Reverse transcriptase variants

Abstract

The present invention provides an MMLV reverse transcriptase with increased thermostability compared to wild-type MMLV reverse transcriptase and wild-type AMV reverse transcriptase. The improved thermostability allows reverse transcription from RNA to cDNA at temperatures higher than 37°C, thereby reducing the error rate introduced during cDNA synthesis. As a result, the reverse transcriptase of the present invention allows increased accuracy in determining the transcriptome of an organism. The present invention provides modified reverse transcriptases with increased thermostability, increased solubility and reduced qPCR inhibition compared to conventional commercially available reverse transcriptases, such as Superscript II reverse transcriptase (prod-dnd, shown herein as SEQ ID NO:2).

Description

[Technical field]

[0001] (Sequence Listing) A "Sequence Listing XML" has been submitted herewith in XML file format, (i) the name of the file is WMG-005-01WO-ST26.xml; (ii) the creation date is March 7, 2023; and (iii) the size of the file is 30,940 bytes, and the material in the XML file is incorporated by reference.

[0002] (Technical field) The present invention relates to reverse transcriptase enzymes. [Background technology]

[0003] (background) The transcriptome refers to the RNA transcripts of a cell or organism at a given time. Knowledge of the transcriptome can reveal active cellular processes and provide information about cellular regulation, proliferation and dysfunction.

[0004] Transcriptome analysis typically involves microarray technology, and more commonly next-generation sequencing technology (e.g., RNA-Seq). The general goal of RNA-Seq is to detect all of the diverse transcripts present (including mRNA and non-coding RNA), and to detect splice variants, mutations, mobile genetic elements, and expression levels during or at various developmental stages. Transcriptomics and RNA-Seq have applications in diagnostics, disease profiling, pathogen detection, evolutionary biology, and other research areas. For example, RNA-Seq can potentially identify genes that involve resistance to environmental stress (e.g., drought resistance in crops). In another example, transcriptomic profiling can provide information about the mechanisms of drug resistance, potentially revealing strategies to combat hospital-acquired antibiotic-resistant infections.

[0005] Typically, in an RNA-Seq workflow, RNA is first used to synthesize a stable complementary DNA (cDNA) copy of the target RNA through a reverse transcription reaction. Reverse transcriptase is the typical enzyme used to synthesize cDNA from RNA. Unlike DNA polymerases, a higher reaction temperature is generally desirable for reverse transcription reactions because reverse transcriptase does not have any proofreading capabilities. This is because the higher temperature reduces off-target primer binding. In addition, the higher temperature reduces secondary structures in the RNA, which can cause steric hindrance to the reverse transcriptase (RT), thus shortening the transcript. However, common commercial reverse transcriptases (e.g., Moloney Murine Leukemia Virus (MMLV), Superscript II, and Avian Myeloblastosis Virus (AMV)) start to lose efficiency above 37°C. As a result, their higher error rate allows them to accumulate mutations at an accelerated rate compared to the proofreading form of synthesis. For example, AMV reverse transcriptase typically has an error rate of 1 in 17,000 bases, and MMLV typically has an error rate of 1 in 30,000 bases. As a result, commercially available reverse transcriptases impede accurate determination of the transcriptome. Summary of the Invention [Means for solving the problem]

[0006] (Abstract) The present invention provides modified reverse transcriptases that have increased thermostability, increased solubility and reduced qPCR inhibition compared to conventional commercially available reverse transcriptases, such as Superscript II reverse transcriptase (prod-dnd, shown herein as SEQ ID NO:2). The improved thermostability allows reverse transcription of RNA to cDNA at temperatures higher than 37° C., thereby reducing the error rate introduced during cDNA synthesis. As a result, the reverse transcriptases of the present invention allow increased accuracy in transcriptomics.

[0007] The reverse transcriptase of the present invention can include a polypeptide comprising a sequence of amino acids having at least about 95% sequence identity to the reverse transcriptase of SEQ ID NO:2 and at least one amino acid substitution at an amino acid position selected from positions 56, 72, 138, 163, 232, 234, 249, 290, 344, 420, 424, 444, and 615.

[0008] At least one amino acid substitution is S at position 56 substituted with G, A, V, L, or I; G at position 138 substituted with S, C, T, or M; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, Q; V at position 444 substituted with G, A, L, or I; and D at position 615 substituted with E, N, or Q may be selected from:

[0009] For example, the at least one amino acid substitution can be S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615E. Additionally, any of the following are examples of mutations in accordance with the present invention: S27T, V43K, A46P, L48I, A54P, S56A, S60W, Q68K, L72Q / R, E123Q, Y133C, G138S, T163K, M177R, D200H, I212T, I218T, T231E, S232T, L234E, Q237E, A242D, N249E, Q265E, K264Q, K267T, L272I T281S, G290K, N335Q P338E, D339E, Y344F, Q345D E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444I, D518E, G524D, L528F, A554P, Q562E, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D, and K658E.

[0010] The reverse transcriptase of the invention further comprises at least one additional amino acid substitution at an amino acid position selected from positions 524, 583, and 562. The additional amino acid substitution may be selected from: D at position 524 substituted with G, AV, L, or I; D at position 583 substituted with E, N, or Q; and E at position 562 substituted with D, N, or Q.

[0011] For example, the at least one additional amino acid substitution may be selected from D524G, D583N, and E562Q.

[0012] In some embodiments, the reverse transcriptase comprises any number of substitutions from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. For example, the reverse transcriptase may comprise substitutions at at least three amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. The reverse transcriptase may comprise substitutions at at least five amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. The reverse transcriptase may comprise substitutions at at least 10 amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. The reverse transcriptase may comprise substitutions at 12 amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, 562.

