Improved thermostable viral reverse transcriptases

Abstract

The present invention provides novel engineered reverse transcriptases that offer beneficial improvements in thermostability, processivity, cDNA yield, and elimination of secondary enzymatic activity. The present invention also provides methods for amplifying template nucleic acids using such reverse transcriptases. The present invention addresses deficiencies in the current state of the art of reverse transcriptases for RNA detection and analysis, including deficiencies in detection sensitivity, specificity, side enzymatic activity, enzyme stability, and synthetic capacity, particularly when using template nucleic acids that vary in length, secondary structure, and nucleotide content.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides novel engineered reverse transcriptases for improved RNA detection and analysis. The present invention also provides methods for amplifying template nucleic acids using such reverse transcriptases and methods for producing said enzymes. The present invention addresses the deficiencies of current state-of-the-art reverse transcriptases in RNA detection and analysis, including deficiencies in detection sensitivity, specificity, side enzyme activity, enzyme stability and synthetic ability, especially when using template nucleic acids of various lengths, secondary structures and nucleotide contents. [Background technology]

[0002] Technical background RNA detection and analysis can provide insight into important biological phenomena such as gene expression, gene silencing, the presence or absence of important pathogens, particularly RNA viruses, and other medically important conditions. The ability to monitor the presence or absence or changes in the amount of messenger RNA, non-coding RNA and viral RNA allows for the diagnosis of important disease states such as cancer or viral infections, and generally facilitates the study of biological processes. For these reasons, there is a strong need to improve the state of the art in these analyses.

[0003] The most common methods of RNA analysis rely on reverse transcriptase to catalyze the synthesis of complementary DNA (cDNA) using an RNA template, a key step in several related analytical and preparative methods (Spiegelman 1971, Telesnitsky 1997). For example, reverse transcription PCR (RT-PCR) (Lee 1989) and its variants quantitative RT-PCR (RT qPCR), real-time quantitative RT-PCR (RT RT-PCR) (Owarzek 1992, Paria 1993, Gibson 1996) and digital RT-PCR (RT dPCR) (Warren 2006, Sanders 2018) are essentially two-step processes in which cDNA is synthesized by reverse transcription and amplified by PCR. In general, these two functions are provided by separate enzymes: reverse transcriptase (RT), often a Moloney Murine Leukemia Virus (MMLV) RT derivative, and a thermostable DNA polymerase (Pol), usually TaqPol. The performance of these methods depends on both activities, but because the PCR step is generally well established and robust, improvements have most commonly focused on the RT enzyme.

[0004] In addition to RT-PCR, the field of RNA-centric genomics, focusing on transcriptomes, non-coding RNAs, and viral genomics and metagenomics, relies on variants of an analytical approach collectively referred to as RNA-Seq (reviewed in Hrdlickova 2017, Boone et al. 2018). Different RNA-Seq strategies are used to address a variety of biological questions related to variation in gene expression and relative abundance of transcripts, diversity of viral genome sequences and their evolution, phenomena such as intron processing and splicing, and other related phenomena. In all cases, enzymatic components are critical for the generation of libraries used for sequence analysis, greatly affecting the overall quality and reliability of the analysis. In addition to RT-PCR and RNA-Seq, cDNA synthesis has traditionally been used more as a preparative method associated with cloning cDNA for more focused analyses on transcripts and translated proteins (Spiegelman 1971, Burrell 1996). Each of these analyses places unique demands on the enzyme for optimal performance, and the choice of RT has a significant impact on the validity and reliability of the method (Okello 2010, Bustin 2015).

[0005] Engineering has improved the performance of RTs in specific applications. Many RTs have an inherent RNase H activity that digests the RNA component of RNA / DNA hybrids (Gerard 1975). This is a positive property for certain applications, but it hampers other common uses (Garces 1991). Disabling RNase H activity has secondary effects. In addition to removing the canonical function as a nuclease, removing RNase H activity by mutagenesis also improves thermostability. This allows synthesis at higher temperatures, thereby facilitating the analysis of highly structured RNA targets. Further work on MMLV RT has shown that certain domains, notably the minor groove binding track (MGBT) and connecting domain (CD), play a role in the interaction with the template (Beard 1994). Modifying either the RNase H activity, the MGBT or the CD or a combination thereof can increase template switching (Garces 1991, Svarovskaia 2000), affinity for the template, and, by extension, the length of the product. These improvements are beneficial to varying degrees in RT-PCR, RNA-Seq library preparation, and cDNA cloning.

[0006] To address the specialized needs of these various applications, numerous natural and engineered RTs have been explored. The most common are derivatives of Moloney Murine Leukemia Virus (MMLV) RT (Gerard 1975) or, less commonly, Avian Myeloblastosis Virus (AMV) RT (Kacian 1971). Other RT enzymes have been derived from alternative retroviral enzymes (e.g., US7560117), phages (Moser 2012) and bacteria (Grabco 1996), but none of them fully address the need for sensitive, highly specific and robust synthesis of cDNAs of targets that vary in length, secondary structure and nucleotide content. Thus, these shortcomings in the state of the art need to be continuously improved.

[0007] References [ka] [ka] [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,560,117 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention The present invention provides novel engineered reverse transcriptases that provide beneficial improvements in thermostability, length of cDNA synthesized using an RNA template, and elimination of secondary enzymatic activity. These improvements were engineered into the parental Equine Infectious Anemia Virus Reverse Transcriptase (EIAV RT) molecule by directed mutagenesis of amino acid residues present in the thumb, connecting, and RNase H domains. These mutations, which collectively eliminate RNase H activity and increase RT activity at high temperatures, were identified from both rational design and random mutagenesis / screening. Combining these mutations allows for significant improvements in performance, such as the ability to synthesize cDNAs of lengths greater than 12 kb at temperatures up to 65°C.

[0010] In a first aspect, the present invention relates to a reverse transcriptase (RT) comprising two subunits, each of which is encoded by a variant of the polynucleotide sequence of SEQ ID NO:1, wherein the amino acid sequence encoded by the variant is at least 90% identical to the amino acid sequence of SEQ ID NO:2 or a fragment thereof, and wherein the variant comprises one or more mutations in the polynucleotide sequence of SEQ ID NO:1 resulting in one or more amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 in the thumb domain of RT and / or in the minor groove binding track (MGBT) of the connecting domain (CD) of RT.

[0011] The one or more amino acid exchanges according to this aspect of the invention may be made within the encoded amino acid sequence at amino acid positions corresponding to 263, 265, 267, 269, and 271 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, and S271Q.

[0012] Additionally or alternatively, one or more amino acid exchanges may be made within the encoded amino acid sequence at amino acid positions corresponding to 356, 360, 362, and 363 of SEQ ID NO:2, where the amino acid exchanges are I356G or I356D or I356N, N360A, G362T or G362N, and W363K or W363N or W363I, preferably I356G, N360A, G362T and W363K.

