models. It also showed inhibition rates of 46.20% and 57.75% against key DNA damage response targets pATM-S1981 and p53-binding protein 1 (53BP1), respectively. Furthermore, it showed inhibition rates of 46.01% and 59.23% against key proteins in the nucleotide excision repair pathway XPA and XPC, respectively. All these indicators are significantly superior to PDRN prepared using wild-type enzymes. Compared with similar products on the market, it can systematically reduce the burden of UVB-induced cellular DNA damage and the pressure of repair response, with significant repair effects; 4. The PDRN product obtained by this invention can be widely used in the preparation of cosmetics, skin care products, and topical skin pharmaceutical preparations. It can effectively repair UVB-induced skin cell DNA damage, improve skin photoaging and barrier damage, and provide a high-performance core raw material for skin repair products. It has broad market application prospects and industrialization value. Attached Figure Description

[0024] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings.

[0025] Figure 1 These are fluorescence images of the experimental wells for each group in the XPA test; Figure 2 A bar chart showing the ratio of XPA relative integrated optical density (IOD) to the average cell number. Figure 3 These are fluorescence images of the experimental wells for each group in the XPC test; Figure 4 A bar chart showing the ratio of relative integrated optical density (IOD) to average cell number in XPC. Figure 5 These are fluorescence images of the experimental wells in each group during the pATM-S1981 test; Figure 6A bar chart showing the relative integrated optical density (IOD) / average cell number of pATM-S1981; Figure 7 These are fluorescence images of the experimental wells in each group during the p53 binding protein 1 assay. Figure 8 The bar chart shows the relative integrated optical density (IOD) of p53 binding protein 1 versus the average cell number. Figure 9 These are fluorescence images of the experimental wells in each group during the γH2AX test; Figure 10 A bar chart showing the relative integrated optical density (IOD) of γH2AX / mean cell number; Figure 11 Images of electrophoretic gels for competitor A, competitor B, and RJMPDRN-850K. Detailed Implementation

[0026] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] Unless otherwise specified, the experimental methods used in the specific embodiments are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified; and the percentages mentioned in the specific embodiments are all mass percentages unless otherwise specified.

[0028] I. Preparation of the mutant DNase-1-E61K-S144W 1. Mutant sequence screening This invention utilizes the Swiss Model to perform homology modeling on wild-type Deoxyribonuclease-1 (amino acid sequence shown in SEQ ID NO. 1). After quality verification of the model, the AutoDock software was used for enzyme-substrate molecule docking and analysis to screen out key sites affecting enzyme-substrate interaction. Different mutation types (single-point or combined mutations) were designed for each site obtained by screening. It was found that by mutating glutamic acid (Glu, E) at position 61 to lysine (Lys, K) and serine (Ser, S) at position 144 to tryptophan (Trp, W) based on the amino acid sequence of wild-type Deoxyribonuclease-1, an enzyme mutant with significantly enhanced enzyme activity was obtained, named DNase-1-E61K-S144W, whose amino acid sequence is shown in SEQ ID NO. 2, and the nucleic acid sequence encoding this amino acid is shown in SEQ ID NO. 3.

[0029] 2. Enzyme activity comparison The recombinant plasmid pET-28a-E61K-S144W obtained from the screening process in item 1 above was introduced into E. coli BL21 competent cells to obtain the engineered strain BL21-pET-28a-E61K-S144W. The strain BL21-pET-28a-E61K-S144W was plated on LB solid medium containing a final concentration of 100 μg / mL kanamycin and cultured at 37°C for 12 h. A single colony was picked and inoculated into 5 mL of LB liquid medium containing a final concentration of 100 μg / mL kanamycin and cultured on a shaker at 37°C and 220 rpm until OD200. 600 =0.6~0.8, add IPTG to a final concentration of 0.1mmol / L, and induce culture at 25℃ for 12h. After induction, collect the bacterial cells by centrifugation at 4℃ and 8000r / min for 10min, resuspend the bacterial cells with lysis buffer, lyse the cells by sonication, centrifuge at 4℃ and 12000r / min for 20min, and collect the supernatant to obtain the enzyme mutant DNase-1-E61K-S144W.

[0030] Wild-type DNase-1 and enzyme mutant A69H-S132R-Q215C were prepared using recombinant plasmid pET-28a-DNase-1 (containing the wild-type DNase-1 gene sequence) and recombinant plasmid pET-28a-A69H-S132R-Q215C (containing the gene sequence of the deoxyribonuclease mutant A69H-S132R-Q215C, which has been disclosed in Chinese patent CN118931877B) according to the above-mentioned enzyme mutant preparation method.