[0013] In an embodiment of the invention, the reverse transcriptase of the invention comprises a polypeptide comprising a sequence of amino acids having at least about 95% sequence identity to the reverse transcriptase of SEQ ID NO:2 and at least one amino acid substitution at an amino acid position selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, and 642.

[0014] At least one amino acid substitution is S at position 56 substituted with G, A, V, L, or I; G at position 138 substituted with S, C, T, or M; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, or Q; G at position 290 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q. is selected from.

[0015] For example, the at least one amino acid substitution may be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D.

[0016] The reverse transcriptase may further comprise at least one additional amino acid substitution at an amino acid position selected from positions 524, 583, 562, 204, and 306.

[0017] At least one additional amino acid substitution is D at position 524 substituted with G, AV, L, or I; D at position 583 substituted with E, N, or Q; E at position 562 substituted with D, N, or Q; H at position 204 substituted with K or R; and T at position 306 substituted with H, K, or R may be selected from:

[0018] For example, the additional amino acid substitutions can be G524D, N583D, Q562E, R204H, or K306T.

[0019] The reverse transcriptase of the present invention exhibits increased thermostability compared to both wild-type reverse transcriptase as well as common commercially available reverse transcriptases (e.g., Superscript II (SEQ ID NO: 2)). For example, the reverse transcriptase of the present invention at 50°C has at least about 50% of the reverse transcription activity of either the wild-type reverse transcriptase or the reverse transcriptase of SEQ ID NO: 2 at 42°C. However, depending on the number of mutations and conditions, at 50°C or higher, the activity of the reverse transcriptase of the present invention may even exceed about 90% of the reverse transcription activity of either the wild-type reverse transcriptase or the reverse transcriptase of SEQ ID NO: 2 at 42°C. For example, when 10 or more mutations are present, the reverse transcription activity exceeds 80% of the reverse transcription activity of the reverse transcriptase of SEQ ID NO: 2 at 42°C.

[0020] Moreover, the activity of the reverse transcriptase of the present invention exceeds the physiological activity of, for example, the reverse transcriptase shown in SEQ ID NO:2 at higher temperatures (e.g., 65°C). For example, the reverse transcriptase of the present invention at temperatures of about 65°C or higher exhibits at least 50% of the reverse transcriptase activity of the reverse transcriptase of SEQ ID NO:2 at 42°C. In contrast, the reverse transcriptase activity of either the wild-type transcriptase or that shown in SEQ ID NO:2 would be significantly inhibited at temperatures higher than about 50°C and would be nearly inactive at temperatures around about 65°C. However, also depending on the number and conditions of mutations, at about 65°C or higher, the reverse transcriptase activity of the reverse transcriptase of the present invention may even exceed 80% of the reverse transcriptase activity of either the reverse transcriptase shown in SEQ ID NO:2 or its wild-type reverse transcriptase at 42°C. For example, when 10 or more mutations are present, the reverse transcriptase activity may exceed 70% of the reverse transcriptase activity of either the wild-type reverse transcriptase or that shown in SEQ ID NO:2 at 42°C.

[0021] The reverse transcriptase of the present invention has improved activity compared to conventional commercial reverse transcriptases (including wild-type reverse transcriptases) in the presence of inhibitors.Various inhibitors are found in various samples, which cause wild-type and commercial reverse transcriptases to function inefficiently.For example, plant samples typically contain polysaccharides, phenols, and flavonoids that inhibit reverse transcription.Sample preparation steps also frequently introduce reverse transcription inhibitors (e.g., detergents, alcohols, salts, heparin, and hematin).

[0022] The invention further provides methods for reverse transcribing cDNA from RNA using the reverse transcriptases of the invention.

[0023] A preferred method of the invention includes providing to a sample containing RNA a reverse transcriptase comprising a polypeptide comprising a sequence of amino acids having at least about 95% sequence identity to the reverse transcriptase of SEQ ID NO:2 and at least one amino acid substitution at an amino acid position selected from positions 56, 138, 234, 290, 344, 420, 424, 444, and 615; and reagents for reverse transcription. The method further includes performing a reverse transcription reaction between the reverse transcriptase and the RNA in the sample to form cDNA.

[0024] At least one amino acid substitution is S at position 56 substituted with G, A, V, L, or I; G at position 138 substituted with S, C, T, or M; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; and D at position 615 substituted with E, N, or Q may be selected from:

[0025] For example, at least one of the amino acid substitutions can be S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615.

[0026] The reverse transcriptase may further comprise at least one additional amino acid substitution at an amino acid position selected from positions 524, 583, and 562. The at least one additional amino acid substitution may be selected from: D at position 524 substituted with G, AV, L, or I; D at position 583 substituted with E, N, or Q; and E at position 562 substituted with D, N, or Q.

[0027] In one example, the additional amino acid substitutions are selected from G524D, N583d, and Q562E.

[0028] The reverse transcriptase may include any number of substitutions from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. For example, the reverse transcriptase may include substitutions at at least three amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. The reverse transcriptase may include substitutions at at least five amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. The reverse transcriptase may comprise substitutions at at least 10 amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, and 562. The reverse transcriptase may comprise substitutions at 13 amino acid positions selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 524, 583, 562.

[0029] In an embodiment of the invention, the reverse transcriptase of the invention comprises a sequence of amino acids having at least about 95% sequence identity to the reverse transcriptase of SEQ ID NO:2, and at least one amino acid substitution at an amino acid position selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 444, 615, and 642.

[0030] At least one amino acid substitution is S at position 56 substituted with G, A, V, L, or I; G at position 138 substituted with S, C, T, or M; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, or Q; G at position 290 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q is selected from.

