Nucleic acid modified protein and application thereof

Abstract

The invention relates to the field of bioengineering technology and biomedicine, and discloses a nucleic acid modified protein and application thereof. The TdpC protein disclosed by the invention not only shows efficient catalytic activity in DNA phosphorus-sulfur acylation modification and specific adenylation of a DNA phosphate skeleton, but also can stably work in a high-temperature, strong-acidity or high-sulfur-enrichment extreme environment, so that the TdpC protein has wide application potential in the extreme environment. Experimental results fully prove that the method has high efficiency and applicability in the fields related to epigenetics research, genomics research and biological medicine.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering technology and biomedicine, and more particularly, to a nucleic acid-modified protein and its applications. Background Art

[0002] Deoxyribonucleic acid (DNA) is the material basis of life and the carrier of genetic information. As is well known, DNA is composed of four nucleotides A, T, C, and G formed by five elements: carbon, hydrogen, oxygen, nitrogen, and phosphorus. Their combination encodes various genes, ultimately giving rise to complex individual lives. Epigenetics is an important branch of genetics that studies heritable changes in gene expression caused by modifications without changes in the gene sequence. As an important component in the field of epigenetics, DNA methylation has been widely and deeply studied for its influence on gene expression and regulation through methylation modification of DNA bases. In recent years, a new type of epigenetics - DNA phosphorothioation modification, that is, the replacement of the non-bridging oxygen atom on the DNA backbone by a sulfur atom to form phosphorothioation, has been identified (Wang, L. et al. 2007. Nature Chemical Biology).

[0003] The DNA phosphorothioation modification of mesophilic bacteria is a complex biochemical process mediated by multiple proteins of the dnd or ssp modification system and involves their cooperative action. In the dnd modification system, DndA is a PLP-dependent cysteine desulfurase that catalyzes the desulfurization of L-cysteine to form L-alanine. In some strains lacking DndA, its function can be replaced by IscS, which has been shown to extract sulfur from free L-cysteine to generate a persulfidated enzyme compound (R-S-SH), and then use the persulfidated sulfur for the biosynthesis of sulfur-containing cofactors (Mueller, E. et al. 2006. Nature Chemical Biology; An, X. et al. 2012. PLOS ONE). The DndC protein containing a [4Fe-4S] cluster has a conserved PAPS reductase domain (i.e., COG0175) and exhibits ATP pyrophosphatase activity in vitro (You, D. et al. 2007. Biochemistry). DndD has ATP hydrolase activity and is thus considered to provide energy for the oxygen-sulfur exchange in the DNA backbone (Yao, F. et al. 2009. FEBS Letters). DndE forms a tetrameric conformation in vitro and shows a binding preference for double-stranded DNA containing a nick (Hu, W. et al. 2012. Cell Research).

[0004] However, the great difficulty in assembling the DndCDE complex and the extremely low efficiency of the in vitro modification reaction are not sufficient to analyze and explain the biochemical reaction mechanism of DNA phosphorothioation modification and thus enable various effective applications of the modification reaction. Summary of the Invention

[0005] To overcome the defects in the prior art such as the great difficulty in assembling the DndCDE complex, the low efficiency of the in vitro modification reaction, and the inability to be applied to high-temperature, acidic, or high-sulfur environments, etc., the present invention provides a new nucleic acid-modifying protein.

[0006] Another object of the present invention is to provide an application of the new nucleic acid-modifying protein.

