MMLV reverse transcriptase mutant, RNA reverse transcription method and application thereof

Abstract

The invention provides an MMLV reverse transcriptase mutant, an RNA reverse transcription method and application of the MMLV reverse transcriptase mutant, and relates to the technical field of biology. Compared with the amino acid sequence of a wild type MMLV reverse transcriptase as shown in SEQ ID NO: 1, the amino acid sequence of the MMLV reverse transcriptase mutant comprises at least one of the following mutations: serine at the 60th site is replaced with phenylalanine, proline at the 89th site is replaced with arginine, and threonine at the 163rd site is replaced with lysine. The MMLV reverse transcriptase mutant provided by the invention endows the MMLV reverse transcriptase with the key capability of keeping high activity in a specific complex chemical environment. The performance is improved, so that the requirements of a single-cell transcriptome sequencing platform based on the dissolvable microbeads can be better met, and the efficiency and the reliability of the whole technical process are improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to MMLV reverse transcriptase mutants, RNA reverse transcription methods, and their applications. Background Technology

[0002] Gene expression analysis is one of the core technologies in molecular biology, genetic engineering, and modern medical research. Gene expression activity is typically reflected in the type and quantity of its transcriptional product, messenger RNA (mRNA). Analyzing all transcripts in an organism (i.e., the transcriptome) can reveal the molecular mechanisms underlying specific physiological states, developmental stages, or disease processes. The key step in transcriptome analysis is the reverse transcription of genetic information from RNA into complementary DNA (cDNA), a process reliant on a core biocatalyst—reverse transcriptase. This enzyme uses RNA as a template to synthesize DNA, transforming unstable RNA information into stable cDNA molecules that are easily amplified and analyzed. Therefore, the catalytic efficiency, stability, and tolerance to the reaction environment of reverse transcriptase directly determine the accuracy and reliability of downstream gene analysis results, forming the cornerstone of the entire technical process.

[0003] To achieve precise quantification and analysis of the transcriptome, various sequencing methods have been developed. Traditional bulk RNA sequencing (Bulk RNA-seq) extracts total RNA from tissues or cell populations to obtain the average gene expression of the population. However, this method has inherent limitations. Because it samples a large number of cells, traditional bulk RNA sequencing cannot reveal the heterogeneity between cells. For example, in the tumor microenvironment, the differences in gene expression among different cell subpopulations are averaged, leading to the loss of key information. At the same time, low-abundance transcripts are often ignored, and the dynamic changes in gene expression over time cannot be analyzed. With the advancement of research, higher-resolution single-cell transcriptome sequencing (scRNA-seq) technology has emerged. This technology can capture the transcriptome information of a single cell with high throughput and high precision. Currently, one of the most widely used single-cell sequencing technology platforms is the 10X Genomics platform, which utilizes microfluidic technology to encapsulate single cells with microbeads carrying molecular barcodes in nanoliter-sized water-in-oil droplets. These microbeads are coated with barcodes (barcode and UMI sequences) to label each cell and each transcript, as well as primers to initiate reverse transcription. Inside the droplet, the microbeads dissolve, triggered by a chemical reagent (such as DTT), releasing the barcodes and primers to initiate a reverse transcription reaction targeting the mRNA within a single cell.

[0004] While single-cell sequencing technology has greatly advanced life science research, its technological implementation path has shortcomings that urgently need to be addressed. Specifically, the technical process itself introduces new challenges, primarily in the introduction of compounds (such as DTT) into reagents to release barcodes and primers from microbeads. DTT can disrupt the microbead structure through thiol-disulfide bond exchange reactions. This fragmentation process inevitably generates byproducts in the tiny reaction system of droplets, including material fragments from the microbeads and small chemical molecules generated after fragmentation, such as (4S, 5S)-1, 2-dithiane-4, 5-disulfides.