[0031] The enzyme activities of mutant DNase-1-E61K-S144W, wild-type DNase-1, and enzyme mutant A69H-S132R-Q215C were compared under the following conditions: 40mM Tris-HCl (pH 8.0), 10mM MgSO4, 1mM CaCl2, 1μg pBR322 plasmid DNA. Enzyme activity definition: One unit of activity is defined as the amount of enzyme required to completely degrade 1μg of pBR322 plasmid DNA within 10 minutes at 37℃.

[0032] The measurement results are shown in Table 1.

[0033] Table 1 Wild-type DNase-1 <![CDATA[1.5×10 6 ]]> / Mutant A69H-S132R-Q215C <![CDATA[4.8×10 6 ]]> 220 Mutant DNase-1-E61K-S144W <![CDATA[6.8×10 6 ]]> 353.3 As shown in Table 1, the enzyme activity of the mutant DNase-1-E61K-S144W was significantly higher than that of the wild-type deoxyribonuclease, reaching 6.8 × 10⁻⁶. 6The activity of the enzyme was increased by 3.53 times compared to that of wild-type deoxyribonuclease, and it was also significantly improved compared to the enzyme mutant A69H-S132R-Q215C in the prior art.

[0034] II. PDRN prepared using the two-enzyme method Preparation of dual-enzyme components: The enzyme mutant DNase-1-E61K-S144W obtained by the aforementioned method was freeze-dried under vacuum to obtain solid enzyme powder with an enzyme activity of 7.5 × 10⁻⁶. 9 U / g, then with DNA exonuclease III (in solution form, Exonuclease III) E. coli The enzyme activity was 100,000 units / ml, purchased from New England Biolabs (NEB). The enzymes were mixed at a mass ratio of 3:0.2 to obtain the dual-enzyme components.

[0035] Materials required for preparation: salmon testes, dual enzyme components, lysis buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 8.0), chloroform-isoamyl alcohol (24:1, v / v), RNase A, anhydrous ethanol, and enzyme digestion buffer (10 mmol / L Tris-HCl, 5 mmol / L MgCl2, 1 mmol / L CaCl2, pH 7.5 calibrated at 37℃).

[0036] The specific steps are as follows: Step 1: Take frozen salmon testes, thaw at 4℃, cut into 1-2 mm tissue blocks, rinse 3 times with sterile physiological saline at 4℃ and drain. Add pre-cooled lysis buffer at a material-to-liquid ratio of 1:5 (g:mL), homogenize at 10000 rpm at 4℃, homogenize for 30 seconds each time with a 1-minute interval, repeat 3 times, and let the homogenate stand at 4℃ for 2 hours to fully release genomic nucleic acids and obtain the solution to be processed. Step 2: Add an equal volume of chloroform-isoamyl alcohol mixture to the solution to be treated, gently invert and mix for 10 min, centrifuge at 4℃ and 8000 rpm for 15 min, collect the upper aqueous phase, repeat the extraction twice until no protein precipitation occurs in the middle, add RNase A to the upper aqueous phase to a final concentration of 50 μg / mL, incubate at 37℃ for 30 min to completely degrade RNA, and extract once more with chloroform-isoamyl alcohol to remove enzymes and residual proteins, obtaining the purified aqueous phase; Step 3: Add 1 / 10 volume of 3mol / L sodium acetate to the purified aqueous phase, mix well, add 2.5 volumes of -20℃ pre-cooled anhydrous ethanol, let stand at -20℃ for 2 hours to precipitate DNA, centrifuge at 4℃ and 12000rpm for 20 minutes and discard the supernatant, wash the precipitate twice with 70% pre-cooled ethanol, vacuum dry at low temperature for 5 minutes, prepare a 4mg / mL DNA substrate solution with enzyme digestion buffer, and filter through a 0.22μm filter membrane for sterilization; Step 4: Add the dual enzyme components to the sterilized DNA substrate at a dosage of 0.1 wt% of the substrate solution. Incubate at 37°C for 1 hour. After inactivation, incubate at 75°C for 12 minutes to deactivate each enzyme. Centrifuge the deactivated enzyme solution at 4°C and 12,000 rpm for 10 minutes. Collect the supernatant and filter it through a 0.45 μm filter membrane. Use an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to ultrafilter the filtrate. Collect the permeate and concentrate it with an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. Discard the permeate and collect the retentate. Freeze-dry the retentate under vacuum to obtain a white, loose PDRN, named RJMPDRN-850K.

[0037] III. Performance Testing 1. In vitro DNA damage repair assay (Report No. G4601-2603140-1) 1.1 Test Basis The detection was conducted according to the "Detection Method for XPA, XPC, pATM-S1981, p53-binding protein 1, and human phosphorylated histone (γH2AX) Content Based on UVB Irradiation of Keratinocytes" and the "Detection Method for 8-hydroxydeoxyguanosine (8-OHdG) and DNA damage cyclobutanepyrimidine dimer (CPD) Content Based on UVB Irradiation of 3D Epidermal Skin Model (EpiKutis®)".

