阅读说明：本技术 具有热稳定性增加的突变型逆转录酶以及涉及其的产物、方法和用途 (Mutant reverse transcriptase having increased thermostability and products, methods and uses relating thereto ) 是由 C.H.贝尔 H.佐贝克 H.瓦尔希 K.安川 M.马场 于 2018-04-10 设计创作，主要内容包括：本发明涉及相对于野生型具有增加的热稳定性的突变型逆转录酶(RT),编码突变型RT的核酸,包含突变型RT或核酸的细胞,包含突变型RT的试剂盒,突变型RT用于cDNA合成的用途,使用突变型RT用于逆转录包含合成cDNA的RNA的方法,以及使用突变型RT检测样品中的RNA标记物的方法。(The present invention relates to mutant Reverse Transcriptases (RTs) having increased thermostability relative to wild type, nucleic acids encoding mutant RTs, cells comprising mutant RTs or nucleic acids, kits comprising mutant RTs, use of mutant RTs for cDNA synthesis, methods for reverse transcription of RNA comprising synthetic cDNA using mutant RTs, and methods for detecting RNA markers in a sample using mutant RTs.)

1. A method of producing a polypeptide relative to SEQ ID NO: 1 a mutant RT having increased thermostability, the mutant RT comprising a wild-type Reverse Transcriptase (RT)

i) Relative to SEQ ID NO: 1 has an amino acid sequence with six amino acid substitutions, wherein

-substitution of Ala at position 32 with Val (a 32V);

-substitution of Leu at position 72 with Arg (L72R);

-substitution of Glu at position 286 with Arg (E286R);

-Glu at position 302 is substituted with Lys (E302K);

-Trp at position 388 is substituted by Arg (W388R); and

-substitution of Leu at position 435 with Arg (L435R), or

ii) an amino acid sequence which is at least 95% identical to the amino acid sequence of i) and which has six amino acid substitutions as defined in i),

wherein the mutant RT exhibits reverse transcriptase activity.

2. The mutant RT of claim 1 wherein the mutant RT is identical to SEQ ID NO: 1, otherwise differs in the amino acid sequence of SEQ ID NO: 1, and/or a deletion of up to five amino acids at the N-terminus of SEQ ID NO: 1, deletion of up to five amino acids at the C-terminus.

3. The mutant RT of claim 1 or 2 wherein the mutant RT comprises or consists of an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 2 is at least 96%, 97%, 98% or 99%, in particular 100%, identical.

4. The mutant RT of any one of claims 1 to 3, wherein the mutant RT has increased thermostability relative to mutant MM3, wherein MM3 has a sequence identical to SEQ ID NO: 1 by an amino acid sequence that differs by only three amino acid substitutions, wherein Glu at position 286 is substituted with Arg (E286R), Glu at position 302 is substituted with Lys (E302K), and Leu at position 435 is substituted with Arg (L435R).

5. Mutant according to any of claims 1-4, wherein the thermostability is determined by measuring the reverse transcriptase activity of the mutant as measured after heat treatment, in particular after incubation for 10 minutes at 60 ℃, and/or wherein the thermostability is increased by at least 10%, 20%, 30% or 40%, preferably by at least 50%, relative to wild type or mutant MM3.

6. The mutant of any of claims 1-5 wherein the reverse transcriptase activity of the mutant RT is at least 50%, particularly at least 60%, more particularly at least 70%, especially at least 80% of the reverse transcriptase activity of the wild type and/or wherein the reverse transcriptase activity is determined by RT-mediated incorporation of dTTP at 37 ℃.

7. The mutant RT of any one of claims 1 to 6, which is fused to a further protein.

8. A nucleic acid encoding the mutant RT of any one of claims 1 to 7.

9. A cell comprising the mutant RT of any one of claims 1 to 7 or the nucleic acid of claim 8.

10. A kit for performing reverse transcription comprising the mutant RT of any one of claims 1 to 7.

11. Use of a mutant RT according to any one of claims 1 to 7 for cDNA synthesis.

12. A method for reverse transcription of RNA comprising synthesizing cDNA from RNA using the mutant RT of any one of claims 1 to 7.

13. A method for detecting an RNA marker in a sample,

a) contacting the sample with a mutant RT of any one of claims 1 to 7 under conditions conducive to the activity of the mutant RT;

b) synthesizing cDNA from the RNA tag using the mutant RT; and

c) detecting the presence of the cDNA synthesized in step b), thereby detecting the RNA marker in said sample.