[0005] These lysis byproducts severely inhibit subsequent biochemical reactions, especially reverse transcription. The catalytic activity of reverse transcriptases responsible for converting single-cell transcriptome information into cDNA, such as the commonly used MMLV reverse transcriptase, is severely affected by these inhibitors, leading to a significant decrease in reverse transcription efficiency. Therefore, the fundamental flaw in existing technologies lies in the microbead lysis step designed to achieve labeling of transcriptome reverse transcription products in single cells (see microbead lysis principle for details). Figure 1 However, this process simultaneously contaminates the environment of key enzymatic reactions. This inherent contradiction in the technology forces reverse transcriptase to operate in an environment filled with inhibitors, making its performance unsuitable for the efficient and high-fidelity capture of single-cell transcriptomes. This directly affects the quality of sequencing libraries and ultimately limits the depth and breadth of single-cell sequencing technology's application in precision medicine and basic research. Therefore, improving the tolerance of reverse transcriptase MMLV to microbead lysates through enzyme modification is a necessary condition for reverse transcriptase performance to match that of single-cell transcriptome sequencing.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an MMLV reverse transcriptase mutant, an RNA reverse transcription method, and their applications. The MMLV reverse transcriptase mutant helps to obtain more accurate and comprehensive single-cell gene expression data, providing a more solid technical foundation for downstream research such as analyzing cellular heterogeneity and conducting precision medicine.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides an MMLV reverse transcriptase mutant, wherein the amino acid sequence of the MMLV reverse transcriptase mutant, compared with the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1, contains at least one of the following mutations: A. Serine at position 60 is replaced with phenylalanine; B. Proline at position 89 is replaced with arginine; C. Threonine at position 163 is replaced with lysine.

[0009] In an optional embodiment, the MMLV reverse transcriptase mutant includes any one of the following mutants: A. The first mutant with the amino acid sequence shown in SEQ ID NO: 2; B. The second mutant with the amino acid sequence shown in SEQ ID NO: 3; C. The third mutant with the amino acid sequence shown in SEQ ID NO: 4.

[0010] Secondly, the present invention provides a nucleic acid molecule encoding the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0011] Thirdly, the present invention provides an expression vector comprising a nucleic acid molecule as described in the foregoing embodiments.

[0012] Fourthly, the present invention provides a recombinant expression transformant comprising a nucleic acid molecule as described in the foregoing embodiments, or an expression vector as described in the foregoing embodiments.

[0013] Fifthly, the present invention provides a detection product comprising the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0014] In an optional implementation, the detection product includes at least one of a kit and a reaction premix.

[0015] In a sixth aspect, the present invention provides a composition for RNA reverse transcription, the composition comprising the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0016] In a seventh aspect, the present invention provides an RNA reverse transcription method, comprising: A reverse transcription reaction system comprising an RNA template, at least one primer, and the MMLV reverse transcriptase mutant as described in the foregoing embodiments is provided; The reverse transcription system was incubated under suitable conditions for reverse transcription to synthesize cDNA.

[0017] Eighthly, the present invention provides the application of the MMLV reverse transcriptase mutant as described in the foregoing embodiments in the preparation of reverse transcription reagents for single-cell transcriptome sequencing.

[0018] This invention provides an MMLV reverse transcriptase mutant, an RNA reverse transcription method, and its applications. The MMLV reverse transcriptase mutant introduces specific amino acid substitutions at positions 60, 89, or 163. The most direct beneficial effect is that it significantly enhances the tolerance of the MMLV reverse transcriptase to the aforementioned microbead lysates. Experimental results show that, under the inhibitory environment of microbead lysates, the activity of the wild-type MMLV enzyme is almost completely inhibited, retaining only about 2.2%. In contrast, the mutants carrying these specific substitutions maintain extremely high catalytic activity, with activity retention rates reaching 77.97%, 93.85%, and 99.60%, respectively. This means that the modified enzyme exhibits a 35- to 45-fold increase in inhibitory capacity compared to the wild type.

[0019] This high tolerance to inhibitors directly translates into significant advantages at the application level. In the actual reaction environment of single-cell transcriptome sequencing, this modified enzyme can work efficiently and stably, unaffected by microbead cleavage byproducts. Therefore, it can more completely and accurately reverse transcribe trace amounts of RNA into cDNA in a single cell, resulting in higher-quality sequencing libraries. This effectively avoids the loss of transcript information due to enzyme activity inhibition, especially for genes with low expression levels, ensuring its capture efficiency.

