Reverse transcriptase for rapid reverse transcription synthesis of cDNA and application of reverse transcriptase

Abstract

The invention discloses reverse transcriptase for rapid reverse transcription synthesis of cDNA (complementary deoxyribonucleic acid) and application of the reverse transcriptase, and belongs to molecular biology. A novel reverse transcriptase is obtained by fusing a thermal stable structural domain of AMV reverse transcriptase and a low RNase H active structural domain of MMLV reverse transcriptase, performing specific site mutation and optimizing surface charges, and the reverse transcriptase has thermal stability, no RNase H activity and high reverse transcription activity, and can be used for preparing a novel reverse transcriptase. And the method also has excellent long fragment synthesis capability and high-efficiency processing capability on a high-GC template. In combination with a matched reaction reagent, the cDNA can be quickly and efficiently synthesized through reverse transcription, and the cDNA has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a reverse transcriptase for rapid reverse transcription and synthesis of cDNA and application thereof. BACKGROUND

[0002] The reverse transcription process is an important supplement to the central dogma of molecular biology, which enables RNA information to be converted into a more stable DNA form for subsequent analysis and manipulation. As the core catalyst of this process, the performance parameters of reverse transcriptase, such as catalytic efficiency, thermal stability, RNase H activity, and ability to handle complex templates, directly affect the quality and efficiency of cDNA synthesis.

[0003] Currently, the reverse transcriptases commonly used in the market are mainly divided into two categories: AMV reverse transcriptase and MMLV reverse transcriptase. AMV reverse transcriptase is isolated from avian myeloblastosis virus, and its optimal reaction temperature is 42℃. Compared with MMLV reverse transcriptase, it has better thermal stability. This thermal stability enables it to overcome the secondary structure in the RNA template to some extent, because RNA molecules, especially those with high GC content, tend to form stable stem-loop structures, which hinder the movement of reverse transcriptase and cause the interruption of cDNA synthesis. The activity of AMV reverse transcriptase at a higher temperature enables it to partially unfold these secondary structures, thereby improving the integrity of reverse transcription. However, a significant disadvantage of AMV reverse transcriptase is its high RNase H activity. RNase H is a nuclease that can specifically recognize and cut the RNA strand in the RNA-DNA hybrid. During cDNA synthesis, when reverse transcriptase synthesizes cDNA with RNA as the template, an RNA-DNA hybrid intermediate is formed, and the RNase H activity of AMV reverse transcriptase will degrade the RNA strand in it. This not only causes the premature termination of cDNA synthesis and the inability to obtain full-length cDNA, but also produces a large amount of RNA fragments that interfere with subsequent experimental results. In addition, the preparation cost of AMV reverse transcriptase is relatively high, which also limits its application in large-scale experiments.

[0004] MMLV reverse transcriptase is derived from Moloney murine leukemia virus, and has lower RNase H activity compared to AMV reverse transcriptase. This feature gives it an advantage in synthesizing long cDNA fragments, as it reduces the degradation of the RNA strand in the RNA-DNA hybrid, thereby increasing the full-length rate of cDNA. However, MMLV reverse transcriptase has poor thermal stability, with an optimal reaction temperature usually around 37℃. When the reaction temperature exceeds 40℃, the activity of the enzyme gradually decreases, and above 45℃, the activity decreases sharply. This thermal instability greatly limits the efficiency of reverse transcription of RNA templates with complex secondary structures, especially for RNA templates with a GC content of more than 60%, as their secondary structures are very stable and difficult to unfold at the optimal temperature of MMLV reverse transcriptase, making it difficult for the reverse transcriptase to pass through, resulting in short and incomplete cDNA synthesis.

[0005] To overcome the inherent defects of these two reverse transcriptases, researchers have attempted to modify them through genetic engineering. Early studies focused on single-point mutations or local domain modifications. However, some mutations were found to slightly reduce the reverse transcription activity while improving thermal stability, which was not desirable in some scenarios where enzyme activity was highly required.

[0006] In addition, with the development of high-throughput experimental techniques, there is an increasing demand for faster reverse transcription reactions. Traditional reverse transcription reactions usually take 30 minutes to 1 hour, which can significantly affect experimental efficiency when dealing with large numbers of samples. Therefore, developing a reverse transcriptase and supporting reagents that can synthesize high-quality cDNA in a short time (such as 5 minutes) has become an important direction of current research.

[0007] Therefore, the existing reverse transcriptases still have many shortcomings in terms of thermal stability, RNase H activity control, handling of complex templates, and reaction speed. There is an urgent need to develop a new type of reverse transcriptase and optimize its supporting reagent formula to meet the needs of modern molecular biology research and applications for efficient and high-quality cDNA synthesis. In view of this, the present application is proposed. SUMMARY

[0008] The present application aims to provide a rapid reverse transcription cDNA synthesis reverse transcriptase and its application, which has high thermal stability, no RNase H activity, and can quickly and efficiently synthesize full-length cDNA.

[0009] The present application is implemented as follows: In a first aspect, the present application provides a reverse transcriptase, which is a fusion protein comprising a first reverse transcriptase and a second reverse transcriptase connected by a flexible linker; the first reverse transcriptase is a thermostable domain derived from an AMV reverse transcriptase; the second reverse transcriptase is a low RNase H activity domain derived from an MMLV reverse transcriptase. The first reverse transcriptase and the second reverse transcriptase in the above reverse transcriptase have at least one mutation compared with a wild-type reverse transcriptase; the amino acid sequence of the first reverse transcriptase is shown in SEQ ID NO: 1; the amino acid sequence of the second reverse transcriptase is shown in SEQ ID NO: 2.

