WO2025118254A1 - Reverse transcriptase and use thereof - Google Patents

Abstract

Provided is a new reverse transcriptase or a biologically active fragment thereof. The reverse transcriptase or biologically active fragment thereof comprises a mutated sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, wherein the mutation comprises at least one of substitution, deletion and insertion.

Description

Reverse transcriptase and its application Technical Field

The present disclosure relates to the field of molecular biology, and in particular to a reverse transcriptase or a biologically active fragment thereof and applications thereof.

Background Art

Reverse transcriptase is a special DNA polymerase that can perform reverse transcription and synthesize complementary DNA based on RNA as a template. The sources of reverse transcriptase are varied, and the main source is RNA viruses. Common reverse transcriptases include Moloney murine leukemia virus reverse transcriptase (MMLV RT), human immunodeficiency virus reverse transcriptase (HIV RT), avian myeloblastosis virus reverse transcriptase (AMV RT), etc. In practical applications, reverse transcriptase is often used to study RNA molecules, such as in reverse transcription PCR (RT-PCR) or RNA sequencing processes based on template conversion.

Although a variety of reverse transcriptases, such as the modified MMLV reverse transcriptase, have been used in template switching-based RNA sequencing, these reverse transcriptases still have shortcomings such as low fidelity, low cDNA yield, low gene capture, and high temperature intolerance, which to some extent limit the research and development of the transcriptome.

Therefore, there is an urgent need to provide a reverse transcriptase with high thermal stability, strong processivity and excellent template switching activity.

Summary of the invention

To this end, embodiments of the present disclosure provide a reverse transcriptase or a biologically active fragment thereof and applications thereof.

An embodiment of the first aspect of the present disclosure provides a reverse transcriptase or a biologically active fragment thereof, wherein the reverse transcriptase or the biologically active fragment thereof comprises a mutant sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, wherein the mutation comprises at least one of a substitution, a deletion and an insertion.

In some embodiments, the reverse transcriptase or a biologically active fragment thereof is derived from Lentibacillus sp.

In some embodiments, the reverse transcriptase or a biologically active fragment thereof comprises a sequence as shown in SEQ ID NO: 1.

An embodiment of the second aspect of the present disclosure provides a polynucleotide encoding a reverse transcriptase or a biologically active fragment thereof or a complementary sequence thereof as described in any embodiment of the first aspect of the present disclosure.

In some embodiments, the polynucleotide comprises a sequence as shown in SEQ ID NO: 2.

An embodiment of the third aspect of the present disclosure provides a vector, wherein the vector comprises the polynucleotide as described in any embodiment of the second aspect of the present disclosure.

An embodiment of the fourth aspect of the present disclosure provides a cell, wherein the cell comprises a polynucleotide as described in any embodiment of the second aspect of the present disclosure or expresses a reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure.

An embodiment of the fifth aspect of the present disclosure provides a composition comprising the reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure and an enzyme buffer,

In some embodiments, the enzyme buffer comprises one or more selected from Tris-HCl, ammonium sulfate, magnesium chloride, potassium chloride, and PBS.

An embodiment of the sixth aspect of the present disclosure proposes a kit comprising at least one of the following: a reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure, a polynucleotide as described in any embodiment of the second aspect of the present disclosure, a vector as described in any embodiment of the third aspect of the present disclosure, a cell as described in any embodiment of the fourth aspect of the present disclosure, and a composition as described in any embodiment of the fifth aspect of the present disclosure.

The embodiments of the seventh aspect of the present disclosure propose the use of the reverse transcriptase or its biologically active fragment as described in any embodiment of the first aspect of the present disclosure, the polynucleotide as described in any embodiment of the second aspect of the present disclosure, the vector as described in any embodiment of the third aspect of the present disclosure, the cell as described in any embodiment of the fourth aspect of the present disclosure and the composition as described in any embodiment of the fifth aspect of the present disclosure in catalyzing reverse transcription reactions.

An embodiment of the eighth aspect of the present disclosure provides a reverse transcription method, comprising:

Mixing the reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure with an RNA template to obtain a reaction mixture;

The reaction mixture is subjected to a reverse transcription reaction to obtain a DNA product, wherein the DNA product is fully or partially complementary to the RNA template.

In some embodiments, the reaction temperature of the reverse transcription reaction is 40-55°C.