[0007] To solve the above technical problems, the technical solution of the present invention is as follows:

[0008] A novel nucleic acid-modifying protein, the nucleic acid-modifying protein includes a TdpC protein encoded by the tdpC gene, a homologous protein that has obvious similarity with the TdpC protein and performs the same or similar functions in an organism or body, and a derivative protein that substitutes, deletes, or adds one or more amino acids to the amino acid sequence of the TdpC protein and performs the same or similar functions in an organism or body; the nucleotide sequence of the tdpC gene is as shown in SEQ ID NO.1, and the amino acid sequence of the TdpC protein is as shown in SEQ ID NO.2:

[0009] SEQ ID NO.1 (tdpC gene): atgaaggtttcccttgaagcgctggccgcggggataggggaaccctttgggtctttcgacctcaaagaggccacagacctggccgagcgcgccctcgaggccgccctcgtccagggggccgaggccttcgtcctcacctactcggggggcaaggactccaccgccaccaccgtcctcaccctggagtggtggaagcgccgggggaagcccgtggagatccacgtggtctacgccgacacggggctggagatccccaccctccacgcccaggccctggccttcctggaggcggtgaagaggctccaccccggggtccacgttcacaccgcccgcccccgccccgaggagagcttctgggtccaaatcatcggcaagggctaccccccgccccacaaccgcttccgctggtgcacccgcaggttgaagatcgcccccatggaccgcctggtccagagccttcccgggaagaaggccatcctcacgggggtgcgcttcggggagtcggacgcccgggaccagaggctcatcctctcttgctcccggggcggggagtgcggccagggggtgctcttccaggaggccaagcgcctgaatgccctctacgtggcccccatcgccttctggcgggagtgcttcgtgtgggactacctgaacttcgtggccccctccctgggctaccccacggagggcctggaggcggtctacgggggccgggacacccgcttcgggtgctggacctgcaccgtggtgcggcgggacaaggccatggcgcgggccctggagaacggccacgcccacctcctccccctctacgagttccgggagtggctttgggcgtggacccgggatccccggacccgggagaagcggaaggacggcaagcccgggaggctcaccctggaggccaggcgggaggtgtaccggaggttgaaggaggtggaggcgaagctcgggatggagttcctcacttccgaggaggaggcgctcattcagaagctgtggcgttccgggaggtacaatggaaaacgcaggtga;

[0010] SEQ ID NO.2 (TdpC protein): MKVSLEALAAGIGEPFGSFDLKEATDLAE RALEAALVQGAEAFVLTYSGGKDSTATTVLTLEWWKRRGKPVEIHVVYADTGLEIPTLHAQALAFLEAVKRLHPGVHVHTARPRPEESFWVQIIGKGYPPPHNRFRWCTRRLKIAPMDRLVQSLPGKKAILTGVRFGESDARDQRLILSCSRGGECG QGVLFQEAKRLNALYVAPIAFWRECFVWDYLNFVAPSLGYPTEGLEAVYGGRDTRFGCWTCTVVRRDKAMARALENGHAHLLPLYEFREWLWAWTRDPRTREKRKDGKPGRLTLEARREVYRRLKEVEAKLGMEFLTSEEEALIQKLWRSGRYNGKRR。

[0011] Preferably, at least one cysteine residue in the TdpC protein is responsible for the sulfur transfer process.

[0012] An application of the nucleic acid modification protein, which is applied to the preparation of drugs for sulfur transfer reactions.

[0013] Preferably, it is applied to the identification of related amino acids responsible for sulfur transfer reactions.

[0014] Furthermore, it is applied to nucleic acid phosphorothioation modification.

[0015] An application of the nucleic acid modification protein, which is applied to the preparation of drugs for activating the nucleic acid phosphate backbone.

[0016] Furthermore, it is applied to the specific adenylation of the nucleic acid phosphate backbone.

[0017] A nucleic acid modification system, the components of the nucleic acid modification system include at least one of the nucleic acid modification protein TdpC or the tdpC gene.

[0018] Furthermore, the components further include buffer, ATP, sulfur source, magnesium ions.

[0019] Preferably, the components further include nucleic acid substrates.

[0020] Preferably, the sulfur source includes Na2S, L-cysteine.

[0021] Furthermore, the nucleic acid modification system can be applied at a temperature of 95 °C or below.

[0022] Preferably, it is applied at 20 - 65 °C.

[0023] A method for nucleic acid phosphorothioation modification, which uses the nucleic acid modification system for nucleic acid phosphorothioation modification.