[0010] In a second aspect, the present application provides a biological material related to the above reverse transcriptase, which is any one of (A1)-(A4) below: (A1) a nucleic acid molecule encoding the above reverse transcriptase; (A2) an expression cassette containing the nucleic acid molecule of (A1); (A3) a recombinant vector containing the nucleic acid molecule of (A1), or a recombinant vector containing the expression cassette of (A2); (A4) a recombinant microorganism containing the nucleic acid molecule of (A1), or a recombinant microorganism containing the expression cassette of (A2), or a recombinant microorganism containing the recombinant vector of (A3).

[0011] In a third aspect, the present application provides a use of the above reverse transcriptase or biological material, which includes any one of (B1)-(B4) below: (B1) a use in preparing a rapid reverse transcription cDNA synthesis product; (B2) a use in preparing an RT-PCR detection product; (B3) a use in preparing a cDNA library construction product; (B4) a use in preparing a long-chain cDNA synthesis product.

[0012] In a fourth aspect, the present application provides a reaction reagent for reverse transcription synthesis of cDNA, which includes the above reverse transcriptase.

[0013] In a fifth aspect, the present application provides a use of the above reverse transcriptase, biological material or reaction reagent, which includes any one of (C1)-(C4) below: (C1) a use in rapid reverse transcription cDNA synthesis; (C2) a use in RT-PCR detection; (C3) a use in cDNA library construction; (C4) a use in long-chain cDNA synthesis.

[0014] In a sixth aspect, the present application provides a method for rapidly reverse transcribing and synthesizing cDNA, comprising: mixing the reverse transcriptase or the reaction reagent described above with a nucleic acid template, and performing a reverse transcription reaction at 55℃. wherein the reaction time of the reverse transcription reaction is 5 min.

[0015] The present application has the following beneficial effects: (1) The present application obtains a new reverse transcriptase by fusing the heat-stable domain of AMV reverse transcriptase and the low RNase H activity domain of MMLV reverse transcriptase, specific site mutation, and surface charge optimization. The reverse transcriptase has heat stability, no RNase H activity, and high reverse transcription activity, and also has excellent long fragment synthesis capability and efficient processing capability for high GC templates.

[0016] (2) The cDNA product synthesized by the reverse transcriptase of the present application can meet the experimental requirements of cDNA library construction, long fragment gene cloning, high GC content gene expression analysis, and other high-quality cDNA requirements.

[0017] (3) The reverse transcriptase of the present application has good reverse transcription effects on RNA templates of different lengths (including long fragments of more than 14 kb) and different GC contents, and is compatible with Random 6 and Oligo dT18 primers, suitable for reverse transcription of various RNAs such as mRNA, rRNA, and tRNA, and widens its application range.

[0018] (4) The reverse transcriptase of the present application is designed with matching reaction reagents, which are highly matched with the characteristics of the reverse transcriptase and can maximize its catalytic performance. Under the synergistic effect of the matching reaction reagents, the reverse transcriptase and the reaction reagents can complete the synthesis of cDNA of long fragments of more than 14 kb in only 5 min at 55-60℃, greatly shortening the experimental time and improving the experimental efficiency, and are especially suitable for high-throughput screening and rapid diagnosis and other time-sensitive application scenarios. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0020] Figure 1 Comparison results of the reaction reagent of the present application and commercial kit A in Example 4; Figure 2Results of comparison between the reaction reagent of the present application and commercialized kit B in Example 4; Figure 3 Results of comparison between the reaction reagent of the present application and commercialized kits A, B and C in Example 4; Figure 4 Results of electrophoresis detection in Example 5. DETAILED DESCRIPTION

[0021] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, the conventional products which can be purchased in the market are adopted.

[0022] The conventional methods of molecular biology and nucleic acid chemistry used in the following examples and experimental examples are all prior art and are introduced in detail in the literature, see "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press; "Oligonucleotide Synthesis", M.J. Gait et al., 1984; "Nucleic Acid Hybridization", B.D. Hames and S.J. Higgins, 1984; "Basic Methods in Molecular Biology", Elsevier; "Current Protocols in Molecular Biology", John Wiley and Sons; "Methods in Enzymology", Academic Press; if the raw materials (including biological materials), reagents, culture medium, instruments and the like are not specially indicated, they are all the conventional products in the art, which can be easily obtained by the public or can be obtained through commercial channels; the terms and abbreviations involved are all the conventional meanings in the art.

[0023] The terms "mutant", "mutation" or "mutant type" and the like in this text mean that one or more mutations exist compared to the wild-type DNA sequence or the wild-type amino acid sequence. Of course, the mutation can occur at the nucleic acid level or at the amino acid level.

[0024] In this document, when referring to mutation sites, the conventional notation in the art is used, namely "abbreviation of the amino acid before mutation + site + abbreviation of the amino acid after mutation", such as "D524A", where "D" represents the amino acid before mutation, "524" is the corresponding mutation site, and "A" represents the amino acid after mutation. Both "D" and "A" are single-letter abbreviations commonly used in the art to represent amino acids. When describing combined mutations, the two mutations are connected by " / ", for example, the mutation site "D524A / E562Q" indicates that compared to the wild type, both amino acids 524 and 562 are altered simultaneously.

[0025] According to embodiments of the present invention, a first aspect provides a novel reverse transcriptase, which is optimized from a wild-type reverse transcriptase as follows: (1) The thermostable domain of AMV reverse transcriptase (i.e., the first reverse transcriptase) is fused with the low RNaseH activity domain of MMLV reverse transcriptase (i.e., the second reverse transcriptase). (2) The thermostable domain of AMV reverse transcriptase is linked to the low RNase H activity domain of MMLV reverse transcriptase by a flexible linker peptide. (3) Mutate a specific site on the low RNase H activity domain of MMLV reverse transcriptase; (4) The fused amino acid sequence was subjected to computer simulation and surface charge analysis to identify concentrated charge regions on the surface that may lead to intermolecular repulsion or attraction, and multiple consecutive positively charged amino acid residues in the region were mutated.