In some embodiments, the reaction temperature of the reverse transcription reaction is 42-50°C.

The ninth aspect of the present disclosure provides a method for preparing a library, comprising:

Mixing the reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure with the RNA to be tested to obtain a library preparation mixture;

The library preparation mixture is subjected to a reverse transcription reaction to obtain the library.

In some embodiments, the library is used for transcriptome sequencing.

In some embodiments, it is used for NGS transcriptome sequencing and/or third-generation transcriptome sequencing.

In some embodiments, the transcriptome sequencing includes single-cell transcriptome sequencing and/or spatial transcriptome sequencing.

In some embodiments, the library preparation is based on template switching.

In some embodiments, the library is used for one or more of Smart-seq, STOmics, Nanopore sequencing, Drop seq, such as 10×Genomics 3’ sequencing.

The embodiments of the present disclosure achieve the following beneficial effects:

The embodiments of the present disclosure propose a new type of reverse transcriptase (abbreviated as Len-RT). Compared with existing reverse transcriptases, the Len-RT provided by the present disclosure has excellent catalytic performance, such as being able to mediate reverse transcription of complex structure RNA at relatively high temperatures, and the obtained reverse transcription product fragments are longer, etc., and is suitable for multiple application scenarios such as reverse transcription reactions (including ordinary cDNA synthesis, RACE, etc.), library preparation based on reverse transcription reactions such as template switching, and transcriptome sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

FIG. 1 is a schematic diagram of the sequence comparison results of the reverse transcriptase Len-RT and similar reverse transcriptases in the embodiments of the present disclosure.

FIG. 2 is a schematic diagram of the SDS-PAGE analysis results of the reverse transcriptase Len-RT in the embodiments of the present disclosure.

FIG. 3 is a schematic diagram of the polymerization activity measurement results of the reverse transcriptase Len-RT in the embodiments of the present disclosure.

FIG4 is a schematic diagram of the Smart-Seq process based on template conversion in an embodiment of the present disclosure.

FIG5 is a schematic diagram of the performance evaluation results of the reverse transcriptase Len-RT in sequencing in an embodiment of the present disclosure.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

The present disclosure is made based on the following knowledge of the inventors:

Reverse transcriptase is a special DNA polymerase that can perform reverse transcription and synthesize DNA using RNA as a template. The sources of reverse transcriptase are varied, and the main source is RNA viruses. Common reverse transcriptases include Moloney murine leukemia virus reverse transcriptase (MMLV RT), human immunodeficiency virus reverse transcriptase (HIV RT), avian myeloblastosis virus reverse transcriptase (AMV RT), etc. In practical applications, reverse transcriptase is often used to study RNA molecules, such as in reverse transcription PCR (RT-PCR) or RNA sequencing processes based on template switching. The properties of reverse transcriptase that directly affect the effect of reverse transcription of RNA into DNA include the thermal stability, processivity, fidelity, and template switching activity of reverse transcriptase.

Thermal stability

The thermal stability of reverse transcriptase, that is, the ability to withstand high temperatures, is an important factor affecting cDNA synthesis. Increasing the reaction temperature helps to denature RNA with a strong secondary structure and/or a high GC content, allowing the reverse transcriptase to read the sequence. The optimum temperature of most reverse transcriptases is about 40°C, while the reverse transcriptase Len-RT provided in the embodiments of the present disclosure is not affected by its reverse transcription activity at 50°C. This is conducive to the synthesis of full-length cDNA and to increasing the yield, thereby enabling better reverse transcription of RNA with a complex structure.

Continuous synthesis capacity

The processivity of a reverse transcriptase refers to the number of nucleotides that bind to a single binding site of the enzyme. A reverse transcriptase with high processivity can synthesize longer cDNA chains in a shorter reaction time. The reverse transcriptase Len-RT provided in the disclosed embodiments can synthesize cDNA bands of a shorter length even when faced with low-quality and low-abundance RNA samples. Longer, and therefore better performing, RNA isolated from microorganisms, plants, animals, and clinical trial samples, since these samples are easily degraded during handling and in RNase-rich environments.