[0031] For example, the amino acid substitutions may be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D.

[0032] The reverse transcriptase may further comprise at least one additional amino acid substitution at an amino acid position selected from positions 524, 583, 562, 204, and 306. Additional amino acid substitutions may also be D at position 524 substituted with G, AV, L, or I; D at position 583 substituted with E, N, or Q; E at position 562 substituted with D, N, or Q; H at position 204 substituted with K or R; and T at position 306 substituted with H, K, or R may be selected from:

[0033] In one alternative, the additional amino acid substitutions are selected from G524D, N583D, Q562E, R204H, and K306T.

[0034] The use of the reverse transcriptase of the present invention allows the reaction to occur at temperatures of 50° C. or higher without loss of enzyme activity that would occur with conventional reverse transcriptases. Thus, the performing process can be carried out at temperatures of about 65° C. or higher with at least about 50% activity of the reverse transcriptase shown in SEQ ID NO:2 at 42° C. Due to the increased thermostability of the reverse transcriptase of the present invention, the elevated reaction temperature allows for both high activity reverse transcriptase activity and reduced nucleotide base addition errors in cDNA synthesis. Furthermore, the reverse transcription reaction can result in reduced incubation times, for example, the incubation times can be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 30 minutes, compared to the typical method requiring 50 minutes.

[0035] An embodiment of the invention provides modified reverse transcriptases that include at least one mutation in at least one domain of the reverse transcriptase of SEQ ID NO: 2. Importantly, the modified reverse transcriptases of the invention exhibit increased thermostability compared to either the wild-type reverse transcriptase or the reverse transcriptase shown in SEQ ID NO: 2, such that the activity of the reverse transcriptase at a temperature of about 50° C. or higher is at least about 90% of the activity of the transcriptase of SEQ ID NO: 2 at 42° C.

[0036] Preferred mutations include amino acid substitutions at positions 27, 138, 200, 204, 212, 218, 231, 232, 234, 237, 242, 249, 264, 265, 267, and / or 272 in the palm domain of SEQ ID NO:2.

[0037] Additionally, the present invention contemplates mutations at positions 43, 46, 48, 54, 56, 72, 163, and / or 177 in the finger domain; positions 280, 281, 290, 306, 335, 338, 339, 344, 345, 346, and / or 349 in the thumb domain; positions 376, 413, 420, 424, 443, and / or 444 in the connection domain; and / or positions 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642, and / or 658 in the RNAse H domain of SEQ ID NO:2.

[0038] The following is a list of preferred substitution mutations for use in the present invention: S at position 56 substituted with G, A, V, L, or I; L at position 72 substituted with Q or R; G at position 138 substituted with S, C, T, or M; H at position 204 substituted with K or R; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; T at position 306 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 524 substituted with G, AV, L, or I; E at position 562 substituted with D, N, or Q; D at position 583 substituted with E, N, or Q; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q.

[0039] The modified reverse transcriptase of the present invention may have substitutions at 1 to 14 amino acid positions selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642.

[0040] Further, preferred amino acid substitutions include S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and / or D615E. In addition to the above, additional amino acid substitutions selected from G524d, N583D, and Q562E may be added. In other embodiments, the amino acid substitutions are selected from L72Q, T163K, N249E, and H642D. In yet other embodiments, the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D. The embodiment may also include at least one additional amino acid substitution selected from D524G, D583N, E562Q, H204R, and T306K.

[0041] In specific embodiments, the amino acid substitutions are S27T, V43K, A46P, L48I, A54P, S56A, L72Q, G138S, T163K, M177R, D200H, I212T, I218T, T231E, S232T, L234E, Q237E, A242D, N249E, Q265E, K264Q, K267T, L272I T281S, G290K, N335Q P338E, D339E, Y344F, Q345D relative to SEQ ID NO:2. Selected from E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444I, D518E, G524D, L528F, A554P, E562Q, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D, and K658E.

[0042] In a preferred embodiment, the modified reverse transcriptase of the invention exhibits an enzymatic activity at a temperature of about 65° C. or higher that is at least about 50% equivalent to the reverse transcriptase activity of the reverse transcriptase shown in SEQ ID NO:2 at about 42° C. The activity of the reverse transcriptase of the invention is improved in the presence of an inhibitor. Additionally, the modified reverse transcriptase of the invention exhibits increased thermostability relative to the reverse transcriptase of SEQ ID NO:2 and exhibits the following properties: E5K; M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H ; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

[0043] Specifically, the activity of the reverse transcriptase of the present invention is improved in the presence of a reverse transcription inhibitor relative to the reverse transcriptase shown in SEQ ID NO: 2, and is selected from the group consisting of M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q ; Y344H; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

[0044] In other embodiments, the reverse transcriptase of the invention exhibits improved activity in formalin-fixed samples relative to the reverse transcriptase of SEQ ID NO:2, and is selected from the group consisting of M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333 It may contain at least one amino acid substitution selected from the group consisting of: Q; Y344H; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

[0045] In yet other embodiments, the reverse transcriptase of the present invention exhibits increased thermostability compared to the reverse transcriptase of SEQ ID NO:2, and the reverse transcriptase of the present invention may contain at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

[0046] The activity of the reverse transcriptase of the present invention in the presence of an inhibitor is also improved compared to the reverse transcriptase of SEQ ID NO: 2, and the activity of the reverse transcriptase of the present invention in the presence of an inhibitor may include at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R. Similarly, the reverse transcriptase activity of the reverse transcriptase of the present invention is improved in formalin-fixed samples compared to the reverse transcriptase of SEQ ID NO: 2, and the reverse transcriptase activity of the reverse transcriptase of the present invention may include at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

[0047] In certain embodiments, the modified reverse transcriptase of the invention has at least about 95% sequence identity to the reverse transcriptase shown in SEQ ID NO:2.