[0038] 1.2 Test Objective This test consists of three parts. The first part, based on keratinocytes, conducts cytotoxicity testing to determine the drug concentration of the sample on keratinocytes. The second part, based on UVB-irradiated keratinocytes, detects changes in the levels of XPA, XPC, pATM-S1981, p53-binding protein 1, and human phosphorylated histone (γH2AX). The third part, based on a UVB-irradiated 3D epidermal skin model (EpiKutis®), detects changes in the levels of 8-hydroxydeoxyguanosine (8-OHdG) and DNA damage cyclobutanepyrimidine dimer (CPD). The overall evaluation assesses the DNA damage repair effect of the tested samples.

[0039] 1.3 Test Materials 1.3.1 Test System The cells used in this test were keratinocytes, batch number: Ep25070703, provided by Guangdong Boxi Shaanxi Branch.

[0040] The 3D epidermal skin model (EpiKutis®) used in this test, batch number: ES260104, was provided by Guangdong Boxi Shaanxi Branch.

[0041] 1.3.2 Sample Information The samples were provided by Inai (Shanghai) Biotechnology Co., Ltd., and are shown below: RJMPDRN-850K: Prepared using the aforementioned method; Competitor B PDRN powder: a commercially available product, prepared using a single enzymatic digestion method; Competitor A PDRN powder: The method described in Section II, "PDRN Preparation Using the Two-Enzyme Method," is followed, except that the enzyme mutant DNase-1-E61K-S144W solid enzyme powder in the two-enzyme component is replaced with wild-type DNase-1 solid enzyme powder. The wild-type DNase-1 solid enzyme powder has an enzyme activity of 9.6 × 10⁻⁶. 8 The U / g of the enzyme was mixed with DNA exonuclease III at a mass ratio of 3:0.2 to obtain a dual-enzyme component, which was used in step 4 for enzymatic hydrolysis of the DNA substrate. At the same time, after collecting the permeate in step 4, the permeate was concentrated using a 10kDa ultrafiltration membrane instead of a 50kDa ultrafiltration membrane. The permeate was discarded, and the retentate was collected and freeze-dried under vacuum. The remaining steps and parameters were the same, and the competitor A PDRN powder was finally prepared.

[0042] 1.3.3 Main Reagents KcGrowth culture medium (Guangdong Boxi Shaanxi Branch), EpiGrowth culture medium (Guangdong Boxi Shaanxi Branch), PBS (Solepro), MTT (Sigma), DMSO (Sigma), Vitamin E (VE, Sigma), XPA antibody (Wuhan Sanying), XPC antibody (Thermofisher), pATM-S1981 antibody (Thermofisher), p53 binding protein 1 antibody (MCE), γH2AX antibody (Abcam), 8-OHdG antibody (Abcam), CPD antibody (CosMoBioCo.LTD.), paraformaldehyde (Biosharp).

[0043] 1.3.4 Main Equipment CO2 incubator (Thermo, 150I), ultra-clean workbench (Sujing Antai, SW-CJ-2F), microplate reader (BioTek, Epoch), UVB irradiator (Philips), fluorescence microscope (Leica, DM2500 LED), upright microscope (Olympus, BX53).

[0044] 1.4 Cytotoxicity Test 1.4.1 Test Method 1) Cell seeding: After cell resuscitation, when the cell seeding rate reaches about 60%, seed the cells into 96-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0045] 2) Experimental Groups: The experiment included a zeroing group, a solvent control group, a positive control group, and a sample group. In the sample group, each sample had 8 concentration gradients, and each concentration gradient had 3 replicate wells.

[0046] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table.

[0047] Table 2 Test Concentration Setting Table

[0048] 4) Drug administration: Drug administration was performed when the cell seeding rate in the 96-well plate reached 50%–60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of the sample was added to each well; for the zeroing group, no cells were seeded, only 200 μL of cell culture medium was added. After drug administration, the 96-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0049] 5) Detection: After culturing cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 150 µL of DMSO to each well, and read the OD value at 490 nm.

[0050] 6) Calculation of relative cell viability: Calculated according to the formula. .

[0051] 1.4.2 Test Results Table 3 MTT test results of sample competitor A

[0052] Based on the MTT results, competitor A, which is based on keratinocytes, did not exhibit significant cytotoxicity at a concentration range of 0.125% (m / m). Table 4 MTT test results of competitor sample B

[0053] Based on the MTT results, it was concluded that competitor B, which is based on keratinocytes, did not exhibit significant cytotoxicity at a concentration range of 0.125% (m / m).