[0026] During the research, it was discovered that the N-terminal sequence of AMV reverse transcriptase contains a key domain related to thermostability. The amino acid composition and spatial structure of this region endow AMV reverse transcriptase with inherent thermostability. A specific region in wild-type MMLV reverse transcriptase contains a complete reverse transcription catalytic active site, including a template binding site, a primer binding site, and a catalytic site. To obtain a reverse transcriptase that simultaneously satisfies the requirements of thermostability and RNase H elimination activity, this invention fuses the thermostable domain of AMV reverse transcriptase with the low RNase H activity domain of MMLV reverse transcriptase. In this fusion enzyme, the fragment derived from AMV reverse transcriptase serves as the basis for thermostability, while the fragment derived from MMLV reverse transcriptase provides the reverse transcription catalytic activity.

[0027] The amino acid sequence of the thermostable domain of AMV reverse transcriptase is shown in SEQ ID NO:1, and the amino acid sequence of the low RNase H activity domain of MMLV reverse transcriptase is shown in SEQ ID NO:2.

[0028] Research has found that fusing the thermostable domain of AMV reverse transcriptase with the low RNase H activity domain of MMLV reverse transcriptase results in an unsuitable connection between the domains of the fusion enzyme. For example, using rigid linkers or direct linkage leads to conformational distortion of the fusion enzyme, disrupting the structure of the catalytic active site and causing the reverse transcription activity to be far lower than expected. Therefore, the inventors have addressed this issue by connecting the thermostable domain of AMV reverse transcriptase with the low RNase H activity domain of MMLV reverse transcriptase using a flexible linker, thus overcoming the problem of low reverse transcription activity caused by conformational distortion of the fusion enzyme.

[0029] In some embodiments, the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:3. The linker peptide is composed of glycine (G) and serine (S), where glycine has the smallest side chain (only one hydrogen atom), providing maximum flexibility, while the hydroxyl group of serine has a certain degree of polarity, which helps to increase the water solubility of the linker peptide. The flexibility of the linker peptide allows the two domains to freely adjust their relative positions in space, thereby forming the correct folded conformation and avoiding the destruction of the catalytic active site caused by rigid linkage.

[0030] The low RNase H activity domain of the MMLV reverse transcriptase is located in a specific region. To eliminate RNase H activity, this invention first completely removes the corresponding RNase H domain from the MMLV sequence. However, removing this domain alone may not completely eliminate residual RNase H activity. Therefore, two key mutations, D524A and E562Q, are further introduced into the MMLV sequence. These two sites are key amino acid residues in the RNase H active site. Mutating aspartic acid (D) to alanine (A) and glutamic acid (E) to glutamine (Q) completely disrupts the catalytic activity of these residual structures, thereby ensuring the complete elimination of RNase H activity in the fusion enzyme. This combined strategy (deletion of the RNase H domain + key site mutation) can more thoroughly eliminate RNase H activity compared to single domain deletion or single-point mutation, ensuring the synthesis of full-length cDNA.

[0031] Building upon this, to further improve the thermostability of reverse transcriptase at 55-60℃, this invention also involves mutations at specific sites on the low RNase H activity domain of the MMLV reverse transcriptase, including mutating methionine (M) at position 66 to leucine (L). Since common Y133F mutations, or mutations combining Y133F with other sites, may slightly reduce the enzyme's reverse transcription activity while improving thermostability, the single mutation at position 66 in this invention enhances the hydrophobic core structure within the enzyme molecule by increasing the hydrophobicity of the amino acid side chains, thereby improving the enzyme's thermostability.

[0032] Furthermore, the fused enzyme molecules are prone to aggregation at high temperatures due to the mismatch in surface charge distribution between different domains. This leads to enhanced intermolecular interactions, forming inactive aggregates and further reducing the effective concentration and activity of the enzyme. To improve this situation, this invention also adjusts the surface charge distribution by mutating specific sites. These specific sites are those that cause electrostatic repulsion between molecules or non-specific binding to negatively charged RNA templates, and include multiple consecutive positively charged amino acid residues in a certain region.

[0033] The inventors discovered that by performing computer simulations and surface charge analysis on the amino acid sequence of fusion enzymes, concentrated charge regions on the surface that may lead to intermolecular repulsion or attraction can be identified. For example, if a region contains multiple consecutive positively charged lysine (K) or arginine (R) residues, it may lead to electrostatic repulsion between molecules or nonspecific binding to negatively charged RNA templates. By mutating some of these residues to neutral amino acids (such as alanine A) or amino acids with opposite charges (such as glutamic acid E), the distribution of surface charge can be adjusted, reducing the tendency of enzyme molecules to aggregate at high temperatures and improving the stability of the enzyme under high-temperature reaction conditions. The specific mutation sites are determined based on the calculated charge distribution. These mutations do not affect the catalytic active site of the enzyme but significantly improve its high-temperature stability.

[0034] In some embodiments, specific mutation sites include, but are not limited to, position 287 of the second reverse transcriptase, and the mutation mode includes T287A; more sites can be determined based on the actual sequence and analysis results.

[0035] In some embodiments, the amino acid sequence of the reverse transcriptase of the present invention is as shown in SEQ ID NO:4.

[0036] After the above modifications and optimizations, the reverse transcriptase mutant of the present invention possesses high reverse transcription activity, complete elimination of RNase H activity, high thermal stability, excellent long fragment synthesis ability, efficient amplification of templates with high GC content, and good anti-aggregation properties, specifically manifested as follows: (1) It incorporates the reverse transcription catalytic active site of MMLV, and through reasonable connection and conformation optimization, it ensures that the enzyme can efficiently catalyze the reaction of synthesizing cDNA using RNA as a template.

[0037] (2) By removing the RNase H domain and introducing key mutations, the RNase H activity was completely eliminated, so that the RNA chain in the RNA-DNA hybrid would not be degraded during the cDNA synthesis process, thus ensuring the synthesis of full-length cDNA.

