Sponge terminal deoxynucleotidyl transferase and application thereof

Abstract

The invention relates to sponge terminal deoxynucleotidyl transferase and application thereof, and belongs to the technical field of genetic engineering. The invention provides sponge terminal deoxynucleotidyl transferase. The amino acid sequence of the sponge terminal deoxynucleotidyl transferase is shown as SEQ ID NO: 2. It is found that AquTdT derived from coral sponge not only has terminal deoxynucleotidyl transferase activity under in vitro conditions, but also has different nucleotide transfer preference from TdT derived from vertebrates. Therefore, the AquTdT has better selectivity for a specific sequence or a terminal structure, and non-specific addition can be reduced in a practical application process, so that complementation with mammal TdT is formed, and the method is suitable for industrial processes needing different preferences.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a sponge-terminal deoxynucleotidyl transferase and its applications. Background Technology

[0002] Terminal deoxyribonucleotidyl transferase (TdT) is a polymerase belonging to the PolX polymerase protein family. TdT is unique in that it can add deoxynucleotides to the 3' end of the DNA strand without a template. This unique biochemical property makes it irreplaceable in various biotechnologies. For example, the TUNEL (terminal deoxynucleotidyl transferased UTP nick-end labeling) assay, developed in 1992, uses TdT to label the ends of DNA double-strand breaks, becoming a classic method for detecting programmed cell death (apoptosis). In RACE (rapid amplification of cDNA ends) technology, TdT is used to add sequences to the ends of cDNA, providing primer binding sites for subsequent PCR amplification. Furthermore, TdT is also an important tool for in vitro DNA synthesis. In 2018, Palluk et al. developed a controlled DNA synthesis strategy based on TdT-dNTP conjugates. This method exhibits highly efficient nucleotide elongation capabilities, providing an important foundation for high-fidelity and high-efficiency DNA synthesis. In addition, TdT plays an important role in DNA information storage and cell lineage tracing.

[0003] Currently, commonly used TdT proteins are mainly derived from mammals, such as cattle and mice. Among the various mammalian TdTs, bovine TdTs have become the preferred choice for nucleic acid tool development and related detection methods due to their more efficient DNA tailing ability acquired during evolution, and commercial kits based on bovine TdTs are widely available on the market. Commercially available TdT proteins are generally obtained by expressing terminal transferase genes in E. coli to obtain active TdT proteins.

[0004] While mammalian-derived TdT possesses strong terminal transferase activity, it exhibits a significant base preference during catalysis. In mammals, the preferred order of TdT for the four deoxynucleotides is typically dGTP > dCTP > dTTP > dATP. This preference affects the catalytic efficiency of TdT in practical applications, altering the relative proportions of different products and thus limiting the application range of mammalian TdT to some extent. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sponge-terminal deoxynucleotidyl transferase and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a sponge-terminal deoxynucleotidyltransferase, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] As a preferred embodiment of the first aspect, the nucleotide sequence of the sponge terminal deoxynucleotidyl transferase is shown in SEQ ID NO: 1.

[0008] To address the issue of mammalian-derived TdT proteins exhibiting a significant preference for polymerizing dGTP and dCTP during catalysis, we systematically screened TdT-like sequences from various invertebrates. Ultimately, we identified an active TdT-like protein (hereinafter referred to as AquTdT) in the invertebrate sponge (Amphimedon queenslandica, Aqu). This protein was successfully expressed and purified in the 293T cell line. In vitro enzyme activity assays confirmed that AquTdT possesses significant terminal transferase activity. Furthermore, we discovered that its base preference is significantly different from that of vertebrate TdTs; AquTdT shows a higher polymerization preference for dATP, exhibiting the order dATP>dTTP>dGTP>dCTP. This characteristic suggests that AquTdT has potential advantages in overcoming the application limitations caused by the base preference of mammalian TdTs, providing new possibilities for developing terminal transferases with higher controllability and broader applicability.

[0009] In a second aspect, the present invention provides a recombinant vector containing the nucleotide sequence as described in the first aspect.

[0010] Thirdly, the present invention provides a recombinant cell containing the recombinant vector as described in the second aspect, and the cell expressing the spongy terminal deoxynucleotidyl transferase as described in the first aspect.

[0011] Fourthly, the present invention provides the application of the sponge-terminal deoxynucleotidyl transferase described in the first aspect and the recombinant vector described in the second aspect in the synthesis of nucleic acid molecules.