Fidelity

The fidelity of reverse transcriptase refers to the sequence accuracy during the reverse transcription of RNA into DNA, which affects the accuracy of RNA sequencing. The reverse transcriptase Len-RT provided by the disclosed embodiments has high fidelity and is suitable for transcriptome sequencing, especially for NGS transcriptome sequencing, third-generation transcriptome sequencing, single-cell transcriptome sequencing and/or spatial transcriptome sequencing.

Template switching activity

The template switching activity of reverse transcriptase refers to the property of converting the template from RNA to template switching oligos (TSO) to continue cDNA synthesis during reverse transcription. During reverse transcription, after the first cDNA chain is reverse transcribed based on the RNA template, the reverse transcriptase will add TSO to the C oligonucleotide at the 5' end of the non-template chain to initiate the replication of the second cDNA chain. The reverse transcriptase Len-RT provided in the embodiments of the present disclosure has excellent template switching activity, which is necessary for obtaining complete cDNA based on RNA and is conducive to the effective amplification of the full-length transcript library.

Bacterial group II introns

Bacterial type II introns are large catalytic RNAs that include intronic ribozymes and intron-encoded reverse transcriptases. Generally, Group II reverse transcriptases derived from type II introns have high fidelity, strong processivity, and special template switching activity (directly connecting RNA sequencing adapters to cDNA), and have important potential application value in RT-PCR and RNA sequencing. The reverse transcriptase Len-RT provided in the present disclosure is a reverse transcriptase derived from a bacterial type II intron of Lentibacillus sp.

In the disclosed embodiments, "naturally occurring" or "wild type" refers to the form found in nature. For example, a naturally occurring or wild type polypeptide or polynucleotide sequence is a sequence present in an organism that has not been intentionally modified by human manipulation. "Mutant" means a sequence that has at least one amino acid change relative to a natural or wild type amino acid sequence. In some embodiments, the change (mutation) includes at least one of a substitution, a deletion, and an insertion.

An embodiment of the first aspect of the present disclosure provides a reverse transcriptase or a biologically active fragment thereof, wherein the reverse transcriptase or the biologically active fragment thereof comprises a mutant sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, wherein the mutation comprises at least one of a substitution, a deletion and an insertion.

In some embodiments, the biologically active fragment may include any number of consecutive amino acid residues of the reverse transcriptase Len-RT. The disclosed embodiments also include polynucleotides encoding any such biologically active fragments.

The amino acid sequence of reverse transcriptase Len-RT (SEQ ID NO: 1) is as follows:

In some embodiments, the reverse transcriptase or a biologically active fragment thereof is derived from Lentibacillus sp.

In some embodiments, the reverse transcriptase or a biologically active fragment thereof comprises a sequence as shown in SEQ ID NO: 1.

The reverse transcriptase Len-RT or its biologically active fragment proposed in the embodiments of the present invention may contain a sequence having at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99% but less than 100% identity with SEQ ID NO: 1, wherein the sequence has one or more amino acid mutations compared with SEQ ID NO: 1.

In the disclosed embodiments, the term "percent identity" with respect to nucleic acid or polypeptide sequences is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to a known polypeptide, after aligning the sequences to obtain the maximum percent identity and introducing gaps (if necessary) to achieve the maximum percent homology. N-terminal or C-terminal insertions or deletions should not be construed as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis, which uses the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res [Nucleic Acids Research]. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 87, 2264-2268), which are customized for sequence similarity searches.

In the disclosed embodiments, the term "biologically active fragment" refers to any fragment, derivative, homologue or analogue of reverse transcriptase Len-RT and its mutants, which has in vivo or in vitro activity specific to biological molecules; including, for example, reverse transcriptase activity. In some embodiments, the biologically active fragment, derivative, homologue or analogue of reverse transcriptase Len-RT has any degree of biological activity of reverse transcriptase Len-RT in any in vivo or in vitro assay of interest.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbon atoms are combined with hydrogen atoms, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain a chemical structure that is essentially the same as that of naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the common chemical structure of amino acids, but act in a manner similar to that of naturally occurring amino acids. The amino acid sequence proposed in the embodiments of the present disclosure (e.g., the amino acid sequence shown in SEQ ID NO: 1) may contain the above-mentioned amino acid analogs and mimetics or related modifications, as long as the basic properties of the corresponding amino acids and the activity of the entire enzyme or its active fragments are not affected.