[0038] (3) After being kept at 55℃ for 30 minutes, it can still retain more than 80% of its activity; after being kept at 60℃ for 30 minutes, it retains about 60% of its activity, which is much higher than wild-type MMLV reverse transcriptase and ordinary fusion enzyme, and can meet the needs of high temperature rapid reverse transcription.

[0039] (4) It can efficiently synthesize cDNA of more than 14kb, thanks to its lack of RNase H activity and high thermal stability, which allows the enzyme to continuously move along the long RNA template and complete the full-length synthesis.

[0040] (5) Due to its high activity at 55℃, it can effectively untangle the secondary structure of RNA templates with high GC content. Therefore, it has better reverse transcription efficiency than MMLV reverse transcriptase for templates with GC content of more than 60%, and the length and yield of synthesized cDNA are significantly improved.

[0041] (6) By optimizing the surface charge, the tendency of enzyme molecules to aggregate at high temperatures is reduced, thereby improving the effective concentration and stability of the enzyme in the reaction system.

[0042] A second aspect of the present invention also provides biological materials related to the above-mentioned reverse transcriptase, including a nucleic acid molecule encoding the above-mentioned reverse transcriptase, an expression cassette containing the nucleic acid molecule, a recombinant vector, and a recombinant microorganism.

[0043] Since the same amino acid can be encoded by several different codons, i.e., synonymous codons, the same amino acid can correspond to different nucleotide sequences. Therefore, the reverse transcriptase of this invention can be encoded by multiple nucleotide sequences. It is understood that the synonymous codon sequence can be a codon synonymous mutation of the mutant amino acid site of the above-mentioned reverse transcriptase, or a codon synonymous mutation of other non-mutant amino acids.

[0044] Those skilled in the art can obtain the reverse transcriptase of the present invention by using existing molecular biology techniques, cDNA cloning and site-directed mutagenesis or other suitable methods based on the amino acid sequence of the reverse transcriptase disclosed in the present invention. Therefore, the nucleotide sequence encoding the reverse transcriptase is not unique. As long as the reverse transcriptase obtained does not have obvious functional differences, it is included within the scope of the present invention.

[0045] The expression cassette, including the aforementioned nucleic acid molecule, is a modular structure obtained by inserting the aforementioned nucleic acid molecule encoding the aforementioned reverse transcriptase between a suitable promoter and terminator.

[0046] For recombinant vectors, including the aforementioned nucleic acid molecules or expression cassettes, the nucleic acid molecule encoding the aforementioned reverse transcriptase is cloned into the expression vector or the aforementioned expression cassette is integrated into the expression vector to obtain the recombinant vector.

[0047] The recombinant microorganisms include the aforementioned nucleic acid molecules or the aforementioned recombinant vectors. The host cell is obtained by transforming the aforementioned recombinant vector into engineered cells.

[0048] The aforementioned reverse transcriptase can be obtained through chemical synthesis or by inducing expression using recombinant engineered cell lines. The reverse transcriptase obtained by inducing expression using recombinant engineered cell lines can be in the form of a crude enzyme extract or a purified enzyme. This invention does not limit the preparation method of this reverse transcriptase.

[0049] A third aspect of this invention also provides the application of the above-mentioned reverse transcriptase or biological material in the preparation of rapid reverse transcription synthesized cDNA products, RT-PCR detection products, cDNA library construction products, and long-chain cDNA synthesis products. The above-mentioned reverse transcriptase exhibits higher reverse transcription activity, higher thermostability, and is completely free of RNase H activity compared to wild-type reverse transcriptase.

[0050] A fourth aspect of the present invention also provides a reaction reagent for reverse transcription synthesis of cDNA, comprising the above-described reverse transcriptase.

[0051] Preferably, the reaction reagents are selected from, depending on the intended use, such as: reverse transcription reaction kits, RT-PCR amplification kits, RT-qPCR kits, cDNA library construction kits, RNA sequencing kits, etc.

[0052] Suitable reaction reagents are crucial for the effective activity of reverse transcriptase. Factors such as pH, ionic strength, and metal ion concentration in the reaction reagents all affect the enzyme's conformation and catalytic efficiency. However, most existing reagent formulations are designed for wild-type or early-modified reverse transcriptases, and these formulations often fail to achieve optimal reaction results for novel fusion mutant reverse transcriptases. To fully utilize the reverse transcriptase of this invention, the composition and ratio of the reaction reagents have been optimized, mainly including: Mg 2+ Trehalose, glycerol, ethylene glycol, primer type and concentration, Oligo dT18 primer. Specifically, Mg... 2+ It is an essential cofactor of reverse transcriptase, participating in the binding of the enzyme to the template-primer complex and playing a role in stabilizing the transition state in the catalytic reaction. Mg 2+Both excessively high and low concentrations of enzymes can reduce their activity. Trehalose, glycerol, and ethylene glycol have been found to improve the thermostability of enzymes by forming hydrogen bonds with enzyme molecules or coating the enzyme molecule surface, reducing the unfolding and aggregation of enzyme molecules at high temperatures. The type and concentration of primers also affect the efficiency and specificity of reverse transcription. Random 6 primers (random hexamers) can randomly bind to any position on the RNA template, making them suitable for the reverse transcription of various RNAs, especially RNAs without a poly(A) tail; while Oligo dT18 primers specifically bind to the 3' poly(A) tail of mRNA, making them suitable for the reverse transcription of mRNA. A proper combination of these two primers can improve the coverage and efficiency of reverse transcription.

[0053] In some embodiments, the components of the above reaction reagents are: reverse transcriptase, Tris-HCl pH 8.3, KCl, MgCl2, BSA, dNTPs, Tween 20, glycerol, ethylene glycol, EDTA, DTT, Random 6, Oligo dT18, trehalose, gelatin, RNase inhibitor and DEPC water.

