Mmlv reverse transcriptase mutants, rna reverse transcription methods and uses thereof

Abstract

The application provides a MMLV reverse transcriptase mutant, an RNA reverse transcription method and application thereof, and relates to the technical field of biology.The amino acid sequence of the MMLV reverse transcriptase mutant comprises at least one of the following mutations compared with the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO:1: the serine at the 60th position is replaced by phenylalanine, the proline at the 89th position is replaced by arginine, and the threonine at the 163rd position is replaced by lysine.The MMLV reverse transcriptase mutant provided by the application endows the MMLV reverse transcriptase with the key ability to maintain high activity in a specific complex chemical environment.The improvement in performance enables the MMLV reverse transcriptase to better adapt to the requirements of a soluble microbead-based single-cell transcriptome sequencing platform, thereby improving the efficiency and reliability of the entire technical process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a mutant of MMLV reverse transcriptase, a method for RNA reverse transcription and application thereof. BACKGROUND

[0002] Gene expression analysis is one of the core technologies in the fields of molecular biology, genetic engineering and modern medical research. The expression activity of a gene is usually reflected by the types and quantities of its transcription product messenger RNA (mRNA), and the analysis of all transcripts in a biological body (i.e., transcriptome) can reveal the molecular mechanisms in a specific physiological state, developmental stage or disease process. The reverse transcription of genetic information from RNA to complementary DNA (cDNA) is a key step for transcriptome analysis, and this process relies on the core biological catalyst, reverse transcriptase. This enzyme can synthesize DNA using RNA as a template, thereby converting unstable RNA information into stable and easily amplified and analyzed cDNA molecules. Therefore, the catalytic efficiency, stability and tolerance to reaction environment of reverse transcriptase directly determine the accuracy and reliability of the downstream gene analysis results, and is the cornerstone of the entire technical process.

[0003] To achieve accurate quantification and analysis of the transcriptome, the prior art has developed various sequencing methods. Traditional transcriptome sequencing (Bulk RNA-seq) obtains the average value of gene expression in a population of cells by extracting total RNA from a tissue or cell population. However, this method has its inherent limitations, i.e., the traditional population cell sequencing method cannot reveal the heterogeneity between cells because it is based on a large number of cells, for example, in a tumor microenvironment, the gene expression differences between different cell subpopulations are averaged, resulting in the loss of key information; at the same time, low-abundance transcripts are often ignored, and the gene expression dynamics of cells over time cannot be analyzed. With the deepening of research, higher resolution single-cell transcriptome sequencing (scRNA-seq) technology has emerged, which can capture the transcriptome information of single cells with high throughput and high precision. At present, one of the most widely used single-cell sequencing technology platforms is the 10X Genomics platform, which is based on microfluidic technology to encapsulate single cells with molecular barcoded microbeads in nanoliter volume water-in-oil droplets. These microbeads are attached with barcodes (barcode and UMI sequences) for labeling each cell and each transcript and primers for initiating reverse transcription. Inside the droplet, the dissolution of the microbeads is triggered by chemical reagents (such as DTT), thereby releasing the barcodes and primers to initiate the reverse transcription reaction for mRNA in single cells.

[0004] Although single-cell sequencing technology greatly promotes life science research, there are defects in the technical implementation path thereof to be solved. That is, the technical process itself introduces new challenges, mainly in that in order to release the barcodes, primers, etc. in the microbeads, a compound (such as DTT) is introduced into the reagent, and DTT can destroy the microbead structure through a thiol disulfide exchange reaction. This lysis process inevitably produces by-products in the droplet, a small reaction system, including material fragments of the microbeads and small chemical molecules generated after lysis, such as (4S, 5S)-1, 2-dithiane-4, 5-disulfide.