[0054] Table 5. MTT test results for sample RJMPDRN-850K

[0055] Based on the MTT results, the RJMPDRN-850K sample, which is based on keratinocytes, did not show significant cytotoxicity at a concentration range of 0.0625% (m / m).

[0056] 1.5 Tests based on UVB irradiation of keratinocytes 1.5.1 Test Method 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 24-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0057] 2) Solution preparation: Prepare working solutions for the test substances according to the test groups.

[0058] Table 6 Test Groups

[0059] 3) Drug administration: According to the test groups, when the cell deposition rate in the 24-well plate reaches 30%–50%, drug administration is performed in groups, with 3 replicates per group. 1 mL of culture medium is added to each well of the blank control group and negative control group, 1 mL of culture medium containing the corresponding positive control is added to each well of the positive control group, and 1 mL of culture medium containing the corresponding test sample is added to each well of the sample group. After drug administration, the 24-well plate is placed in a CO2 incubator (37℃, 5% CO2) for 24 hours.

[0060] 4) Irradiation: According to the test groups, except for the blank control group, all other groups were subjected to UVB irradiation with an irradiation dose of 300 mJ / cm². 2 After irradiation, the samples were placed in a CO2 incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0061] 5) Immunofluorescence assay: The model used for detection was fixed with 4% paraformaldehyde for 24 hours, embedded and sectioned, and then subjected to immunofluorescence detection. The images were photographed and analyzed under a microscope.

[0062] 6) Inhibition rate calculation: .

[0063] 7) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0064] 1.5.2 Test Results 1.5.2.1 XPA Protein Test Results For detailed XPA immunofluorescence results, please refer to... Figure 1-2 Table 7-8; Figure 1 These are fluorescence images of the experimental wells in each group. Figure 1 Note: Images were taken using a fluorescence microscope (Leica, DM2500LED) at 200× magnification. Blue fluorescence represents cell nuclei, and green fluorescence represents XPA. The stronger the green fluorescence, the higher the XPA content. Table 7 shows the average relative integrated optical density (IOD) of XPA per cell number, which reflects the XPA content. Table 8 shows the difference analysis among competing products A-0.03%, competing product B-0.03%, and RJMPDRN-850K-0.03% in the group; Figure 2 This is a bar chart showing the average relative integrated optical density (IOD) of XPA versus the average number of cells.

[0065] Table 7 Results of XPA Immunofluorescence Analysis BC 1.00 0.03 / / NC 2.76 0.10 0.000## / PC 1.30 0.02 0.000** 52.90% Competitor A - 0.03% 2.14 0.09 0.001** 22.46% Competitor B - 0.03% 1.73 0.09 0.000** 37.32% RJMPDRN-850K-0.03% 1.49 0.05 0.000** 46.01% Note: Relative integrated optical density (IOD) / average cell number reflects the XPA content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0066] Table 8 Summary of Results of Analysis of Significant Differences Between Samples Competitor A - 0.03% / / Competitor B powder -0.03% 0.005▲▲ / RJMPDRN-850K-0.03% 0.000▲▲ 0.016▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0067] Compared with group BC, the XPA content in group NC increased significantly, indicating that the stimulation conditions in this test were effective. Compared with group NC, the XPA content in group PC decreased significantly, indicating that the positive control in this test was effective. Compared with group NC, the XPA content of competitor A-0.03%, competitor B powder-0.03%, and RJMPDRN-850K-0.03% decreased significantly, with inhibition rates of 22.46%, 37.32%, and 46.01%, respectively, indicating that the PDRN prepared in this invention can significantly downregulate the XPA content in UVB-irradiated keratinocytes.

[0068] 1.5.2.2 XPC protein test results For detailed XPC immunofluorescence results, please refer to... Figure 3-4 Table 9-10; Figure 3The images show fluorescence images of the experimental wells in each group. Note: The images were taken using a fluorescence microscope (Leica, DM2500LED) at a magnification of 200×. Blue fluorescence represents cell nuclei, and green fluorescence represents XPC. The stronger the green fluorescence, the higher the XPC content.

[0069] Table 9 shows the average relative integrated optical density (IOD) of XPC per cell number, which reflects the content of XPC. Table 10 shows the difference analysis among the following groups: competitor A-0.03%, competitor B-0.03%, and RJMPDRN-850K-0.03%. Figure 4 This is a bar chart showing the ratio of XPC relative integrated optical density (IOD) to the average cell number.