[0012] As a preferred embodiment of the fourth aspect, the synthesis involves adding a deoxynucleotide to the 3' end of the starting nucleic acid chain.

[0013] As a preferred embodiment of the fourth aspect, the preference for adding deoxynucleotides is dATP>dTTP>dGTP>dCTP.

[0014] Fifthly, the present invention provides a method for synthesizing nucleic acid molecules, the method comprising the following steps: contacting a starting nucleic acid chain with at least one deoxynucleotide in the presence of the sponge-terminal deoxynucleotidyltransferase described in the first aspect.

[0015] In a sixth aspect, the present invention provides a kit comprising the spongy terminal deoxynucleotidyl transferase as described in the first aspect, one or more nucleotides, and at least one starting nucleic acid strand.

[0016] In a preferred embodiment of the sixth aspect, the starting nucleic acid fragment is single-stranded DNA.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: We discovered that AquTdT derived from coral sponges not only possesses nucleotide terminal transferase activity under in vitro conditions, but also exhibits a different nucleotide preference compared to vertebrate TdT. This allows AquTdT to have better selectivity for specific sequences or terminal structures, and reduces non-specific addition during practical applications, thus complementing mammalian TdT and making it suitable for industrial processes requiring different preferences. Attached Figure Description

[0018] Figure 1 A schematic diagram of pFXB plasmid construction (a is a schematic diagram of pFXB plasmid map, b is the restriction enzyme site sequence that replaced the intermediate sequence from T7 primer to SP6 primer in pcDNA3.0 vector). Figure 2 Electrophoresis image of purified AquTdT protein; Figure 3 A schematic diagram illustrating the principle of in vitro detection of AquTdT protein activity on single-stranded DNA. Figure 4 A schematic diagram showing the results of in vitro detection of AquTdT protease activity and preference (A represents AquTdT protein in Mn). 2+ Under certain conditions, it exhibits significant terminal transferase activity; B represents the AquTdT protein's most preferred polymerase for dATP, and MmuTdTS is the mouse TdT; both figures use single-stranded DNA as the substrate. Detailed Implementation

[0019] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0020] Example 1: Construction, expression, and purification of the AquTdT eukaryotic expression vector 1. Construction of AquTdT eukaryotic expression vector (1) Constructing the pFXB plasmid: This plasmid was modified by the applicant based on the commercial eukaryotic expression plasmid pcDNA3.0, and its plasmid map is shown below. Figure 1 As shown in a. Replace the intermediate sequence from T7 primer to SP6 primer in the pcDNA3.0 vector with the following: Figure 1 The enzyme cleavage site sequence shown in b is provided to offer more cleavage sites.

[0021] (2) Construction of AquTdT eukaryotic expression vector: Artificially synthesized coral sponges in the NCBI database ( Amphimedon queenslandica The TdT gene (XM_019996299) was extracted and inserted between the BamHI and XhoI restriction sites of plasmid pFXB. The sequence of the AquTdT gene fragment is shown in SEQ ID NO: 1:

[0022] The amino acid profile of the AquTdT protein is shown in SEQ ID NO: 2. MDSKRRKQDEGECDHVSKRHQGEKEDEGRRSEQVLYLVKNKLTAKHCNHLKSLASKNGISVTETFNNNVTHIVTVLPSLERVKEVLGKSDFGSTDVVTLDWLTACFIEGRYVQVTDQYRLKEVRTEAV QTSEKKESAANVAEITAYECQRPTLLKHHNPHITEALELLEKYYQFLDHKQGDTRALAFRQASCTIKALPKRVTRVEEVRNLHRIGKHSISVIEDIVLHGTSEEVEDIRQSDWFKCMELFTSVYGCGPA TADKWYKKGFRTIDEIKTSETLELRELQKLGLLYYEDLSKSIPRDEVEVIIEIIKKEVSECCPETQVEAVGGYRRGKSHSHDVDLLLTHKDSSITATLLESVVSHLKAKDMIIHASVYVGQNTLSRVS ETHDSSAQPTYTSEGQQIKRVQFDHLDKAFCIMKLTRSHDKTPLIRRVDLIVTPPDQYPFSLVSWTGSKQFNRSLRRYSVKECSKTVTAHGIFDIIERKFSTAKTERDIFDILKLNYLEPWERNC (SEQ ID NO: 2).