[0013] According to one embodiment, the variant of SEQ ID NO:1 further comprises one or more mutations in the polynucleotide sequence of SEQ ID NO:1, which cause one or more amino acid exchanges with respect to SEQ ID NO:2 of the RNase H domain of RT. The one or more amino acid exchanges according to this aspect of the invention can be caused in the encoded amino acid sequence at amino acid positions corresponding to 443, 470, 476, 491, 526 and 553 of SEQ ID NO:2, where the amino acid exchanges are D443G, V470F, E476Q, Q491R, R526H and K553R. In a preferred embodiment, the amino acid exchange is D443G and / or E476Q. In a highly preferred embodiment, the amino acid exchange is D443G.

[0014] According to another embodiment, the one or more mutations in the polynucleotide sequence of SEQ ID NO:1 cause one or more amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 263, 265, 267, 269, 271 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q and D443G. In a preferred embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 cause amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q and D443G.

[0015] According to further embodiments, the one or more mutations in the polynucleotide sequence of SEQ ID NO:1 result in one or more amino acid exchanges within the encoded amino acid sequence at amino acid positions corresponding to 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K and D443G. In a preferred embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K and D443G. Alternatively, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363N and D443G. Further alternatively, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356D, N360A, G362T, W363K and D443G.Further alternatively, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356N, N360A, G362N, W363I and D443G.

[0016] According to a further embodiment, the one or more mutations in the polynucleotide sequence of SEQ ID NO: 1 cause one or more amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 263, 265, 267, 269, 271, 491, 526 and 443 of SEQ ID NO: 2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, Q491R, R526H and D443G. In a preferred embodiment, the mutations in the polynucleotide sequence of SEQ ID NO: 1 cause amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 491, 526 and 443 of SEQ ID NO: 2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, Q491R, R526H and D443G. Alternatively, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 491, 526 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, Q491R, K553R and D443G. Further alternatively, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 491, 526 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, Q491R, R526H, K553R and D443G.

[0017] According to further embodiments, the one or more mutations in the polynucleotide sequence of SEQ ID NO:1 result in one or more amino acid exchanges within the encoded amino acid sequence at amino acid positions corresponding to 263, 265, 267, 269, 271, 356, 360, 362, 363, 491, 526 and 443 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K, Q491R, R526H and D443G. In a preferred embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363, 491, 526 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K, Q491R, R526H and D443G.

[0018] According to a preferred embodiment, a first of the two subunits comprises the complete amino acid sequence encoded by a variant of SEQ ID NO:1, and a second of the two subunits comprises a proteolytic fragment of the complete amino acid sequence encoded by a variant of SEQ ID NO:1.

[0019] In another aspect, the present invention refers to a method for amplifying a template nucleic acid comprising contacting the template nucleic acid with a RT of the present invention. In one embodiment, the method is reverse transcription (RT) PCR.

[0020] In a further aspect, the present invention provides a kit comprising the RT of the present invention and a buffer.

[0021] Another aspect of the invention relates to a polynucleotide encoding the RT of the invention. A further aspect of the invention relates to a vector comprising a polynucleotide according to the invention. In another aspect, the invention relates to a transformed host cell comprising said vector.

[0022] In another aspect, the present invention provides a RT obtained by expressing the polynucleotide or vector according to the present invention in a host cell. In a preferred embodiment, the host cell is E. coli.

[0023] In yet another embodiment, the invention relates to a method for producing an RT of the invention, comprising isolating RT from a transformed host cell.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS The present disclosure will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows the results of thermostability assays using various RTs, where a preincubation step at the indicated temperatures was performed prior to the RT reaction at 42° C.

[0026] [Diagram 2] Figure 2 shows the results of a thermostability assay measuring cDNA synthesis at elevated temperatures using various RTs. Panel (a) shows the results of a cDNA synthesis reaction at 50°C. Panel (b) shows the results of a two-step RT-PCR reaction in which cDNA was generated at 50°C (top panel) and 60°C (bottom panel).

[0027] [Diagram 3] FIG. 3 shows the effect of temperature on cDNA yield in a one-step RT-qPCR assay using a state-of-the-art MMLV RT with reduced RNase H activity (EnzScript) and an RNase H neg EIAV RT variant according to the invention, where EIAV RT V3 additionally contains a point mutation within MGBT.

[0028] [Figure 4]FIG. 4 shows the effect of temperature on the yield of cDNA of various lengths in a two-step RT-qPCR assay using the state-of-the-art MMLV RT (SuperScript IV) and the EIAV RT variant according to the invention.

[0029] [Diagram 5] Figure 5 shows the time course of 9 kb cDNA synthesis at 55°C by EIAV RT V3 and the state-of-the-art MMLV RT SuperScript IV.

[0030] [Figure 6] FIG. 6 shows the effect of temperature on cDNA yield in a two-step RT-qPCR assay using different EIAV RT variants according to the invention.

[0031] [Figure 7] FIG. 7 shows the effect of temperature on cDNA yield in a long-range end-point PCR assay using different EIAV RT variants according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry).

[0033] In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA may be used. These techniques are well known and are published, for example, in Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (FMAusubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; ed.);Oligonucleotide Synthesis,1984(MLGait ed.);Nucleic Acid Hybridization,1985(Hames and Higgins);Transcription and Translation,1984(Hames and Higgins eds.);Animal Cell Culture,1986(RIFreshney ed.);Immobilized Cells and Enzymes,1986(IRL Press);Perbal,1984,A Practical Guide to Molecular Cloning;the series,Methods In Enzymology(Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (JH Miller and MPCalos eds., Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively).

[0034] As used herein, the term "comprising" should be interpreted as embracing both "comprising" and "consisting of," both meanings being specifically intended, and thus including individually disclosed embodiments in accordance with the present invention.

[0035] As used herein, the term "nucleic acid sequence, nucleotide sequence" or "polynucleotide sequence" refers to an oligonucleotide, nucleotide or polynucleotide and fragments and portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of both.

[0036] A "variant" of a polynucleotide sequence (e.g., RNA or DNA) contains one or more mutations in the polynucleotide sequence relative to another polynucleotide sequence in which one or more nucleic acid residues are inserted, deleted and / or substituted into the nucleic acid sequence. The one or more mutations may cause one or more amino acid exchanges in the amino acid sequence encoded by the variant compared to another amino acid sequence (i.e., "non-silent mutations"). Variants also include nucleic acid sequences in which one or more codons are replaced with synonyms that do not cause an amino acid exchange, and thus are referred to as "silent mutations."

[0037] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. In these calculations, alignment gaps (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SI AM J. Applied Math. 48:1073.

[0038] When calculating percent identity, the sequences to be compared are usually aligned in such a way as to bring about maximum matching between sequences.Computer programs that can be used to determine percent identity are, for example, reviewed by Pearson (Pearson, 2013, Curr.Protoc.Bioinform.42:3.1.1-3.1.8).Such computer algorithms are used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined.Sequences are aligned for the best match of their respective amino acids or nucleotides ("matched span" as determined by the algorithm).

[0039] A particular alignment scheme for aligning two sequences may result in matching of only a short region of the two sequences, and this small aligned region may show very high sequence identity despite the lack of significant relationship between the two full-length sequences. Thus, the alignment method selected may be adjusted as necessary to result in an alignment spanning at least 50 or any other number of consecutive nucleotides or amino acids.