14. The method according to claim 13, wherein the sample is selected from the group consisting of a bodily fluid, blood, plasma, serum, urine, bile, cerebrospinal fluid, swab, clinical specimen, organ sample and tissue sample, and/or wherein the sample has been obtained from a cell culture, a source suspected of being contaminated or a subject, particularly wherein the subject is selected from the group consisting of a human, an animal and a plant, especially a human.

15. The method of claim 13 or 14, wherein the RNA marker is indicative of a microorganism, a cell, a virus, a bacterium, a fungus, a mammalian species, a genetic condition, or a disease.

Suitable methods for determining increased thermal stability are detailed in the examples. An exemplary condition for the stress condition may be a preincubation at 48 to 65 ℃ C (particularly 60 ℃ C.) for 10 minutes, followed by use of [2 ]³H]-reverse transcription assay of dTTP, reverse transcription assay using fluorescent dye PicoGreen, or cDNA synthesis preferably by real-time PCR.

The RT of the invention is derived from MMLV RT, which is a 75-kDa monomer. It consists of finger, palm, thumb, junction and rnase H domain. The active site for the DNA polymerase reaction is located in the finger/palm/thumb domain, while the active site for the rnase H reaction is located in the rnase H domain.

The amino acid sequence of the RT, referred to as wild-type RT, includes the numbering of the amino acids as follows:

the corresponding nucleic acid sequences are as follows:

the term "mutant reverse transcriptase" (RT) relates to a reverse transcriptase whose amino acid sequence is identical to SEQ ID NO: 1 differ in amino acid sequence by at least six mutated RT enzymes. As detailed above, relative to SEQ ID NO: 1, mutant RT of the invention has six mandatory amino acid substitutions, wherein

-substitution of Ala at position 32 with Val (a 32V);

-substitution of Leu at position 72 with Arg (L72R);

-substitution of Glu at position 286 with Arg (E286R);

-Glu at position 302 is substituted with Lys (E302K);

-Trp at position 388 is substituted by Arg (W388R); and

-substitution of Leu at position 435 with Arg (L435R).

And SEQ ID NO: 1 differs only by the amino acid sequence of the six compulsory mutated versions of the above mentioned RT, which is designated SEQ ID NO: 2, and the following:

the six mandatory mutations are indicated by bold letters with underlining and their positions are designated by the respective amino acid numbers.

The corresponding nucleic acid sequences are as follows:

however, the mutant RT may have one or more further amino acid substitutions, additions, deletions, or combinations thereof. According to the invention, the mutant RT of the invention may further comprise a sequence identical to SEQ ID NO: 2 is at least 95% identical and has an amino acid sequence with six mandatory amino acid substitutions as defined above (a 32V/L72R/E286R/E302K/W388R/L435R).

In one embodiment of the invention, the mutant RT according to the invention may comprise one or more amino acid substitutions, in particular a limited number of substitutions (e.g. up to 30, 20 or especially 10 amino acid substitutions), in particular conservative substitutions. "conservative amino acid substitutions" refer to the substitution of a residue with a different residue having a similar side chain, and thus generally involve the substitution of an amino acid in a polypeptide with an amino acid within the same or similarly defined class of amino acids. By way of example and not limitation, an amino acid having an aliphatic side chain may be substituted with another aliphatic amino acid, such as alanine, valine, leucine, and isoleucine; the amino acid having a hydroxyl side chain is substituted with another amino acid having a hydroxyl side chain, such as serine and threonine; the amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, such as phenylalanine, tyrosine, tryptophan, and histidine; the amino acid having a basic side chain is substituted with another amino acid having a basic side chain, such as lysine and arginine; the amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain, such as aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is substituted with another hydrophobic or hydrophilic amino acid, respectively. Examples of conservative amino acid substitutions include those listed below:

in one embodiment of the invention, the mutant RT according to the invention may comprise one or more amino acid additions, in particular small (e.g. up to 30, 20 or especially 10 amino acids) internal or terminal amino acid additions.

In one embodiment of the invention, the mutant RT according to the invention may comprise one or more amino acid deletions, in particular N-and/or C-terminal deletions. Deletions may be small (e.g. up to 5, 4, 3, 2, especially 1 amino acid at each end). In a preferred embodiment, the mutant RT is identical to SEQ ID NO: 1 differs in the amino acid sequence of SEQ id no: 1, and/or a deletion of up to five amino acids at the N-terminus of SEQ ID NO: 1-deletion of up to five amino acids at the C-terminus-in addition to the mandatory mutations defined above.