[0023] 2. Expression and purification of AquTdT protein (1) The eukaryotic expression vector pFXB containing the AquTdT gene was transfected into the 293T cell line using the jetPRIME kit from Polyplus. Cell samples were collected 48 hours after transfection.

[0024] (2) Wash cells with sterile filtered and pre-cooled 1×PBS, add 1 mL of lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1 mM EDTA; 1×cocktail protease inhibitor) to a large dish, and lyse at 4°C for 30 min. Aspirate the lysis products, centrifuge at 12000×g at 4°C for 10 min, and retain the supernatant to obtain the lysis buffer.

[0025] (3) Take 30 μL of resuspended ANTI-FLAG M2 Affinity Gel, add pre-cooled lysis buffer to wash, centrifuge at 3000×g for 2 min at 4℃, remove the supernatant, and repeat 3 times. Add cell lysis supernatant and incubate at 4℃ for 4 h.

[0026] (4) Centrifuge at 3000×g for 3 min at 4℃ to collect the beads. Wash five times with 1 mL of pre-cooled lysis buffer (containing 1 mM PMSF) to remove impurities. Centrifuge at 3000×g for 1 min at 4℃ and discard the supernatant.

[0027] (5) Add 100 μL of lysis buffer, then add 60 μg of 3×FLAG(TBS) peptide, to a final concentration of 600 ng / μL. Incubate at 4℃ for 30-40 min by rotation, centrifuge at 3000×g at 4℃ for 4 min, and collect the supernatant to obtain the protein supernatant.

[0028] (6) Dialyze the protein sample supernatant using an activated 10 kDa dialysis tube. Replace the supernatant solution with dialysis buffer (25 mM Tris, pH 7.5; 150 mM KCl; 2 mM DTT; 10% glycerol). Centrifuge at 14000×g for 30 min at 4℃. Dialyze three times and aliquot and store at -80℃.

[0029] (7) The protein staining results showed that... Figure 2 As shown, a distinct band appears at a position slightly larger than 60 kDa, consistent with the expected size of the AquTdT protein, confirming it as the AquTdT protein. These results demonstrate that our cloned AquTdT protein can be expressed and successfully purified in the 293T cell line.

[0030] Example 2: In vitro activity and preference verification of AquTdT protein Detection principle: In vitro activity assay: AquTdT protein is a template-independent DNA polymerase that uses fluorescently labeled single-stranded oligonucleotides as substrates. Under metal ion mediation, it specifically recognizes the 3'-OH end of single-stranded DNA and sequentially incorporates free deoxyribonucleotides (dNTPs) into this end, elongating the nucleotide chain and increasing the molecular weight of the original DNA substrate. The reaction product and unreacted substrate are then separated by urea-polyacrylamide gel electrophoresis (Urea-PAGE). Enzyme activity is determined by the migration rate of the fluorescent bands (a hysteresis band appears if there is activity; band brightness / area can semi-quantitatively determine enzyme activity). The dT20 substrate consists of 20 consecutive dTTPs and does not have the conditions to form complementary strands; therefore, it is a standard system for evaluating template-independent end-transfer activity.

[0031] Preferentiality detection: Preferentiality detection was performed using single-component dNTP reaction systems (i.e., each reaction system contained only one type of dNTP, divided into four parallel systems: dATP, dTTP, dCTP, and dGTP). AquTdT exhibits inherent differences in catalytic efficiency for different dNTPs—for preferred nucleotides, it can rapidly and continuously incorporate them into the substrate terminus, generating products with larger molecular weights and longer tail lengths, resulting in stronger hysteresis / fluorescence signal intensity on electrophoresis bands; for unpreferred nucleotides, the incorporation efficiency is low, the sustained tailing ability is weak, the product molecular weight changes are small, and the detection signal is weak or even nonexistent. Preferentiality determination: By quantitatively / qualitatively comparing the hysteresis of the electrophoretic bands or the differences in product signals in the four parallel systems, the tailing preference order of AquTdT for the four dNTPs was determined.