[0040] The nucleic acid sequences of the present invention can be made using standard techniques well known to those skilled in the art. The term "encoding" or "coding" refers to the inherent property of a particular sequence of nucleotides in a nucleic acid, such as a gene in a chromosome or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (i.e., rRNA, tRNA, other RNA molecules) or amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein when transcription and translation of the mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA. Protein-encoding nucleic acids include nucleic acids that have different nucleotide sequences but code for the same protein amino acid sequence due to the degeneracy of the genetic code. Protein-encoding nucleic acids and nucleotide sequences may include introns.

[0041] The term "polypeptide" is used interchangeably with "amino acid sequence" or "protein" and refers to a polymer of amino acids of any length. These terms also include proteins that are post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation, or protein processing. Modifications and changes, such as fusion to other proteins, substitutions, deletions, or insertions of amino acid sequences, can be made in the structure of a polypeptide while the molecule retains its biological functional activity. For example, specific amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with similar properties. Amino acid modifications can be made, for example, by performing site-directed mutagenesis or polymerase chain reaction-mediated mutagenesis on the underlying nucleic acid sequence.

[0042] The term "expressed" as used herein refers to the transcription and / or translation of a heterologous nucleic acid sequence in a host cell. "Production" includes both transcription and translation. The amount of expression in a host cell may be determined based on either the amount of corresponding mRNA present in the cell, with production being based on the amount of the desired polypeptide. For example, mRNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA or PCR (see Sambrook et al. (1989), supra; Ausubel et al. (updated 1994), supra). The protein encoded by the selected sequence can be quantified by a variety of methods, such as ELISA, Western blotting, radioimmunoassay, immunoprecipitation, protein bioactivity assays, or protein immunostaining followed by FACS analysis PCR (see Sambrook et al. (1989), supra; Ausubel et al. (updated 1994), supra).

[0043] The term "PCR" refers to the polymerase chain reaction, a standard method in molecular biology for DNA amplification.

[0044] "RT-PCR" refers to reverse transcription polymerase chain reaction, a variant of PCR commonly used for the detection and quantification of RNA. RT-PCR involves two steps: synthesis of complementary DNA (cDNA) by reverse transcription from RNA, and amplification of the generated cDNA by PCR. Variants of RT-PCR include quantitative RT-PCR (RT-qPCR), real-time RT-PCR, digital RT-PCR (dRT-PCR) or digital droplet RT-PCR (ddRT-PCR).

[0045] The present invention will be described in more detail below with reference to the accompanying drawings. It will be apparent to those skilled in the art that these embodiments and items are merely examples of multiple possibilities. Therefore, the embodiments shown herein should not be understood as being limited to these features and configurations. Any possible combination and configuration of the described features can be selected according to the scope of the present invention.

[0046] Polymerases / Enzymes Equine infectious anemia virus reverse transcriptase (EIAV RT) is active in vivo as a heterodimer containing 66 kDa and 51 kDa subunits derived from a single open reading frame. The smaller 51 kDa subunit is a proteolytic fragment of the larger 66 kDa subunit. Expression of the open reading frame of SEQ ID NO:1 in E. coli yields both the 66 kDa and 51 kDa subunits that can form homodimers (i.e., p66 / p66 and p51 / p51) and heterodimers (i.e., p66 / p51). Some commercial preparations of RT are mixtures of homodimers and heterodimers. Both types of dimers are active in DNA synthesis, but the heterodimers show higher stability and processivity than the homodimers.

[0047] Thus, a preferred embodiment of the RT according to the invention is an enzyme preparation enriched in heterodimers. The enrichment can be carried out using any method known to the skilled artisan, such as, for example, chromatography with heparin, hydrophobic interaction chromatography (HIC), and ion exchange. The enzyme preparation is enriched in heterodimers if the content of heterodimers is at least 25% higher compared to the content of heterodimers in the enzyme preparation before enrichment. In some embodiments of this aspect of the invention, the content of heterodimers is enriched at least 30% compared to the content of heterodimers in the enzyme preparation before enrichment. In another embodiment, the content of heterodimers is enriched at least 50% compared to the content of heterodimers in the enzyme preparation before enrichment. In a preferred embodiment of this aspect of the invention, the content of heterodimers is enriched at least 75% compared to the content of heterodimers in the enzyme preparation before enrichment. In a more preferred embodiment of this aspect of the invention, the content of heterodimers is enriched at least 100% compared to the content of heterodimers in the enzyme preparation before enrichment. In a particularly preferred embodiment of this aspect of the invention, the heterodimers are purified to apparent homogeneity.

[0048] In a first aspect, the present invention provides a reverse transcriptase (RT) comprising two subunits, wherein the two subunits are each encoded by a variant of the polynucleotide sequence of SEQ ID NO:1, wherein the amino acid sequence encoded by the variant is at least 90% identical to the amino acid sequence of SEQ ID NO:2 or a fragment thereof, wherein the variant comprises one or more mutations in the polynucleotide sequence of SEQ ID NO:1 resulting in one or more amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 in the thumb domain of RT and / or the minor groove binding track (MGBT) of the connecting domain (CD) of RT.

[0049] A variant of SEQ ID NO:1 can include any number of codons replaced by their synonyms (see Table 1) that do not result in an amino acid exchange within the amino acid sequence relative to SEQ ID NO:2. Additionally, the variant includes at least one mutation that results in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the variant includes at least two mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant includes at least three mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant includes at least four mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant includes at least five mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant includes at least six mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant includes at least eight mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant includes at least ten mutations that result in an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. Table 1: Genetic code [Table 1-1] [Table 1-2]

[0050] In some embodiments according to this aspect of the invention, the variant of SEQ ID NO:1 comprises one mutation that causes an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises two mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises three mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises four mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises five mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises six mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises eight mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises ten mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. In another embodiment, the variant comprises twelve mutations that cause an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2.

[0051] The one or more amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 can be located in the minor groove binding track (MGBT) of the thumb domain of RT spanning amino acid residues 255-298 of SEQ ID NO:2, the connecting domain (CD) of RT spanning amino acid residues 320-414 of SEQ ID NO:2, and / or the RNase H domain of RT spanning amino acid residues 438-553 of SEQ ID NO:2. In some embodiments according to this aspect of the invention, at least one amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 is located in the MGBT of the thumb domain of RT. In other embodiments according to this aspect of the invention, at least one amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 is located in the CD of RT. In further embodiments according to this aspect of the invention, at least one amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 is located in the RNase H domain of RT. In a preferred embodiment, at least two amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 are located in the MGBT of the thumb domain of RT and the CD of RT. In another preferred embodiment, at least two amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 are located in the MGBT of the thumb domain of RT and the RNase H domain of RT. In a particularly preferred embodiment, the at least three amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 are located in the thumb domain of RT, the RNase H domain of RT and the MGBT of the CD of RT.