In another embodiment, the sequence of the mutant RT according to the invention may comprise a combination of one or more deletions, substitutions or additions as defined above, in addition to the mandatory mutation (substitution). However, mutant RT comprises a sequence identical to SEQ id no: 2, and an amino acid sequence at least 95% identical to the amino acid sequence of 2.

As used herein, the term "at least 95% identical" or "at least 95% sequence identity" means that the sequence of the mutant RT according to the invention has an amino acid sequence characterized in that within a stretch of 100 amino acids, at least 95 amino acid residues are identical to SEQ ID NO: 2 are identical in sequence. Other percentages of sequence identity are defined accordingly.

Sequence identity according to the invention can be determined, for example, by means of sequence alignment methods in the form of sequence comparisons. Methods of sequence alignment are well known in the art and include various programs and alignment algorithms. In addition, the NCBI Base Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Mutants according to the invention are compared to e.g. SEQ ID NO: 2, are typically characterized using NCBI Blast blastp with standard settings. Alternatively, software GENEious with standard settings can be used to determine sequence identity. The alignment results can be derived, for example, from Software Geneious (version R8), using a global alignment scheme with a free end gap as the alignment type and Blosum62 as the cost matrix.

As detailed above, the mutant RT of the invention comprises a sequence identical to SEQ ID NO: 2, and an amino acid sequence at least 95% identical to the amino acid sequence of 2. In a preferred embodiment, the mutant RT comprises or consists of an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 2 is at least 96%, 97%, 98% or 99%, in particular 100%, identical. Sequence identity may be determined as described above.

In another preferred embodiment, the mutant RT has equal or even increased thermostability relative to mutant MM3, wherein MM3 has a sequence identical to SEQ ID NO: 1 by an amino acid sequence that differs by only three amino acid substitutions, wherein Glu at position 286 is substituted with Arg (E286R), Glu at position 302 is substituted with Lys (E302K), and Leu at position 435 is substituted with Arg (L435R). MM3 (E286R/E302K/L435R) is a thermostable MMLV RT triple variant, which was generated by introducing three mutations aimed at increasing the positive charge into the wild-type MMLV RT (Yasukawa et al, 2010).

Preferably, thermostability is determined by measuring the reverse transcriptase activity of the mutant form measured after heat treatment, in particular after incubation at 60 ℃ for 10 minutes. Additionally or alternatively, thermostability is increased by at least 10%, 20%, 30% or 40%, preferably at least 50% relative to wild type RT or mutant MM3. Details regarding these embodiments are given above.

It is further preferred that the reverse transcriptase activity of the mutant RT (unstressed) is at least 50%, in particular at least 60%, more in particular at least 70%, especially at least 80% of the reverse transcriptase activity of the wild type. Additionally or alternatively, reverse transcriptase activity was determined by RT-mediated dTTP incorporation at 37 ℃ (see examples). Details regarding the enzyme activity assay are given above.

In another embodiment, the mutant RT may be fused to a further protein. Fusion proteins are proteins produced by linking two or more initially separate proteins or peptides. This procedure results in polypeptides having functional properties derived from each of the original proteins. Accordingly, depending on the intended use of the RT, it may be combined with further peptides or proteins into fusion proteins. Proteins can be fused via a linker or spacer, which increases the likelihood that the proteins fold independently and behave as expected. Especially in cases where the linker allows for protein purification, the linker in the protein or peptide fusion is sometimes engineered with a cleavage site for a protease or chemical agent that allows for the release of two separate proteins. Dimeric or multimeric fusion proteins can be made by genetic engineering of fusions to the original protein (e.g., streptavidin or leucine zipper) of a peptide domain that induces dimerization or multimerization of the artificial protein. Fusion proteins can also be made with toxins or antibodies attached thereto. Other fusions include addition signal sequences such as lipidation signal sequences, secretion signal sequences, glycosylation signal sequences, translocation signal peptides, and the like.

Preferably, the fusion protein of the invention comprises a tag. Tags are attached to proteins for various purposes, for example, to facilitate ease of purification, to aid in proper folding of the protein, to prevent precipitation of the protein, to alter chromatographic properties, to modify or label the protein or to tag the protein. Examples of tags include Arg-tag, His-tag, Strep-tag, Flag-tag, T7-tag, V5-peptide-tag, GST-tag and c-Myc-tag. The preferred tag in the present invention is a His-tag consisting of six histidine residues.

In a further aspect, the invention relates to a nucleic acid encoding a mutant RT of the invention.