[0032] Based on the above detection principle, firstly, we reacted the AquTdT protein with a single-stranded DNA substrate (dT20) labeled with a Cy5 fluorescent group at its 5' end and dNTPs at 30°C for 30 minutes. After the reaction, we terminated the reaction by high temperature and ensured complete dissociation of the hydrogen bonds between DNA molecules, guaranteeing that it would be in single-stranded form for electrophoresis. Subsequently, we used 20% urea-denaturing polyacrylamide gel electrophoresis (Urea-PAGE) to separate and analyze the reaction products. Figure 3 Under these conditions, Urea-PAGE can distinguish differences in the length of individual nucleotides, thus accurately reflecting the terminal tailing activity of AquTdT.

[0033] Furthermore, the tailing preference of the AquTdT protease for different nucleotides was further evaluated by adding four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP, and dGTP) to the reaction system. Figure 3 The specific steps are as follows: 1. Synthesize DNA substrates carrying fluorescent groups: A DNA substrate labeled with Cy5 at the 5' end was synthesized by Sangon Biotech (Shanghai) Co., Ltd.: PAA_S1:5′-Cy5-TTTTTTTTTTTTTTTTTTTT-3′.

[0034] 2. In vitro activity and preference assay of AquTdT protein: (1) Prepare Urea-PAGE adhesive according to the following system: Table 1. Preparation of 20% Urea-PAGE Dissolve and mix the first four reagents in Table 1. After mixing and preparing the glass plate and comb, add the subsequent reagents. After the PAGE gel solidifies, perform pre-electrophoresis at 400 V for 60 min using 0.5×TBE buffer.

[0035] (2) Prepare a 10 μL TdT terminal deoxyribonucleic acid reaction system (50 mM Tris-HCl; 50 μM dNTP; 1 mM DTT; 100 ng / mL BSA; 10 mM MnCl2; 4% glycerol; 300 nM TdT; 0.1 μM DNA; ddH2O to make up to 10 μL), and react at 30 ℃ for 30 min.

[0036] Table 2.10 μL TdT-terminal deoxyribonucleotide reaction system (3) After the reaction is complete, add an equal volume of stop solution (95% formamide, 50 mM EDTA) and mix well to terminate the reaction. Denature the sample in a PCR instrument at 100℃ for 5 min, then remove it and place it on ice.

[0037] (4) Spot the system after the reaction in step (3) and perform electrophoresis. Perform electrophoresis at 400V in the dark for 3-4 hours and perform fluorescence imaging.

[0038] 3. Verification results: The results are as follows Figure 4 As shown in Figure A, in vitro DNA polymerization results revealed that AquTdT exhibited significant terminal transferase activity against the single-stranded substrate PAA_S1. Further experimental results are as follows... Figure 4 B shows that the bands in the dATP channel have the strongest lag and the largest synthesized DNA molecular weight, followed by dTTP. The bias shown is dATP>dTTP>dGTP>dCTP, which is significantly different from the bias of vertebrate TdT (MmuTdTS) (dGTP>dTTP>dCTP>dATP).

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A sponge-terminal deoxynucleotidyl transferase, characterized in that, The amino acid sequence of the spongy terminal deoxynucleotidyl transferase is shown in SEQ ID NO:

2.

2. The spongy terminal deoxynucleotidyl transferase as described in claim 1, characterized in that, The nucleotide sequence of the spongy terminal deoxynucleotidyl transferase is shown in SEQ ID NO:

1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleotide sequence as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells contain the recombinant vector as described in claim 3, and the cells express the spongy terminal deoxynucleotidyl transferase as described in claim 1 or 2.

5. The application of the sponge-terminal deoxynucleotidyl transferase as described in claim 1 or 2, and the recombinant vector as described in claim 3, in the synthesis of nucleic acid molecules.

6. The application as described in claim 5, characterized in that, The synthesis involves adding a deoxynucleotide to the 3' end of the starting nucleic acid strand.

7. The application as described in claim 6, characterized in that, The preference for adding deoxynucleotides is dATP > dTTP > dGTP > dCTP.

8. A method for synthesizing nucleic acid molecules, characterized in that, The method includes the following steps: contacting the starting nucleic acid chain with at least one deoxynucleotide in the presence of the sponge-terminal deoxynucleotide transferase of claim 1 or 2.

9. A reagent kit, characterized in that, The kit comprises the sponge-terminal deoxynucleotidyltransferase as described in claim 1 or 2, one or more nucleotides, and at least one starting nucleic acid strand.

10. The kit according to claim 9, characterized in that, The starting nucleic acid chain is a single-stranded DNA.

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