[0005] These lysis by-products constitute a serious inhibition to subsequent biochemical reactions, especially reverse transcription reactions. The reverse transcriptase responsible for converting single-cell transcriptome information into cDNA, such as the commonly used MMLV reverse transcriptase, will be seriously affected by these inhibitors, resulting in a significant decrease in reverse transcription efficiency. Therefore, the fundamental defect of the prior art is that the microbead lysis step designed to achieve labeling of the transcriptome reverse transcription product in single cells (the principle of microbead lysis is referred to in Figure 1 ), has simultaneously contaminated the environment of the key enzymatic reaction. This intrinsic contradiction of the technology makes the reverse transcriptase work in an environment full of inhibitors, and its performance cannot meet the efficient and faithful single-cell transcriptome capture requirements, which directly affects the quality of the sequencing library and ultimately limits the depth and breadth of the application of single-cell sequencing technology in precision medicine and basic research. Therefore, improving the tolerance of reverse transcriptase MMLV to microbead lysis through enzyme modification is a necessary condition for the performance of reverse transcriptase to match single-cell transcriptome sequencing.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to provide a MMLV reverse transcriptase mutant, an RNA reverse transcription method and application thereof. The MMLV reverse transcriptase mutant helps to obtain more accurate and comprehensive single-cell gene expression data, and provides a more solid technical foundation for analyzing cell heterogeneity and conducting precision medicine and other downstream research.

[0008] In order to achieve the above purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides a MMLV reverse transcriptase mutant, wherein the amino acid sequence of the MMLV reverse transcriptase mutant comprises at least one of the following mutations compared with the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1:

[0010] A. the serine at position 60 is replaced by phenylalanine;

[0011] B. the proline at position 89 is replaced by arginine;

[0012] C. the threonine at position 163 is replaced by lysine.

[0013] In an alternative embodiment, the MMLV reverse transcriptase mutant comprises any one of the following mutants:

[0014] A. a first mutant having an amino acid sequence as set forth in SEQ ID NO: 2;

[0015] B. a second mutant having an amino acid sequence as set forth in SEQ ID NO: 3;

[0016] C. a third mutant having an amino acid sequence as set forth in SEQ ID NO: 4.

[0017] In a second aspect, the present application provides a nucleic acid molecule encoding the MMLV reverse transcriptase mutant as described in the preceding embodiments.

[0018] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule as described in the preceding embodiments.

[0019] In a fourth aspect, the present application provides a recombinant expression transformant comprising the nucleic acid molecule as described in the preceding embodiments, or the expression vector as described in the preceding embodiments.

[0020] In a fifth aspect, the present application provides a detection product comprising the MMLV reverse transcriptase mutant as described in the preceding embodiments.

[0021] In an alternative embodiment, the detection product comprises at least one of a kit and a reaction premix.

[0022] In a sixth aspect, the present application provides a composition for RNA reverse transcription, comprising the MMLV reverse transcriptase mutant as described in the preceding embodiments.

[0023] In a seventh aspect, the present application provides a method for RNA reverse transcription, comprising:

[0024] providing a reverse transcription reaction system comprising an RNA template, at least one primer, and the MMLV reverse transcriptase mutant as described in the preceding embodiments;

[0025] incubating the reverse transcription reaction system under conditions suitable for reverse transcription to synthesize cDNA.

[0026] In an eighth aspect, the present application provides use of the MMLV reverse transcriptase mutant as described in the preceding embodiments in the preparation of a reverse transcription reagent for single-cell transcriptome sequencing.

[0027] The present application provides a mutant of MMLV reverse transcriptase, a method for RNA reverse transcription and its application. The mutant of MMLV reverse transcriptase introduces specific amino acid substitutions at positions 60, 89 or 163. The most direct benefit is that the MMLV reverse transcriptase has significantly enhanced tolerance to the above-mentioned microbead lysate. Experimental results show that in the presence of the microbead lysate, the activity of the wild-type MMLV enzyme is almost completely inhibited, retaining only about 2.2%. In contrast, the mutant carrying these specific substitutions can maintain very high catalytic activity, with activity retention rates of 77.97%, 93.85% and 99.60%, respectively. This means that the modified enzyme has been improved by 35 to 45 times or more in terms of inhibition.