[0052] In some embodiments according to this aspect of the invention, the variant of SEQ ID NO:1 comprises one or more mutations resulting in a single amino acid exchange relative to the amino acid sequence of SEQ ID NO:2. This single amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 can be located in the minor groove binding track (MGBT) of the thumb domain of RT spanning amino acid residues 255-298 of SEQ ID NO:2, in the connecting domain (CD) of RT spanning amino acid residues 320-414 of SEQ ID NO:2, and / or in the RNase H domain of RT spanning amino acid residues 438-553 of SEQ ID NO:2. In certain embodiments, this single amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 is located in the MGBT of the thumb domain of RT. In other embodiments, this single amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 is located in the CD of RT. In further embodiments, this single amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 is located in the RNase H domain of RT. In preferred embodiments, the variant comprises mutations resulting in two amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2. These two amino acid exchanges relative to the amino acid sequence of SEQ ID NO:2 are located in the MGBT of the thumb domain of RT and the CD of RT. In another preferred embodiment, these two amino acid exchanges relative to the amino acid sequence of SEQ ID NO: 2 are located in the thumb domain of RT and the MGBT of the RNase H domain of RT. In a particularly preferred embodiment, the variant comprises a mutation resulting in three amino acid exchanges relative to the amino acid sequence of SEQ ID NO: 2. These three amino acid exchanges relative to the amino acid sequence of SEQ ID NO: 2 are located in the thumb domain of RT, the RNase H domain of RT and the MGBT of the CD of RT.

[0053] In some embodiments according to this aspect of the invention, the amino acid sequence encoded by the variant of SEQ ID NO:1 is at least 90% identical to the amino acid sequence of SEQ ID NO:2, or a fragment thereof. In other embodiments, the amino acid sequence encoded by the variant is at least 95% identical to the amino acid sequence of SEQ ID NO:2, or a fragment thereof. In preferred embodiments according to this aspect of the invention, the amino acid sequence encoded by the variant is at least 97% identical to the amino acid sequence of SEQ ID NO:2, or a fragment thereof. An amino acid sequence that is at least 90%, at least 95% or at least 97% identical to the amino acid sequence of SEQ ID NO:2, or a fragment thereof, retains the biological function of the amino acid sequence of SEQ ID NO:2, or a fragment thereof. More specifically, an amino acid sequence that is at least 90%, at least 95% or at least 97% identical to the amino acid sequence of SEQ ID NO:2, or a fragment thereof, retains the reverse transcriptase activity of EIAV-RT.

[0054] According to one embodiment, one or more amino acid exchanges according to this aspect of the invention may be made in the encoded amino acid sequence at amino acid positions corresponding to positions 263, 265, 267, 269, and 271 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, and S271Q. Preferably, the EIAV RT comprises at least two of said amino acid exchanges in its MGBT. More preferably, the EIAV RT comprises at least three of said amino acid exchanges in its MGBT. In some further embodiments, the EIAV RT comprises two of said amino acid exchanges in its MGBT. More preferably, the EIAV RT comprises three of said amino acid exchanges in its MGBT. Particularly preferably, the EIAV RT comprises all of said amino acid exchanges in its MGBT.

[0055] In addition to or instead of at least one amino acid exchange in the MGBT of the RT, one or more amino acid exchanges may be made in the encoded amino acid sequence at amino acid positions corresponding to positions 356, 360, 362, and 363 of SEQ ID NO:2, where the amino acid exchanges are I356G or I356D or I356N, N360A, G362T or G362N, and W363K or W363N or W363I, preferably I356G, N360A, G362T, and W363K. Preferably, the EIAV RT comprises at least two of said amino acid exchanges in its CD. More preferably, the EIAV RT comprises at least three of said amino acid exchanges in its CD. In some further embodiments, the EIAV RT comprises two of said amino acid exchanges in its CD. More preferably, the EIAV RT comprises three of said amino acid exchanges in its CD. Particularly preferably, the EIAV RT comprises all of said amino acid exchanges in its CD.

[0056] In addition to or instead of at least one amino acid exchange in the MGBT of the RT, one or more amino acid exchanges may be made in the encoded amino acid sequence at amino acid positions corresponding to positions 443, 470, 476, 491, 526 and 553 of SEQ ID NO:2, where the amino acid exchanges are D443G, V470F, E476Q, Q491R, R526H and K553R. According to one embodiment, the EIAV RT comprises an amino acid exchange corresponding to positions D443G and / or E476Q of SEQ ID NO:2. According to one embodiment, the EIAV RT comprises one amino acid exchange corresponding to position D443G of SEQ ID NO:2. The EIAV RT has the amino acid sequence of SEQ ID NO:4 encoded by the nucleic acid sequence of SEQ ID NO:3 or a synonymous variant thereof. According to another embodiment, the EIAV RT comprises two amino acid exchanges corresponding to positions D443G and E476Q of SEQ ID NO:2. Said EIAV RT has the amino acid sequence of SEQ ID NO: 6 encoded by the nucleic acid sequence of SEQ ID NO: 5 or a synonymous variant thereof. In a preferred embodiment, one of the amino acid exchanges is D443G. In another embodiment, EIAV RT comprises at least two of the aforementioned amino acid exchanges in its RNase H domain. According to a further embodiment, EIAV RT comprises at least three of the aforementioned amino acid exchanges in its RNase H domain. In some further embodiments, EIAV RT comprises two of the aforementioned amino acid exchanges in its RNase H domain. According to a further embodiment, EIAV RT comprises three of the aforementioned amino acid exchanges in its RNase H domain. Particularly preferably, EIAV RT comprises the amino acid exchanges D443G, Q491R, and R526H.

[0057] In some embodiments according to this aspect of the invention, the one or more mutations in the polynucleotide sequence of SEQ ID NO:1 cause one or more amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 263, 265, 267, 269, 271 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q and D443G. Preferably, the EIAV RT comprises at least two of the aforementioned amino acid exchanges, where at least one of the amino acid exchanges is located in the RNase H domain and where at least one of the amino acid exchanges is located in MGBT. More preferably, the EIAV RT comprises at least three of the aforementioned amino acid exchanges, where at least one of the amino acid exchanges is located in the RNase H domain and where at least two of the amino acid exchanges are located in MGBT. According to another preferred embodiment, the EIAV RT comprises at least four of the aforementioned amino acid exchanges, where at least one of the amino acid exchanges is located in the RNase H domain and where at least three of the amino acid exchanges are located in MGBT. According to a further preferred embodiment, the EIAV RT comprises at least five of the aforementioned amino acid exchanges, where at least one of the amino acid exchanges is located in the RNase H domain and at least four of the amino acid exchanges are located in MGBT. In some further embodiments, the EIAV RT comprises two of the aforementioned amino acid exchanges, where one of the amino acid exchanges is located in the RNase H domain and one of the amino acid exchanges is located in MGBT. Preferably, the EIAV RT comprises three of the aforementioned amino acid exchanges, where one of the amino acid exchanges is located in the RNase H domain and two of the amino acid exchanges are located in MGBT. According to another preferred embodiment, the EIAV RT comprises four of the aforementioned amino acid exchanges, where one of the amino acid exchanges is located in the RNase H domain and three of the amino acid exchanges are located in MGBT. According to a further preferred embodiment, the EIAV RT comprises five of the aforementioned amino acid exchanges, where one of the amino acid exchanges is located in the RNase H domain and four of the amino acid exchanges are located in MGBT. Particularly preferably, the EIAV RT comprises all of the aforementioned amino acid exchanges.The latter EIAV RT has the amino acid sequence of SEQ ID NO: 8 encoded by the nucleic acid sequence of SEQ ID NO: 7 or a synonymous variant thereof.