[0070] Table 9 Summary of XPC Immunofluorescence Analysis Results BC 1.00 0.10 / / NC 4.44 0.05 0.000## / PC 1.75 0.07 0.000** 60.59% Competitor A - 0.03% 3.83 0.08 0.000** 13.74% Competitor B - 0.03% 2.49 0.08 0.000** 43.92% RJMPDRN-850K-0.03% 1.81 0.06 0.000** 59.23% Note: Relative integrated optical density (IOD) / mean cell number reflects the XPC content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0071] Table 10 Summary of Results of Analysis of Significant Differences Between Samples Competitor A - 0.03% / / Competitor B powder -0.03% 0.000▲▲ / RJMPDRN-850K-0.03% 0.000▲▲ 0.000▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0072] Compared with the BC group, the XPC content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the XPC content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the XPC content of competitor samples A-0.03%, competitor B-0.03%, and RJMPDRN-850K-0.03% was significantly decreased, with inhibition rates of 13.74%, 43.92%, and 59.23%, respectively, indicating that the PDRN prepared in this invention can significantly downregulate the XPC content in UVB-irradiated keratinocytes.

[0073] 1.5.2.3, pATM-S1981 Test Results For detailed results of pATM-S1981 immunofluorescence, please refer to... Figure 5-6 Tables 11-12; Figure 5The images show fluorescence images of the experimental wells in each group. Note: The images were taken using a fluorescence microscope (Leica, DM2500LED) at a magnification of 200×. Blue fluorescence represents cell nuclei, and green fluorescence represents pATM-S1981. The stronger the green fluorescence, the higher the content of pATM-S1981.

[0074] Table 11 shows the average relative integrated optical density (IOD) / cell number of pATM-S1981, which reflects the content of pATM-S1981. Table 12 shows the difference analysis among competing products A-0.03%, competing product B-0.03%, and RJMPDRN-850K-0.03% in each group; Figure 6 This is a bar chart showing the relative integrated optical density (IOD) / average cell number for pATM-S1981.

[0075] Table 11 Summary of pATM-S1981 Immunofluorescence Analysis Results BC 1.00 0.04 / / NC 3.68 0.05 0.000## / PC 1.93 0.13 0.000** 47.55% Competitor A - 0.03% 3.61 0.05 0.148 / Competitor B - 0.03% 2.46 0.09 0.000** 33.15% RJMPDRN-850K-0.03% 1.98 0.09 0.000** 46.20% Note: The relative integrated optical density (IOD) / average cell number reflects the pATM-S1981 content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0076] Table 12 Summary of Results of Significant Difference Analysis Between Samples Competitor A - 0.03% / / Competitor B - 0.03% 0.000▲▲ / RJMPDRN-850K-0.03% 0.000▲▲ 0.003▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0077] Compared with the BC group, the pATM-S1981 content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the pATM-S1981 content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the pATM-S1981 content of competitor samples B-0.03% and RJMPDRN-850K-0.03% was significantly decreased, with inhibition rates of 33.15% and 46.20%, respectively; the pATM-S1981 content of competitor sample A-0.03% did not change significantly, indicating that the PDRN prepared in this invention can significantly downregulate the pATM-S1981 content in UVB-irradiated keratinocytes.

[0078] 1.5.2.4, p53-binding protein 1 test results For details of the p53-binding protein 1 immunofluorescence results, please refer to [link to details]. Figure 7-8 Tables 13-14; Figure 7 The images show fluorescence images of the experimental wells in each group. Note: The images were taken using a fluorescence microscope (Leica, DM2500LED) at a magnification of 200×. Blue fluorescence represents cell nuclei, and green fluorescence represents p53-binding protein 1. The stronger the green fluorescence, the higher the content of p53-binding protein 1.

[0079] Table 13 shows the average relative integrated optical density (IOD) of p53-binding protein 1 per cell number, which reflects the content of p53-binding protein 1. Table 14 shows the difference analysis among competing products A-0.03%, competing product B-0.03%, and RJMPDRN-850K-0.03% in the group; Figure 8 This is a bar chart showing the relative integrated optical density (IOD) of p53 binding protein 1 versus the average number of cells.

[0080] Table 13 Summary of immunofluorescence analysis results for p53-binding protein 1 BC 1.00 0.05 / / NC 2.13 0.08 0.000## / PC 1.22 0.03 0.000** 42.72% Competitor A - 0.03% 1.44 0.09 0.001** 32.39% Competitor B - 0.03% 1.25 0.09 0.000** 41.31% RJMPDRN-850K-0.03% 0.90 0.05 0.000** 57.75% Note: Relative integrated optical density (IOD) / mean cell number reflects the content of p53-binding protein 1. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0081] Table 14 Summary of Results of Analysis of Significant Differences Between Samples Competitor A - 0.03% / / Competitor B - 0.03% 0.049▲ / RJMPDRN-850K-0.03% 0.001▲▲ 0.002▲▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0082] Compared with the BC group, the p53-binding protein 1 content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the p53-binding protein 1 content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the p53-binding protein 1 content of competitor samples A-0.03%, competitor B-0.03%, and RJMPDRN-850K-0.03% was significantly decreased, with inhibition rates of 32.39%, 41.31%, and 57.75%, respectively. This indicates that the PDRN prepared in this invention can significantly downregulate the p53-binding protein 1 content in UVB-irradiated keratinocytes, and the effect is better than that of the positive control.

