Complex enzyme and use thereof

Abstract

A complex enzyme and the use thereof. The complex enzyme comprises: (1) a double-stranded RNA ligase; and (2) a protein having substrate affinity, wherein the protein is one or more selected from a DNA double-strand binding domain, an RNA / DNA hybrid-stranded binding domain, a single-stranded DNA covalent binding protein or a dsRNA binding protein, the one or more proteins having substrate affinity being ligated to the N terminus and / or the C terminus of the double-stranded RNA ligase by means of a first fusion protein linker; and the substrate is a first single-stranded RNA fragment, a second single-stranded RNA fragment and a splint oligonucleotide. The complex enzyme can improve the ligation efficiency to 80-90% or even higher, which is far higher than that of common RNA ligases.

Description

A composite enzyme and its application Technical Field

[0001] The present invention relates to synthetic biology, and in particular to a composite enzyme and applications thereof. Background Art

[0002] In the technical field to which this invention relates, the current state of the art involves chemically synthesizing small nucleic acid fragments (DNA, RNA, or analogs) to form precursor nucleic acids (precursor RNAs), which are then spliced ​​into longer nucleic acids (RNAs) through enzymatic synthesis. However, a technical problem is that available enzymes, such as T4 RNA ligase, have low ligation activity, achieving a maximum ligation efficiency of only around 50%, which results in excessively high overall raw material costs. Therefore, there is an urgent need for a highly efficient RNA ligase.

[0003] Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a composite enzyme and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a composite enzyme comprising:

[0007] (1) Double-stranded RNA ligase;

[0008] (2) a protein with substrate affinity, wherein the protein is selected from one or more of a DNA double-strand binding domain, an RNA / DNA composite chain binding domain, a single-stranded DNA covalent binding protein, or a dsRNA binding protein;

[0009] One or more of the substrate-affinity proteins are connected to the N-terminus and / or C-terminus of the double-stranded RNA ligase via a first fusion protein linker;

[0010] The substrates are a first single-stranded RNA fragment, a second single-stranded RNA fragment and a splint oligonucleotide (adaptor);

[0011] The first single-stranded RNA segment comprises a 3' terminal region containing a hydroxyl group;

[0012] The second single-stranded RNA segment comprises a 5' terminal region containing a phosphate group;

[0013] The splint oligonucleotide is a single-stranded DNA and / or single-stranded RNA oligonucleotide, and the splint oligonucleotide (adaptor) includes a first part and a second part, the first part is complementary to the 5' end sequence of the second single-stranded RNA fragment, and the second part is complementary to the 3' end sequence of the first single-stranded RNA fragment; the first part starts from the 5' end of the splint oligonucleotide sequence and accounts for 30% to 70% (for example, 35%, 40%, 45%, 50%, 55%, 60%, 65%) of the total length of the splint oligonucleotide.

[0014] The complex enzyme of the present invention can connect the first single-stranded RNA fragment and the second single-stranded RNA fragment to form an RNA of medium length.

[0015] In the above-mentioned complex enzyme, as a preferred embodiment, the double-stranded RNA ligase is T4 RNA ligase 2 (T4RL2), which is a protein composed of the amino acid sequence as shown in SEQ ID NO: 8, or a protein in which the amino acid sequence as shown in SEQ ID NO: 8 is substituted, deleted or added with one or more amino acids and has T4 RNA ligase 2 activity.

[0016] In the above complex enzyme, as a preferred embodiment, the splint oligonucleotide (adaptor) consists of a first part and a second part, wherein the first part starts from the 5' end of the splint oligonucleotide sequence and accounts for 40% to 60% of the total length of the splint oligonucleotide.

[0017] In the above complex enzyme, as a preferred embodiment, multiple identical or different proteins with substrate affinity are connected through a second fusion protein linker and then connected to the N-terminus and / or C-terminus of the double-stranded RNA ligase through a first fusion protein linker.

[0018] In the above-mentioned complex enzyme, as a preferred embodiment, the first fusion protein linker or the second fusion protein linker is a plurality of repeated GGGGS amino acid sequences; preferably, the first fusion protein linker or the second fusion protein linker is 3 to 4 repeated GGGGS amino acid sequences.

[0019] In the above-mentioned complex enzyme, as a preferred embodiment, the length of the splint oligonucleotide is 20 to 100 (eg, 30, 40, 50, 60, 70, 80, 90) nucleotides.

[0020] The splint oligonucleotides of the present invention can, through sequence pairing, form a substrate capable of being ligated by a double-stranded RNA enzyme by combining the first single-stranded RNA fragment (P1) and the second single-stranded RNA fragment (P2). Furthermore, through covalent or non-covalent binding of the structural domains, the double-stranded RNA enzyme is spatially brought into close proximity with the first single-stranded RNA fragment (P1) and the second single-stranded RNA fragment (P2), further improving the ligation efficiency of the double-stranded RNA enzyme.

[0021] In the above complex enzyme, as a preferred embodiment, the lengths of the first and second single-stranded RNA fragments are 40 to 70 (eg, 45, 50, 55, 60, 65) nucleotides respectively.