[0048] An embodiment of the invention includes a method of reverse transcribing cDNA from RNA. Such a method includes utilizing modified reverse transcriptases provided herein. Such modified enzymes exhibit improved thermostability compared to existing reverse transcriptases, such that the activity of those modified enzymes at temperatures of about 50° C. or higher is at least about 90% of the reverse transcription activity of conventional reverse transcriptases at 42° C.; The sample is provided with reagents for reverse transcription, and a reverse transcription reaction is carried out between the reverse transcriptase and the RNA in the sample. The reverse transcription reaction is carried out at a temperature of 50° C. or higher to form cDNA.

[0049] The modified enzymes of the invention may be modified to include positions 27, 138, 200, 204, 212, 218, 231, 232, 234, 237, 242, 249, 264, 265, 267, and 272 in the palm domain of the reverse transcriptase shown in SEQ ID NO:2, positions 43, 46, 48, 54, 56, 72, 163, and 177 in the finger domain of SEQ ID NO:2, positions 280, 281, 290, 306, 335, 338, 339, 344, 345, 346, and 349 in the thumb domain of SEQ ID NO:2, positions 376, 413, 420, 424, 443, and 444 in the connection domain of SEQ ID NO:2, and positions 450, 451, 452, and 453 in the RNAse domain of the reverse transcriptase shown in SEQ ID NO:2. At least one amino acid substitution at a position selected from positions 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642, and 658 in the H domain.

[0050] For example, amino acid substitutions include S at position 56 substituted with G, A, V, L, or I; L at position 72 substituted with Q or R; G at position 138 substituted with S, C, T, or M; H at position 204 substituted with K or R; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; T at position 306 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 524 substituted with G, AV, L, or I; E at position 562 substituted with D, N, or Q; D at position 583 substituted with E, N, or Q; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q may be selected from:

[0051] The modified reverse transcriptase of the present invention can include substitutions at at least three amino acid positions, and up to at least 12 amino acid positions, selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642. Specifically, the amino acid substitutions can be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615E. In certain embodiments, the modified reverse transcriptase can include at least one additional amino acid substitution selected from G524D, N583D, and Q562E. The at least one amino acid substitution can be selected from L72Q, T163K, N249E, and H642D. Additionally and / or alternatively, the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D. This embodiment may further comprise additional amino acid substitutions selected from D524G, D583N, E562Q, H204R, and T306K.

[0052] The methods of the invention can further include any known reagents and methods for reverse transcription. For example, the providing step can include providing the sample with a capture oligo that captures a target RNA molecule. [Brief description of the drawings]

[0053] [Figure 1A] FIG. 1A shows qPCR results indicating the thermo-activity of the reverse transcription (RT) enzyme of the present disclosure. [Figure 1B] FIG. 1B shows the efficiency scores for the enzymes. [Figure 1C] FIG. 1C shows RT-qPCR yields at high temperatures using the reverse transcription (RT) enzymes of the disclosure.

[0054] [Figure 2A] FIG. 2A shows the enhanced thermo-activity of the reverse transcription (RT) enzyme of the present disclosure. [Figure 2B] FIG. 2B shows the processivity of the reverse transcription (RT) enzyme of an embodiment of the present disclosure at high temperature.

[0055] [Figure 3A] FIG. 3A shows the thermostability of reverse transcriptase (RT) variant enzymes of the present disclosure. [Figure 3B] FIG. 3B shows melting curves indicating thermal stability. [Figure 3C] FIG. 3C shows the results of a thermal ramp assay of an embodiment of a reverse transcription (RT) enzyme of the disclosure. [Figure 3D] FIG. 3D shows a table of results for the thermal ramp assay of FIG. 3C. [Figure 3E] FIG. 3E shows an isothermal reaction incubation for an embodiment of a reverse transcription (RT) enzyme of the disclosure.

[0056] [Figure 4A] FIG. 4A shows the resistance of an embodiment of the reverse transcriptase (RT) enzyme of the present disclosure to inhibition by heparin. [Figure 4B] FIG. 4B shows the change in Ct value with increasing heparin concentration for the reverse transcriptase (RT) enzyme of the present disclosure. [Figure 4C] FIG. 4C shows the resistance of an embodiment of the reverse transcriptase (RT) enzyme of the present disclosure to inhibition by guanidine isothiocyanate. [Figure 4D] FIG. 4D shows the change in Ct value with increasing guanidine isothiocyanate concentrations for the reverse transcriptase (RT) enzyme of the disclosure. [Figure 4E] FIG. 4E shows the resistance of embodiments of the reverse transcriptase (RT) enzyme of the present disclosure to inhibition by ethanol. [Figure 4F] FIG. 4F shows the change in Ct value with increasing ethanol concentrations for the reverse transcription (RT) enzyme of the present disclosure.

[0057] [Diagram 5] FIG. 5 shows the cDNA yield of the reverse transcription (RT) enzyme of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] (Detailed Description) The present invention provides reverse transcriptases that have increased thermostability compared to conventional reverse transcriptases (e.g., Superscript II (SEQ ID NO:2) or the wild-type reverse transcriptase shown in SEQ ID NO:1). The improved thermostability allows reverse transcription of RNA to cDNA at temperatures above about 42°C, thereby reducing the error rate introduced during cDNA synthesis. As a result, the reverse transcriptases of the present invention allow increased accuracy in determining the transcriptome of an organism.