[0054] In some embodiments, the concentrations of each component in the reaction reagent are as follows: 50-150 mM Tris-HCl pH 8.3, 150-250 mM KCl, 5-15 mM MgCl2, 0.01-0.1 mg / mL BSA, 1-2 mM dNTPs, 0.1%-1% Tween 20, 1%-10% glycerol, 10%-20% ethylene glycol, 1-5 mM EDTA, 0.1-1 mM DTT, 10-50 nM Random 6, 1-50 nM Ligo dT18, 50-200 mM Trehalose, 0.1%-1% Gelatin, 1-10 U / μL RNase inhibitor, 1-200 U / μL reverse transcriptase, and the remainder being DEPC water.

[0055] Tris-HCl at pH 8.3 provides a stable alkaline environment for the reaction. pH 8.3 is the optimal pH determined through multiple experiments, under which the reverse transcriptase mutant exhibits the highest activity. As a commonly used buffer, Tris-HCl effectively maintains the pH stability of the reaction system, preventing pH fluctuations during the reaction from affecting enzyme activity.

[0056] KCl provides potassium ions, regulating the ionic strength of the reaction system. An appropriate K⁺ concentration promotes the annealing of primers to the RNA template, while stabilizing the binding of the enzyme to the template-primer complex, thus improving catalytic efficiency. After optimization, a KCl concentration of 250 mM was determined to be the optimal concentration; concentrations that are too high or too low will affect enzyme activity.

[0057] Mg in MgCl2 2+ It is an essential cofactor of reverse transcriptase, participating in the formation of the enzyme's active site and playing a crucial role in catalyzing dNTP incorporation. A 10 mM MgCl2 concentration, determined through multiple gradient experiments, can maximally activate the reverse transcriptase mutant's activity while avoiding the adverse effects of Mg... 2+ Excessive concentration can lead to enzyme activity inhibition or non-specific binding.

[0058] BSA, as a stabilizer, can protect enzymes from degradation by proteases by binding to enzyme molecules, while reducing enzyme adsorption loss on the container surface and increasing the effective concentration of the enzyme.

[0059] dNTPs provide the raw materials for cDNA synthesis, containing four deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, and dTTP, with a balanced concentration of the four nucleotides. A total concentration of 1.67 mM is sufficient to meet the raw material requirements for long-fragment cDNA synthesis, avoiding synthesis interruption due to insufficient dNTPs.

[0060] Tween 20 is a nonionic surfactant that can reduce the surface tension of enzyme molecules, reduce the aggregation between enzyme molecules, and improve the dispersibility and stability of enzymes in the reaction system, especially at high temperatures.

[0061] As a polar molecule, glycerol can form hydrogen bonds with enzyme molecules, stabilizing the enzyme's three-dimensional structure and lowering its freezing point, thus facilitating its low-temperature preservation. During the reaction process, glycerol can also reduce the damage to the enzyme structure caused by high temperatures, improving the enzyme's thermal stability.

[0062] Ethylene glycol has a stabilizing effect similar to glycerol, but its smaller molecule allows it to penetrate deeper into the enzyme molecule's structure, forming hydrogen bonds with key amino acid residues and further enhancing the enzyme's stability at high temperatures. In synergy with glycerol, it can significantly improve the activity retention rate of reverse transcriptase mutants at 55°C.

[0063] EDTA is a metal chelating agent that can chelate trace amounts of heavy metal ions (such as Fe) that may be present in the reaction system. 3 + Cu 2+ These ions (such as ions) may damage the structure of enzymes or inhibit their activity through oxidation. The addition of EDTA can eliminate these interfering factors.

[0064] DTT (dithiothreitol) is a reducing agent that can maintain the reduced state of the sulfhydryl group (-SH) in the enzyme molecule, preventing abnormal enzyme conformation caused by incorrect formation of disulfide bonds, thereby ensuring the active conformation of the enzyme.

[0065] Random 6, a random hexamer primer, can bind to any position on the RNA template to initiate the reverse transcription reaction. Its concentration has been optimized; a concentration of 40 nM ensures sufficient primer binding to the template without causing non-specific binding or primer dimer formation due to excessive primer.

[0066] Oligo dT18, an oligothymidine primer, specifically binds to the 3' poly(A) tail of mRNA. When used in conjunction with the Random 6 primer, it improves the efficiency and coverage of reverse transcription of the mRNA template. The low concentration of 20 nM is designed to avoid competition for binding sites with the Random 6 primer while ensuring specific recognition of the poly(A) tail.

[0067] Trehalose is a non-reducing disaccharide that can encapsulate enzyme molecules at high temperatures, forming a protective film that reduces thermal denaturation and aggregation of enzyme molecules. Studies have shown that trehalose has a significant protective effect on thermostable enzymes; a concentration of 125 mM can provide effective protection for reverse transcriptase mutants during reactions at 55°C.

[0068] As a natural high-molecular polymer, benzoic acid can increase the viscosity of the reaction system, reduce the diffusion and collision of enzyme molecules, thereby reducing the probability of aggregation at high temperatures and further stabilizing enzyme activity.

[0069] RNase inhibitors can suppress potentially contaminating RNases (such as RNase A and RNase H) in the reaction system, protecting the RNA template from degradation. A concentration of 3.33 U / μL provides sufficient inhibitory activity to ensure the integrity of the RNA template during reverse transcription.

[0070] The concentration of the reverse transcriptase shown in SEQ ID NO:4 was determined based on the enzyme's specific activity and reaction time. A concentration of 16.7 U / μL can complete the reverse transcription of long RNA templates within 5 minutes.

[0071] DEPC water: As a solvent, water treated with DEPC (diethyl pyrocarbonate) can inactivate RNase, prevent the degradation of RNA template, and ensure an RNase-free environment in the reaction system.