[0024] An application of a nucleic acid modification system, which is applied to fields including bioengineering, enzyme engineering, fermentation engineering, synthetic biology, and biomedicine.

[0025] Preferably, it is applied to biotechnology development, in vitro and in vivo nucleic acid modification and transformation, engineering bacteria transformation, synthetic biology, nucleic acid drugs, mRNA vaccines, and phage therapy technologies.

[0026] Preferably, it is applied to sulfur transfer reactions.

[0027] The novel phosphorothioation modification gene cluster specific to thermophilic microorganisms of the present invention has a unique gene composition and distinct functional proteins. The novel thermophilic nucleic acid phosphorothioation modification system of the present invention contains the tdpC gene or the protein TdpC encoded by it. The TdpC protein encoded by the tdpC gene can endow related hosts with the ability of nucleic acid phosphorothioation modification, and help related thermophilic microorganisms adapt to complex extreme environments such as high temperature, strong acidity, or high sulfur enrichment. The present invention innovatively uses the novel thermophilic nucleic acid phosphorothioation modification system to identify the molecular mechanism of sulfur atom transfer to the nucleic acid backbone under the participation of the special sulfur metabolism pathway of thermophilic bacteria, as well as the functions and biochemical activities of key proteins in the system.

[0028] The gene sequence and protein information of tdpC of the present invention provide a basis for genetic transformation and can be applied to many fields such as biotechnology development, in vitro and in vivo nucleic acid modification and transformation, engineering bacteria transformation, synthetic biology, nucleic acid drugs, mRNA vaccine development, and phage therapy technology development.

[0029] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0030] 1. Application of a novel nucleic acid modification protein. The TdpC protein of the present invention, as a novel nucleic acid modification protein, has unique catalytic activity and can specifically modify DNA under specific conditions, providing a powerful tool for nucleic acid drug modification and mRNA vaccines, etc.

[0031] 2. Application of DNA phosphorothioation modification. The present invention provides a novel method for DNA phosphorothioation modification, which realizes the sulfurization modification of the DNA phosphate backbone by using the TdpC protein. Phosphorothioation modification not only affects the chemical structure of DNA but also plays an important role in gene expression regulation, DNA repair, etc., providing a new tool for biotechnology and phage therapy technology development.

[0032] 3. DNA specific adenylation. The TdpC protein in the present invention also has the catalytic function of specifically adenylating the DNA phosphate backbone. Adenylation is a reaction in which the adenyl group of ATP is transferred to the DNA phosphate backbone. This property makes the TdpC protein more diverse in its application in DNA modification, and may affect the physicochemical properties of DNA, thereby regulating gene expression and the interaction between DNA and other biomolecules.

[0033] 4. Tolerance to extreme environments. The TdpC protein is derived from Thermus antranikianii JCM19900 and can work stably in complex environments with high temperature, strong acidity or high sulfur enrichment. This makes the protein have great potential in applications under extreme environments, especially in biocatalytic reactions under high temperature, acidic or sulfur-rich conditions, with significant advantages. The high-temperature resistance of this technology not only expands its application fields in synthetic biology and engineering bacteria transformation, but also enhances its adaptability in extreme industrial environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows the gene composition and arrangement of the tdpABC gene cluster in Thermus antranikianii JCM19900.

[0035] Figure 2 is a schematic diagram of plasmid pTH, with pUC19 as the original backbone, which contains the repA fragment responsible for autonomous replication on the endogenous plasmid pTT8 from Thermus thermophilus HB8, and a high-temperature kanamycin resistance gene HTK.

[0036] Figure 3 is a schematic diagram of plasmid pTH-tdpABC, with pTH as the initial backbone, which contains the tdpABC gene cluster from Thermus antranikianii JCM19900.