[0022] In the present invention, the manner in which a protein with substrate affinity binds to a substrate is related to the function of the protein itself. The RNA / DNA complex chain binding domain can directly bind to an RNA / DNA complex chain formed by a first or second single-stranded RNA molecule and a single-stranded DNA oligonucleotide (splint oligonucleotide); the single-stranded DNA covalent binding protein Halotag can be covalently linked to the single-stranded DNA oligonucleotide (splint oligonucleotide); the DNA double-stranded binding domain binds to the splint oligonucleotide via an additional DNA double strand formed on the splint DNA oligonucleotide (the additional DNA double strand can be a fully complementary nucleotide sequence added to the middle position, 3' end, or 5' end of the single-stranded DNA splint oligonucleotide, or a stem-loop structure extending from the 3' end or 5' end of the single-stranded DNA splint oligonucleotide), thereby pulling T4RL2 to the interface position required for connection; and the dsRNA binding protein can bind to the RNA double strand formed by the first or second single-stranded RNA molecule and the single-stranded RNA oligonucleotide (splint oligonucleotide).

[0023] In the above complex enzyme, as a preferred embodiment, the DNA double-strand binding domain includes: one or more of Sso7d, NF-κB p50, and HBD.

[0024] In the above-mentioned complex enzyme, as a preferred embodiment, the RNA / DNA complex chain binding domain includes one or more of ScFV of monoclonal antibody S9.6, RNaseH1 (D210N), HBD, and Sso7d.

[0025] In the above complex enzyme, as a preferred embodiment, the single-stranded DNA covalent binding protein is Halotag.

[0026] In the present invention, the RNaseH1 (D210N) is an RNaseH1 in which asparagine (N) is replaced by aspartic acid (D) at position 210 of the amino acid sequence, i.e., an RNaseH1 with inactivated catalytic activity; the source of the Halotag can be the Halotag in the commercial plasmid pFN19A.

[0027] In the above-mentioned complex enzyme, as a preferred embodiment, the dsRNA binding protein (DRBP) includes one or more of PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.

[0028] All dsRNA binding proteins known in the art are suitable for the present invention, such as the dsRNA binding proteins listed in Table 1 in Saunders LR, Barber GN. The dsRNA binding protein family: critical roles, diverse cellular functions [J]. The FASEB Journal, 2003, 17 (9).

[0029] In the above-mentioned complex enzyme, as a preferred embodiment, the Sso7d is a protein consisting of the amino acid sequence as shown in SEQ ID NO: 9, or a protein having a DNA double-stranded binding domain activity in which the amino acid sequence as shown in SEQ ID NO: 9 is substituted, deleted, or added with one or more amino acids;

[0030] Preferably, the HBD is a protein consisting of the amino acid sequence shown in SEQ ID NO: 10, or a protein having RNA / DNA complex binding activity with one or more amino acids substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 10;

[0031] Preferably, the NF-κB p50 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, or a protein having a DNA double-stranded binding domain activity in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 11;

[0032] Preferably, the Halotag is a protein consisting of the amino acid sequence shown in SEQ ID NO: 12, or a protein having single-stranded DNA covalent binding activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 12;

[0033] Preferably, the RNaseH1 (D210N) is a protein consisting of the amino acid sequence as shown in SEQ ID NO: 14, or a protein having RNA / DNA complex binding activity with one or more amino acids substituted, deleted or added to the amino acid sequence as shown in SEQ ID NO: 14;

[0034] Preferably, the ScFV of the monoclonal antibody S9.6 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 13, or a protein having the amino acid sequence shown in SEQ ID NO: 13 with one or more amino acids substituted, deleted or added and having RNA / DNA complex chain binding activity.

[0035] In the above-mentioned complex enzyme, as a preferred embodiment, the nucleotide sequence of the complex enzyme is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0036] In a second aspect, the present invention provides a method for enzymatically synthesizing RNA based on the above-mentioned complex enzyme, the method comprising method A, method B or method C:

[0037] The method A comprises the following steps in sequence:

[0038] S1, adding the first single-stranded RNA fragment, the second single-stranded RNA fragment and the single-stranded DNA splint oligonucleotide into an enzyme-linked buffer to obtain a mixed solution;

[0039] S2, adding the complex enzyme to the mixed solution to perform enzyme ligation reaction;

[0040] The method B comprises the following steps in sequence:

[0041] Sa, mixing the single-stranded DNA splint oligonucleotide with the complex enzyme to prepare a single-stranded DNA-protein complex;

[0042] Sb. Adding the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the single-stranded DNA-protein complex into an enzyme ligation buffer to perform an enzyme ligation reaction.

[0043] The method C comprises the following steps in sequence:

[0044] Si, the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the single-stranded RNA splint oligonucleotide are added to an enzyme-linked buffer to obtain a mixed solution;

[0045] Sii. Adding the complex enzyme into the mixed solution to carry out enzyme-linked reaction.

[0046] In the above-mentioned enzymatic RNA synthesis method, as a preferred embodiment, the protein with substrate affinity of the complex enzyme in method A is one or more of a double-stranded DNA binding domain and an RNA / DNA composite strand binding domain. When the protein with substrate affinity is a double-stranded DNA binding domain, the single-stranded DNA splint oligonucleotide is a single-stranded DNA splint oligonucleotide with additional double-stranded DNA.

[0047] In the above-mentioned method for synthesizing RNA by enzymatic method, as a preferred embodiment, the protein with substrate affinity of the complex enzyme in method B is a single-stranded DNA covalent binding protein.

[0048] In the above-mentioned method for synthesizing RNA by enzymatic method, as a preferred embodiment, the protein with substrate affinity of the complex enzyme in method C is a dsRNA binding protein.