[0059] Specifically disclosed herein are modified reverse transcriptases that include a substitution at least one position in SEQ ID NO:2 selected from positions 56, 72, 138, 163, 234, 249, 290, 344, 420, 424, 444, 615, 642, 524, 583, 562, 204, and 306. For example, the amino acid substitution is S at position 56 substituted with G, A, V, L, or I; G at position 138 substituted with S, C, T, or M; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, or Q; G at position 290 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 615 substituted with E, N, or Q; H at position 642 substituted with D, E, N, or Q. D at position 524 substituted with G, AV, L, or I; D at position 583 substituted with E, N, or Q; E at position 562 substituted with D, N, or Q; H at position 204 substituted with K or R; and T at position 306 substituted with H, K, or R It could be.

[0060] The present invention recognizes that elevated temperature during reverse transcription reaction reduces non-target primer binding, error rate in cDNA synthesis, and reduces RNA secondary structure.Thus, the improved thermostability of the reverse transcriptase of the present invention allows reverse transcription of cDNA from RNA at temperatures higher than 37°C (the optimum temperature for wild-type reverse transcriptase and other conventional reverse transcriptases).By allowing elevated reaction temperatures, the reverse transcriptase of the present invention results in reduced error rate in cDNA synthesis.

[0061] Moreover, the reverse transcriptases of the present invention provide additional advantages over wild-type reverse transcriptases and other conventional reverse transcriptases, including improved efficiency in the presence of inhibitors or reverse transcriptase, and improved efficiency in formalin-fixed samples.

[0062] (Reverse Transcription) Reverse transcription is the synthesis of DNA from an RNA template, producing cDNA. Using an RNA template and a short primer complementary to the 3' end of the RNA, reverse transcriptase directs the synthesis of a first strand cDNA, which can be used directly as a template for polymerase chain reaction (PCR). This combination of reverse transcription and PCR allows for the detection of RNA in a sample and the generation of the corresponding cDNA. Ribonuclease (RNase) H activity (either derived from the reverse transcriptase or supplied exogenously) can be used to separate the RNA / cDNA hybrids. Additional components can also include a buffer, one or more primers, a dNTP mix (a mixture of nucleotides dATP, dCTP, dGTP and dTTP), agents required for PCR, agents required for the synthesis of the second DNA strand, or amplification reagents.

[0063] Reverse transcription may be performed by a capture oligo with a free 3' poly-T region. The 3' portion of the capture oligo may contain a target-specific sequence or a target-specific oligomer (e.g., a capture primer for reverse transcribing an RNA molecule containing the capture sequence). The oligomer may be a random oligomer or a "not-so-random" (NSR) oligomer (NSRO) (e.g., a random hexamer or an NSR hexamer). The oligos may contain one or more handles (e.g., a primer binding sequence cognate to a PCR primer used in the amplification step or a sequence of a next-generation (NGS) sequencing adapter). The reverse transcription primer may contain a template switching oligo (TSO), which may contain a poly-G sequence that hybridizes to and captures a poly-C segment added during reverse transcription.

[0064] Reverse transcription of RNA may include the use of a capture sequence and a capture primer or capture probe. The primer sequence may include a binding site (e.g., a primer sequence that is expected to hybridize to a complementary sequence in a nucleic acid molecule). The primer sequence may also be a "universal" primer sequence (i.e., a sequence that is complementary to a nucleotide sequence that is very common to a particular set of nucleic acid fragments). The primer sequence may be the P5 primer and P7 primer provided by Illumina, Inc., San Diego, California. The primer sequence may also allow the capture probe to bind to a solid support (e.g., a template particle).

[0065] This process may involve hybridizing the reverse transcription primer to the probe, followed by a reverse transcription reaction. The complement of the nucleic acid need not be perfect when aligned; stable duplexes may contain mismatched base pairs or unmatched bases.

[0066] Those skilled in the art of nucleic acid technology can determine duplex stability empirically, taking into consideration many variables, including, for example, the length of the oligonucleotide, the percentage concentration of cytosine and guanine bases in the oligonucleotide, ionic strength, and the occurrence of mismatched base pairs.

[0067] Reverse transcription can also be used to add a barcode, a unique molecular identifier (UMI), a sequencing adapter, or any additional nucleotide sequence to the final cDNA product. For example, the reverse transcription primer can contain additional bases that add a barcode or UMI to the cDNA product.

[0068] A barcode is any nucleotide sequence that can be used to derive information about a nucleic acid product, for example, after sequencing. For example, the same sample barcode can be attached to each nucleic acid from a single sample source. The sample barcode can then be used to identify nucleic acids from separate samples after sequencing the nucleic acids and the sample barcode.

[0069] UMI is a kind of barcode that can be provided to a sample to make each nucleic acid molecule that is combined with the barcode unique or nearly unique.This can be achieved by adding one or more UMIs to one or more capture probes of the present invention.By selecting an appropriate number of UMIs, every nucleic acid molecule in a sample is unique or nearly unique when combined with the UMI.

[0070] UMIs are advantageous in that they can be used to correct errors (e.g., amplification bias or incorrect base pairing during amplification) that are generated during amplification.For example, when using UMIs, every nucleic acid molecule in a sample is unique or nearly unique with its UMI or UMIs, so that after amplification and sequencing, molecules with identical sequences can be considered to refer to the same starting nucleic acid molecule, thereby reducing amplification bias.Methods for error correction using UMIs are described in Karlsson et al., 2016, "Counting Molecules in cell-free DNA and single cells RNA", Karolinska Institute, Stockholm, Sweden (incorporated herein by reference).