An embodiment of the second aspect of the present disclosure provides a polynucleotide encoding a reverse transcriptase or a biologically active fragment thereof or a complementary sequence thereof as described in any embodiment of the first aspect of the present disclosure.

In some embodiments, the polynucleotide comprises a sequence as shown in SEQ ID NO: 2.

The polynucleotide sequence encoding Len-RT reverse transcriptase (SEQ ID NO: 2) is shown below:

It should be noted that due to the principle of codon degeneracy, the polynucleotide sequence for translating the amino acid sequence is not the only constant sequence, and any nucleotide sequence that can encode the same amino acid sequence is within the scope of protection of this patent.

As used herein, "nucleic acid" refers to DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs and any chemical modifications thereof. Modifications include, but are not limited to, those modifications of chemical groups that provide for the introduction of other charges, polarizability, hydrogen bonds, electrostatic interactions, connection points and action points with nucleic acid ligand bases or nucleic acid ligands as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNA), phosphodiester group modifications (e.g., phosphorothioate, methylphosphonate), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amine, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylation, unusual base pairing combinations such as isobases, isocytidines and isoguanidines, etc. Nucleic acids may also contain non-natural bases, such as nitroindole. Modifications may also include 3' and 5' modifications, such as capping with fluorophores (e.g., quantum dots) or other moieties. The nucleic acid sequence proposed in the embodiments of the present disclosure (for example, the nucleic acid sequence shown in SEQ ID NO: 2) may contain the above-mentioned non-natural bases or related modifications as long as they do not affect the basic properties of the corresponding nucleotides.

An embodiment of the third aspect of the present disclosure provides a vector, wherein the vector comprises the polynucleotide as described in any embodiment of the second aspect of the present disclosure.

"Vector" refers to a polynucleotide that is capable of replicating in a host organism independent of the host chromosome. Preferred vectors include plasmids and usually have a replication origin. Vectors may include, for example, transcription and translation terminators, transcription and translation initiation sequences, and promoters for regulating expression of a particular nucleic acid.

An embodiment of the fourth aspect of the present disclosure provides a cell, wherein the cell comprises a polynucleotide as described in any embodiment of the second aspect of the present disclosure or expresses a reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure.

Len-RT of the present invention can be expressed in a variety of host cells, including Escherichia coli, other bacterial hosts, yeast, filamentous fungi and a variety of higher eukaryotic cells such as COS, CHO and HeLa cell lines and myeloma cell lines. The technology of gene expression in microorganisms is described in, for example, Smith, Gene Expression in Recombinant Microorganisms (Gene Expression in Recombinant Microorganisms) (Bioprocess Technology ("Bioprocess Technology"), Volume 22), Marcel Dekker, 1994. Examples of bacteria that can be used for expression include, but are not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobium, Vitreoscilla and Paracoccus. Filamentous fungi that can be used as expression hosts include, for example, the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Agromyces, Podospora, Mucor, Coccidioides and Pyrospora. See, for example, U.S. Pat. No. 5,679,543 and Stahl and Tudzynski, eds., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992. Synthesis of heterologous proteins in yeast is well known and described in the literature. Sherman, F et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well-known work describing various methods that can be used to produce enzymes in yeast.

There are many expression systems for producing polypeptides known to those of ordinary skill in the art. (See, for example, Gene Expression Systems, ed. Fernandex and Hoeffler, Academic Press, 1999; Sambrook and Russell, supra; and Ausubel et al., supra). Typically, the polynucleotide encoding the polypeptide is under the control of a promoter that is functional in the desired host cell. Many different promoters are available and known to those skilled in the art, and can be used in the expression vectors of the present invention, depending on the specific application. Typically, the promoter selected depends on the cells in which the promoter will be active. Other expression control sequences, such as ribosome binding sites, transcription termination sites, etc., may also be optionally included. A construct comprising one or more of these control sequences is referred to as an "expression cassette." Thus, the nucleic acid encoding the joined polypeptide is integrated for high-level expression in the desired host cell.

An embodiment of the fifth aspect of the present disclosure provides a composition comprising the reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure and an enzyme buffer,

In some embodiments, the enzyme buffer comprises one or more selected from Tris-HCl, ammonium sulfate, magnesium chloride, potassium chloride, and PBS.