[0049] In the above-mentioned method for enzymatically synthesizing RNA, as a preferred embodiment, step S1 of method A and / or step S1 of method C further include subjecting the mixed solution to thermal denaturation treatment, and naturally cooling or gradient cooling to 20°C to 30°C (e.g., 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C) after the thermal denaturation treatment. The denaturation treatment temperature is 94°C to 96°C (e.g., 94.5°C, 95°C, 95.5°C), and the denaturation treatment time is 3 to 5 minutes (e.g., 3.5 minutes, 4 minutes, 4.5 minutes); preferably, the gradient cooling is to cool to 20-30°C at a rate of 0.05-0.2°C / s (e.g., 0.075°C / s, 0.1°C / s, 0.125°C / s, 0.15°C / s, 0.175°C / s).

[0050] In the above-mentioned enzymatic RNA synthesis method, as a preferred embodiment, in method A and method C, the final concentrations of the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the splint oligonucleotide in the enzyme ligation reaction system are all 10 to 30 μM.

[0051] In the above-mentioned enzymatic method for synthesizing RNA, as a preferred embodiment, in method A and method C, the final concentration of the above-mentioned complex enzyme in the enzyme-linked reaction system is 1nM to 10μM (for example, 10nM, 50nM, 100nM, 500nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM).

[0052] In the above-mentioned enzymatic method for synthesizing RNA, as a preferred embodiment, in method B, the final concentrations of the first single-stranded RNA fragment and the second single-stranded RNA fragment in the enzyme-linked reaction system are both 10 to 30 μM (e.g., 15 μM, 20 μM, 25 μM), and the final concentration of the single-stranded DNA-protein complex is 10 to 30 μM (e.g., 15 μM, 20 μM, 25 μM).

[0053] In the above-mentioned method for enzymatically synthesizing RNA, as a preferred embodiment, in method A, method B and method C, the pH of the enzyme-linked buffer is 7.5, and the enzyme-linked buffer includes DTT at a final concentration of 0.1 to 50 mM (e.g., 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM), ATP at a final concentration of 10 to 1000 μM (e.g., 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM), and Tris-HCl at a final concentration of 10 to 500 mM (e.g., 50 mM, 100 mM, 200 mM, 300 mM, 400 mM).

[0054] In the above-mentioned enzymatic RNA synthesis method, as a preferred embodiment, in method A, method B and method C, the temperature of the enzymatic reaction is 36-38°C (e.g., 36.5°C, 37°C, 37.5°C).

[0055] In the above-mentioned enzymatic method for synthesizing RNA, as a preferred embodiment, in method A, method B and method C, the enzymatic reaction time is 10 to 720 min (e.g., 50 min, 100 min, 150 min, 200 min, 300 min, 400 min, 500 min, 600 min, 700 min).

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] Compared with the existing commercial T4 RNA ligase 2, the engineered T4 RNA ligase can increase the ligation efficiency to 80-90%, or even higher, which is far higher than the wild-type T4 RNA ligase 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1. Schematic diagram of the mechanism of action of a complex enzyme composed of a fusion of T4RL2 (T4 RNA ligase 2) and an RNA / DNA complex chain binding domain. The fused complex enzyme (DNA / RNA binding domain-T4RL2) can bind to an RNA / DNA double-stranded substrate, which is an RNA / DNA complex double-stranded formed by two RNA molecules, P1 (the first single-stranded RNA fragment) and P2 (the second single-stranded RNA fragment), and an adaptor (splint oligonucleotide). T4RL2 can form a covalent phosphate backbone between P1 and P2, thereby connecting P1 (the first single-stranded RNA fragment) and P2 (the second single-stranded RNA fragment) into a single RNA molecule.

[0059] Figure 2. Schematic diagram of the mechanism of action of a complex enzyme composed of a fusion of T4RL2 (T4 RNA ligase 2) and Halotag. The fused complex enzyme can covalently link with the Adaptor (splint oligonucleotide) to form a splint oligonucleotide-Halotag-T4RL2 complex structure, which has the function of both binding to the substrate and catalyzing the substrate connection. The substrate is an RNA / DNA complex double-stranded structure formed by two RNA molecules, P1 (the first single-stranded RNA fragment) and P2 (the second single-stranded RNA fragment), and the splint molecule Adaptor (splint oligonucleotide). T4RL2 can form a covalent phosphate backbone between P1 and P2, thereby connecting P1 (the first single-stranded RNA fragment) and P2 (the second single-stranded RNA fragment) into a single RNA molecule.

[0060] Figure 3. A complex enzyme composed of T4RL2 (T4 RNA ligase 2) fused with a DNA double-stranded binding domain. The complex enzyme can non-covalently bind to an adaptor molecule (splint oligonucleotide) to form a T4RL2-splint oligonucleotide-DNA binding domain complex. This complex structure not only binds to the substrate but also catalyzes substrate ligation. The substrate is an RNA / DNA duplex formed by two RNA molecules, P1 (the first single-stranded RNA fragment) and P2 (the second single-stranded RNA fragment), and the adaptor molecule (splint oligonucleotide). T4RL2 forms a covalent phosphate backbone between P1 (the first single-stranded RNA fragment) and P2 (the second single-stranded RNA fragment), thereby linking P1 and P2 into a single RNA molecule. The splint oligonucleotide is terminated by an additional DNA duplex (either a duplex formed by the two splint oligonucleotides or a stem-loop structure extending from the splint oligonucleotide), which can bind to DNA double-stranded binding domains within the complex enzyme, such as Sso7d and p50.