[0037] Figure 4 shows the content of d(G PS A) phosphorothioation modification of genomic DNA after heterologous expression of pTH-tdpABC and its derivative plasmids in HB8. The relative d(G PS A) content of the pTH-tdpABC heterologous expression strain was set to 100%. Error bars represent the standard deviation of three independent repeated experiments, and ns indicates not significant.

[0038] Figure 5 shows the results of the gene knockout experiment of JCM19900, and the method is set the same as Figure 4 .

[0039] Figure 6 These are the results of the phosphorothioation modification reaction completed by the TdpC protein using two different sulfur sources in vitro and the identification of essential factors.

[0040] Figure 7 These are the results of the identification of the intermediate product of the phosphorothioation modification reaction catalyzed by TdpC detected by radioactively labeled [α- 32 P]ATP.

[0041] Figure 8 These are the results of the proteomic identification of TdpC. Specific Embodiments

[0042] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0044] Example 1

[0045] 1. Establishment of a novel phosphorothioation modification system

[0046] In order to specifically establish a system mediating novel phosphorothioation modification, first, a total of 31,478 sequenced bacterial and archaeal genomes were downloaded from the NCBI Reference Sequence Genome Database. All genomes containing the COG0175 domain were retrieved from them, and 15 open reading frames upstream and downstream of the COG0175 gene were extracted to establish a protein sequence database. The functional annotations and domains of the coding products of a total of 30 genes upstream and downstream of the COG0175 gene in the database were analyzed by sequence homology alignment and classified.

[0047] According to the classification results, among the 31,478 complete microbial genomes, there are 31,146 COG0175 genes. Among them, 20.7% of the coding products are CysH, which belongs to the PAPS reductase family and is responsible for degrading PAPS into 3'-phosphoadenosine-5'-phosphate (Table 1), but these genes are not the target genes of the novel phosphorothioation modification system of the present invention. Usually, the cysH gene is arranged in a cluster with cysDN (genes encoding two subunits of ATP sulfurylase), cysJ (gene encoding sulfite reductase flavoprotein), and cysI (gene encoding sulfite reductase hemoglobin), and they jointly participate in the key enzymatic reaction of synthesizing sulfide from sulfate in the sulfate assimilation process.

[0048] COG0175 genes adjacent to cysDNJI in complete microbial genomes

[0049]

[0050] Note: Only 10 representative strains are listed.

[0051] In addition, dndD and sspC genes were identified near 776 and 463 COG0175 genes respectively, indicating that they may belong to the dnd and ssp phosphorothioation modification systems respectively, and they are not the target genes of the novel phosphorothioation modification system of the present invention (Table 2, Table 3).

[0052] COG0175 genes adjacent to dndD in complete microbial genomes

[0053]

[0054] Note: Only 10 representative strains are listed.

[0055] COG0175 genes adjacent to sspC in complete microbial genomes

[0056]

[0057] Note: Only 10 representative strains are listed.

[0058] Finally, it was found that 26 COG0175 genes formed a highly conserved gene cluster arrangement with two other genes. This gene cluster specifically exists in thermophilic bacteria and hyperthermophilic archaea, including genera such as Thermus, Sulfolobus, Metallosphaera, Acidianus, Thermocrinis, etc. In addition to the COG0175 gene, the gene cluster also contains a gene encoding a HerA-like ATP hydrolase and a gene encoding a NurA-like nuclease, which are similar to the functions of some proteins in the dnd and ssp modification systems, further increasing the possibility of this gene cluster being a novel phosphorothioation modification system. Further searching of 246,040 draft genomes in the NCBI RefSeq database found that this conserved gene cluster composed of three genes also exists in another 55 thermophilic microbial genomes.

[0059] 2. Identification of the modification function of the tdp phosphorothioation modification system

[0060] Co-transcription analysis was performed on the three genes tdpC, tdpB, and tdpA in the genome of Thermus antranikianii JCM19900. JCM19900 was cultured in the laboratory and genomic DNA was extracted, followed by enzymatic digestion and mass spectrometry detection.