As used herein, the term "nucleic acid" generally relates to any nucleotide molecule that encodes a mutant RT of the present invention and may have a variable length. Examples of nucleic acids of the invention include, but are not limited to, plasmids, vectors, or any kind of DNA and/or RNA fragments that can be isolated by standard molecular biology procedures including, for example, ion exchange chromatography. The nucleic acids of the invention may be used for transfection or transduction of specific cells or organisms.

The nucleic acid molecules of the invention may be in the form of RNA, such as mRNA or cRNA, or may be in the form of DNA, including, for example, cDNA and genomic DNA, obtained, for example, by cloning or produced by chemical synthesis techniques or a combination thereof. The DNA may be triplex, double stranded or single stranded. The single-stranded DNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand. As used herein, a nucleic acid molecule also refers to, inter alia, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-or triple-stranded, or a mixture of single-and double-stranded regions. In addition, as used herein, a nucleic acid molecule refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA.

Alternatively, the nucleic acid may contain one or more modified bases. Such nucleic acids may also contain modifications, for example, in the ribose phosphate backbone, to increase the stability and half-life of such molecules in physiological environments. Thus, a DNA or RNA having a backbone modified for stability or for other reasons is a "nucleic acid molecule" as contemplated herein. Furthermore, DNA or RNA comprising a rare base such as inosine or a modified base such as a tritylated base are nucleic acid molecules in the context of the present invention, to name only two examples. It will be appreciated that a wide variety of modifications have been made to DNA and RNA which serve many useful purposes known to those skilled in the art. As it is used herein, the term nucleic acid molecule includes such chemically, enzymatically or metabolically modified forms of nucleic acid molecules, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including particularly simple and complex cells.

In addition, nucleic acid molecules encoding mutant RTs of the invention can be functionally linked to any desired sequence, e.g., a regulatory sequence, a leader sequence, a heterologous marker sequence, or a heterologous coding sequence, using standard techniques, e.g., standard cloning techniques, to produce fusion proteins.

In general, the nucleic acids of the invention may be initially formed in vitro or in cultured cells by manipulating the nucleic acids by endonucleases and/or exonucleases and/or polymerases and/or ligases and/or recombinases or other methods known to the skilled person to produce nucleic acids.

The nucleic acid of the invention may be comprised in an expression vector, wherein the nucleic acid is operably linked to a promoter sequence capable of promoting expression of the nucleic acid in a host cell.

As used herein, the term "expression vector" generally refers to any kind of nucleic acid molecule that can be used to express a protein of interest in a cell (see also details above for nucleic acids of the invention). In particular, the expression vector of the present invention may be any plasmid or vector known to those skilled in the art that is suitable for expressing a protein in a particular host cell, including but not limited to mammalian cells, bacterial cells, and yeast cells. The expression construct of the invention may also be a nucleic acid encoding the RT of the invention and used for subsequent cloning into a separate vector to ensure expression. Suitable carriers are described in the examples and are shown in fig. 2. Plasmids and vectors for protein expression are well known in the art and may be purchased commercially from various suppliers including, for example, Promega (Madison, WI, USA), Qiagen (Hilden, germany), Invitrogen (Carlsbad, CA, USA) or MoBiTec (germany). Methods for protein expression are well known to those skilled in the art and are described, for example, in Sambrook et al, 2000 (Molecular Cloning: A laboratory, third edition).

The vector may additionally comprise nucleic acid sequences which allow it to replicate in the host cell, for example an origin of replication, one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art, for example regulatory elements which direct the transcription, translation and/or secretion of the encoded protein. The vectors may be used to transduce, transform, or infect cells, thereby causing the cells to express nucleic acids and/or proteins that are different from those native to the cells. The vector optionally includes materials that aid in achieving entry of the nucleic acid into the cell, such as viral particles, liposomes, protein capsids, and the like. Numerous types of suitable expression vectors for protein expression are known in the art by standard molecular biology techniques. Such vectors are selected from conventional vector types, including insect, e.g., baculovirus, expression, or yeast, fungal, bacterial or viral expression systems. Other suitable expression vectors of which many types are known in the art may also be used for this purpose. Methods for obtaining such expression vectors are well known (see, e.g., Sambrook et al, supra).

As detailed above, the nucleic acid encoding the mutant RT of the invention is operably linked to sequences suitable for driving protein expression in a host cell in order to ensure expression of the protein. However, it is contemplated in the present invention that the claimed expression constructs may represent intermediates that are subsequently cloned into suitable expression vectors to ensure expression of the protein. The expression vectors of the present invention may further comprise all kinds of nucleic acid sequences including, but not limited to, polyadenylation signals, splice donor and splice acceptor signals, insertion sequences, transcriptional enhancer sequences, translational enhancer sequences, drug resistance genes, or the like. Optionally, the drug resistance gene may be operably linked to an Internal Ribosome Entry Site (IRES), which may be cell cycle specific or cell cycle independent.