[0072] The concentrations of the above components were determined through extensive single-factor and orthogonal experiments. They work synergistically to provide the optimal reaction environment for reverse transcription, enabling rapid and efficient synthesis of cDNA at 55°C.

[0073] The fifth aspect of the present invention also provides the application of the above-mentioned reverse transcriptase, biological materials or reaction reagents in rapid reverse transcription synthesis of cDNA, RT-PCR detection, cDNA library construction and long-chain cDNA synthesis.

[0074] A sixth aspect of this invention also provides a method for rapid reverse transcription synthesis of cDNA, comprising: mixing the aforementioned reverse transcriptase or reaction reagent with a nucleic acid template, and performing a reverse transcription reaction at 55-60°C. Verification has shown that a clear 14kb cDNA band can be detected after 5 minutes of reaction. Therefore, using the reverse transcriptase or reaction reagent of this invention, rapid synthesis of long-chain cDNA can be achieved.

[0075] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0076] Example 1 This example demonstrates the gene synthesis and expression of a reverse transcriptase mutant (SEQ ID NO:4). 1. Gene sequence design and synthesis Based on the provided amino acid sequences of the AMV (SEQ ID NO:1), MMLV (SEQ ID NO:2), and the flexible linker peptide GGSGG (SEQ ID NO:3), a nucleotide sequence encoding a reverse transcriptase mutant (SEQ ID NO:4) was designed. During the design process, the following mutations were introduced: M66L, D524A, and E562Q in the MMLV sequence, and the T287A surface charge adjustment mutation determined based on surface charge optimization results. Simultaneously, to facilitate subsequent cloning and expression, an NdeⅠ restriction endonuclease site (catatg) was introduced at the 5' end of the gene sequence, an XhoⅠ restriction endonuclease site (ctcgag) was introduced at the 3' end, and a 6×His tag coding sequence was added after the start codon to facilitate protein purification.

[0077] The designed nucleotide sequence is submitted to the Biotech Department for whole-genome synthesis. After the synthesized gene fragment is verified by sequencing, it is used for subsequent vector construction.

[0078] 2. Construction of recombinant expression vectors (1) Enzyme digestion: The synthesized target gene fragment and pET-28a(+) expression vector were double-digested with NdeⅠ and XhoⅠ restriction endonucleases, respectively. The enzyme digestion reaction system (50 μL) consisted of 5 μL of 10×CutSmart buffer, target gene / vector DNA (approximately 2 μg), 1 μL of NdeⅠ (10 U / μL), 1 μL of XhoⅠ (10 U / μL), and ddH2O to a final volume of 50 μL. The reaction was carried out in a water bath at 37℃ for 3 h.

[0079] (2) Gel recovery: The enzyme digestion products were separated by 1% agarose gel electrophoresis. The target gene fragment and the enzyme-digested pET-28a(+) vector fragment were recovered using a laboratory-developed DNA gel recovery kit. The operation was performed according to the kit instructions.

[0080] (3) Ligation: The recovered target gene fragment was ligated to the pET-28a(+) vector fragment at a molar ratio of 3:1. Ligation reaction system (20 μL): 2 μL 10×T4 DNA ligase buffer, target gene fragment (approximately 0.3 μg), vector fragment (approximately 0.1 μg), 1 μL T4 DNA ligase (5 U / μL), and ddH2O to a final volume of 20 μL. Ligation was carried out overnight at 16℃.

[0081] (4) Transformation and screening: The ligation product was transformed into E. coli DH5α competent cells. 5 μL of the ligation product was added to 50 μL of competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, immediately incubated on ice for 2 min, and then 450 μL of LB liquid medium was added. The cells were cultured at 37℃ with shaking at 200 rpm for 1 h. 100 μL of the culture was spread on LB solid medium containing antibiotics and cultured overnight at 37℃. Single colonies were picked and inoculated into LB liquid medium containing antibiotics and cultured overnight with shaking at 37℃. The plasmid was extracted using a self-developed plasmid mini-prep kit, and the correctness of the recombinant plasmid (named pET-28a-RTmut) was verified by NdeⅠ / XhoⅠ double enzyme digestion and sequencing.

[0082] 3. Expression and purification of recombinant proteins (1) Expression: The verified recombinant plasmid pET-28a-RTmut was transformed into Escherichia coli BL21(DE3) competent cells. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 220 rpm. The next day, the seed culture was inoculated into 1 L of LB liquid medium (containing 50 μg / mL kanamycin) at a ratio of 1:100 and cultured at 37°C with shaking at 220 rpm until OD. 600nm When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mM, and induce expression at 30℃ and 180 rpm for 6 h.

[0083] (2) Collecting bacterial cells: After induction, collect bacterial cells by centrifugation at 4℃ and 8000 rpm for 10 min, discard the supernatant, wash the bacterial cells twice with pre-cooled PBS buffer (pH 7.4), centrifuge again, and resuspend the bacterial cell pellet in Binding Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole). Add 10 mL Binding Buffer per gram of bacterial cells.

[0084] (3) Ultrasonic disruption: The resuspended bacterial cells were ultrasonically disrupted under ice bath conditions (power 300 W, working time 3 s, interval 5 s, total time 30 min) until the bacterial solution changed from turbid to clear. Centrifuge at 4℃ and 12000 rpm for 30 min, collect the supernatant (crude enzyme solution), and filter it with a 0.45 μm filter membrane.

[0085] (4) Nickel column affinity purification: The filtered crude enzyme solution was loaded onto a Ni column pre-equilibrated with Binding Buffer. 2 + -NTA affinity chromatography column (column volume 5 mL). After sample loading, wash with 5 column volumes of Binding Buffer to remove contaminating proteins, then wash with 10 column volumes of Washing Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 50 mM imidazole), and finally elute the target protein with Elution Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 300 mM imidazole). Collect the elution peak.