[0083] 1.5.2.5 γH2AX protein test results For detailed results of γH2AX immunofluorescence, please refer to [link / reference]. Figure 9-10 Tables 15-16; Figure 9 The images show fluorescence images of the experimental wells in each group. Note: The images were taken using a fluorescence microscope (Leica, DM2500LED) at a magnification of 200×. Blue fluorescence represents cell nuclei, and green fluorescence represents γH2AX. The stronger the green fluorescence, the higher the γH2AX content.

[0084] Table 15 shows the average relative integrated optical density (IOD) of γH2AX per cell number, which reflects the content of γH2AX. Table 16 shows the difference analysis among competing products A-0.03%, competing product B-0.03%, and RJMPDRN-850K-0.03% in the group; Figure 10 This is a bar chart showing the relative integrated optical density (IOD) of γH2AX / average cell number.

[0085] Table 15 Summary of γH2AX Immunofluorescence Analysis Results BC 1.00 0.11 / / NC 2.49 0.02 0.000 ## / PC 1.67 0.08 0.000 ** 32.93% Competitor A - 0.03% 2.11 0.14 0.010 * 15.26% Competitor B - 0.03% 1.46 0.07 0.000 ** 41.37% RJMPDRN-850K-0.03% 1.01 0.10 0.000 ** 59.44% Note: Relative integrated optical density (IOD) / mean cell number reflects the content of γH2AX. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0086] Table 16 Summary of Results of Analysis of Significant Differences Between Samples Competitor A - 0.03% / / Competitor B - 0.03% 0.002▲▲ / RJMPDRN-850K-0.03% 0.000▲▲ 0.003 ▲▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0087] Compared with the BC group, the γH2AX content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the γH2AX content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the γH2AX content of competitor samples A-0.03%, competitor B-0.03%, and RJMPDRN-850K-0.03% was significantly decreased, with inhibition rates of 15.26%, 41.37%, and 59.44%, respectively. This indicates that the PDRN prepared in this invention can significantly downregulate the γH2AX content in UVB-irradiated keratinocytes, and the effect is significantly better than the positive control.

[0088] 1.6 Testing based on a 3D epidermal skin model (EpiKutis®) irradiated with UVB. 1.6.1 Test Method 1.6.1.1 Test Grouping Table 17 Test Groups

[0089] 1.6.1.2. Drug administration and irradiation 1) Drug administration: According to the test group, the model was transferred to a 6-well plate and randomly divided into a blank control group (BC), a negative control group (NC), a positive control group (PC), and a sample group, with 3 replicates in each group. 0.9 mL of model culture medium was added to each well of the BC and NC groups, 0.9 mL of culture medium containing the corresponding positive control was added to each well of the PC group, and the sample group was administered the drug on the surface.

[0090] 2) UVB irradiation: After the drug administration was completed, all groups except the blank control group underwent UVB irradiation at a dose of 600 mJ / cm². 2 .

[0091] 3) Model incubation: After irradiation, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) for 24 hours.

[0092] 4) Cleaning: After incubation, clean the surface of the model with sterile PBS to remove any remaining test material, and wipe away any residual liquid inside and outside the model with sterile cotton swabs.

[0093] 1.6.1.3 Immunohistochemical test The model used for testing was fixed with 4% paraformaldehyde for 24 hours. Immunohistochemical detection was performed, and images were taken and analyzed under a microscope.

[0094] 1.6.1.4 Inhibition Rate Calculation ; 1.6.1.5 Statistical Analysis of Results GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0095] 1.6.2 Test Results 1.6.2.1 8-OHdG Test Results Table 18 Summary of 8-OHdG Immunohistochemical Analysis Results BC 1.00 0.08 / / NC 1.85 0.18 0.002## / PC 1.01 0.06 0.002** 45.41% Competitor A - 0.03% 1.40 0.06 0.014* 24.32% Competitor B - 0.03% 1.31 0.04 0.007** 29.19% RJMPDRN-850K-0.03% 1.06 0.04 0.002** 42.70% Note: The average relative integrated optical density (IOD) reflects the content of 8-OHdG. t When performing statistical analysis using the -test method, significance compared to group BC is indicated by #. P -Value < 0.05 is represented by #. P -Value < 0.01 is represented by ##; significance compared to the NC group is represented by *. P -value < 0.05 indicates as *. P -value < 0.01 is represented as **.