[0028] This high tolerance to inhibitors directly brings significant advantages in application. In the actual reaction environment of single-cell transcriptome sequencing, this modified enzyme can work efficiently and stably without being disturbed by the microbead lysis byproducts. Therefore, it can more completely and accurately reverse transcribe the trace amount of RNA in a single cell into cDNA, thereby obtaining a higher-quality sequencing library. This effectively avoids the loss of transcript information due to inhibited enzyme activity, especially for low-expression genes, and ensures their capture efficiency.

[0029] In summary, specific amino acid substitutions endow MMLV reverse transcriptase with the key ability to maintain high activity in a specific complex chemical environment. This performance improvement enables it to better adapt to the needs of soluble microbead-based single-cell sequencing platforms, thereby improving the efficiency and reliability of the entire technical process. Ultimately, this helps to obtain more accurate and comprehensive single-cell gene expression data, providing a more solid technical foundation for analyzing cell heterogeneity and conducting precision medicine and other downstream research. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0031] Figure 1 Chemical reaction equation for microbead lysis;

[0032] Figure 2 MMLV crystal structure diagram;

[0033] Figure 3RT-PCR of MMLV wild type and variant crude enzyme in the absence / presence of Dynabeads reverse transcription system. DETAILED DESCRIPTION

[0034] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by purchase.

[0035] In the embodiments of the present application, a MMLV reverse transcriptase mutant is provided, which has an amino acid sequence comprising at least one of the following mutations compared with the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1:

[0036] A, the serine at position 60 is replaced by phenylalanine;

[0037] B, the proline at position 89 is replaced by arginine;

[0038] C, the threonine at position 163 is replaced by lysine.

[0039] In the embodiments of the present application, a modified protein, i.e., a mutant of MMLV (Moloney Murine Leukemia Virus) reverse transcriptase, is provided. Biologically, MMLV reverse transcriptase is a tool enzyme, whose basic function is to catalyze the synthesis of complementary DNA (cDNA) from RNA templates. The substance defined by this expression is an artificial protein obtained by specific modification of the amino acid sequence of the natural, wild-type MMLV reverse transcriptase.

[0040] Among them, the basic component of the substance is amino acid. The amino acid sequence of the main part is the same as that of the wild-type MMLV reverse transcriptase shown in SEQ ID NO: 1. The key "part" or "component" that distinguishes it from the wild-type enzyme is that the amino acid sequence has changed at a specific position.

[0041] The wild-type MMLV gene amino acid sequence (SEQ ID NO: 1) is as follows:

[0042] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI.

[0043] Specifically, the amino acid at least one of the following three positions is replaced:

[0044] A: The naturally occurring Serine at the 60th position from the N-terminal is replaced with Phenylalanine.

[0045] B: The naturally occurring Proline at the 89th position is replaced with Arginine.

[0046] C: The naturally occurring Threonine at the 163rd position is replaced with Lysine.

[0047] The MMLV reverse transcriptase mutant is designed based on the principle of protein engineering. The amino acid sequence of a protein determines its spatial structure, and the spatial structure determines its function and characteristics. By replacing the amino acids at specific sites, the local or overall conformation of the enzyme can be changed, thereby optimizing its performance, such as increasing its tolerance to inhibitors.

[0048] It should be noted that the single-cell sequencing platform uses dissolvable 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 will undergo a thiol-disulfide exchange reaction with the disulfide bonds in the polymer network that makes up the microbeads, breaking the cross-linking structure and causing the microbeads to lyse.

[0049] After the cross-linked polymer containing disulfide bonds (-S-S-) reacts with DTT, the disulfide bonds are reduced to two thiol groups (-SH), causing the polymer to disintegrate. This process produces inhibitory byproducts, such as (4S, 5S)-1, 2-dithiane-4, 5-disulfide and other chemicals and material fragments. These byproducts can severely inhibit the activity of wild-type MMLV reverse transcriptase. The principle of this substance is that by introducing specific amino acid substitutions at positions 60, 89, or 163 (or multiple sites, such as 60+89, 60+89+163), the structure of the enzyme is changed, making it less susceptible to binding or being affected by these inhibitors, thereby enabling it to maintain its catalytic activity in an environment full of inhibitors.