[0061] Figure 4 is a schematic diagram of the T4RL2 complex enzyme structure and a schematic diagram of the T4RL2 enzyme structure of Examples 1-3 of the present invention; wherein, A is a schematic diagram of the unmodified T4RL2 enzyme structure, B is a schematic diagram of the Sso7d-Linker-T4RL2 complex enzyme structure, C is a schematic diagram of the T4RL2-Linker-HBD-Linker-HBD complex enzyme structure, and D is a schematic diagram of the Halotag-Linker-T4RL2 complex enzyme structure.

[0062] Figure 5 shows the SDS-PAGE detection results of T4RL2 enzyme, Sso7d-Linker-T4RL2 complex enzyme, T4RL2-Linker-HBD-Linker-HBD complex enzyme, and Halotag-Linker-T4RL2 complex enzyme.

[0063] Figure 6 is an analysis diagram of the enzymatic ligation efficiency of Sso7d-Linker-T4RL2, wherein A is the Urea-PAGE detection result of the enzymatic products of the complex enzyme at different concentrations, and B is the Michaelis-Menten regression curve fitting the ligation efficiency of the Sso7d-Linker-T4RL2 complex enzyme at different concentrations.

[0064] Figure 7 is a diagram showing the analysis of the enzymatic ligation efficiency of T4RL2-Linker-HBD-Linker-HBD, wherein A is the Urea-PAGE detection result of the enzymatic products of the complex enzyme at different concentrations, and B is the Michaelis-Menten regression curve fitting the ligation efficiency of the T4RL2-Linker-HBD-Linker-HBD complex enzyme at different concentrations.

[0065] FIG8 is a diagram showing the analysis of the enzymatic ligation efficiency of Halotag-Linker-T4RL2, wherein A is the Urea-PAGE detection result of the enzymatic products of the complex enzyme at different concentrations, and B is the Michaelis-Menten regression curve fitting the ligation efficiency of the complex enzyme at different concentrations.

[0066] FIG9 is an analysis diagram of the enzymatic ligation efficiency of the unmodified wild-type T4RL2 enzyme, wherein A is the Urea-PAGE detection result of the enzymatic products of T4RL2 enzyme at different concentrations, and B is the Michaelis-Menten regression curve of the ligation efficiency fitting of T4RL2 enzyme at different concentrations.

[0067] Figure 10 is a unified analysis diagram of the ligation efficiency fitting curves of T4RL2 enzyme, Sso7d-Linker-T4RL2 complex enzyme, T4RL2-Linker-HBD-Linker-HBD complex enzyme, and Halotag-Linker-T4RL2 complex enzyme. DETAILED DESCRIPTION

[0068] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0069] The present application improves the efficiency of T4 RNA ligase 2 in connecting RNA substrates by engineering the enzyme. The principle of the transformation is to fuse the domains of T4 RNA ligase 2 that can specifically bind to the substrate to increase the probability of T4 RNA ligase 2 binding to the substrate, thereby improving the connection efficiency. These domains include but are not limited to RNA / DNA complex chain binding domains, such as the ScFV of monoclonal antibody S9.6 and the RNA / DNA binding domain of RNase H (RNaseH1 (D210N)); DNA double-stranded binding domains such as NF-κB p50 and Sso7d; single-stranded DNA covalent binding protein Halotag, etc., respectively achieve enhanced binding to the substrate.

[0070] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0071] Information on the first single-stranded RNA fragment (P1), the second single-stranded RNA fragment (P2), and the splint oligonucleotide (adaptor) used in the following examples and comparative examples can be found in Table 1 below.

[0072] Table 1

[0073] Example 1

[0074] (1) The complex enzyme used in this example is a complex enzyme with a domain Sso7d added to the N-terminus of T4RL2 that can enhance the affinity for RNA / DNA double-stranded substrates. It is named Sso7d-Linker-T4RL2 complex enzyme. The schematic diagram of its structure is shown in FIG4B . The mechanism of action of this complex enzyme in promoting substrate ligation is shown in FIG1 . The expression and purification steps are as follows:

[0075] The nucleotide sequence of the complex enzyme is shown in the sequence listing as SEQ ID NO:1 (Sso7d-Linker-T4RL2). The nucleic acid sequence encoding the fusion protein of T4RL2 and the RNA / DNA binding functional domain (Sso7d-Linker-T4RL2 sequence shown in the sequence listing as SEQ ID NO:1) was sent to a recombinant protein expression company. The service provided included gene synthesis of the complex enzyme and cloning into an E. coli expression vector for prokaryotic expression in E. coli. A 6×His tag was added to the N-terminus of the complex enzyme to facilitate purification. If the protein purity on SDS-PAGE after the single-step affinity purification exceeds 90%, further purification is not required. If it is less than 90%, further purification is required through molecular sieves. The SDS-PAGE analysis results of the Sso7d-Linker-T4RL2 complex enzyme are shown in Figure 5. The purified protein was stored in a buffer solution (the buffer solution included Tris-HCl at a final concentration of 10 mM, KCl at a final concentration of 50 mM, (NH4)2SO4 at a final concentration of 35 mM, DTT at a final concentration of 0.1 mM, EDTA at a final concentration of 0.1 mM, and Glycerol at a final concentration of 50% by volume) at -20°C.