[0061] In the genome of Thermus antranikianii JCM19900, the three genes tdpC, tdpB, and tdpA are arranged in a tandem cluster in the same direction ( Figure 1 ). Co-transcription analysis found that the three genes formed an operon and were co-transcribed, indirectly reflecting the correlation of their physiological functions. After mass spectrometry detection, it was found that the genome contained d(G PS A) phosphorothioation modification.

[0062] Example 2

[0063] Construction of Thermus thermophilus HB8-Escherichia coli shuttle plasmid series vectors

[0064] Based on the Escherichia coli plasmid pUC19, first, the repA fragment responsible for autonomous replication on the endogenous plasmid pTT8 of T. thermophilus HB8 was amplified by PCR and ligated to the PciI digestion site of pUC19. Then, the obtained pUC19-repA vector was double-digested with KpnI and XbaI, and a DNA fragment of the heat-resistant kanamycin resistance gene HTK was inserted as a screening marker to obtain the pTH vector. By amplifying the tdpABC gene cluster on the JCM19900 genome and its own promoter fragment and inserting it between the XbaI and HindⅢ digestion sites of the pTH vector, the heterologous expression vector pTH-tdpABC was obtained for natural transformation of HB8, and a kanamycin resistance plate was spread to screen for transformants. Then, the transformants were picked for bacterial liquid culture, genomic DNA was enzymatically digested, and mass spectrometry detection was performed.

[0065] Thermus thermophilus HB8 is a model Thermus strain whose genome does not contain the tdp gene cluster and is often used in the study of fermentation products of thermophilic microorganisms. To heterologously express the gene cluster from Thermus antranikianii JCM19900 in T. thermophilus HB8, this project designed and constructed a Thermus-Escherichia coli shuttle vector pTH( Figure 2 ). The heterologous expression vector pTH-tdpABC is as Figure 3 shown.

[0066] Mass spectrometry detection found that the tdp gene cluster from JCM19900 conferred a new d(GPS A) Modification, with a modification frequency similar to that of the source bacterium. By performing mass spectrometry identification on the genomes of heterologous expression strains with single-gene deletions, it was found that the deletions of tdpA and tdpB did not affect the content of phosphorothioation modification, and the d(G PS A) content produced by the heterologous expression of the pTH-tdpC plasmid expressing tdpC alone and its own promoter was also not significantly different from that of pTH-tdpABC ( Figure 4 ).

[0067] Example 3

[0068] tdpC is the only essential gene for DNA phosphorothioation modification

[0069] Using the principle of homologous recombination double exchange and the kanamycin resistance gene for high-temperature as the screening marker, the tdp gene in the JCM19900 genome was constructed with a frame deletion to obtain a gene knockout strain. The construction process of the gene knockout strain of Thermus thermophilus JCM19900 is as follows: According to the principle of double exchange homologous recombination, the tdp gene in the JCM19900 genome was subjected to a frame deletion. The 500bp sequences upstream and downstream of the gene to be knocked out were selected as the left and right arms, and the kanamycin resistance gene for high-temperature HTK was used as the screening marker. After PCR amplification of each fragment, fusion PCR was performed in the form of left arm-HTK-right arm to obtain the fusion product. The fusion fragment was inserted into the pUC19 plasmid to obtain the Thermus thermophilus knockout vector. The correctly sequenced knockout vector was introduced into JCM19900 cells by natural transformation. Since this plasmid vector cannot replicate autonomously in Thermus thermophilus, transformants that have successfully undergone double exchange homologous recombination can be screened using kanamycin resistance plates. Finally, by extracting the genomic DNA of the transformants and designing corresponding verification primers for PCR identification, the PCR products were sent for sequencing to confirm the correct gene knockout strain.

[0070] The mass spectrometry detection results of the mutant strains showed that after the complete tdpABC gene cluster was deleted, JCM19900 lost the phosphorothioation modification phenotype, while the mutant strains with single deletions of tdpA or tdpB had a phosphorothioation modification frequency equivalent to that of the wild-type strain ( Figure 5 ). The above results indicate that in the tdp phosphorothioation modification system of Thermus thermophilus, tdpC is the essential gene mediating sulfur modification, and tdpA and tdpB have no significant effect on the modification frequency.