[0050] The tolerance to the inhibitory effect of microbead lysis products is significantly improved. In the reaction system with added microbead lysis products, the activity of wild-type MMLV enzyme is severely inhibited, retaining only about 2.20%. In contrast, the mutants carrying one of the A, B, and C substitutions can retain up to 99.60%, 93.85%, and 77.97% of their 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. In addition, due to its high tolerance to inhibitors, the substance can efficiently reverse transcribe RNA to cDNA in the actual reaction environment of single-cell sequencing, ensuring the efficiency and fidelity of the first step of sequencing library construction, thereby obtaining more accurate and comprehensive single-cell gene expression data.

[0051] In some embodiments, the MMLV reverse transcriptase mutant includes any one of the following mutants:

[0052] A, the first mutant with an amino acid sequence as 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:

[0053] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVFIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI.

[0054] C. A second mutant having an amino acid sequence as set forth in SEQ ID NO: 3 (designated MMLV-P89R in this example). The mutant MMLV-P89R amino acid sequence (SEQ ID NO: 3) is:

[0055] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVRCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI.

[0056] B. a third mutant having an amino acid sequence as shown in SEQ ID NO: 4 (named MMLV-T163K in this example); the amino acid sequence of mutant MMLV-T163K (SEQ ID NO: 4) is:

[0057] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPKSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI.

[0058] The present application provides a nucleic acid molecule encoding the mutant MMLV reverse transcriptase as described in the preceding embodiments.

[0059] The present application provides an expression vector comprising the nucleic acid molecule as described in the preceding embodiments.

[0060] The present application provides a recombinant expression transformant comprising the nucleic acid molecule as described in the preceding embodiments, or the expression vector as described in the preceding embodiments.

[0061] In this embodiment, a "recombinant expression transformant" is defined. In the field of biotechnology, a recombinant expression transformant can be a genetically engineered host cell, an expression cell, etc., which contains artificially introduced exogenous genetic material inside. The host cell is an E. coli BL21 DE3 strain that can be used to produce MMLV reverse transcriptase mutants. The core use of this transformant is as a miniature biomanufacturing plant for efficiently and controllably producing target proteins, i.e., the MMLV reverse transcriptase mutants described in the foregoing embodiments.

[0062] In this embodiment, a detection product is provided, which includes the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0063] In some embodiments, the detection product includes at least one of a kit and a reaction premix.

[0064] In this embodiment, a composition for RNA reverse transcription is provided, which includes the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0065] In this embodiment, a method for RNA reverse transcription is provided, which includes:

[0066] Step S1, providing a reverse transcription reaction system containing an RNA template, at least one primer, and the MMLV reverse transcriptase mutant as described in the foregoing embodiments.

[0067] The above steps describe the preparation stage of the reverse transcription reaction. In essence, in a reaction container (such as a PCR tube), several core chemical components necessary for reverse transcription are mixed together to form a uniform liquid reaction system. The process is to mix the RNA template as the source of genetic information, the primer as the starting point of the reaction, and the MMLV reverse transcriptase mutant as the catalyst according to a specific formula.

[0068] After this step is completed, a ready-to-use reverse transcription reaction mixture containing all reactants is obtained. At this time, various molecules already exist in the solution, but since the optimal reaction temperature has not been provided, large-scale enzymatic reactions have not yet begun.

[0069] In this step, by actively selecting and adding the MMLV reverse transcriptase mutant with inhibitor tolerance, the entire reaction system constructed from the beginning has the ability to resist potential inhibitors. This is a forward-looking setting that ensures that even if there are interfering substances (such as microbead lysates) in the reaction environment, the subsequent reaction can proceed smoothly.

[0070] Step S2, incubating the reverse transcription reaction system under conditions suitable for reverse transcription to synthesize cDNA.

[0071] 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 accurately controlling the temperature, and a temperature and time suitable for the catalytic activity of the enzyme are set to allow the reaction to proceed. The core process here is 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 to continuously connect deoxynucleotides (dNTPs, usually contained in the reaction buffer) from the primer binding site to synthesize a long DNA chain complementary to the RNA sequence. After the reaction is completed, the system contains the newly generated cDNA product.