[0096] Table 19 Summary of Results of Analysis of Significant Differences Between Samples Competitor A - 0.03% / / Competitor B - 0.03% 0.083 / RJMPDRN-850K-0.03% 0.001▲▲ 0.001▲▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0097] Compared with the BC group, the 8-OHdG content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the 8-OHdG content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the 8-OHdG content of competitor samples A-0.03%, competitor B-0.03%, and RJMPDRN-850K-0.03% was significantly decreased, with inhibition rates of 24.32%, 29.19%, and 42.70%, respectively, indicating that the PDRN prepared in this invention can significantly downregulate the 8-OHdG content in the UVB-irradiated 3D epidermal skin model.

[0098] 1.6.2.2 CPD Test Results Table 20 Summary of CPD Immunohistochemical Analysis Results BC 1.00 0.20 / / NC 88.70 8.20 0.000## / PC 31.41 3.49 0.000** 64.59% Competitor A - 0.03% 66.91 7.99 0.030* 24.57% Competitor B - 0.03% 63.83 4.60 0.010* 28.04% RJMPDRN-850K-0.03% 38.36 5.77 0.001** 56.75% Note: The average relative integrated optical density (IOD) reflects the CPD content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0099] Table 21 Summary of Results of Significant Difference Analysis Between Samples Competitor A - 0.03% / / Competitor B - 0.03% 0.593 / RJMPDRN-850K-0.03% 0.007▲▲ 0.004▲▲ Note: Significance is indicated by ▲, P-value < 0.05 is indicated by ▲, P-value < 0.01 is indicated by ▲▲.

[0100] Compared with the BC group, the CPD content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the CPD content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the CPD content of competitor samples A-0.03%, competitor B-0.03%, and RJMPDRN-850K-0.03% was significantly decreased, with inhibition rates of 24.57%, 28.04%, and 56.75%, respectively, indicating that the PDRN prepared in this invention can significantly downregulate the CPD content in the UVB-irradiated 3D epidermal skin model.

[0101] 1.7 Conclusion Compared with the control group, keratinocytes irradiated with UVB showed significantly decreased levels of XPA, XPC, p53-binding protein 1, and γH2AX in sample A at a concentration of 0.03% (m / m), with inhibition rates of 22.46%, 13.74%, 32.39%, and 15.26%, respectively. Sample B also showed significantly decreased levels of XPA, XPC, pATM-S1981, p53-binding protein 1, and γH2AX in sample B at a concentration of 0.03% (m / m), with inhibition rates of 37.32%, 43.92%, 33.15%, 41.31%, and 41.37%, respectively. The contents of XPA, XPC, pATM-S1981, p53 binding protein 1, and γH2AX in sample RJMPDRN-850K at a concentration of 0.03% (m / m) were significantly reduced, with inhibition rates of 46.01%, 59.23%, 46.20%, 57.75%, and 59.44%, respectively.

[0102] Based on a 3D epidermal skin model (EpiKutis®) irradiated with UVB, compared to the control group, competitor sample A showed a significant decrease in 8-OHdG and CPD content at a concentration of 0.03% (m / m), with inhibition rates of 24.32% and 24.57%, respectively. Competitor sample B also showed a decrease in 8-OHdG and CPD content at a concentration of 0.03% (m / m), with inhibition rates of 29.19% and 28.04%, respectively. Sample RJMPDRN-850K showed a significant decrease in 8-OHdG and CPD content at a concentration of 0.03% (m / m), with inhibition rates of 42.70% and 56.75%, respectively.

[0103] In summary, this indicates that competitor A, competitor B, and RJMPDRN-850K all possess DNA damage repair capabilities. Among them, RJMPDRN-850K showed the best performance in CPD, 8-OHdG, pATM-S1981, p53-binding protein 1, γH2AX, XPA, and XPC, suggesting that it has a higher proportion of functional fragment populations, better end-base integrity, and lower stress residual burden, thus making it easier for skin cells to effectively process and utilize. Competitor B has a certain ability to improve DNA damage and good cell compatibility, but its improvement effect on each indicator is weaker than that of RJMPDRN-850K, suggesting that its effective fragment window enrichment degree and process refinement level are still limited. Competitor A performed the weakest in CPD, 8-OHdG, 53BP1, XPA and XPC, and showed no significant improvement in pATM-S1981, suggesting that it has complex quality defects such as fragment window deviation, poor end group status or high residual burden, and has the lowest overall biological function quality.