[0058] In a further embodiment according to this aspect of the invention, the one or more mutations in the polynucleotide sequence of SEQ ID NO:1 result in one or more amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K and D443G. Preferably, the EIAV RT comprises at least three of the foregoing amino acid exchanges, where at least one of said amino acid exchanges is located in the RNase H domain, at least one of said amino acid exchanges is located in the MGBT and at least one of said amino acid exchanges is located in the CD. According to another preferred embodiment, the EIAV RT comprises at least four of the aforementioned amino acid exchanges, wherein at least one of the amino acid exchanges is located in the RNase H domain, at least one of the amino acid exchanges is located in MGBT, and at least one of the amino acid exchanges is located in the CD. According to a further preferred embodiment, the EIAV RT comprises at least five of the aforementioned amino acid exchanges, wherein at least one of the amino acid exchanges is located in the RNase H domain, at least two of the amino acid exchanges are located in MGBT, and at least two of the amino acid exchanges are located in the CD. According to another preferred embodiment, the EIAV RT comprises at least six of the aforementioned amino acid exchanges, wherein at least one of the amino acid exchanges is located in the RNase H domain, at least two of the amino acid exchanges are located in MGBT, and at least two of the amino acid exchanges are located in the CD. According to a more preferred embodiment, the EIAV RT comprises at least seven of the aforementioned amino acid exchanges, wherein at least one of the amino acid exchanges is located in the RNase H domain, at least three of the amino acid exchanges are located in MGBT, and at least three of the amino acid exchanges are located in the CD.According to an even more preferred embodiment, the EIAV RT comprises at least eight of the aforementioned amino acid exchanges, wherein at least one of the amino acid exchanges is located in the RNase H domain, at least three of the amino acid exchanges are located in MGBT, and at least three of the amino acid exchanges are located in CD. According to other more preferred embodiments, the EIAV RT comprises at least nine of the aforementioned amino acid exchanges, wherein at least one of the amino acid exchanges is located in the RNase H domain, at least four of the amino acid exchanges are located in MGBT, and at least four of the amino acid exchanges are located in CD. In some further embodiments, the EIAV RT comprises three of the aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, one of the amino acid exchanges is located in MGBT, and one of the amino acid exchanges is located in CD. According to another embodiment, the EIAV RT comprises four of the aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, and one or two of the amino acid exchanges are located in MGBT and CD, respectively. According to a further embodiment, the EIAV RT comprises five of the aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, two of the amino acid exchanges are located in MGBT, and two of the amino acid exchanges are located in CD. According to another embodiment, the EIAV RT comprises six of the aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, and two or three of the amino acid exchanges are located in MGBT and CD, respectively. According to a more preferred embodiment, the EIAV RT comprises seven of the aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, three of the amino acid exchanges are located in MGBT, and three of the amino acid exchanges are located in CD. According to an even more preferred embodiment, the EIAV RT comprises eight of the aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, and three or four of the amino acid exchanges are located in MGBT and CD, respectively.According to another more preferred embodiment, the EIAV RT comprises the nine aforementioned amino acid exchanges, wherein one of the amino acid exchanges is located in the RNase H domain, four of the amino acid exchanges are located in the MGBT, and four of the amino acid exchanges are located in the CD. Particularly preferred, the EIAV RT comprises all of the aforementioned amino acid exchanges. The latter EIAV RT has the amino acid sequence of SEQ ID NO: 10, which is encoded by the nucleic acid sequence of SEQ ID NO: 9, or a synonymous variant thereof.

[0059] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363N and D443G.

[0060] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363N and D443G.

[0061] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356D, N360A, G362T, W363K and D443G.

[0062] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356N, N360A, G362N, W363I and D443G.

[0063] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 443, 491 and 526 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, D443G, Q491R and R526H. The EIAV RT has the amino acid sequence of SEQ ID NO:12 encoded by the nucleic acid sequence of SEQ ID NO:11 or a synonymous variant thereof.

[0064] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 443, 491 and 526 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, D443G, Q491R and K553R.

[0065] According to another embodiment, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 443, 470, 491, 526 and 553 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, D443G, V470F, Q491R, R526H and K553R.

[0066] According to further embodiments, the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges in the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363, 443, 491 and 526 of SEQ ID NO:2, where the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K, D443G, Q491R and R526H. The EIAV RT has the amino acid sequence of SEQ ID NO:14 encoded by the nucleic acid sequence of SEQ ID NO:13 or a synonymous variant thereof.

[0067] Table 2 below summarizes the amino acid sequences of preferred embodiments according to the invention and shows one nucleic acid sequence encoding same. Those skilled in the art will know that the listed amino acid sequences can also be encoded by nucleic acid sequences synonymous with those shown in Table 2. The present invention also encompasses any of said synonymous nucleic acid sequences. Table 2: Preferred embodiments and their nucleic acid / amino acid sequences [Table 2]

[0068] The EIAV RT according to the invention comprises two subunits that are both encoded by variants of the polynucleotide sequence of SEQ ID NO:1. The variants according to the invention are described in more detail herein above. Preferably, the RT is a heterodimer. According to some embodiments, a first of the two subunits comprises the complete amino acid sequence encoded by the variant, and a second of the two subunits comprises a proteolytic fragment of the complete amino acid sequence encoded by the variant, i.e., a proteolytic fragment of the first subunit. The molecular weight of the first subunit is 66 kDa and the molecular weight of the second subunit is 51 kDa. Expression of the open reading frame of SEQ ID NO:1 in a host cell such as E. coli results in both the 66 kD and 51 kD subunits that can form homodimers (i.e., p66 / p66 and p51 / p51) and heterodimers (i.e., p66 / p51). Some commercially available preparations of RT are a mixture of homodimers and heterodimers. Although both types of dimers are active in DNA synthesis, the heterodimers exhibit greater stability and processivity than the homodimers, and therefore, in a preferred embodiment, the heterodimeric form of EIAV RT is purified to homogeneity.

[0069] In another aspect, the invention provides a RT obtained by expressing a variant of the polynucleotide sequence of SEQ ID NO: 1 or a vector comprising said variant in a host cell. Host cells that can be used for the production of EIAV RT according to the invention are mammalian cells, insect cells, yeast cells or bacterial cells. In a preferred embodiment, the host cell is a mammalian cell, such as a HEK293 or CHO cell, or a bacterial cell, such as E. coli. A preferred host cell is E. coli.