[0072] In this step, due to the use of a strong tolerance mutant enzyme, the synthesis of cDNA is efficient and complete. Even if there are inhibitors in the reaction system, the incubation step can still produce a high amount of cDNA, thereby truly and accurately converting the original RNA information into stable DNA information, providing high-quality starting material for subsequent amplification and sequencing analysis.

[0073] In the embodiments of the present application, the use of the MMLV reverse transcriptase mutant as described in the foregoing embodiments in the preparation of a reverse transcription reagent for single-cell transcriptome sequencing is provided.

[0074] The present application will be further described below through specific examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the present application in any form.

[0075] Example 1

[0076] In this embodiment, site-directed mutagenesis and generation of saturation mutagenesis clone library were performed.

[0077] Experimental method:

[0078] In this embodiment, rational design of MMLV reverse transcriptase was first performed.

[0079] The crystal structure of MMLV reverse transcriptase (see Figure 2 , the blue area is the surface of the enzyme active pocket) was rationally analyzed, and site-directed mutagenesis of three sites S60F, P89R and T163K was performed on the wild-type sequence of MMLV reverse transcriptase (amino acid sequence see SEQ ID NO: 1).

[0080] 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.

[0081] (1) According to the wild type MMLV gene sequence (SEQ ID NO: 1), MMLV-F and MMLV-R primers containing NheI and XhoI enzyme cutting sites of pET28a were designed.

[0082] Table 1 MMLV variant construction primers

[0083]

[0084] (2) According to Table 2, the PCR amplification system was configured, and the MMLV gene was amplified according to the amplification program in Table 3 to form an MMLV gene fragment library.

[0085] Table 2, MMLV variant construction rolling circle PCR amplification system

[0086]

[0087] Table 3, MMLV variant construction rolling circle PCR amplification program

[0088]

[0089] (3) NheI and XhoI enzyme cutting linearized plasmid vector pET28a. After purification and recovery of the MMLV gene fragment library and the enzyme cutting linearized plasmid vector pET28a, according to the Takara DNA Ligation Kit Ver.2.1 kit instructions, according to the fragment / carrier=(0.03 pmol ~0.3 pmol):0.03 pmol, 16℃ incubation 2 h for ligation, the ligation reaction system is then transformed into E. coli competent cells BL21 DE3.

[0090] (4) Take out 100 μl of BL21 DE3 competent cells from -80℃ refrigerator, melt on ice. Add 5 μL of each ligation reaction solution to the competent cells, mix gently, and incubate on ice for 30 min. Then heat shock at 42℃ water bath for 90 s, immediately cool in ice water bath, add 750 μL LB liquid medium after 3 min, recover at 37℃ shaker for 50 min. After recovery, centrifuge at 4000 rpm for 2 min. Remove the supernatant, resuspend the bacterial cells in the remaining 100 μL liquid, and evenly spread on LB Kan solid plate. Incubate at 37℃ for 12 h or so, pick single colonies, and extract plasmids for use after sequencing verification.

[0091] Design rolling circle PCR primers S60F, P89R, T163K, configure the amplification system according to Table 2, perform PCR amplification according to Table 3, digest the amplification product with DpnI enzyme, take 10 μL, and plate culture to pick single colonies according to the above method, and save for use after sequencing verification.

[0092] Example 2

[0093] In this embodiment, the culture induction and lysis of MMLV and its mutants are carried out.

[0094] Experimental method: MMLV and its mutant culture induction

[0095] (1) Primary culture

[0096] MMLV and its mutants are inoculated into 1.5 ml EP tubes (500 μL LB medium, 50 μg / mL Kan antibiotic) respectively, and cultured at 37°C, 250 rpm overnight to obtain the culture solution.

[0097] (2) Secondary induction

[0098] 10 μL of MMLV and its mutant culture solution is transferred from the 1.5 ml EP tube to a 200 ml conical flask (50 mL LB medium, 50 μg / mL Kan antibiotic) respectively, and cultured at 37°C, 250 rpm until OD600 is about 0.6. A certain amount of IPTG (final concentration 1 mM) is added to induce enzyme expression, and the induction is carried out at 37°C, 250 rpm overnight.