[0071] Example 4

[0072] TdpC protein catalyzes the DNA phosphorothioation modification reaction under in vitro conditions

[0073] Using the Thermus antranikianii JCM19900 genome as a template, PCR reactions were carried out with primer pairs 5’-GTGCCG CGCGGCAGCCATATGATGAAGGTTTCCCTTGAA-3’ and 5’-CTCGAGTGCGG CCGCAAGCTTTCACCTGCGTTTTCCATT-3’. Homologous recombination was performed with the pET28a plasmid that had been digested with NdeI and HindIII and recovered, to obtain the circular plasmid pET28a-19900tdpC, which was used for the expression and purification of the TdpC protein. Subsequently, pET28a-19900tdpC was transformed into competent E. coli BL21(DE3) cells and spread on LA plates containing kanamycin resistance, and cultured overnight at 37°C. After monoclonal colonies grew out, the transformants were picked and placed in 50 mL centrifuge tubes for liquid overnight culture. The next day, the overnight culture was transferred at a ratio of 1:100 to 1 L of LB medium containing kanamycin resistance, and cultured at 37°C with shaking at 220 rpm for 3 - 4 h. When the OD600 of the bacterial solution reached 0.8 - 1.0, the culture was transferred to a shaker pre-cooled at 16°C. After complete cooling, an IPTG solution with a final concentration of 0.2 mM was added, and the bacteria were cultured at 16°C with shaking at 180 rpm for 16 - 18 h to induce the expression of the target protein. To prepare the holo-TdpC protein with intact iron-sulfur clusters, 250 μM ammonium ferric citrate and 100 μM L-cysteine were added simultaneously to the LB medium during IPTG induction, and the iron-sulfur cluster reconstruction system in E. coli was used to reconstruct the iron-sulfur clusters of the overexpressed TdpC. After the IPTG induction ended, the bacterial solution was centrifuged at 4°C and 4000 rpm for 20 min to collect the cell pellet. The pellet was quickly frozen with liquid nitrogen and immediately transferred into an anaerobic glove box containing 95% nitrogen, 5% hydrogen, and an oxygen concentration lower than 1 ppm. The cell pellet was resuspended with 25 mL of pre-deoxygenated nickel column equilibration buffer, protease inhibitor PMSF was added after thoroughly pipetting and mixing evenly with a pipette, and ultrasonic disruption was carried out. Under ice bath conditions, disruption was continued until the bacterial solution became clear and no longer viscous. The disrupted solution was centrifuged at 4°C and 14,000 rpm for 1 h to remove cell debris, and the supernatant was collected. Then the supernatant was incubated in a metal bath at 50°C for 20 - 30 min for heat treatment, and the supernatant was collected again by centrifugation. Subsequently, nickel column affinity chromatography was carried out on the supernatant under anaerobic conditions, and both the equilibration solution and the elution solution used were pretreated by deoxygenation. The gradient elution fractions were collected, and the holo-TdpC protein showed an obvious brownish-black color. The eluate obtained in the previous step was passed through Sephadex TMDesalination was performed using a G-25PD-10 column, and the buffer was exchanged to the heparin column equilibrium buffer. The protein was further purified using a heparin affinity chromatography column. After loading the sample, the column was repeatedly loaded 2 - 3 times. Then, it was washed with the equilibrium buffer for 5 column volumes to remove the miscellaneous proteins that non-specifically bound to the heparin column. Subsequently, it was rinsed with the elution buffer containing a high concentration of NaCl for 3 column volumes, and the eluate was collected. The eluate was again exchanged to the heparin column equilibrium buffer, concentrated to approximately 2 mg / mL, and stored in a 4°C refrigerator in an anaerobic glove box for later use.