[0020] In summary, specific amino acid substitutions endow MMLV reverse transcriptase with the crucial ability to maintain high activity in specific complex chemical environments. This performance improvement allows it to better adapt to the needs of single-cell sequencing platforms based on soluble microbeads, thereby enhancing the efficiency and reliability of the entire technical process. Ultimately, this contributes to obtaining more accurate and comprehensive single-cell gene expression data, providing a more solid technical foundation for downstream research such as analyzing cellular heterogeneity and conducting precision medicine. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The diagram shows the chemical reaction of microbead pyrolysis. Figure 2 Here is the crystal structure diagram of MMLV; Figure 3 RT-PCR of crude enzymes of wild-type and variant MMLV in reverse transcription systems with and without microbeads. Detailed Implementation

[0023] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] This application provides an MMLV reverse transcriptase mutant, wherein the amino acid sequence of the MMLV reverse transcriptase mutant, compared with the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1, contains at least one of the following mutations: A. Serine at position 60 is replaced with phenylalanine; B. Proline at position 89 is replaced with arginine; C. Threonine at position 163 is replaced with lysine.

[0025] This embodiment provides a modified protein, namely a mutant of MMLV (Moloney Murine Leukemia Virus) reverse transcriptase. Biologically, MMLV reverse transcriptase is a tool enzyme whose basic function is to catalyze the synthesis of complementary DNA (cDNA) using RNA as a template. The substance described herein is an artificial protein obtained by specifically modifying the amino acid sequence of natural, wild-type MMLV reverse transcriptase.

[0026] The basic component of this substance is amino acids. Its main amino acid sequence is identical to that of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1. The key difference between this and the wild-type enzyme lies in the alteration of its amino acid sequence at specific positions.

[0027] The amino acid sequence of the wild-type MMLV gene (SEQ ID NO: 1) is as follows: .

[0028] Specifically, at least one of the following three positions contains an amino acid that is replaced: A: At position 60, counting from the N-terminus, the naturally occurring serine is replaced with phenylalanine.

[0029] B: At position 89, the naturally occurring proline is replaced with arginine.

[0030] C: At position 163, the naturally occurring threonine is replaced with lysine.

[0031] MMLV reverse transcriptase mutants are designed based on the principles of protein engineering. The amino acid sequence of a protein determines its spatial structure, which in turn determines its function and properties. By substituting amino acids at specific sites, the local or overall conformation of an enzyme can be altered, thereby optimizing its performance, such as improving its resistance to inhibitors.

[0032] It should be noted that the single-cell sequencing platform uses soluble microbeads to encapsulate barcodes and primers. The dissolution principle of these microbeads is based on a chemical reaction: DTT (dithiothreitol) in the reverse transcription reagent undergoes a thiol-disulfide bond exchange reaction with the disulfide bonds in the polymer network that makes up the microbeads, thereby breaking the cross-linking structure and causing the microbeads to lyse.

[0033] When cross-linked polymers (microbeads) containing disulfide bonds (-SS-) react with DTT, the disulfide bonds are reduced to two thiol groups (-SH), leading to polymer disintegration. This process produces inhibitory byproducts, such as (4S, 5S)-1, 2-dithiane-4, 5-disulfides and other chemical substances and material fragments. These byproducts severely inhibit the activity of wild-type MMLV reverse transcriptase. The principle behind this approach is that by introducing specific amino acid substitutions (or combined mutations at multiple sites, such as 60+89 or 60+89+163) at positions 60, 89, or 163, the enzyme's structure is altered, making it less susceptible to binding to or being affected by these inhibitors, thus maintaining its catalytic activity in an environment filled with inhibitors.

[0034] The inhibitory effect on microbead lysis products is significantly enhanced. In reaction systems with added microbead lysis products, the activity of wild-type MMLV enzyme was severely inhibited, retaining only about 2.20%. In contrast, mutants carrying one of the three substitutions, A, B, and C, retained up to 99.60%, 93.85%, and 77.97% of the activity, respectively. This means that the tolerance of these mutants is 45.1 times, 42.7 times, and 35.4 times that of the wild type, respectively. Furthermore, due to its high tolerance to inhibitors, this substance can efficiently reverse transcribe RNA into cDNA in the actual reaction environment of single-cell sequencing, ensuring the efficiency and fidelity of the first step in sequencing library construction, thereby obtaining more accurate and comprehensive single-cell gene expression data.