As used herein, the term "operably linked" generally means that the genetic elements are arranged such that they act synergistically for their intended purpose, e.g., transcription is initiated by a promoter and proceeds through a DNA sequence encoding a protein of the invention. That is, the sequence encoding the fusion protein is transcribed into mRNA by RNA polymerase, which is then spliced and translated into protein.

As used in the context of the present invention, the term "promoter sequence" generally refers to any kind of regulatory DNA sequence operably linked to a downstream coding sequence, wherein the promoter is capable of binding RNA polymerase and initiating transcription of the encoded open reading frame in a cell, thereby driving expression of the downstream coding sequence. The promoter sequence of the present invention may be any kind of promoter sequence known to those skilled in the art, including but not limited to constitutive promoters, inducible promoters, cell cycle specific promoters, and cell type specific promoters.

In a further aspect, the invention relates to a cell comprising a mutant RT of the invention or a nucleic acid of the invention. The cell is preferably a host cell. "host" of the inventionThe cell "may be any kind of organism suitable for application in recombinant DNA technology and includes, but is not limited to, all classes of bacteria and yeast strains suitable for expression of one or more recombinant proteins. Examples of the host cell include, for example, various Bacillus subtilis (B.) (Bacillus subtilis) Or an E.coli strain. Various E.coli bacterial host cells are known to those skilled in the art and include, but are not limited to, strains such as DH 5-alpha, HB101, MV1190, JM109, JM101, or XL-1 blue, which are commercially available from various suppliers including, for example, Stratagene (CA, USA), Promega (WI, USA), or Qiagen (Hilden, Germany). A particularly suitable host cell, i.e.E.coli BL21 (DE 3) cell, is also described in the examples. Bacillus subtilis strains that can be used as host cells are commercially available.

The cultivation of the host cells according to the invention is a routine procedure known to the skilled worker. That is, nucleic acids encoding the mutant RT of the present invention can be introduced into a suitable host cell by recombinant means to produce the corresponding protein. These host cells may be obtained by any kind of suitable cells, preferably bacterial cells, such as e.g. e. In a first step, the method may comprise cloning the respective genes into a suitable plasmid vector. Plasmid vectors are widely used for gene cloning and can be easily introduced, i.e. transformed into bacterial cells that have been made competent. After the protein has been expressed in the respective host cell, the cells may be disrupted by means of chemical or mechanical cell lysis, which are well known to those skilled in the art and include, but are not limited to, for example, hypotonic salt treatment, detergent treatment, homogenization or sonication.

The invention also provides kits for performing reverse transcription comprising the mutant RT of the invention. Reverse transcription is the synthesis of DNA from an RNA template, which is usually mediated by reverse transcriptase, and produces complementary DNA (cdna). Reverse transcriptase uses an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of first strand cDNA, which can be used directly as a template for Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNA in a sample, as well as the production of corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first strand cDNA may be made double stranded using DNA polymerase I and DNA ligase. These reaction products can be used directly for cloning without amplification. In this case, RNase H activity from RT or an exogenous supply is required. Depending on the intended use, the kit may comprise further components, such as a buffer, one or more primers and a dNTP mix, in addition to the mutant RT of the invention. The kit may also comprise reagents required for further reactions, such as PCR, synthesis of the second DNA strand or reagents required for amplification (e.g. primers, probes, polymerase or labels). In addition, the kit may contain an instruction manual.

In a further aspect, the invention relates to the use of the mutant RT of the invention for cDNA synthesis. A common technique for studying gene expression in, for example, living cells is the production of DNA copies (cdnas) of the complement of the RNA-producing cell. This technique provides a means of studying RNA from living cells, which avoids direct analysis of intrinsically unstable RNA. After optional mRNA isolation (using, for example, methods such as affinity chromatography using oligo dT), the oligonucleotide sequence is typically annealed to the mRNA molecule, and an enzyme with reverse transcriptase activity can be used to generate cDNA copies of the RNA sequence using the RNA/DNA primers as templates. Therefore, reverse transcription of mRNA is a critical step in many forms of gene expression analysis. Typically, mRNA is reverse transcribed into cDNA for subsequent analysis by primer extension or polymerase chain reaction. In the use of the invention, RNA is contacted with the mutant RT of the invention and a typical primer sequence that anneals to the RNA template to initiate DNA synthesis from the 3' OH of the primer. The primer may be selected to be complementary or substantially complementary to the sequence present at the 3' end of each strand of the target nucleic acid sequence. In an exemplary embodiment, the reverse transcription reaction is performed using an annealing temperature in the reverse transcriptase reaction, which is typically about 42 to 65 ℃. The reverse transcription reaction is preferably carried out at about 50 ℃ to 60 ℃ or 60 ℃ to 65 ℃.