[0076] (2) The Sso7d-Linker-T4RL2 complex enzyme of this example was used to connect the P1 and P2 single-stranded RNAs in Table 1. To facilitate calculation of the complex enzyme's ligation efficiency, the 5' end of the P1 single-stranded RNA sequence was connected to a FAM fluorescent group. The splint oligonucleotide used was the single-stranded DNA splint oligonucleotide Adaptor 1. The RNA ligation reaction conditions were as follows:

[0077] The concentrations of the first single-stranded RNA fragment P1, the second single-stranded RNA fragment P2, and the single-stranded splint oligonucleotide Adaptor 1 are all 100 μM, and the solvent is water.

[0078] The first single-stranded RNA fragment, the second single-stranded RNA fragment, and the single-stranded DNA splint oligonucleotide were mixed at room temperature in 1× RNA ligase buffer (pH 7.5, containing DTT at a final concentration of 1 mM, ATP at a final concentration of 500 μM, and Tris-HCl at a final concentration of 50 mM) to form a mixed solution. The final concentrations of the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the single-stranded DNA splint oligonucleotide in the mixed solution were all 20 μM. The mixed solution was denatured at 95°C for 5 minutes and then annealed to 25°C.

[0079] The Sso7d-Linker-T4RL2 RNA complex is then added to the mixed solution to form reaction systems with final concentrations of 1μM, 2μM, 4μM, 8μM, 10μM, 16μM, and 20μM, respectively. The reaction is incubated at 37°C for 30 minutes. A control without the addition of the complex is also set up, i.e., the final concentration of the RNA complex in the reaction system is 0. The incubation product is concentrated, desalted, and then purified and analyzed for RNA molecules. Purification analysis can be performed by PAGE or HPLC.

[0080] Methods for detecting and calculating the efficiency of complex enzyme ligation

[0081] The product of the RNA ligation reaction is concentrated, desalted, and then purified by PAGE. The PAGE gel is a urea PAGE gel, the general gel concentration is 6-20%, and the buffer is 1×TBE. The operating voltage is 300V, and 8cm×10cm vertical electrophoresis is used to separate the ligation product (Production) and unreacted P1, P2, and Adaptor1. Since the FAM fluorescent sequence is labeled at the 5' end of P1, when P1 and P2 are connected to form a complete RNA molecule, it can be judged by fluorescence imaging. The imaging detection results under different complex enzyme concentrations are shown in Figure 6A. Image J software is used to calculate the gray value of the band to obtain the ligation efficiency. The calculation of the ligation efficiency is based on P1: ligation efficiency = Production gray value / (P1 gray value + Production gray value) × 100%. The Michaelis-Menten regression curve is fitted to the ligation efficiency of different Sso7d-Linker-T4RL2 concentrations. The results are shown in Figure 6B. The maximum ligation efficiency is 95.52%, K m 1.389 μM, K cat 0.5186(S -1 ).

[0082] Example 2

[0083] (1) The complex enzyme used in this example is a complex enzyme with a domain HBD added to the C-terminus of T4RL2 that can enhance the affinity for double-stranded DNA substrates. It is named T4RL2-Linker-HBD-Linker-HBD complex enzyme. The schematic diagram of its structure is shown in FIG4C . The mechanism of action of this complex enzyme in promoting substrate ligation is shown in FIG3 . The expression and purification steps are as follows:

[0084] The nucleotide sequence of the complex enzyme is shown in the sequence listing as SEQ ID NO:3 (T4RL2-Linker-HBD-Linker-HBD). The nucleotide sequence encoding the fusion protein of the T4RL2 and HBD functional domains (SEQ ID NO:3) was sent to a recombinant protein expression company. The service includes gene synthesis of the complex enzyme and cloning into an E. coli expression vector for prokaryotic expression in E. coli. A 6×His tag is added to the N-terminus of the complex enzyme to facilitate purification. If the SDS-PAGE purity of the protein after the single-step affinity purification exceeds 90%, further purification is not required. If it is less than 90%, further purification is required through molecular sieves. The SDS-PAGE analysis results of the T4RL2-Linker-HBD-Linker-HBD complex enzyme are shown in Figure 5. The purified protein was stored in a buffer solution (the buffer solution included Tris-HCl at a final concentration of 10 mM, KCl at a final concentration of 50 mM, (NH4)2SO4 at a final concentration of 35 mM, DTT at a final concentration of 0.1 mM, EDTA at a final concentration of 0.1 mM, and Glycerol at a final concentration of 50% by volume) at -20°C.

[0085] (2) The T4RL2-Linker-HBD-Linker-HBD complex enzyme of this example was used to link the P1 and P2 single-stranded RNAs in Table 1. To facilitate calculation of the complex enzyme's linking efficiency, the 5' end of the P1 single-stranded RNA sequence was linked to a FAM fluorescent group in this example. The splint oligonucleotide used was a splint oligonucleotide Adaptor2 containing a partially double-stranded DNA sequence, which was formed by SEQ ID NO: 18 and SEQ ID NO: 19 in Table 1. The underlined sequence at the 5' end of the single-stranded oligonucleotide shown in SEQ ID NO: 18 would complementarily pair with the single-stranded oligonucleotide shown in SEQ ID NO: 19, thereby forming a partially double-stranded structure of the splint oligonucleotide Adaptor2.