An embodiment of the sixth aspect of the present disclosure proposes a kit comprising at least one of the following: a reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure, a polynucleotide as described in any embodiment of the second aspect of the present disclosure, a vector as described in any embodiment of the third aspect of the present disclosure, a cell as described in any embodiment of the fourth aspect of the present disclosure, and a composition as described in any embodiment of the fifth aspect of the present disclosure.

The embodiments of the seventh aspect of the present disclosure provide the reverse transcriptase or biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure, the polynucleotide as described in any embodiment of the second aspect of the present disclosure, the vector as described in any embodiment of the third aspect of the present disclosure, the cell as described in any embodiment of the fourth aspect of the present disclosure, and the cell as described in any embodiment of the fifth aspect of the present disclosure. An embodiment provides a method for catalyzing a reverse transcription reaction.

An embodiment of the eighth aspect of the present disclosure provides a reverse transcription method, comprising:

Mixing the reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure with an RNA template to obtain a reaction mixture;

The reaction mixture is subjected to a reverse transcription reaction to obtain a DNA product, wherein the DNA product is fully or partially complementary to the RNA template.

In some embodiments, the reaction temperature of the reverse transcription reaction is 40-55°C. In some embodiments, the reaction temperature of the reverse transcription reaction is 42-50°C. The reverse transcription activity of the reverse transcriptase Len-RT provided in the disclosed embodiments is not affected under the condition of 50°C. This is conducive to the synthesis of full-length cDNA and the increase of yield, thereby enabling better reverse transcription of RNA with complex structure.

The ninth aspect of the present disclosure provides a method for preparing a library, comprising:

Mixing the reverse transcriptase or a biologically active fragment thereof as described in any embodiment of the first aspect of the present disclosure with the RNA to be tested to obtain a library preparation mixture;

The library preparation mixture is subjected to a reverse transcription reaction to obtain the library.

In some embodiments, the library is used for transcriptome sequencing.

In some embodiments, it is used for NGS transcriptome sequencing and/or third-generation transcriptome sequencing.

In some embodiments, the transcriptome sequencing includes single-cell transcriptome sequencing and/or spatial transcriptome sequencing.

In some embodiments, the library preparation is based on template switching.

In some embodiments, the library is used for one or more of Smart-seq, STOmics, Nanopore sequencing, Drop seq, such as 10×Genomics 3’ sequencing.

In summary, the new reverse transcriptase Len-RT proposed in the embodiments of the present invention has excellent catalytic performance compared to existing reverse transcriptases, such as the ability to mediate reverse transcription of complex structure RNA at relatively high temperatures, the obtained reverse transcription products have high fidelity and long fragment length, etc., and is suitable for multiple application scenarios such as reverse transcription reactions (including ordinary cDNA synthesis, RACE, etc.), library preparation based on reverse transcription reactions such as template switching, and transcriptome sequencing.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

Example 1 Identification of a novel reverse transcriptase Len-RT

The disclosed embodiment identified a new reverse transcriptase Len-RT by performing metagenomic sequencing and subsequent analysis on samples from deep-sea hydrothermal sediments in the western Pacific Ocean.

(1) Use MGIEasy Microbial DNA Extraction Kit (Cat. No. 1000027955) to extract metagenomic DNA from deep-sea hydrothermal sediment samples, and obtain metagenomic sequence data through library construction and sequencing;

(2) Assemble the obtained metagenomic sequence data and perform species and functional annotation;

(3) Compare the sequence of interest with that of similar enzymes (Clustal Omega online sequence comparison website) and construct a structural model.

It was preliminarily identified that the reverse transcriptase Len-RT provided by the present disclosure may be derived from Lentibacillus sp., specifically from bacterial type II introns. Bacterial type II introns have mobile reverse transcription elements, so the sequence of Len-RT is considered to be a sequence of interest.

The sequence alignment results of Len-RT amino acid sequence (SEQ ID NO: 1) with similar reverse transcriptases marathon RT and TGI RT are shown in Figure 1. The sequence identities of Len-RT with similar enzymes marathon RT and TGI RT are 39.76% and 42.82%, respectively.

The sequence of marathon RT is shown in SEQ ID NO: 3:

The sequence of TGI RT is shown in SEQ ID NO: 4:

The above results indicate that the reverse transcriptase Len-RT provided in the embodiments of the present disclosure is a new type of reverse transcriptase with a novel sequence and a broad modification space. It can provide a new enzyme skeleton for the modification needs of different application scenarios and has excellent application potential.