[0071] For example, in the context of RNA analysis, it may be advantageous to analyze the relative or absolute amount of each RNA molecule in a sample (e.g., a single-cell sample). However, typical RNA analysis methods first require reverse transcription of the RNA molecule into a cDNA copy, followed by amplification of the cDNA molecule. The cDNA molecule must then be amplified to generate sufficient nucleic acid to be analyzed. As a result, the absolute number of each original RNA molecule present in the sample is lost, and multiple copies of each molecule now present in the prepared sample are analyzed. Furthermore, due to amplification bias that results in uneven amplification of some cDNA molecules to a greater extent than others, information regarding the relative amount of each RNA molecule in the original sample is also lost. By providing each cDNA molecule with a UMI prior to amplification, each cDNA molecule (corresponding to each RNA molecule in the sample) is made unique or nearly unique. After amplifying each cDNA molecule (with its UMI), the amplified cDNA molecule can later be sequenced, and a sequence read is generated for each molecule. Multiple sequence reads with both the same base sequence and UMI can be collapsed into a single sequence read and considered to be reads derived from a single RNA molecule in the original sample. Sequence reads with the same base sequence but different UMIs are not collapsed and each sequence read is considered to be a separate instance of an RNA molecule present in the original sample.

[0072] After reverse transcription, biotinylated capture baits or biotinylated capture probes can also be used for targeted enrichment of specific cDNA molecules of interest. Biotinylated capture probes can include RNA, DNA, or hybrids of RNA and DNA nucleotides. The biotinylated RNA capture probes can be added to a cDNA library and incubated at a temperature and for a time sufficient for the biotinylated RNA capture probes to hybridize to their cDNA target molecules based on Watson-Crick base pairing. For example, a mixture containing the cDNA and probes can be incubated at 65 degrees Celsius for 24 hours. After hybridization, the biotinylated RNA capture probes hybridized to the target cDNA molecules can be captured and separated using streptavidin or antibodies. The target cDNA molecules can then be amplified and sequenced.

[0073] Any known sequencing technology can be used to analyze the cDNA reverse transcribed by the reverse transcriptase and method of the present invention. An example of a sequencing technology that can be used is Illumina sequencing. Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchor primers. Genomic DNA is fragmented and attached to the surface of a flow cell channel. Four reversibly terminated nucleotides labeled with fluorophores are used to perform sequential sequencing. After nucleotide incorporation, a laser is used to excite the fluorophores, an image is captured, and the intensity of the first base is recorded. Sequencing according to this technique is described in U.S. Patent Application Publication No. 2011 / 0009278, U.S. Patent Application Publication No. 2007 / 0114362, U.S. Patent Application Publication No. 2006 / 0024681, U.S. Patent Application Publication No. 2006 / 0292611, U.S. Patent No. 7,960,120, U.S. Patent No. 7,835,871, U.S. Patent No. 7,232,656, U.S. Patent No. 7,598,035, U.S. Patent No. 6,306,597, U.S. Patent No. 6,210,891, U.S. Patent No. 6,828,100, U.S. Patent No. 6,833,246, and U.S. Patent No. 6,911,345, each of which is incorporated by reference. For example, an Illumina Mi-Seq sequencer can be used to generate multiple sequence reads, which can be uploaded to a web portal for analysis, for example, by the Genome Analysis Toolkit (GATK), and / or analyzed using the methods set forth in U.S. Pat. No. 8,209,130 ​​(incorporated by reference).

[0074] Analyzing the sequence reads can be performed according to a multi-step procedure known in the art using known software. For example, first, the quality of each sequence read (i.e., FASTQ sequence) can be evaluated using software FASTQC. Then, the reads can be trimmed, for example, by Trimmomatic software. The trimmed sequence reads can then be mapped to the human genome using HISAT2 software. HISAT2 outputs a file in SAM (sequence alignment / map) format, which can be compressed into a binary sequence alignment / map file using SAMtools version before sequence read quantification. The mapped reads can then be counted using feature Counts software.

[0075] (Reverse Transcriptase) As discussed above, reverse transcriptase is an enzyme used to generate cDNA from an RNA template (a process called reverse transcription). It is primarily associated with retroviruses; however, some non-retroviruses also use reverse transcriptase. Retroviral reverse transcription has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H, and DNA-dependent DNA polymerase activity. These activities are used by retroviruses to convert single-stranded genomic RNA into double-stranded cDNA. The same sequence of reactions is used in the laboratory to convert RNA into cDNA for use in molecular cloning, RNA sequencing, polymerase chain reaction, or genomic analysis.

[0076] Reverse transcriptase is a member of a specific enzyme family within the DNA polymerase superfamily. Enzymes of this superfamily share a similar three-dimensional structure, despite relatively low amino acid sequence homology and distinct origins, in which DNA polymerases consist of three distinct domains (palm, thumb, and fingers). Furthermore, reverse transcriptases with ribonuclease (RNase) H activity display a unique RNase H domain with its active site, and a connection domain.

[0077] Moloney murine leukemia virus (MMLV) is a retrovirus known to cause cancer in mice, and its associated reverse transcriptase can be used in the laboratory to convert RNA into cDNA. The MMLV wild-type reverse transcriptase is a 75 kDa monomer with optimal activity at 37°C (approximately the median body temperature of mice or humans). The MMLV reverse transcriptase contains domains called the fingers, palm, thumb, connection, and RNase H domains. The active site for the DNA polymerase reaction is in the fingers / palm / thumb domains, while the active site for the ribonuclease (RNase) H reaction is in the RNase H domain.