[0035] In some embodiments, the MMLV reverse transcriptase mutant includes any one of the following mutants: A. The first mutant with the amino acid sequence shown in SEQ ID NO: 2 (named MMLV-S60F in this embodiment); the amino acid sequence of mutant MMLV-S60F (SEQ ID NO: 2) is as follows: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVFIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI。

[0036] C. The second mutant (designated as MMLV - P89R in this embodiment) with an amino acid sequence as shown in SEQ ID NO: 3. The amino acid sequence of mutant MMLV - P89R (SEQ ID NO: 3) is: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVRCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI。

[0037] B. The third mutant (named MMLV-T163K in this embodiment) with the amino acid sequence shown in SEQ ID NO: 4; The amino acid sequence of mutant MMLV-T163K (SEQ ID NO: 4) is: .

[0038] This application provides a nucleic acid molecule that encodes the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0039] This application provides an expression vector comprising the nucleic acid molecule as described in the foregoing embodiments.

[0040] This application provides a recombinant expression transformant, which comprises a nucleic acid molecule as described in the foregoing embodiments, or an expression vector as described in the foregoing embodiments.

[0041] This embodiment defines a "recombinant expression transformant". In the field of biotechnology, a recombinant expression transformant can be a genetically engineered host cell, expression cell, etc., containing artificially introduced exogenous genetic material. The host cell is an *E. coli* BL21 DE3 strain capable of producing the MMLV reverse transcriptase mutant. The core purpose of this transformant is as a miniature biofactory for the efficient and controllable production of the target protein, namely the MMLV reverse transcriptase mutant described in the preceding embodiments.

[0042] This application provides a detection product, which includes the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0043] In some embodiments, the detection product includes at least one of a kit and a reaction premix.

[0044] This application provides a composition for RNA reverse transcription, the composition comprising the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0045] This application provides an RNA reverse transcription method, including: Step S1: Provide a reverse transcription reaction system comprising an RNA template, at least one primer, and the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0046] The steps described above describe the preparation stage of the reverse transcription reaction. Essentially, it involves mixing several core chemical components necessary for reverse transcription together in a reaction vessel (e.g., a PCR tube) to form a homogeneous liquid reaction system. The process involves mixing substances such as the RNA template (the source of genetic information), primers (the starting point of the reaction), and the MMLV reverse transcriptase mutant (the catalyst) according to a specific formula.

[0047] After this step, you get a ready reverse transcription reaction mixture containing all the reactants. At this point, all the molecules are present in the solution, but since the optimal reaction temperature has not yet been reached, the large-scale enzymatic reaction has not yet begun.

[0048] This step involves actively selecting and adding MMLV reverse transcriptase mutants with inhibitor tolerance, ensuring that the entire reaction system is resistant to potential inhibitors from the outset. This proactive setup ensures that subsequent reactions can proceed smoothly even in the presence of interfering substances (such as bead lysates) in the reaction environment.

[0049] Step S2: Incubate the reverse transcription reaction system under suitable conditions for reverse transcription to synthesize cDNA.

[0050] This step describes the execution phase of the reverse transcription reaction. The reaction system prepared in the previous step is placed in a device capable of precise temperature control, and a suitable temperature and time are set for the enzyme to exert its catalytic activity, allowing the reaction to proceed. The core processes here are temperature control and time maintenance, i.e., "incubation." The direct result of this step is the synthesis of complementary DNA (cDNA). During incubation, the MMLV reverse transcriptase mutant uses RNA as a template, starting from the primer binding site, continuously linking deoxyribonucleotides (dNTPs, usually contained in the reaction buffer) to synthesize a long DNA chain complementary to the RNA sequence. After the reaction, the system contains the newly generated cDNA product.

[0051] This step utilizes a highly resistant mutant enzyme, ensuring efficient and complete cDNA synthesis. Even with inhibitors in the reaction system, the incubation step still yields a high quantity of cDNA, accurately converting the raw RNA information into stable DNA information and providing high-quality starting material for subsequent amplification and sequencing analysis.

[0052] This application provides an example of the application of the MMLV reverse transcriptase mutant as described in the foregoing embodiments in the preparation of reverse transcription reagents for single-cell transcriptome sequencing.