[0086] RNA ligation reaction:

[0087] The concentrations of the first single-stranded RNA fragment P1, the second single-stranded RNA fragment P2, the single-stranded oligonucleotide sequence shown in SEQ ID NO: 18, and the single-stranded oligonucleotide sequence shown in SEQ ID NO: 19 were all 100 μM.

[0088] The first single-stranded RNA fragment, the second single-stranded RNA fragment, the single-stranded oligonucleotide sequence set forth in SEQ ID NO: 18, and the single-stranded oligonucleotide sequence set forth in SEQ ID NO: 19 were mixed at room temperature in 1× RNase buffer (pH 7.5, containing DTT at a final concentration of 1 mM, ATP at a final concentration of 500 μM, and Tris-HCl at a final concentration of 50 mM) to produce a mixed solution. The final concentrations of the first single-stranded RNA fragment, the second single-stranded RNA fragment, the single-stranded oligonucleotide sequence set forth in SEQ ID NO: 18, and the single-stranded oligonucleotide sequence set forth in SEQ ID NO: 19 in the mixed solution were all 20 μM. The mixed solution was denatured at 95° C. for 5 minutes and then annealed to 25° C.

[0089] T4RL2-Linker-HBD-Linker-HBD RNA enzyme complex is then added to the mixed solution to form reaction systems with final concentrations of 1μM, 2μM, 4μM, 8μM, 10μM, 16μM, and 20μM, respectively. The reaction systems are incubated at 37°C for 30 minutes. A control without the addition of the enzyme complex is also set up, i.e., the final concentration of the RNA enzyme complex in the reaction system is 0. The incubation product is concentrated, desalted, and then purified and analyzed for RNA molecules. Purification analysis can be performed by PAGE or HPLC.

[0090] The detection and calculation methods of the complex enzyme ligation efficiency were the same as those in Example 1. The imaging detection results at different complex enzyme concentrations are shown in FIG7A . The Michaelis-Menten regression curve was fitted to the ligation efficiency at different Sso7d-Linker-T4RL2 concentrations. The results are shown in FIG7B . The maximum ligation efficiency was 96.94%, and K m 1.152 μM, K cat 0.5473(S -1 ).

[0091] Example 3

[0092] The complex enzyme of this example consists of a fusion of T4RL2 (T4 RNA ligase 2) and Halotag. The fused complex enzyme can covalently bind to the adaptor molecule (splint oligonucleotide) to form a splint oligonucleotide-Halotag-T4RL2 complex structure, which not only binds to the substrate but also catalyzes substrate ligation. The specific implementation and mechanism of action are shown in Figure 2.

[0093] (1) The complex enzyme used in this example is a complex enzyme to which the Halotag domain that can enhance the affinity for single-stranded DNA substrates is added to the N-terminus of T4RL2. It is named Halotag-Linker-T4RL2 complex enzyme. The schematic diagram of its structure is shown in FIG4D . The expression and purification steps are as follows:

[0094] The nucleotide sequence of the complex enzyme is shown in the sequence listing as SEQ ID NO:5 (Halotag-Linker-T4RL2). The nucleotide sequence (SEQ ID NO:5) encoding the fusion protein of T4RL2 and Halotag functional domains is sent to the recombinant protein expression company. The service content includes gene synthesis of the complex enzyme and cloning into an E. coli expression vector for prokaryotic expression in E. coli. A 6×His tag is added to the N-terminus of the complex enzyme to facilitate purification. If the SDS-PAGE purity of the protein after one-step affinity purification exceeds 90%, further purification is not required. If it is less than 90%, further purification through molecular sieve is required. The SDS-PAGE detection results of the Halotag-Linker-T4RL2 complex enzyme are shown in Figure 5. The purified protein was stored in a buffer solution (the buffer solution included Tris-HCl at a final concentration of 10 mM, KCl at a final concentration of 50 mM, (NH4)2SO4 at a final concentration of 35 mM, DTT at a final concentration of 0.1 mM, EDTA at a final concentration of 0.1 mM, and Glycerol at a final concentration of 50% by volume) at -20°C.

[0095] (2) The Halotag-Linker-T4RL2 complex enzyme of this example was used to link the P1 and P2 single-stranded RNAs in Table 1. To facilitate calculation of the complex enzyme's linking efficiency, the 5' end of the P1 single-stranded RNA sequence was linked to a FAM fluorescent group. The splint oligonucleotide used was the single-stranded oligonucleotide Adaptor1 in Table 1. To enhance the binding of the splint oligonucleotide to the Halotag, Cl was added to the 3' end of the nucleotide sequence shown in SEQ ID NO: 17 in Table 1.

[0096] RNA ligation reaction:

[0097] The concentrations of the first single-stranded RNA fragment and the second single-stranded RNA fragment are both 100 μM.

[0098] First, the splint oligonucleotide was further complexed with the complex enzyme of this example (Halotag-Linker-T4RL2) to form a single-stranded DNA-protein complex at a concentration of 100 μM.

[0099] The first single-stranded RNA fragment, the second single-stranded RNA fragment, and the single-stranded DNA-protein complex are then mixed at room temperature in 1× RNase buffer (pH 7.5, containing DTT at a final concentration of 1 mM, ATP at a final concentration of 500 μM, and Tris-HCl at a final concentration of 50 mM) to form a mixed solution reaction system. Multiple reactions containing different concentrations of the single-stranded DNA-protein complex are then set up. In each mixed solution reaction system, the final concentrations of the first and second single-stranded RNA fragments are 20 μM, and the final concentrations of the single-stranded DNA-protein complex are 0, 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, 16 μM, and 20 μM, respectively. The mixed solution is incubated at 37°C for 30 minutes. The incubated product is concentrated, desalted, and then purified for RNA analysis. Purification analysis can be performed by PAGE or HPLC.