The invention further provides a method for reverse transcription of RNA comprising synthesizing cDNA from RNA using the mutant RT of the invention. The method can be performed as detailed for the use of the mutant RT of the invention for cDNA synthesis.

In addition, the present invention provides a method for detecting an RNA marker in a sample,

a) contacting the sample with a mutant RT of the invention under conditions conducive to the activity of the mutant RT;

b) synthesizing cDNA from the RNA tag using the mutant RT of the present invention; and

c) detecting the presence of the cDNA synthesized in step b), thereby detecting the RNA marker in the sample.

RNA can be used as a marker in various applications. The detected RNA may be indicative of itself, or it may be indicative of the presence of DNA or expression of a target gene, which in turn is indicative of a disease, the presence of a pathogen, or the like. The RNA itself may indicate the presence of viral RNA, in particular retroviral RNA. Retroviruses cause a variety of diseases such as cancer, AIDS, autoimmunity, and diseases of the central nervous system, bone and joints, such as myeloid leukemia, erythroid leukemia, lymphoid leukemia, lymphoma, sarcoma, breast cancer, renal cancer, aplastic anemia, hemolytic anemia, autoimmune diseases, immunodeficiency, osteopetrosis, arthritis (arthritis), peripheral neuropathy (perpheral neuropathy), encephalopathy, neurodegeneration, dementia, pneumonia, and adenomatosis. Viruses that induce such diseases include Human Immunodeficiency Virus (HIV), human T-lympho virus (HTLV), Rous Sarcoma Virus (RSV), and Murine Mammary Tumor Virus (MMTV). However, RNA markers may indicate gene expression. Many genes are expressed only under specific conditions (including disease conditions) or by specific species. Accordingly, the presence of a protein (or corresponding mRNA) may be indicative of a disease state, cell or pathogen-to mention just a few. For example, cancer cells are characterized by specific markers whose nucleic acids can be used for their detection and quantification. Examples which may be mentioned are: especially oncogenes and tumor suppressor genes, such as p53, genes of the ras family erb-B2, c-myc, mdm2, c-fos, DPC4, FAP, nm23, RET, WT1 and the like, such as LOH for p53, DCC, APC, Rb and the like, and microsatellite instability of BRCA1 and BRCA2, MSH2, MLH1, WT1 and the like in hereditary tumors; and tumor RNA, such as CEA, cytokeratins such as CK20, BCL-2, MUC1, particularly tumor-specific splice variants thereof, MAGE3, Muc18, tyrosinase, PSA, PSM, BA46, Mage-1, and the like, or other morphogenic RNA, such as mammary silk inhibin, hCG, GIP, motilin, hTG, SCCA-1, AR, ER, PR, various hormones, and the like; in addition, especially the expression of RNAs and proteins which influence the metastatic profile, i.e.molecules involved in angiogenesis, motility, adhesion and matrix degradation, such as bFGF, bFGF-R, VEGF-Rs, for example VEGF-R1 or VEGF-R2, E-cadherin, integrins, selectins, MMP, TIMP, SF-R and the like, cell cycle profiles or proliferation profiles such as cyclins (for example the expression ratio of cyclins D, E and B), Ki67, p120, p21, PCNA and the like, or apoptosis profiles such as FAS (L + R), TNF (L + R), perforin, granzyme B, BAX, bcl-2, caspase 3 and the like. Alternatively, the RNA may be indicative of DNA of a pathogen other than a retrovirus.

In the first step of the method, the sample is contacted with the mutant RT of the invention under conditions conducive to the activity of the mutant RT. Suitable conditions are detailed herein and are well known to those skilled in the art. The contacted sample may be any sample suspected of containing the RNA in question, including samples from a subject. A sample is a limited amount of material that is expected to be the same as and representative of a larger amount of that material. The act of obtaining a sample may be performed by an individual or automatically. The sample may be obtained or provided for testing, analysis, examination, investigation, demonstration or experimental use. Sometimes, the sampling may be continued. The sample may comprise or consist of a solid, liquid or gas; it may be a material with some intermediate characteristics, such as a gel or sputum, a tissue, an organism, or a combination of these. Preferably, the sample is a liquid or suspension that allows for easy dispensing.