[0078] The fingers (41-124, 160-192 amino acids), palm (1-40, 125-159, 193-275 amino acids), and thumb (276- 361) domains constitute the N-terminus of MMLV reverse transcriptase (RT), while the connection domain (362-496 amino acids) and RNase H (497-671 amino acids) are located in the C-terminal part. The fingers domain is proposed to provide an intermediate binding site for the template-primer during the phosphonucleotidyl transfer reaction. The thumb domain plays an important role in substrate binding and processivity. In the palm domain, the regions I125-F155, L220-E233, and K257-E275 are located on the surface and interact with the template. The connection domain includes P360-K373, Y394-A436, and S453-A462 (which interact with the template) on its surface, and five consecutive hydrophobic residues L432-V433-I434-L435-A436. The RNase H domain can change its conformation, and is thought to be involved in processive DNA synthesis.

[0079] Superscript II reverse transcriptase (ThermoFisher) is a mutant MMLV reverse transcriptase that has reduced ribonuclease (RNase) H activity and increased thermostability compared to the wild-type MMLV reverse transcriptase. The Superscript II reverse transcriptase is the commonly used commercial form of the enzyme and is the primary standard for comparison of the reverse transcriptases of the present invention below. The sequence of the Superscript II reverse transcriptase is shown in SEQ ID NO:2.

[0080] The reverse transcriptase of the present invention has at least one amino acid substitution compared to the reverse transcriptase of SEQ ID NO:2.

[0081] The modified reverse transcriptases of the invention have increased thermostability compared to both the wild-type and Superscript II versions of the enzyme. Increased thermostability of one reverse transcriptase compared to another means that the reverse transcriptase with increased thermostability is less prone to lose activity at high temperatures (e.g., greater than 37° C.). Increased stability of an enzyme can also include avoidance of denaturation mechanisms to realize the full potential of the enzyme as a catalyst.

[0082] In addition, a higher reaction temperature is desirable for cDNA synthesis because it reduces RNA secondary structures and non-specific primer binding. Stability between two enzymes can be determined by comparing the remaining activity of both enzymes after exposure to a particular condition (e.g., high temperature, in the presence of an inhibitor of the enzyme, or in a fixed sample). Alternatively, the stability can be determined by comparing the remaining or residual acidity of the enzyme after incubation at a given condition (e.g., time or temperature) to the initial activity before incubation.

[0083] Increased efficiency of one reverse transcriptase compared to another means that under a given set of conditions, the more efficient enzyme produces fewer base-calling errors over time.

[0084] Increased activity of one enzyme compared to another means that the enzyme with increased activity converts its substrate at a higher rate (for reverse transcription, this means the rate at which RNA (the substrate) is used to synthesize cDNA). The SI unit for enzyme activity is the katal (1 katal = 1 mol s -1 ). A more practical and commonly used value is enzyme unit (U) = 1 μmol min -1One U corresponds to 16.67 nanokatals and is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute. The specific activity of an enzyme is the activity of the enzyme per milligram of total protein (μmol min -1 mg -1 (expressed as:

[0085] Reverse transcriptase activity can be determined in an assay that measures the formation of cDNA over time. For example, reverse transcriptase can be incubated with an RNA template, a primer, and an appropriate dNTP mixture, and the production of cDNA or consumption of dNTPs can be monitored.

[0086] Disclosed herein are reverse transcriptases comprising an amino acid substitution at least one amino acid substitution at an amino acid position selected from positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 444, 615, 642, 524, 583, 562, 204, and 306 relative to SEQ ID NO:2. For example, the amino acid substitution is S at position 56 substituted with G, A, V, L, or I; G at position 138 substituted with S, C, T, or M; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 615 substituted with E, N, or Q; D at position 524 substituted with HG, AV, L, or I at position 642 substituted with D, E, N, or Q; D at position 583 substituted with E, N, or Q; E at position 562 substituted with D, N, or Q; H at position 204 substituted with K or R; and T at position 306 substituted with H, K, or R It could be.

[0087] It is understood that the reverse transcriptases of the present invention can contain additional mutations that do not alter the function (ie, increased thermostability) of the reverse transcriptase.

[0088] For example, the reverse transcriptase may contain additional conservative mutations. A conservative amino acid substitution is the replacement of one amino acid with another amino acid that has similar properties, and thus typically involves replacing an amino acid in a polypeptide with an amino acid that is within the same or a similar defined class of amino acids. By way of example, and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is substituted with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); a hydrophobic amino acid is substituted with another hydrophobic amino acid, or a hydrophilic amino acid is substituted with another hydrophilic amino acid, respectively.

[0089] In embodiments of the invention, the reverse transcriptase does not include one or more of the following mutations: E5K; M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; L671P, A32V, E286R, E302K, W388R, and L435R. For example, the reverse transcriptase comprises wild-type amino acids at those specified positions or comprises conservative substitutions at the wild-type amino acid positions.

[0090] A reverse transcriptase with at least X% sequence identity means that the enzyme has an amino acid sequence, characterized in that in a stretch of 100 amino acids, at least X amino acid residues are identical to the sequence of the corresponding sequence. Sequence identity can be determined by sequence alignment in the form of sequence comparison. Methods of sequence alignment are well known in the art, and include a variety of programs and alignment algorithms. Basic Local Alignment Search Tool (BLAST) can also be used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

[0091] For example, since wild-type MMLV is 671 amino acids long, the enzyme can tolerate many substitutions (e.g., conservative substitutions) without losing the reverse transcription properties of the enzyme. However, it is understood that mutations at certain positions may reduce the effectiveness of the wild-type enzyme compared to the wild-type, while other, less common substitutions may increase the effectiveness of the wild-type enzyme. Thus, the present invention has discovered that amino acid substitutions at amino acid positions 56, 72, 138, 163, 234, 249, 290, 344, 420, 424, 444, 615, 444, 615, 642, 524, 583, 562, 204, and 306 may enhance the properties of the wild-type enzyme. It is understood that the enzyme containing substitutions at each of the 20 positions identified by the present invention has 97.32% sequence identity with the wild-type enzyme. The enzymes of the invention may contain additional substitutions that do not alter the properties of the invention, such as an additional 15 amino acid substitutions (e.g., conservative substitutions). Thus, the enzymes of the invention may have 95% sequence identity with the wild-type MMLV enzyme. The same principle applies to the Superscript II version.