Example 2 Determination of polymerization activity of reverse transcriptase Len-RT

The disclosed embodiment synthesized, expressed, and purified the Len-RT gene sequence to prepare the reverse transcriptase Len-RT (SEQ ID NO: 1), and then measured its polymerization activity.

2.1 Preparation of reverse transcriptase Len-RT

(1) Synthesize the Len-RT gene sequence (SEQ ID NO: 1) and clone the gene into the pET-28a expression vector (Changzhou Xinyisheng Biotechnology Co., Ltd.), in which the cloning sites were Nde I and Xho I, and the protein purification tag SUMO was added to the vector using the In-Fusion method;

(2) The above vector was transformed into competent Escherichia coli BL21 (DE3) cells, inoculated on a plate, and incubated at 37°C overnight;

(3) Pick 3-6 single colonies from the plate and inoculate them into a conical flask containing 50 ml of liquid culture medium (Cat. No. A507002-0250, purchased from Biotechnology) and culture at 37°C for 5-7 h until the OD600 of the bacterial solution reaches 0.6-4.0;

(4) Inoculate the above bacterial solution into 2 LLB medium at a 1% inoculum volume and culture at 37°C for 2-4 h until the OD600 of the bacterial solution reaches 0.8-1.0;

(5) adding isopropyl-β-D-thiogalactoside (IPTG, catalog number: A600168-0025, purchased from BBI) to the above bacterial solution to a final concentration of 0.5 mM, and then placing in a shaker precooled to 16°C and culturing at 220 rpm for 12-16 h to induce the cells to express reverse transcriptase Len-RT;

(6) Centrifuge the mixed solution obtained in (5) at 8000 g for 30 min to collect the precipitated bacteria;

(7) Add affinity solution A to the precipitate at a ratio of 1:20 to resuspend the bacteria, and then use ultrasound to disrupt the bacteria in an ice bath environment;

(8) The resuspension after ultrasonic disruption was centrifuged at 12000 rpm at 4°C for 60 min, and the supernatant was filtered through a 0.22 μM filter membrane as the sample loaded on the purification column;

(9) After adding the above sample to the pretreated affinity chromatography column (HisTrap FF) at a rate of 3 ml/min, it was rinsed with Ni column affinity A solution (20CV, 20 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, 5% Glycerol, pH 7.8);

(10) Use Ni column affinity solution B (10.5CV, 0-70%, 20mM Tris-HCl, 300mM NaCl, 500mM Imidazole, 5% Glycerol, pH 7.8) for linear elution, and collect the eluted protein when the UV absorption peak reaches 50mAu, and stop collecting when the UV absorption peak drops to 100mAu;

(11) Add the collected eluate to a pretreated anion exchange chromatography column (HiTrap Q HP 5 ml) at a flow rate of 5 ml/min, and collect the flow-through;

(12) Dilute the collected flow-through by 9 times with diluent (20 mM Tris-HCl, 5% Glycerol, pH 8.0) and add it to the pretreated cation exchange chromatography column (HiTrap SP HP 5 ml);

(12) Use Q column A solution (10CV, 20mM Tris-HCl, 50mM NaCl, 5% Glycerol, pH 8.0) at a flow rate of 5ml/min until the baseline is stable;

(13) Use Q column B solution (0-100%, 10CV, 20mM Tris-HCl, 1M NaCl, 5% Glycerol, pH 8.0) for gradient elution at a flow rate of 5ml/min to collect the target protein;

(14) performing SDS-PAGE analysis on the collected target protein to determine the protein purity of the target protein reverse transcriptase Len-RT;

(15) The purified reverse transcriptase Len-RT was dialyzed with a dialysate (40 mM Tris-HCl, 200 mM KCl, 2 mM DTT, 0.2 mM EDTA, 5% Glycerol, pH 8.0) and stored in an enzyme storage solution (10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 8.0) for subsequent analysis.

The SDS-PAGE analysis of the reverse transcriptase Len-RT is shown in FIG2 . The target protein reverse transcriptase Len-RT has a clear band and high purity, and can be used for subsequent activity determination.