Even if a sample of material cannot be counted as an item, the number of samples can still be described in terms of its volume, mass, size, or other such dimension. The solid sample may be divided into one or several discrete pieces, or may be in the form of chips, granules or powder.

In this context, a sample is a quantity of material suspected of containing one or more nucleic acids to be detected or measured and quantified. As used herein, the term includes, but is not limited to, a sample (e.g., biopsy or medical sample), a culture (e.g., microbial culture), or an environmental sample such as water or soil. The sample may be from a subject, such as an animal or human, which may be a fluid, a solid (e.g., stool), a suspension, or a tissue. The term "sample from a subject" includes all biological fluids, excreta and tissues isolated from any given subject. Preferably, the subject is an animal, more preferably a mammal, or even more preferably a human. Samples can be obtained from all the various families of domesticated animals as well as from non-domesticated or wild animals, including but not limited to such animals as ungulates, bears, fish, rodents, and the like.

As detailed above, "sample" means a quantity of material suspected of containing the target nucleic acid to be quantified. As used herein, the term includes a sample (e.g., biopsy or medical sample) or a culture (e.g., microbial culture). The sample may be from a plant or animal, including a human, and it may be a fluid, a solid (e.g. faeces) or a tissue. The sample may include material taken from the patient including, but not limited to, culture, blood, saliva, cerebrospinal fluid, pleural fluid, milk, lymph, sputum, sperm, needle aspirates, and the like. Samples can be obtained from all the various families of domesticated animals as well as from non-domesticated or wild animals, including but not limited to such animals as ungulates, bears, fish, rodents, and the like. By human sample or "tissue sample" or "patient cell or tissue sample" or "specimen", respectively, is meant a collection of similar cells or biological or biochemical compounds obtained from a subject or tissue of a patient. The source of the tissue sample may be solid tissue, e.g. from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituent; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or from a cell at any time during pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

Examples of samples include, but are not limited to, cell or tissue culture, blood, serum, plasma, needle aspirates, urine, sperm, semen, seminal plasma, prostatic fluid, feces, tears, saliva, sweat, biopsy, ascites fluid, cerebrospinal fluid, pleural fluid, amniotic fluid, peritoneal fluid, interstitial fluid, sputum, milk, lymph, bronchial and other lavage fluid samples, or tissue extract samples. The source of the sample may be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; or from a cell at any time during pregnancy or development of the subject. In a preferred embodiment of the method, the sample is selected from the group consisting of body fluids, blood, plasma, serum, urine, bile, cerebrospinal fluid, swabs, clinical specimens, organ samples and tissue samples, in particular human, animal or plant, especially human. Alternatively or additionally, the sample has been obtained from a cell culture, a source suspected of being contaminated or a subject, particularly wherein the subject is selected from the group consisting of a human, an animal and a plant, especially a human.

After step a), cDNA is synthesized from the RNA tag using the mutant RT of the invention. Detailed information on this step is given above. Thereafter, the presence of the synthesized cDNA is detected, thereby detecting the RNA marker in the sample. Methods for detecting DNA are well known in the art and include PCR methods, the use of specific probes or intercalators with labels (e.g., radioactive or fluorescent). In a preferred embodiment, reverse transcriptase is used in combination with real-time PCR for detection of RNA markers.

The methods and uses of the invention are particularly advantageous in the medical field, e.g. in diagnostics or therapy monitoring, and may be used for the detection and/or quantification of target nucleic acids indicative of a particular microorganism, cell, virus, bacterium, fungus, mammalian species, genetic condition or disease. Accordingly, the method can be used for detection of pathogens. Pathogens have the potential to cause disease. Typically, pathogens are used to describe infectious agents, such as a virus, a bacterium, a prion, a fungus, or even another microorganism. Of course, the method of the invention can also be used for the detection of non-pathogenic microorganisms. Accordingly, in another preferred embodiment of the method, the RNA marker is indicative of a microorganism, a cell, a virus, a bacterium, a fungus, a mammalian species, a genetic condition or a disease.