[0070] Another aspect of the present invention relates to a polynucleotide encoding the RT of the present invention. Said polynucleotide is a variant of the polynucleotide sequence of SEQ ID NO: 1, as described in more detail herein above. A further aspect of the present invention relates to a vector comprising a polynucleotide according to the present invention. Said polynucleotide is a variant of the polynucleotide sequence of SEQ ID NO: 1, as described in more detail herein above. In another aspect, the present invention relates to a transformed host cell comprising said vector.

[0071] In yet another embodiment, the invention relates to a method for producing an RT of the invention, comprising isolating RT from a transformed host cell.

[0072] Thus, the present invention provides novel engineered reverse transcriptases that provide beneficial improvements in thermostability, processivity and thus the length of cDNA synthesized using an RNA template, cDNA yield, and secondary enzymatic activity, i.e., elimination of RNase H activity. These improvements were engineered into the parent (EIAV RT) molecule by directed mutagenesis of amino acid residues present in the thumb domain, connecting domain, and / or RNase H domain. These mutations, which collectively eliminate RNase H activity and increase RT activity at high temperatures, were identified from both rational design and random mutagenesis followed by screening. The combination of these mutations allows for significant improvements in performance, including the ability to synthesize cDNAs of lengths greater than 12 kb at temperatures up to 65° C., resulting in superior performance even with the most challenging RNA samples.

[0073] method In another aspect, the invention refers to a method for amplifying a template nucleic acid comprising contacting the template nucleic acid with an RT according to the invention.

[0074] The template nucleic acid according to the present invention can be any kind of nucleic acid, such as RNA, DNA, or RNA:DNA hybrid. The template nucleic acid can be artificially generated (e.g., by molecular or enzymatic manipulation or synthetically) or can be naturally occurring DNA or RNA. In some preferred embodiments, the template nucleic acid is an RNA sequence, such as a transcription product, an RNA virus, or rRNA.

[0075] In some embodiments, the method referred to herein is RT-PCR. RT-PCR may be quantitative RT-PCR (RT-qPCR), real-time RT-PCR, digital RT-PCR (dRT-PCR) or digital droplet RT-PCR (ddRT-PCR).

[0076] In some preferred embodiments, the method of the present invention comprises the steps of: a) generating cDNA using the RT of the present invention; and b) amplifying the cDNA using a DNA polymerase, such as Taq DNA polymerase; Includes.

[0077] In some embodiments, serum albumin is added during amplification, preferably recombinant human, at a concentration of 1 mg / ml.

[0078] In some embodiments, the method of the present invention further comprises detecting and / or quantifying the amplified nucleic acid. Quantification / detection of the amplified nucleic acid may be performed, for example, using non-sequence-specific fluorescent dyes (e.g., SYBR® Green, EvaGreen®) that intercalate into double-stranded DNA molecules in a sequence-nonspecific manner, or sequence-specific DNA probes (e.g., oligonucleotides labeled with fluorescent reporters) that can only be detected after hybridization with the DNA target, synthesis-dependent hydrolysis, or incorporation into PCR products.

[0079] kit The reagents necessary to carry out the methods of the present invention can be included in a kit.

[0080] In some embodiments, the present invention relates to a kit for amplifying a template nucleic acid, wherein the kit comprises the RT of the present invention and a buffer. Optionally, the kit further comprises Taq DNA polymerase and / or serum albumin. The buffer included in the kit can be a conventional buffer containing magnesium. EXAMPLES

[0081] The invention is illustrated by the following examples.

[0082] Example 1: Expression and purification of EIAV RT The open reading frame of SEQ ID NO:1 or variants thereof was expressed in E. coli to produce a 66 kDa polypeptide and its proteolytic fragment, i.e., a 51 kDa polypeptide. The 66 kDa polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2. The 66 kDa and 51 kDa polypeptides form homodimers (i.e., p66 / p66 and p51 / p51) and heterodimers (i.e., p66 / p51). In one preparation, referred to as the "homogeneous" preparation, the heterodimeric form (p66 / p51) was separated from either the homodimeric form (p66 / p66 and p51 / p51) by multiple chromatographic steps, including heparin, hydrophobic interaction, and ion exchange chromatography; in the second preparation, referred to as the "heterogeneous" preparation, the homodimers and heterodimers were present in approximately equal amounts. Although both homodimers and heterodimers were active in DNA synthesis, homogenous preparations of heterodimers provide greater stability and processivity and improved performance than heterogenous mixtures of homodimers and heterodimers (Figures 1 and 2).Unless otherwise stated, all RT variants in the following examples were tested as homogenous preparations of heterodimers.

[0083] Example 2: Generation of EIAV RT mutants RNase H neg mutation Based on commonly studied homologous mutations that eliminated RNase H activity in RT, i.e., MMLV and HIV-1 RT (Mizuno 2010; Gerard 2002), mutations in the EIAV RT RNase H domain were predicted to increase thermostability. neg The variants (EIAV RT V1 and V2; see Table 3 below) were generated by site-directed mutagenesis. Neither EIAV RT V1 nor EIAV RT V2 exhibited detectable RNase H activity when up to 250 ng of sample was tested (data not shown).

[0084] Thumb domain mutation The minor groove-binding track of the thumb domain (MGBT) is a highly conserved structural element among retroviral RTs (Beard 1998, Bebenek 1997). Biochemical and molecular modeling studies of HIV-1 RT revealed that this element is important for maintaining the reading frame, fidelity, and processivity by enhancing template-primer binding affinity (Beard 1994, Bebenek 1995). Based on sequence alignments of the thumb domains of other lentiviral RTs, five point mutations (M263V / N265K / T267N / M269A / S271Q) in the MGBT (EIAV RT V3) were introduced into EIAV RT V1 by site-directed mutagenesis.

[0085] Random mutations and screening in the junction and RNase H domains.

[0086] Key mutations that improve the thermostability of RT have been identified in the connecting domain and RNase H domain (Matamoros 2013). To further improve thermostability and processivity, random mutation libraries of the connecting domain and RNase H domain were constructed and screened. Based on the crystal structure and modeling studies of HIV-1 RT in complex with template / primer (Huang 1998), a region containing 10 amino acids (355–364) within the connecting domain (CD) was selected for randomization. The CD library was generated by cross-linking dsDNA with a mixture of 10 ssDNA oligos containing one random amino acid at each position. A random library of RNase H domain variants was generated by error-prone PCR. The screening assay of the library was performed by measuring the RT activity of heat-treated (50 °C for 10 min) and 60 °C crude cell lysates. These results identified four variants from the CD library and three variants from the RNase H domain library that exhibited higher RT activity than the parent EIAV RT V3 (data not shown). The best performing CD variant was EIAV RT V4, and the best performing RNase H domain variant was EIAV RT V5. Mutations from EIAV RT V4 and EIAV RT V5 were combined to generate variant EIAV RT V6. Table 3: Constructs of optimal EIAV configuration [Table 3-1] [Table 3-2]

[0087] Example 3: RNase H neg Assessment of thermal stability and thermoactivity of variants The thermostability and thermoactivity of RNase H variants EIAV RT V1 and V2 were assessed by heat inactivation assays (Figure 1) and first-strand cDNA synthesis (Figure 2). For heat inactivation assays, RT was preincubated with substrate (poly(A) template primed with oligo(dT)20) at 42–60 °C for 10 min. Following preincubation, the RT reaction was initiated by adding MgCl2, and activity was measured by monitoring the relative rate of dT incorporation at 42 °C. All RNase H neg The variants (EIAV RT V1 and V2) retained full activity after preincubation at 50°C, whereas the heterogeneous EIAV RT WT and homogeneous EIAV RT WT showed a 50% and 30% reduction in activity, respectively (Figure 1). neg The mutant EIAV RT V2 retained 80% of its activity even after preincubation at 55°C.