2.2 Determination of polymerization activity of reverse transcriptase Len-RT

(1) synthesizing oligo(dT16) and 50 nt Poly(rA), wherein the sequence of oligo(dT16) is TTTTTTTTTTTTTTTT (SEQ ID NO: 5), and the sequence of Poly(rA) is AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6);

(2) Reverse transcription was performed at 37°C. After 10 min, 1 μL 0.5 M EDTA was added to terminate the reaction. The specific reaction system is shown in Table 1, where the concentration of the reverse transcriptase Len-RT was 0.57 mg/mL and the concentration of the commercial reverse transcriptase α-RT used as a control was 1 mg/mL.

Table 1 Reverse transcription reaction system

(3) The concentration of the generated dsDNA was measured using the Qubit kit.

The polymerization activity measurement results of the reverse transcriptase Len-RT are shown in Figure 3. When the reverse transcriptase Len-RT provided by the present disclosure is used, the dsDNA concentration produced by the reverse transcription reaction is as high as 24.3ng/μL, while the dsDNA concentration produced by the control commercial reverse transcriptase α-RT enzyme is only 21.8ng/μL. And the initial concentration of the reverse transcriptase Len-RT is only 0.57mg/mL, which is much lower than the concentration of the α-RT enzyme (1mg/mL). The above results show that the reverse transcriptase Len-RT provided by the present disclosure has excellent polymerization activity, and it can also play a reverse transcription function at low concentrations, with high polymerization efficiency and low application cost, and has good application potential.

Example 3 Performance evaluation of reverse transcriptase Len-RT in sequencing

The present disclosure uses Smart-Seq, a transcriptome sequencing method based on template switching to construct a library, to evaluate the performance of the reverse transcriptase Len-RT in actual sequencing. The process can be referred to Picelli et al. (Picelli S, Faridani OR, AK,Winberg G,Sagasser S,Sandberg R.Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014;9(1):171-181.doi:10.1038/nprot.2014.006).

3.1 cDNA synthesis based on template switching

First, cDNA was synthesized based on template conversion. The reverse transcription reaction system used was shown in Table 2. The reaction was carried out in a PCR instrument with the following program settings: 42°C/50°C, 1h; 85°C, 15min; 4°C, ∞.

Table 2 Reverse transcription reaction system based on template switching

Among them, the Smart-Seq process is shown in Figure 4. First, the mRNA with a poly A tail is used as a template, and the added Oligo-dT30VN primer will complement its poly A tail, and reverse transcribe to synthesize the first-chain cDNA under the action of reverse transcriptase. Subsequently, due to the 5'Cap structure of the mRNA template and the terminal transferase performance of the reverse transcriptase, when the first-chain cDNA extends to the end of the mRNA template, the 3' end of the cDNA chain is added with a non-template CCC base. The rGrG+G on the added template switching oligomer (TSO) will complement the CCC at the 3' end of the first-chain cDNA. At this time, under the action of the template switching activity of the reverse transcriptase, the template of the reaction is converted from the original mRNA to TSO, and extended along it to complete the synthesis of cDNA.

The specific sequences of each molecule are as follows:

TSO:AAGCAGTGGTATCAACGCAGAGTACATrGrG+G(SEQ ID NO: 7);

Oligo-dT30VN:AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN (SEQ ID NO: 8).

3.2 cDNA enrichment

After the reverse transcription reaction was completed, 5 μL of the reaction product (synthesized cDNA) was added to 25 μL of the reaction system (Table 3) for PCR enrichment was performed. The reaction was carried out in a PCR instrument with the following program settings: 95°C pre-denaturation for 5 min, 98°C for 20 s, 58°C for 20 s, 72°C for 3 min, 15 cycles, and finally a supplementary reaction at 72°C for 5 min.

Table 3 Enrichment PCR reaction system

It should be noted that Oligo-dT30VN and TSO carry some of the same sequences, so the cDNA can be PCR enriched using ISPCR primers (AAGCAGTGGTATCAACGCAGAGT, SEQ ID NO: 9) to amplify the cDNA to the ng level.

(3) After the reaction, the enriched cDNA was quantified using Qubit dsDNA HS Assay Kit (Cat. No.: Q32854, purchased from Invitrogen) and agarose gel electrophoresis.