Exemplary pathogens include, but are not limited to:

-bacteria: streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Pseudomonas (Pseudomonas), Burkholderia (Burkholderia), Mycobacterium (Mycobacterium), Chlamydophila (Chlamydophila), Elekoniella (Ehrlichia), Rickettsia (Rickettsia), Salmonella (Salmonella), Neisseria (Neisseria), Brucella (Brucella), Mycobacterium, Nocardia (Nocardia), Listeria (Lista), Francisella (Francisella), Legionella (Leginella) and Yersinia (Yersinia)

-virus: adenovirus, herpes simplex virus, varicella zoster virus, cytomegalovirus, papilloma virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, poliovirus, yellow fever virus, dengue fever virus, West Nile virus, TBE virus, HIV, influenza virus, lassa virus, rotavirus and Ebola virus

-fungi: candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys

-parasites: protozoan parasites, helminth parasites and arthropod parasites

It is apparent that reliable detection and optionally quantification of pathogens may be of high relevance in diagnosing the presence and severity of disease.

Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Definitions of common terms in molecular biology can be found in: benjamin Lewis, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); kendrew et al (ed.) by Blackwell Science Ltd, 1994 (ISBN 0-632-02182-9), The Encyclopedia of Molecular Biology; and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-.

The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise", "comprising" and "includes" are to be construed as inclusive rather than exclusive. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" means two or more.

The following figures and examples are intended to illustrate various embodiments of the present invention. As such, the specific modifications discussed should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is therefore to be understood that such equivalent embodiments are included herein.

Drawings

FIG. 1: the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 2) of mutant RT MM 3.14. Six mandatory mutations for the wild type are indicated (substitutions a32V, L72R, E286R, E302K, W388R and L435R).

FIG. 2: expression plasmid for MMLV-RT. Asterisks indicate stop codons.

FIG. 3: activity and stability of individual variants. (A) Specific activity. The dTTP incorporation reaction was carried out at 37 ℃. One unit is defined as 1 nmol of dTTP incorporated into poly (rA) -p (dT) in 10 minutes₁₅The amount of (b). Relative activity is defined as the ratio of the specific activity of the variant to the specific activity of the WT. (B, C) thermal stability. In poly (rA) -p (dT)₁₅(28 μ M) were incubated at 46 ℃ (B) or 49 ℃ (C) for 10 minutes at 100 nM RT. Then, dTTP incorporation reaction was performed at 37 ℃. Relative activity is defined as the ratio of the initial reaction rate with RT incubated at 46 or 49 ℃ for 10 minutes to the initial reaction rate without incubation.

FIG. 4: activity and stability of multiple variants. (A, B) specific activity. (A) dTTP incorporation reactions were performed at 37 ℃ with 5 nM RT. One unit is defined as 1 nmol of dTTP incorporated into poly (rA) -p (dT) in 10 minutes₁₅The amount of (b). Relative activity is defined as the ratio of the specific activity of the variant to the specific activity of the WT. (B) PicoGreen incorporation reactions were performed at 37 ℃ with 5 nM RT. Calculating the initial reaction Rate (. DELTA.)FIPer minute) and normalized with the initial reaction rate of the WT of 1.0. (C-F) thermal stability. In poly (rA) -p (dT)₁₅(28 μ M) was incubated at 49 or 51 ℃ for 10 minutes at 100 nM RT. Then, either the dTTP incorporation reaction (C, E) or the PicoGreen incorporation reaction (D, F) was performed at 37 ℃ with 10 nM RT. Relative activity is defined as the ratio of the initial reaction rate of RT with 10 min heat treatment to RT without 10 min heat treatment.

FIG. 5: temperature dependence of cDNA synthesis by WT, MM3 or MM 3.14. The cDNA synthesis reaction was performed at 50 (A), 55 (B, C), 60 (D) or 65 ℃ (E) for 10 minutes using RT that had received 5 minutes of heat incubation at 55 ℃ (B) or RT that did not have heat incubation (A, C-E). Then, PCR was performed. Fluorescence of real-time PCR using cDNA synthesis products is shown. The Crossover Point (CP) was 28.01, 25.22 and 25.67 minutes (a) for WT, MM3 and MM3.14, respectively, 24.95 and 26.74 minutes (B) for MM3 and MM3.14, respectively, 30.52 minutes (C) for MM3.14, 28.48 and 29.14 minutes (D) for MM3 and MM3.14, respectively, and 32.51 minutes (E) for MM 3.14.

FIG. 6: stability of WT, MM3 or MM3.14 as assessed by cDNA synthesis. In poly (rA) -p (dT)₁₅(28 μ M) was incubated at 48, 54, 57, 60 or 63 ℃ for 10 minutes at 100 nM RT. Then, 16 pg was used for cDNA synthesiscesARNA, 0.5 μ M RV-R26 primer was performed at 45 ℃ for 30 minutes. PCR was performed with primer combinations of RV and F5. The amplification products were applied to a 1% agarose gel followed by staining with ethidium bromide (1 μ g/ml).