[0088] The thermostability of these variants was more rigorously evaluated by measuring cDNA synthesis at elevated temperatures. First-strand cDNA synthesis reactions were carried out at 50 °C using a mixture of RNAs ranging in length from 0.5 to 9 kb as templates (poly(A)-tailed RNA ladders). The first-stranded cDNAs were separated by alkaline electrophoresis and visualized by staining with SYBR Gold. As shown in Figure 2(a), all RNase H neg The variants efficiently synthesized full-length cDNA up to 9 kb, whereas the EIAV RT WT was highly resistant to RNase H. neg The heterogeneous EIAV RT WT cDNA yields were lower than those of the homogeneous EIAV RT variants (lane 1: heterogeneous EIAV RT, lane 2: homogeneous EIAV RT WT, lane 3: EIAV RT V1, lane 4: EIAV RT V2). In contrast, the full-length products from the heterogeneous EIAV RT WT were all close to the limit of visual detection at all sizes. On the other hand, the yield of the homogeneous EIAV RT WT was significantly higher than that of the RNase H variants. neg It was slightly lower than the variant.

[0089] In the two-step RT-PCR assay, the first cDNA synthesis reaction was carried out for 30 min at 50°C (Figure 2(b), upper panel) or 60°C (Figure 2(b), lower panel) using 10 ng of human total RNA as template. Then, a 2 kb fragment of the APC gene was amplified. All RNase H neg The variants produced amplifiable cDNA at both 50 and 60°C, whereas both homogeneous and heterogeneous EIAV RTs failed to produce cDNA at either 50 or 60°C (Figure 2(b)).

[0090] Taken together, these results collectively support the interpretation that abolishing the RNase H activity of RT increases its thermostability.

[0091] Example 4: RNase H with additional mutations within the MGBT of the thumb domain neg Assessment of thermal stability and thermoactivity of EIAV RT variant (EIAV RT V3) The effect of the additional mutations on thermostability and thermoactivity was assessed using one-step RT-qPCR based on FAM probes. First, a region of the ACTB gene was reverse transcribed from human total RNA (2 pg) using EIAV RT V1, EIAV RT V3 or EnzScript RT as indicated, then amplified and quantified. The results show that EIAV RT V1 and EIAV RT V3 both retain most of their activity around 70 °C and show optimal activity at temperatures around 55 °C. However, the decrease in activity of EIAV RT V1 becomes more pronounced at temperatures higher than 65 °C. In contrast, as the temperature increases, a significant increase in Ct values ​​indicating a decrease in cDNA yield was observed with the widely used typical comparator, EnzScript (Moloney murine leukemia virus (MMLV) RT with reduced RNase H activity) (Figure 3).

[0092] Additionally, a two-step RT-qPCR assay was performed using EIAV RT V1, EIAV RT V3 and SuperScript IV. Using human total RNA (50 ng) and a gene-specific primer (MAP4), cDNA was reverse transcribed at the indicated temperatures, and cDNAs of various lengths were quantified by qPCR targeting the MAP4 gene. The results show that EIAV RT V3 produces higher cDNA yields, as indicated by lower Ct values ​​than EIAV RT V1. The difference in yield between these enzymes dramatically increases at higher temperatures or longer cDNA synthesis, indicating improved thermostability and processivity of EIAV RT V3 compared to EIAV RT V1 (Figure 4).

[0093] The synthesis rate and product length of cDNA generated by EIAV RT V3 were compared to SuperScript IV, a widely used commercial RT that is considered state of the art. The reaction was performed at 55 °C using 50 ng of human total RNA as input and oligo(dT)20 as a primer. After heat inactivating the RT at the indicated times for 5 min at 85 °C, the reaction was quenched by amplifying the 9 kb APC gene. Both enzymes generated an amplifiable 9 kb product within 10 min. However, the yield was significantly increased with EIAV RT V3 (Figure 5).

[0094] Example 5: RNase H with additional mutations in the connecting domain and / or RNase H domain neg / MGBT mut Assessment of thermostability and thermoactivity of EIAV RT variants The effect of additional mutations within the CD and / or RNase H domains on thermostability and thermoactivity was tested by two-stage RT-qPCR (Figure 6) and long-term end-point PCR (Figure 7).

[0095] For two-step RT-qPCR, cDNA synthesis using human total RNA (50 ng) and gene-specific primers (MAP4) was performed at the indicated temperatures. Then, cDNAs of various lengths were quantified by amplifying the MAP4 gene using a primer set designed for a specific region of the gene. For long-range two-step RT-PCR (12.3 kb), rat brain total RNA (50 ng) and gene-specific primers for rat dynein were used for cDNA synthesis at various temperatures (Figure 7; lane 1: EIAV RT V3, lane 2: EIAV RT V4, lane 3: EIAV RT V5, lane 4: EIAV RT V6).