The performance evaluation results of the reverse transcriptase Len-RT in sequencing are shown in Figure 5. The cDNA fragment length and Smart-Seq yield generated by Len-RT reverse transcription are significantly higher than those of MMLV reverse transcriptase. It shows that the reverse transcriptase Len-RT provided by the embodiments of the present disclosure is particularly suitable for transcriptome sequencing, especially for NGS transcriptome sequencing, third-generation transcriptome sequencing, single-cell transcriptome sequencing and/or spatial transcriptome sequencing.

In addition, under the condition of 50°C, the reverse transcription activity of the reverse transcriptase Len-RT provided in the embodiment of the present disclosure is not affected, indicating that it has good thermal stability, which is better than the optimal temperature of about 40°C for most reverse transcriptase reactions. This is conducive to the reverse transcription of complex structure RNA, indicating that the Len-RT provided in the embodiment of the present disclosure is suitable for reverse transcription and sequencing of full-length transcriptomes.

In summary, the reverse transcriptase Len-RT provided in the embodiments of the present disclosure has high thermal stability, strong processivity and excellent template switching activity, and is a new type of reverse transcriptase with important application value.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

It should be noted that, in this document, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "include" or any other variation thereof is intended to cover non-exclusive The term "inclusion" refers to the process, method, article, or device that includes a series of elements, so that the process, method, article, or device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article, or device. In the absence of more restrictions, the elements defined by the sentence "including a..." do not exclude the existence of other identical elements in the process, method, article, or device that includes the elements.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (14)

A reverse transcriptase or a biologically active fragment thereof, comprising a mutant sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, wherein the mutation comprises at least one of a substitution, a deletion and an insertion. The reverse transcriptase or its biologically active fragment according to claim 1, wherein the reverse transcriptase or its biologically active fragment is derived from Lentibacillus sp. The reverse transcriptase or its biologically active fragment according to claim 1, wherein the reverse transcriptase or its biologically active fragment comprises the sequence shown in SEQ ID NO: 1. A polynucleotide encoding a reverse transcriptase or a biologically active fragment thereof or a complementary sequence thereof as described in any one of claims 1 to 3, optionally, the polynucleotide comprises a sequence as shown in SEQ ID NO: 2. A vector comprising the polynucleotide according to claim 4. A cell comprising the polynucleotide according to claim 4 or expressing the reverse transcriptase or a biologically active fragment thereof according to any one of claims 1 to 3. A composition comprising a reverse transcriptase or a biologically active fragment thereof as claimed in any one of claims 1 to 3 and an enzyme buffer, Optionally, the enzyme buffer comprises one or more selected from Tris-hydrochloric acid, ammonium sulfate, magnesium chloride, potassium chloride, and PBS. A kit comprising at least one of the following: the reverse transcriptase or biologically active fragment thereof according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector according to claim 5, the cell according to claim 6 and the composition according to claim 7. Use of the reverse transcriptase or biologically active fragment thereof according to any one of claims 1 to 3, the polynucleotide according to claim 3, the vector according to claim 5, the cell according to claim 6, the kit according to claim 7 or the composition according to claim 7 in catalyzing a reverse transcription reaction. A reverse transcription method comprising: The reverse transcriptase or biologically active fragment thereof according to any one of claims 1 to 3 is mixed with an RNA template to obtain to the reaction mixture; The reaction mixture is subjected to a reverse transcription reaction to obtain a DNA product, wherein the DNA product is fully or partially complementary to the RNA template. The reverse transcription method according to claim 10, wherein the reaction temperature of the reverse transcription reaction is 40-55°C, preferably, 42-50°C. A library preparation method comprising: Mixing the reverse transcriptase or biologically active fragment thereof according to any one of claims 1 to 3 with the RNA to be tested to obtain a library preparation mixture; The library preparation mixture is subjected to a reverse transcription reaction to obtain the library. The library preparation method according to claim 12, wherein the library is used for transcriptome sequencing, preferably, for NGS transcriptome sequencing and/or third-generation transcriptome sequencing, Preferably, the transcriptome sequencing includes single-cell transcriptome sequencing and/or spatial transcriptome sequencing. The method for preparing a library according to claim 12, wherein the library preparation is based on template switching, Optionally, the library is used for one or more of Smart-seq, STOmics, Nanopore sequencing, Drop seq, such as 10×Genomics 3′ sequencing.