[0074] In vitro phosphorothioation modification reconstruction was carried out using the TdpC protein, and the entire reaction was performed in a strictly anaerobic glove box. The standard reaction system contained 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl 2 , 1 mM ATP, 5 mM Na 2 S, 20 μM DNA substrate (30 bp, containing a GATC site), and 1 μM TdpC protein. Among them, L-cysteine can replace Na 2 S as the sulfur source. At this time, 10 μM 5'-pyridoxal phosphate (PLP) and 10 μM cysteine desulfurase (IscS or SufS, derived from JCM19900) were additionally added to the system. After mixing the reaction system, it was incubated in a metal bath at 50°C for 1 h, then taken out of the glove box, 1 / 10 volume of sodium acetate (3 M) and 2 volumes of absolute ethanol were added, mixed well, and ethanol precipitation was carried out at -40°C overnight to recover the DNA. The DNA precipitate was washed with 70% ethanol, air-dried, and dissolved in 96 μL ddH 2 O. Finally, NPI nuclease digestion and alkaline phosphatase treatment were carried out. After passing through the column and drying by rotation, the sample was loaded onto LC-MS / MS for detecting the production amount of d(G PS A)R P .

[0075] As Figure 6 shown, under the condition that Na 2 S is used as the sulfur source, TdpC can independently complete the DNA phosphorothioation modification reaction in vitro, and its essential factors include magnesium ions and ATP. The in vitro modification activity of TdpC is relatively high. Roughly estimated, the production amount of d(G PS A) generated by incubating 1 μM TdpC protein with 20 μM DNA oligo substrate at 50°C for 1 h is approximately 10 times that of the enzymatic digestion product of 20 μg genomic DNA. L-cysteine alone cannot be used as a sulfur source by TdpC. Only when IscS or SufS is added to the system and in the presence of cysteine desulfurase activity, L-cysteine can be used as a sulfur donor for the biosynthesis of phosphorothioation modification ( Figure 6)。In addition, the apo-TdpC protein after stripping the iron-sulfur cluster under the action of EDTA can still complete the phosphorothioation modification reaction using organic sulfur sources and inorganic sulfur sources, but its activity decreases, about half of that of holo-TdpC.

[0076] Example 5

[0077] TdpC activates the DNA phosphate backbone through specific adenylation

[0078] Use radioactive α- 32 P-labeled ATP to detect whether there is an adenylated DNA intermediate during the phosphorothioation reaction. According to the aforementioned standard system for the TdpC protein to catalyze the DNA phosphorothioation modification reaction in vitro, the following reaction system was prepared under anaerobic conditions, including 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl 2 , 10 μM DNA substrate (30 bp, containing a GATC site), 5 μM TdpC protein, and 10 μCi [α- 32 P]ATP (Perkin-Elmer), with a total volume of 20 μL. After incubating the system at 50 °C for 90 min, 2×RNA deionized formamide gel loading buffer was added to terminate the reaction, and then it was loaded onto a urea-denaturing polyacrylamide gel for electrophoresis detection. The formula for the 10% urea gel is as follows: total volume 10 mL, 30% acrylamide 3.3 mL, urea 4.8 g, 5×TBE buffer 1 mL, 10% APS 20 μL, TEMED 5 μL, made up with ddH 2 O. The electrophoresis buffer is 0.5×TBE. Other matters that need attention are: (1) Before loading, use a pipette tip to suck the electrophoresis buffer and blow out the urea in the gel wells; (2) Pre-cool the electrophoresis buffer at 4 °C; (3) The electrophoresis time should be long enough to allow the unreacted ATP to run out of the gel as much as possible to avoid affecting the detection of the radioactive signal of the intermediate. Electrophoresis is generally carried out at a constant voltage of 300 V for 50 min. After completion, remove the gel, wrap it with plastic wrap, and place it in a phosphor screen and press it tightly. Place the phosphor screen in the refrigerator overnight. The next day, discard the gel and develop it with a phosphor screen imaging system (Fujifilm).