[0086] (5) Dialysis and concentration: The eluted protein solution was placed in a dialysis bag (molecular weight cutoff 30 kDa) and dialyzed overnight at 4°C in dialysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT, 50% glycerol). The dialysis buffer was changed 3 times during the process to remove imidazole and high concentrations of salt. The dialyzed protein was concentrated using an Amicon Ultra-15 centrifugal ultrafiltration tube (30 kDa molecular weight cutoff), and the protein concentration was determined using a BCA protein quantification kit. The concentration was adjusted to 200 U / μL, aliquoted, and stored at -80°C for later use.

[0087] (6) Purity identification: Take 10 μL of purified protein solution, add 2×SDS loading buffer, boil for 5 min, and then perform 10%SDS-PAGE electrophoresis. After Coomassie brilliant blue staining, observe the protein purity. The results show that the purity of the target protein is above 95%.

[0088] Example 2 This example describes the activity assay of a reverse transcriptase mutant (SEQ ID NO:4). (1) Reverse transcriptase (RT) activity assay RT activity was determined using a radioactive isotope incorporation method. The reaction system (20 μL) contained: 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 0.5 mM dNTPs (containing α- 32P-dTTP (10 μCi), 0.5 μg poly(A)-oligo(dT)18 (template-primer complex), and enzyme solutions of different dilutions were used. The reaction was carried out at 37℃ for 30 min, and then 5 μL of stop solution (200 mM EDTA, 1% SDS) was added to terminate the reaction.

[0089] Take 10 μL of the reaction product and spot it onto DE81 filter paper. After drying at room temperature, wash it three times with 2×SSC solution (5 minutes each time) to remove unincorporated free nucleotides. Then wash it once with anhydrous ethanol. After drying, measure the radioactivity intensity (cpm) on the filter paper using a liquid scintillation counter (such as Beckman LS6500).

[0090] Under the above reaction conditions, one unit of activity (U) is defined as the amount of enzyme that catalyzes the incorporation of 1 nmol dTTP into cDNA per minute. The results show that the specific activity of the reverse transcriptase mutant of this invention is 80,000-100,000 U / mg, indicating that it has high RT activity.

[0091] (2) RNase H activity assay RNase H activity was determined using RNA-DNA hybrids as substrates. First, the substrate was prepared: 0.5 μg poly(A) and 0.5 μg oligo(dT)18 were mixed in annealing buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl), heated at 95°C for 5 min, and then slowly cooled to room temperature to form RNA-DNA hybrids.

[0092] The reaction system (20 μL) contained: 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NaCl, 0.1 μg RNA-DNA hybrid substrate, and 5 U of the test enzyme solution (or 5 U AMV reverse transcriptase as a positive control and 5 U RNase H-deficient MMLV as a negative control). After incubating the reaction at 37°C for 30 min, 5 μL of 5× loading buffer (250 mM EDTA, 50% glycerol, 0.5% bromophenol blue) was added to terminate the reaction.

[0093] The reaction products were separated by 1% agarose gel electrophoresis, and the results were observed under UV light after EB staining. The positive control (AMV reverse transcriptase) showed a free oligo(dT)18 band in its lane (indicating RNA degradation), while the negative control and the reverse transcriptase mutant of this invention showed only RNA-DNA hybrid bands in their lanes, with no free oligo(dT)18 band, indicating that the mutant of this invention completely eliminated RNase H activity.

[0094] (3) Thermal stability determination The purified reverse transcriptase mutant (SEQ ID NO:4) was incubated in water baths at 50℃, 55℃, 60℃, and 65℃. Samples were taken at 0, 5, 10, 20, and 30 min, and the remaining activity was determined using the RT activity assay method described above. The activity at 0 min was taken as 100%, and the relative activity at each time point was calculated.

[0095] The results showed that after incubation at 50℃ for 30 min, the enzyme activity was retained by more than 90%; after incubation at 55℃ for 30 min, the activity was retained by 85%; after incubation at 60℃ for 30 min, the activity was retained by 65%; and after incubation at 65℃ for 30 min, the activity was retained by 30%. In contrast, the activity of the wild-type MMLV reverse transcriptase, used as a control, dropped to below 50% after incubation at 55℃ for 10 min, indicating that the mutant of this invention has significantly improved thermostability.

[0096] Example 3 This embodiment describes a reaction reagent for reverse transcription to synthesize cDNA, and its components are as follows: 80 mM Tris-HCl pH 8.3, 250 mM KCl, 10 mM MgCl2, 0.083 mg / mL BSA, 1.67 mM dNTPs, 0.33% Tween 20, 7.67% glycerol, 12.5% ​​ethylene glycol, 5 mM EDTA, 0.83 mM DTT, 40 nM Random6, 20 nM Oligo dT18, 125 mM trehalose, 0.83% gelatin, 3.33 U / μL RNase inhibitor, 16.7 U / μL reverse transcriptase, and the remainder was DEPC water.

[0097] Example 4 Performance verification of the reaction reagents in Example 3 (1) Effects of different temperatures on cDNA synthesis Using the same 293 cell RNA as a template, the mutant and reagent formulation of this invention were used to react at 37℃, 42℃, and 55℃ for 5 minutes, respectively, and the cDNA synthesis was detected.

[0098] Quantitative fluorescence results showed that the Ct value was obtained earlier at 55℃, indicating that 55℃ is the optimal reaction temperature for this system.

[0099] (2) Comparison with commercially available kits The commercially available SuperScript III reverse transcription kit (Invitrogen) was compared with the reagent (mutant + formulation) of this invention. Using RNA from 293 cells as a template, the cDNA yield and length were compared according to their respective recommended conditions (this invention: 55℃ for 5 min; SuperScript III: 50℃ for 30 min).