[0100] The detection and calculation methods of the complex enzyme ligation efficiency are the same as those in Example 1. The imaging detection results at different complex enzyme concentrations are shown in FIG8A. The Michaelis-Menten regression curve is fitted to the ligation efficiency of different Halotag-Linker-T4RL2 concentrations. The results are shown in FIG8B. The maximum ligation efficiency is 98.35%, and K m 0.7660 μM, K cat 0.6065(S -1 ).

[0101] Comparative Example

[0102] The RNA ligase used in this comparative example is wild-type T4 RNA ligase 2 (T4RL2 enzyme), whose amino acid sequence is shown in SEQ ID NO: 8, its structural schematic is shown in FIG4A , and the SDS-PAGE detection results of the enzyme are shown in FIG5 .

[0103] The ligase was used to connect the first single-stranded RNA fragment and the second single-stranded RNA fragment in Example 1. The sequence of the splint oligonucleotide was also the same as that in Example 1. The reaction system was as follows:

[0104] The first single-stranded RNA fragment, the second single-stranded RNA fragment, and the splint oligonucleotide were mixed at room temperature in 1× RNase buffer (pH 7.5, containing DTT at a final concentration of 1 mM, ATP at a final concentration of 500 μM, and Tris-HCl at a final concentration of 50 mM) to produce a mixed solution. The final concentrations of the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the splint oligonucleotide in the mixed solution were all 20 μM. The mixed solution was denatured at 95°C for 5 minutes and then annealed to 25°C.

[0105] Wild-type T4RL2 enzyme is then added to the mixed solution to form reaction systems with final concentrations of 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, 16 μM, and 20 μM, respectively. The reaction systems are incubated at 37°C for 30 minutes. A control without enzyme is also set up, i.e., the final enzyme concentration in the reaction system is 0. The incubation product is concentrated, desalted, and then purified and analyzed for RNA molecules. Purification analysis can be performed by PAGE or HPLC.

[0106] The detection and calculation methods of the ligation efficiency of wild-type T4 RNA ligase 2 were the same as those in Example 1. The imaging detection results at different enzyme concentrations are shown in FIG9A . The Michaelis-Menten regression curves were fitted to the ligation efficiency of different enzyme concentrations. The results are shown in FIG9B . The maximum ligation efficiency was 49.6%, and K m 0.6029 μM, K cat 0.3181(S -1 ).

[0107] The connection efficiency fitting curves of the enzymes of Examples 1-3 and the comparative example were uniformly analyzed. The results are shown in FIG10 . Halotag-Linker-T4RL2 has higher connection efficiency and higher affinity with the substrate than the other two complex enzymes. K cat It is twice that of T4RL2 enzyme and also higher than the other two complex enzymes. Therefore, the enzymatic activity of Halotag-Linker-T4RL2 is better than that of T4RL2 and the other two complex enzymes.

[0108] The above embodiments are merely examples for illustrative purposes only and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications may be made based on the above descriptions. It is not necessary and impossible to enumerate all implementation methods here. Therefore, any obvious variations or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A composite enzyme, characterized in that: The complex enzyme comprises: (1) Double-stranded RNA ligase; (2) a protein with substrate affinity, wherein the protein is selected from one or more of a DNA double-strand binding domain, an RNA / DNA composite strand binding domain, a single-stranded DNA covalent binding protein, or a dsRNA binding protein; One or more of the proteins with substrate affinity are connected to the N-terminus and / or C-terminus of the double-stranded RNA ligase via a first fusion protein linker; The substrates are a first single-stranded RNA fragment, a second single-stranded RNA fragment and a splint oligonucleotide; The first single-stranded RNA fragment comprises a 3' terminal region containing a hydroxyl group; The second single-stranded RNA fragment comprises a 5' terminal region containing a phosphate group; The splint oligonucleotide is a single-stranded DNA and / or single-stranded RNA oligonucleotide, and the splint oligonucleotide includes a first part and a second part, the first part is complementary to the 5' terminal sequence of the second single-stranded RNA fragment, and the second part is complementary to the 3' terminal sequence of the first single-stranded RNA fragment; the first part starts from the 5' end of the sequence of the splint oligonucleotide and accounts for 30% to 70% of the total length of the splint oligonucleotide.

2. The complex enzyme according to claim 1, characterized in that The double-stranded RNA ligase is T4 RNA ligase 2, which is a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, or a protein having T4 RNA ligase 2 activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 8; And / or, the splint oligonucleotide consists of a first part and a second part, wherein the first part starts from the 5' end of the sequence of the splint oligonucleotide and accounts for 40% to 60% of the total length of the splint oligonucleotide; And / or, a plurality of identical or different proteins with substrate affinity are connected via a second fusion protein linker and then connected to the N-terminus and / or C-terminus of the double-stranded RNA ligase via a first fusion protein linker; And / or, the first fusion protein linker or the second fusion protein linker is a plurality of repeated GGGGS amino acid sequences.