[0096] Both two-step RT-qPCR (Figure 6) and long-term end-point PCR (Figure 7) assays at various temperatures confirmed that all of these variants (EIAV RT V4, EIAV RT V5, and EIAV RT V6) showed improved thermostability and processivity compared to the original EIAV RT V3. One of the constructs, EIAV RT V4, was able to synthesize a 12.3 kb cDNA at a high temperature of 65°C (Figure 7), and this variant appears to be the optimal form for most applications. In certain embodiments, for example, the following items are provided: (Item 1) A reverse transcriptase (RT) comprising two subunits, each of which is encoded by a variant of the polynucleotide sequence of SEQ ID NO:1; The amino acid sequence encoded by the variant is at least 90% identical to the amino acid sequence of SEQ ID NO:2 or a fragment thereof; The variant comprises a mutation in the polynucleotide sequence of SEQ ID NO:1 that causes an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 in the minor groove binding track (MGBT) of the thumb domain of the RT; The mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges within the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, and 271 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, and S271Q. (Item 2) 2. The RT of item 1, wherein the variant comprises one or more additional mutations in the polynucleotide sequence of SEQ ID NO: 1 that result in one or more additional amino acid exchanges relative to SEQ ID NO: 2 in the connecting domain (CD) of the RT. (Item 3) 3. The RT according to item 2, wherein the one or more further mutations in the polynucleotide sequence of SEQ ID NO:1 cause one or more further amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 356, 360, 362 and 363 of SEQ ID NO:2, wherein the amino acid exchanges are I356G / I356D / I356N and / or N360A and / or G362T / G362N and / or W363K / W363N / W363I, preferably I356G and / or N360A and / or G362T and / or W363K, more preferably I356G, N360A, G362T and W363K. (Item 4) The RT of any one of the preceding items, wherein the variant comprises one or more additional mutations in the polynucleotide sequence of SEQ ID NO:1 that result in one or more additional amino acid exchanges relative to SEQ ID NO:2 in the RNase H domain of the RT. (Item 5) 5. The RT according to item 4, wherein the further mutation in the polynucleotide sequence of SEQ ID NO: 1 causes one or more further amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 443 and 476 of SEQ ID NO: 2, wherein the amino acid exchanges are D443G and / or E476Q, preferably D443G. (Item 6) RT according to any one of the preceding items, wherein the variant comprises an additional mutation in the polynucleotide sequence of SEQ ID NO: 1 resulting in an additional amino acid exchange relative to SEQ ID NO: 2, wherein the additional mutation in the polynucleotide sequence of SEQ ID NO: 1 results in an additional amino acid exchange in the encoded amino acid sequence at an amino acid position corresponding to position 491 of SEQ ID NO: 2, wherein the amino acid exchange is Q491R. (Item 7) 7. The RT according to item 6, wherein the variant comprises one or more further mutations in the polynucleotide sequence of SEQ ID NO: 1 resulting in one or more further amino acid exchanges relative to SEQ ID NO: 2, wherein the further mutations in the polynucleotide sequence of SEQ ID NO: 1 result in one or more further amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 470, 526 and 553 of SEQ ID NO: 2, wherein the amino acid exchanges are V470F and / or R526H and / or K533R, preferably R526H and / or K553R, more preferably R526H. (Item 8) 13. The RT of any one of the preceding items, wherein the mutations in the polynucleotide sequence of SEQ ID NO:1 result in amino acid exchanges within the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, and the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K and D443G. (Item 9) 10. The RT of any one of the preceding items, wherein a first of the two subunits comprises the complete amino acid sequence encoded by the variant and a second of the two subunits comprises a proteolytic fragment of the complete amino acid sequence encoded by the variant. (Item 10) 13. A method for amplifying a template nucleic acid comprising contacting said template nucleic acid with a RT according to any one of the preceding items, preferably said method being a reverse transcription (RT) PCR. (Item 11) a) a RT according to any one of items 1 to 9; and b) Buffer solution Kit including: (Item 12) A polynucleotide encoding the RT according to any one of items 1 to 9. (Item 13) A vector comprising the polynucleotide according to item 12. (Item 14) A transformed host cell comprising the vector according to item 13. (Item 15) RT obtained by expressing the polynucleotide according to item 12 or the vector according to item 13 in a host cell, preferably E. coli.

Claims

1. A reverse transcriptase (RT) comprising two subunits, each of which is encoded by a variant of the polynucleotide sequence of SEQ ID NO:1; the amino acid sequence encoded by said variant is at least 90% identical to the amino acid sequence of SEQ ID NO:2; The variant comprises a mutation in the polynucleotide sequence of SEQ ID NO:1 that causes an amino acid exchange relative to the amino acid sequence of SEQ ID NO:2 in the minor groove binding track (MGBT) of the thumb domain of the RT; The mutations in the polynucleotide sequence of SEQ ID NO:1 cause amino acid exchanges within the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, and 271 of SEQ ID NO:2, wherein the amino acid exchanges are M263V, N265K, T267N, M269A, and S271Q.

2. The RT of claim 1, wherein the variant comprises one or more additional mutations in the polynucleotide sequence of SEQ ID NO: 1 that result in one or more additional amino acid exchanges relative to SEQ ID NO: 2 within the connecting domain (CD) of the RT.

3. The RT of claim 2, wherein the one or more further mutations in the polynucleotide sequence of SEQ ID NO:1 cause one or more further amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to 356, 360, 362, and 363 of SEQ ID NO:2, and the amino acid exchanges are I356G / I356D / I356N and / or N360A and / or G362T / G362N and / or W363K / W363N / W363I.

4. The RT of claim 3, wherein the amino acid exchange is I356G and / or N360A and / or G362T and / or W363K.

5. The RT of claim 4, wherein the amino acid exchanges are I356G, N360A, G362T and W363K.

6. The RT of any one of claims 1 to 5, wherein the variant comprises one or more additional mutations in the polynucleotide sequence of SEQ ID NO: 1 that result in one or more additional amino acid exchanges relative to SEQ ID NO: 2 in the RNase H domain of the RT.

7. The RT of claim 6, wherein the further mutation in the polynucleotide sequence of SEQ ID NO:1 causes one or more further amino acid exchanges in the encoded amino acid sequence at amino acid positions corresponding to positions 443 and 476 of SEQ ID NO:2, the amino acid exchanges being D443G and / or E476Q.

8. The RT of claim 7, wherein the amino acid exchange is D443G.

9. The RT of any one of claims 1 to 8, wherein the variant comprises a further mutation in the polynucleotide sequence of SEQ ID NO: 1 that causes a further amino acid exchange relative to SEQ ID NO: 2, wherein the further mutation in the polynucleotide sequence of SEQ ID NO: 1 causes a further amino acid exchange in the encoded amino acid sequence at an amino acid position corresponding to position 491 of SEQ ID NO: 2, wherein the amino acid exchange is Q491R.

10. The RT of claim 9, wherein the variant comprises one or more additional mutations in the polynucleotide sequence of SEQ ID NO: 1 that result in one or more additional amino acid exchanges relative to SEQ ID NO: 2, wherein the additional mutations in the polynucleotide sequence of SEQ ID NO: 1 result in one or more additional amino acid exchanges within the encoded amino acid sequence at amino acid positions corresponding to positions 470, 526 and 553 of SEQ ID NO: 2, and wherein the amino acid exchanges are V470F and / or R526H and / or K533R.

11. The RT of claim 10, wherein the amino acid exchange is R526H and / or K553R.

12. The RT of claim 11, wherein the amino acid exchange is R526H.

13. The RT of any one of claims 1 to 12, wherein the mutation in the polynucleotide sequence of SEQ ID NO:1 causes amino acid exchanges within the encoded amino acid sequence at all amino acid positions corresponding to positions 263, 265, 267, 269, 271, 356, 360, 362, 363 and 443 of SEQ ID NO:2, and the amino acid exchanges are M263V, N265K, T267N, M269A, S271Q, I356G, N360A, G362T, W363K and D443G.

14. 14. The RT of claim 1, wherein the first of the two subunits comprises the complete amino acid sequence encoded by the variant.

15. A method for amplifying a template nucleic acid, comprising contacting the template nucleic acid with an RT according to any one of claims 1 to 14.

16. The method of claim 15, wherein the method is reverse transcription (RT) PCR.

17. a) a RT according to any one of claims 1 to 14; and b) Buffer A kit for amplifying a template nucleic acid, comprising:

18. A polynucleotide encoding the RT of any one of claims 1 to 14.

19. A vector comprising the polynucleotide of claim 18.

20. 20. A transformed host cell comprising the vector of claim 19.

21. 20. An RT obtained by expressing the polynucleotide of claim 18 or the vector of claim 19 in a host cell.

22. 22. The RT of claim 21 , wherein the host cell is E. coli.

Images