[0104] 2. Molecular weight determination Samples A, B, and RJMPDRN-850K were subjected to gel electrophoresis for molecular weight determination. The results are as follows: Figure 11 As shown in the results, competitor B, using a single enzyme digestion method, exhibits longer high molecular weight tails; competitor A, treated with a combination of wild-type DNase-1 and DNA exonuclease III, shows a diffuse distribution of bands with medium molecular weight tails; and RJMPDRN-850K, concentrated in the mid-range window (250-500bp), also exhibits certain medium and high molecular weight tails. This indicates that the dual enzyme digestion method provided by this invention does not simply break down DNA, but rather controls the fragment distribution to a target window more suitable for efficacy, avoiding excessive low molecular weight breakdown while reducing excessively long high molecular weight tails.

[0105] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for preparing polydeoxyribonucleotides, characterized in that, The preparation method includes enzymatic hydrolysis of genomic DNA substrate using a dual-enzyme component; The genomic DNA substrate was derived from salmon testes; The dual-enzyme component is a mixture of the deoxyribonuclease mutant DNase-1-E61K-S144W and DNA exonuclease III; The amino acid sequence of the deoxyribonuclease mutant DNase-1-E61K-S144W is shown in SEQ ID NO.

2.

2. The preparation method according to claim 1, characterized in that, In the dual-enzyme component, the mass ratio of the deoxyribonuclease mutant DNase-1-E61K-S144W to DNA exonuclease III is (2-3):(0.1-0.3).

3. The preparation method according to claim 1 or 2, characterized in that, The preparation method specifically includes the following steps: Step 1: Take frozen salmon testes, thaw at 4℃ and cut into 1-2 mm tissue blocks. Rinse three times with sterile physiological saline at 4℃ and drain. Add pre-cooled lysis buffer at a material-to-liquid ratio of 1g:5mL. Homogenize at 4℃ and 10000rpm for 30s each time with a 1min interval, repeating three times. Let the homogenate stand at 4℃ for 2 hours to swell and obtain the solution to be processed. Step 2: Add an equal volume of chloroform-isoamyl alcohol mixture to the solution to be treated, gently invert and mix for 10 min, centrifuge at 8000 rpm for 15 min at 4 °C, collect the upper aqueous phase, and repeat the extraction twice; add RNase A to the upper aqueous phase to a final concentration of 50 μg / mL, incubate at 37 °C for 30 min, and then extract once more with chloroform-isoamyl alcohol to remove enzymes and residual proteins, obtaining a purified aqueous phase; Step 3: Add 1 / 10 volume of 3 mol / L sodium acetate solution to the purified aqueous phase, mix well, add 2.5 volumes of pre-cooled anhydrous ethanol at -20℃, let stand at -20℃ for 2 hours to precipitate DNA, centrifuge at 12000 rpm at 4℃ for 20 minutes and discard the supernatant, wash the precipitate twice with 70% pre-cooled ethanol, vacuum dry at low temperature for 5 minutes, prepare a 4 mg / mL DNA substrate solution with enzyme digestion buffer, and filter through a 0.22 μm filter membrane for sterilization; Step 4: Add the dual enzyme components to the DNA substrate solution at a dosage of 0.1 wt% of the substrate solution, and incubate at 37°C for 1 hour. After inactivation, inactivate each enzyme by bathing at 75°C for 12 minutes. Centrifuge the inactivated enzyme solution at 4°C and 12,000 rpm for 10 minutes. Collect the supernatant and filter it through a 0.45 μm filter membrane. First, ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and collect the permeate. Then, concentrate the permeate through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and discard the permeate. Collect the retentate and freeze-dry it under vacuum to obtain polydeoxyribonucleotides.

4. The preparation method according to claim 3, characterized in that, The lysis buffer mentioned in step 1 is a buffer solution of 10 mmol / L Tris-HCl, 1 mmol / L EDTA, and pH 8.

0.

5. The preparation method according to claim 3, characterized in that, In step 2, the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixture is 24:

1.

6. The preparation method according to claim 3, characterized in that, The enzymatic digestion buffer described in step 3 is a buffer solution containing 10 mmol / L Tris-HCl, 5 mmol / L MgCl2, 1 mmol / L CaCl2, and pH 7.5 calibrated at 37°C.

7. A polydeoxyribonucleotide, characterized in that, The polydeoxyribonucleotide is prepared by the preparation method according to any one of claims 1-6.

8. The polydeoxyribonucleotide according to claim 7, characterized in that, The molecular weights of the polydeoxyribonucleotide fragments are concentrated in the 250-500 bp range.

9. The use of the polydeoxyribonucleotide according to claim 7 or 8 in the preparation of products with DNA damage repair function.

10. The application according to claim 9, characterized in that, The product is a cosmetic, skin care product, or topical pharmaceutical preparation.

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