3. The complex enzyme according to claim 2, characterized in that The first fusion protein linker or the second fusion protein linker is 3 to 4 repeated GGGGS amino acid sequences; and / or, the length of the splint oligonucleotide is 20 to 100 nucleotides; and / or, the lengths of the first and second single-stranded RNA fragments are 40 to 70 nucleotides respectively; And / or, the double-stranded DNA binding domain includes: one or more of Sso7d, NF-κB p50, and HBD; And / or, the RNA / DNA composite chain binding domain includes one or more of ScFV, RNaseH1 (D210N), HBD, and Sso7d of the monoclonal antibody S9.6; And / or, the single-stranded DNA covalent binding protein is Halotag; And / or, the dsRNA binding protein includes one or more of PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.

4. The complex enzyme according to claim 3, characterized in that The Sso7d is a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or a protein having a DNA double-stranded binding domain activity in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 9; and / or, the HBD is a protein consisting of the amino acid sequence as shown in SEQ ID NO: 10, or a protein having RNA / DNA complex chain binding activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence as shown in SEQ ID NO: 10; And / or, the NF-κB p50 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, or a protein having a DNA double-stranded binding domain activity in which the amino acid sequence shown in SEQ ID NO: 11 is substituted, deleted or added with one or more amino acids; And / or, the Halotag is a protein consisting of the amino acid sequence as shown in SEQ ID NO: 12, or a protein having single-stranded DNA covalent binding activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence as shown in SEQ ID NO: 12; And / or, the RNaseH1 (D210N) is a protein consisting of the amino acid sequence as shown in SEQ ID NO: 14, or a protein having RNA / DNA complex chain binding activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence as shown in SEQ ID NO: 14; And / or, the ScFV of the monoclonal antibody S9.6 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 13, or the amino acid sequence shown in SEQ ID NO: 13 is substituted, A protein that is missing or has one or more amino acids added and has RNA / DNA complex binding activity.

5. The complex enzyme according to any one of claims 1 to 4, characterized in that The nucleotide sequence of the complex enzyme is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:

7.

6. A method for synthesizing RNA enzymatically using the composite enzyme according to any one of claims 1 to 5, characterized in that: The method includes method A, method B or method C, The method A comprises the following steps in sequence: S1, adding the first single-stranded RNA fragment, the second single-stranded RNA fragment and the single-stranded DNA splint oligonucleotide into an enzyme-linked buffer to obtain a mixed solution; S2, adding the complex enzyme to the mixed solution to perform enzyme linkage reaction; The method B comprises the following steps in sequence: Sa, mixing the single-stranded DNA splint oligonucleotide with the complex enzyme to prepare a single-stranded DNA-protein complex; Sb, adding the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the single-stranded DNA-protein complex into an enzyme-linked buffer to perform an enzyme-linked reaction; The method C comprises the following steps in sequence: Si, the first single-stranded RNA fragment, the second single-stranded RNA fragment and the single-stranded RNA splint oligonucleotide are added to an enzyme-linked buffer to obtain a mixed solution; Sii. Adding the complex enzyme into the mixed solution to carry out enzyme linkage reaction.

7. The method for enzymatically synthesizing RNA according to claim 6, characterized in that: The protein with substrate affinity of the complex enzyme in method A is one or more of a DNA double-stranded binding domain and an RNA / DNA composite strand binding domain. When the protein with substrate affinity is a DNA double-stranded binding domain, the single-stranded DNA splint oligonucleotide is a single-stranded DNA splint oligonucleotide with additional double-stranded DNA; And / or, the protein with substrate affinity of the complex enzyme in method B is a single-stranded DNA covalently bound protein; And / or, the protein with substrate affinity in the complex enzyme in method C is a dsRNA binding protein.

8. The method for enzymatically synthesizing RNA according to claim 6 or 7, characterized in that: Step S1 of method A and / or step Si of method C further include subjecting the mixed solution to thermal denaturation treatment, naturally cooling or gradient cooling to 20°C to 30°C after the thermal denaturation treatment, the temperature of the denaturation treatment is 94 to 96°C, and the time of the denaturation treatment is 3 to 5 minutes; preferably, the gradient cooling is to cool to 20 to 30°C at a rate of 0.05 to 0.2°C / s.

9. The method for enzymatically synthesizing RNA according to any one of claims 6 to 8, characterized in that: In method A and method C, the final concentrations of the first single-stranded RNA fragment, the second single-stranded RNA fragment, and the splint oligonucleotide in the enzyme-linked reaction system are all 10 to 30 μM; And / or, in method A and method C, the final concentration of the complex enzyme in the enzyme-linked reaction system is 1 nM to 10 μM; And / or, in method B, the final concentrations of the first single-stranded RNA fragment and the second single-stranded RNA fragment in the enzyme-linked reaction system are both 10-30 μM, and the final concentration of the single-stranded DNA-protein complex is 10-30 μM.

10. The method for enzymatically synthesizing RNA according to any one of claims 6 to 9, characterized in that: In method A, method B and method C, the pH of the enzyme-linked buffer is 7.5, and the enzyme-linked buffer comprises DTT with a final concentration of 0.1 to 50 mM, ATP with a final concentration of 10 to 1000 μM, and Tris-HCl with a final concentration of 10 to 500 mM; And / or, in method A, method B and method C, the temperature of the enzyme ligation reaction is 36-38°C; And / or, in method A, method B and method C, the time of the enzyme linkage reaction is 10 to 720 minutes.

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