[0053] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0054] Example 1 In this embodiment, site-directed mutagenesis and saturation mutagenesis clonal libraries were generated.

[0055] Experimental methods: In this embodiment, a rational design of MMLV reverse transcriptase was first performed.

[0056] Crystal structure of MMLV reverse transcriptase (see) Figure 2 Rational analysis was conducted on the surface of the enzyme activity pocket (blue area). Modifications were made to the wild-type sequence of MMLV reverse transcriptase (amino acid sequence see SEQ ID NO: 1), and a total of three site-directed mutations were designed at S60F, P89R and T163K.

[0057] The nucleic acid encoding the MMLV gene was constructed between the NheI and XhoI restriction endonuclease sites of the commonly used pET28a plasmid, and the plasmid was subjected to rolling circle PCR.

[0058] (1) Based on the wild-type MMLV gene sequence (SEQ ID NO: 1), MMLV-F and MMLV-F primers containing NheI and XhoI restriction sites of pET28a were designed.

[0059] Table 1 Primers for MMLV variant construction

[0060] (2) Configure the PCR amplification system according to Table 2, and amplify the MMLV gene according to the amplification program in Table 3 to form an MMLV gene fragment library.

[0061] Table 2. Rolling Loop PCR Amplification System for MMLV Variant Construction

[0062] Table 3. Rolling Loop PCR Amplification Procedure for MMLV Variant Construction

[0063] (3) The linearized plasmid vector pET28a was digested with NheI and XhoI. After purification and recovery of the MMLV gene fragment library and the linearized plasmid vector pET28a, ligation was performed according to the instructions of Takara DNA Ligation Kit Ver.2.1, with the fragment / vector ratio of (0.03 pmol ~ 0.3 pmol): 0.03 pmol, and incubation at 16℃ for 2 h. The ligation reaction system was then transformed into E. coli competent cells BL21 DE3.

[0064] (4) Take 100 µl of BL21 DE3 competent cells from the -80℃ freezer and thaw them on ice. Add 5 μL of ligation reaction solution to each competent cell, gently pipette to mix, and let stand on ice for 30 min. Then heat shock in a 42℃ water bath for 90 s, immediately cool in an ice-water bath, and add 750 μL of LB liquid medium after 3 min. Recover at 37℃ on a shaker for 50 min. After recovery, centrifuge at 4000 rpm for 2 min. Remove the supernatant, resuspend the cells in the remaining 100 μL of liquid, and spread evenly on LB Kan solid plates. Incubate upside down at 37℃ for about 12 h. Pick single clones, sequence to verify correctness, and then extract plasmids for later use.

[0065] Design rolling circle PCR primers S60F, P89R, and T163K. Configure the amplification system according to Table 2 and perform PCR amplification according to Table 3. Digest the amplification product with DpnI enzyme. Take 10 µL and transform and plate culture according to the above method to pick single clones. After sequencing to verify that it is correct, store it for later use.

[0066] Example 2 In this embodiment, MMLV and its mutants were cultured, induced, and lysed.

[0067] Experimental methods: Culture and induction of MMLV and its mutants: (1) Primary training: MMLV and its mutants were inoculated into 1.5 ml EP tubes (500 μL LB medium, 50 μg / mL Kan antibiotic) and cultured overnight at 37°C and 250 rpm to obtain the culture medium.

[0068] (2) Secondary induction: 10 μL of MMLV and its mutant culture medium were transferred from a 1.5 ml EP tube to a 200 ml Erlenmeyer flask (50 mL LB medium, 50 μg / mL Kan antibiotic), and cultured at 37°C and 250 rpm until the OD600 reached approximately 0.6. A certain amount of IPTG (final concentration 1 mM) was added to induce enzyme expression, and the induction was carried out overnight at 37°C and 250 rpm.

[0069] (3) Purification: The cultured bacterial culture was centrifuged (4000 rpm, 10 min), and the bacterial cells were collected and sonicated. After centrifugation at 8000 rpm for 20 min at 4℃, the cells were further separated, purified, and dialyzed through a Ni-NTA resin purification column to obtain the thermostable reverse transcriptase mutant proenzyme.