[0100] Figures 1-3 The results show a comparison between the reaction reagents in Example 3 and a commercially available kit. Figure 1 In the diagram, purple represents the amplification effect of the reaction reagent in Example 3, and blue represents the amplification effect of commercial kit A; Figure 2 In the diagram, blue indicates the amplification effect of the reaction reagent in Example 3, and yellow indicates the amplification effect of commercial kit B; Figure 3 In the diagram, sky blue represents the amplification effect of the reaction reagent in Example 3, while dark blue, gray, and yellow represent the amplification effects of commercial kits A, B, and C.

[0101] The results showed that using RNA from human 293 cells as a template, this kit could still achieve efficient reversal even with RNA input as low as pg, and was superior to other brands. Furthermore, when using reverse-transcribed cDNA as a template, the Ct value was earlier in the downstream qPCR experiment, and the reaction time was only 1 / 6 of that of other kits, indicating that the reagent of this invention has higher efficiency.

[0102] Example 5 This example is for the verification of long-fragment cDNA synthesis. Using RNA from 293 cells as a template, the reverse transcriptase mutant of the present invention (SEQ ID NO:4) and the reagent formulation were reacted at 55°C for 5 min.

[0103] The reverse transcription products were detected by agarose gel electrophoresis, and the results are as follows: Figure 4 As shown.

[0104] Depend on Figure 4 It can be seen that reverse transcription of RNA from human 293 cells using B690034 for 5 minutes, followed by amplification of the product using B690024 high-fidelity mix, can synthesize 14 kb of cDNA within 5 minutes, and can completely detect RIF1 gene expression.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A reverse transcriptase, characterized in that, The reverse transcriptase is a fusion protein comprising a first reverse transcriptase and a second reverse transcriptase that are flexibly linked. Compared to wild-type reverse transcriptase, the second reverse transcriptase in the reverse transcriptase has at least one mutation; The amino acid sequence of the first reverse transcriptase is shown in SEQ ID NO:1; the amino acid sequence of the second reverse transcriptase is shown in SEQ ID NO:

2.

2. The reverse transcriptase according to claim 1, characterized in that, The mutations include mutations at positions 66, 524, and 562 of the second reverse transcriptase; Preferably, the mutation at position 66 is a mutation from methionine to leucine; Preferably, the mutation at position 524 is a mutation from aspartic acid to alanine. Preferably, the mutation at position 562 is a mutation from glutamic acid to glutamine.

3. The reverse transcriptase according to claim 1, characterized in that, The reverse transcriptase also adjusts the surface charge distribution by mutating specific sites; The specific site is a site that causes electrostatic repulsion between molecules or nonspecific binding to a negatively charged RNA template; Preferably, the specific site comprises a plurality of consecutive positively charged amino acid residues in a certain region; Preferably, the specific site includes position 287 of the second reverse transcriptase.

4. The reverse transcriptase according to claim 1, characterized in that, The amino acid sequence of the reverse transcriptase is shown in SEQ ID NO:

4.

5. A biomaterial relating to the reverse transcriptase according to any one of claims 1-4, characterized in that, It can be any one of the following (A1)-(A4): (A1) A nucleic acid molecule encoding the reverse transcriptase according to any one of claims 1-4; (A2) An expression cassette containing the nucleic acid molecule described in (A1); (A3) A recombinant vector containing the nucleic acid molecule described in (A1), or a recombinant vector containing the expression cassette described in (A2); (A4) A recombinant microorganism containing the nucleic acid molecule described in (A1), or a recombinant microorganism containing the expression cassette described in (A2), or a recombinant microorganism containing the recombinant vector described in (A3).

6. The use of the reverse transcriptase according to any one of claims 1-4 or the biomaterial according to claim 5, characterized in that, The use includes any one of the following (B1)-(B4): (B1) Application in the preparation of rapid reverse transcription synthesized cDNA products; (B2) Application in the preparation of RT-PCR detection products; (B3) Application in the preparation of cDNA library construction products; (B4) Application in the preparation of long-chain cDNA synthetic products.

7. A reaction reagent for reverse transcription synthesis of cDNA, characterized in that, It includes the reverse transcriptase as described in any one of claims 1-4.

8. The reaction reagent according to claim 7, characterized in that, The components of the reaction reagent are: the reverse transcriptase as described in any one of claims 1-4, Tris-HCl pH 8.3, KCl, MgCl2, BSA, dNTPs, Tween 20, glycerol, ethylene glycol, EDTA, DTT, Random 6, Oligo dT18, trehalose, gelatin, RNase inhibitor and DEPC water; Preferably, the concentrations of each component in the reaction reagent are as follows: 50-150 mM Tris-HCl pH 8.3, 150-250 mM M KCl, 5-15 mM MgCl2, 0.01-0.1 mg / mL BSA, 1-2 mM dNTPs, 0.1%-1% Tween 20, 1%-10% glycerol, 10%-20% ethylene glycol, 1-5 mM EDTA, 0.1-1 mM DTT, 10-50 nM Random 6, 1-50 nM Oligo dT18, 50-200 mM trehalose, 0.1%-1% gelatin, 1-10 U / μL RNase inhibitor, 1-200 U / μL reverse transcriptase, and the remainder is DEPC water.

9. The use of the reverse transcriptase according to any one of claims 1-4, the biomaterial according to claim 5, or the reaction reagent according to claim 7 or 8, characterized in that, The use includes any one of the following (C1)-(C4): (C1) Application in rapid reverse transcription synthesis of cDNA; (C2) Application in RT-PCR detection; (C3) Application in cDNA library construction; Application of (C4) in long cDNA synthesis.

10. A method for rapid reverse transcription synthesis of cDNA, characterized in that, include: The reverse transcriptase according to any one of claims 1-4 or the reaction reagent according to claim 7 or 8 is mixed with the nucleic acid template, and a reverse transcription reaction is carried out at 55-60°C. Preferably, the reaction time for the reverse transcription reaction is 5 minutes.

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