[0079] As Figure 7 shown, after TdpC reacts with the 30 bp DNA oligo containing 5'-GATC-3' and [α- 32 P]ATP, a radioactive signal band was observed at the corresponding position of the substrate DNA, indicating that TdpC transferred the α- 32 P in the radioactive ATP to the DNA substrate, that is, an AMP-adenylated DNA intermediate was generated, further proving that TdpC indeed activates the DNA phosphate backbone through specific adenylation.

[0080] Example 6

[0081] TdpC protein profiling identifies key cysteine ​​residues responsible for sulfur transfer

[0082] The anaerobically purified TdpC protein was run on a gel under denaturing and non-reducing conditions. The position of the TdpC protein on the gel was then determined based on the molecular weight of the protein and the gel was cut. The cut gel strip was dissolved and then subjected to protein spectrum identification to find out whether a disulfide bond was formed inside the TdpC protein and its location.

[0083] In order to explore how the sulfur atom in L-cysteine ​​or sulfide is transferred to the DNA backbone by TdpC to form phosphorothioate modification, protein spectrum identification was performed. The results showed that in anaerobically purified TdpC, C179 and C185 formed a disulfide bond. The m / z identified by mass spectrometry was 1106.05, which was consistent with the trypsin hydrolyzed peptide fragment. 175 LILSCSR 181 and 182 GGECGQGVLFQEAK 195 [M+2H] linked by disulfide bonds 2+ Precursor ions are consistent ( Figure 8 ). Based on the results, we propose a catalytic model for TdpC: First, the persulfide state of IscS attacks the C179-C185 disulfide bond, generating the persulfide state of TdpC on C179 or C185, while making another cysteine ​​residue a free sulfhydryl group. Subsequently, the sulfide ion generated by the deprotonation of the free sulfhydryl group attacks the persulfide bond, thereby releasing the persulfide sulfur. When the persulfide sulfur nucleophilically attacks the adenylated DNA intermediate and generates a phosphorothioate modification, the disulfide bond between C179 and C185 of TdpC is reformed.

[0084] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A nucleic acid modifying protein, characterized in that The nucleic acid modified protein includes the TdpC protein encoded by the tdpC gene, a homologous protein that has obvious similarity to the TdpC protein and performs the same or similar functions in an organism or body, and a derivative protein that replaces, deletes, adds one or more amino acids to the amino acid sequence of the TdpC protein and performs the same or similar functions in an organism or body; the tdpC gene nucleotide sequence is shown in SEQ ID NO.1, and the TdpC protein amino acid sequence is shown in SEQ ID NO.

2.

2. An application of the nucleic acid modified protein according to claim 1, characterized in that: Used in the preparation of drugs for sulfur transfer reactions.

3. The use of the nucleic acid modified protein according to claim 2, characterized in that: Applied to phosphorothioate modification of nucleic acids.

4. An application of the nucleic acid modified protein according to claim 1, characterized in that: Used in the preparation of drugs that activate nucleic acid phosphate backbones.

5. The use of the nucleic acid modified protein according to claim 4, characterized in that: Application to the specific adenylation of the phosphate backbone of nucleic acids.

6. A nucleic acid modification system, characterized in that: The components of the nucleic acid modification system include at least one of the nucleic acid modification protein or the tdpC gene according to claim 1.

7. The nucleic acid modification system according to claim 6, characterized in that: The components of the nucleic acid modification system also include a buffer, ATP, a sulfur source, and magnesium ions.

8. The nucleic acid modification system according to claim 6, characterized in that: The nucleic acid modification system can be used at a temperature of 95°C or below.

9. A method for phosphorothioate modification of nucleic acid, characterized in that: The nucleic acid modification system according to claim 6 is used to carry out nucleic acid phosphorothioate modification.

10. An application of a nucleic acid modification system, characterized in that: Applications include bioengineering, enzyme engineering, fermentation engineering, synthetic biology, and biomedicine.

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