[0099] (3) Purification

[0100] The cultured bacterial solution is centrifuged (4000 rpm, 10 min), and the bacterial body is collected and broken by ultrasonic. Centrifugation is carried out at 4°C, 8000 rpm for 20 min, and the heat-stable reverse transcriptase mutant enzyme is obtained by further separation and purification, dialysis, and purification through Ni-NTA resin purification column.

[0101] Example 3

[0102] In this embodiment, the detection of the reverse transcription ability of MMLV variants and wild type is carried out.

[0103] 1. Detection of reverse transcription ability of MMLV variants and wild type

[0104] (1) Preparation of reverse transcription system and amplification system

[0105] Table 4, MMLV variant and wild type reverse transcription system

[0106]

[0107] The reverse transcription system is prepared in the PCR tube according to the proportion in Table 4, and after preparation, it is vortexed for 10 s in a vortex mixer and centrifuged for a few seconds in a microcentrifuge.

[0108] Table 5, MMLV variant and wild type amplification system

[0109]

[0110] Prepare the amplification system in the PCR tube according to the proportion of Table 5. After preparation, vortex for 10 s in a vortex mixer and centrifuge for a few seconds in a microcentrifuge.

[0111] (2) Amplification reaction:

[0112] Table 6, MMLV crude enzyme reverse transcription procedure

[0113]

[0114] Table 7, MMLV crude enzyme amplification procedure

[0115]

[0116] Place the PCR tube in the PCR instrument to perform the above procedure.

[0117] 2. Gel migration experiment of amplification reaction product:

[0118] After amplification, add 5 μl of reaction product to 1 μl of 6x loading buffer, mix, and load on an agarose gel electrophoresis well. Use 1x TAE electrophoresis solution, run the gel at 120 V for 25 min. Take a photo of the product under ultraviolet conditions using ChampGel 5000 imaging system (Sai Zhi), and analyze the brightness (gray value analysis) of the MMLV variant and wild type amplification target bands by ImageJ.

[0119] 3. Experimental results:

[0120] The experimental results are shown in Figure 3 , and the gray scale analysis results are shown in Table 8. Figure 3

[0121] Table 8, statistics Figure 3 of MMLV variants and wild type resistance to microbead inhibition

[0122]

[0123] Table 8 shows that the reverse transcription activity of the three MMLV enzyme variants has no significant difference from the wild type. As shown in Figure 3 , the percentage of gray value of the target band of MMLV mutants S60F, P89R and T163K in the system with / without microbeads is 99.60%, 93.85% and 77.97%, respectively, which is 45.1 times, 42.7 times and 35.4 times of the wild type MMLV resistance to microbead inhibition, respectively. Therefore, the new MMLV enzyme after modification has significantly improved resistance to microbead inhibition, which lays a foundation for the application of MMLV in single cell sequencing.​

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A mutant of MMLV reverse transcriptase, characterized in that, The MMLV reverse transcriptase mutant is any one of the following mutants: A. a first mutant having an amino acid sequence as set forth in SEQ ID NO: 2; B. a second mutant having an amino acid sequence as set forth in SEQ ID NO:

3.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule of claim 2.

3. An expression vector, characterized by, The recombinant expression transformant comprises the nucleic acid molecule of claim 2, or the expression vector of claim 3.

4. A recombinant expression transformant, characterized by, The composition comprises the MMLV reverse transcriptase mutant of claim 1.

5. A composition for reverse transcription of RNA, characterized by, Providing a reverse transcription reaction system comprising an RNA template, at least one primer, and the MMLV reverse transcriptase mutant of claim 1; 6. A method of reverse transcription of RNA, characterized in that, Incubating the reverse transcription reaction system under conditions suitable for reverse transcription to synthesize cDNA.

7. Use of the MMLV reverse transcriptase mutant of claim 1 in the preparation of a reverse transcription reagent for single-cell transcriptome sequencing. ​ ​

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