[0070] Example 3 In this embodiment, the reverse transcription capacity of MMLV variants and wild-type variants was detected.

[0071] 1. Detection of reverse transcription ability of MMLV variants and wild-type: (1) Preparation of reverse transcription system and amplification system Table 4. MMLV variants and wild-type reverse transcription systems

[0072] Prepare the reverse transcription system in the PCR tube according to the proportions in Table 4. After preparation, vortex the mixture for 10 seconds and centrifuge for a few seconds.

[0073] Table 5. Amplification systems for MMLV variants and wild-type

[0074] Prepare the amplification system in the PCR tube according to the proportions in Table 5. After preparation, vortex the mixture for 10 seconds and centrifuge for a few seconds.

[0075] (2) Amplification reaction: Table 6. MMLV crude enzyme reverse transcription program

[0076] Table 7. MMLV crude enzyme amplification program

[0077] Place the PCR tubes in the PCR instrument and perform the above procedure.

[0078] 2. Gel migration experiment of amplification reaction products: After amplification, 5 µl of the reaction product was added to 1 µl of 6× loading buffer and mixed before being loaded onto the agarose gel electrophoresis wells. The gel was run at 120 V for 25 min using 1× TAE electrophoresis buffer. The products were then photographed under UV conditions using a ChampGel 5000 imaging system (Saizhi), and the brightness (grayscale analysis) of the amplified target bands for the MMLV variant and wild type was analyzed using ImageJ.

[0079] 3. Experimental Results: Experimental results are as follows Figure 3 As shown, and for Figure 3 The results of the grayscale analysis are shown in Table 8.

[0080] Table 8. Statistics Figure 3 MMLV variants and wild-type tolerance to microbead inhibition

[0081] Table 8 shows that the reverse transcription activities of the three MMLV enzyme variants were not significantly different from those of the wild type. Figure 3 As shown, the percentages of gray values ​​of the target bands of MMLV mutants S60F, P89R, and T163K in systems with and without microbeads were 99.60%, 93.85%, and 77.97%, respectively, which are 45.1 times, 42.7 times, and 35.4 times the microbead-resistant inhibition ability of wild-type MMLV. Therefore, the microbead-resistant inhibition ability of the modified new MMLV enzyme is significantly improved, laying the foundation for the application of MMLV in single-cell sequencing.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An MMLV reverse transcriptase mutant, characterized in that, The amino acid sequence of the MMLV reverse transcriptase mutant contains at least one of the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1: A. Serine at position 60 is replaced with phenylalanine; B. Proline at position 89 is replaced with arginine; C. Threonine at position 163 is replaced with lysine.

2. The MMLV reverse transcriptase mutant as described in claim 1, characterized in that, The MMLV reverse transcriptase mutant includes any one of the following mutants: A. The first mutant with the amino acid sequence shown in SEQ ID NO: 2; B. The second mutant with the amino acid sequence shown in SEQ ID NO: 3; C. The third mutant with the amino acid sequence shown in SEQ ID NO:

4.

3. A nucleic acid molecule, characterized in that, Encodes the MMLV reverse transcriptase mutant as described in claim 1 or 2.

4. An expression carrier, characterized in that, Including the nucleic acid molecules as described in claim 3.

5. A recombinant expression transformant, characterized in that, The recombinant expression transformant comprises the nucleic acid molecule as described in claim 3, or the expression vector as described in claim 4.

6. A testing product, characterized in that, The test product includes the MMLV reverse transcriptase mutant as described in claim 1 or 2.

7. The test product as described in claim 6, characterized in that, The detection product includes at least one of a reagent kit and a reaction premix.

8. A composition for RNA reverse transcription, characterized in that, The composition comprises the MMLV reverse transcriptase mutant as described in claim 1 or 2.

9. A method for reverse transcription of RNA, characterized in that, include: A reverse transcription reaction system comprising an RNA template, at least one primer, and the MMLV reverse transcriptase mutant as described in claim 1 or 2 is provided; The reverse transcription system was incubated under suitable conditions for reverse transcription to synthesize cDNA.

10. The use of an MMLV reverse transcriptase mutant as described in claim 1 or 2 in the preparation of reverse transcription reagents for single-cell transcriptome sequencing.

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