[0092] An embodiment of the invention provides a modified reverse transcriptase comprising at least one mutation in at least one domain of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase of SEQ ID NO: 2. Importantly, the modified reverse transcriptase of the invention exhibits increased thermostability compared to the wild-type reverse transcriptase such that at temperatures of about 50° C. or greater, its activity is at least about 90% of the activity of the wild-type reverse transcriptase at 42° C. Preferred amino acid substitutions are at positions 27, 138, 200, 204, 212, 218, 231, 232, 234, 237, 242, 249, 264, 265, 267, and / or 272 in the palm domain of MMLV reverse transcriptase, at positions 43, 46, 48, 54, 56, 72, 163, and / or 177 in the finger domain of MMLV reverse transcriptase, at positions 280, 281, 290, 306, 335, 338, 339, 344, 345, 346, and / or 349 in the thumb domain of MMLV reverse transcriptase, at positions 376, 413, 420, 424, 443, and 444 in the connection domain of MMLV reverse transcriptase, and at positions 413, 420, 424, 443, and 444 in the RNAse domain of MMLV reverse transcriptase. Occurs at positions 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642, and / or 658 in the H domain.

[0093] Preferred substitutions are S at position 56 substituted with G, A, V, L, or I; L at position 72 substituted with Q or R; G at position 138 substituted with S, C, T, or M; H at position 204 substituted with K or R; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; T at position 306 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 524 substituted with G, AV, L, or I; E at position 562 substituted with D, N, or Q; D at position 583 substituted with E, N, or Q; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q is selected from.

[0094] Additionally, the modified reverse transcriptase can have substitutions at at least one to at least 14 amino acid positions selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642.

[0095] In some embodiments, the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615E. The modified enzyme may include additional substitutions selected from G524D, N583D, and Q562E. In other embodiments, the amino acid substitutions are selected from L72Q, T163K, N249E, and H642D. In yet other embodiments, the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D. This embodiment may also include at least one additional amino acid substitution selected from D524G, D583N, E562Q, H204R, and T306K.

[0096] In specific embodiments, the amino acid substitutions are S27T, V43K, A46P, L48I, A54P, S56A, L72Q, G138S, T163K, M177R, D200H, I212T, I218T, T231E, S232T, L234E, Q237E, A242D, N249E, Q265E, K264Q, K267T, L272I T281S, G290K, N335Q P338E, D339E, Y344F, Q345D may be selected from E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444I, D518E, G524D, L528F, A554P, E562Q, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D, and K658E.

[0097] The modified reverse transcriptases of the invention generally have an activity at temperatures of 65° C. or higher that is at least about 50% of the reverse transcriptase activity of the reverse transcriptase of SEQ ID NO:2 at about 42° C. Preferably, the activity of the reverse transcriptase is improved compared to the reverse transcriptase of SEQ ID NO:2 in the presence of a reverse transcription inhibitor. Additionally, modified reverse transcriptases of the invention exhibit increased thermostability relative to, for example, a reverse transcriptase of SEQ ID NO:2, E5K; M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q ; Y344H; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

[0098] (Samples and sample preparation) Any sample suspected to contain one or more nucleic acids to be detected or measured and quantified can be analyzed using the reverse transcriptase of the present invention.For example, the sample can be a biopsy, a culture, or an environmental sample (e.g., water or soil).The sample can be derived from a subject (e.g., an animal or human), and the sample can be a fluid, solid, suspension, or tissue.

[0099] Examples of samples include cell or tissue culture, blood, serum, plasma, fine needle aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excreta, tears, saliva, sweat, biopsy, peritoneal fluid, cerebrospinal fluid, pleural fluid, amniotic fluid, peritoneal fluid, interstitial fluid, sputum, milk, lymph, bronchial lavage sample and other washing sample, or tissue extract sample. The source of the sample can be fresh, frozen, and / or preserved, organ sample or tissue sample or solid tissue such as from biopsy or aspirate; or cells from any time point in the subject's pregnancy or development. In a preferred embodiment of the method, the sample is selected from the group consisting of body fluid, blood, plasma, serum, urine, bile, cerebrospinal fluid, swab, clinical specimen, organ sample and tissue sample (particularly human, animal or plant, especially human).

[0100] The sample may contain compounds with which it is naturally mixed in nature, as well as compounds that are not naturally mixed with the sample in nature (e.g., preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.).

[0101] Notably, many samples contain both natural and non-natural inhibitors of reverse transcriptase. For example, humic / fulvic acids, polysaccharides, phenols, and flavonoids in plant samples inhibit reverse transcriptase activity. In addition, proteins and fats from tissue samples, bile salts from stool samples, collagen from connective tissue samples, melanin and eumelanin in hair and skin samples, myoglobin from muscle tissue samples, complex polysaccharides in feces samples, proteinases and calcium ions in milk and bone samples, and urea in urine samples all inhibit transcriptase activity. In addition, heme, hemoglobin, lactoferrin, and antibodies in blood samples all inhibit transcriptase activity.

[0102] Sample preparation steps also frequently introduce products that inhibit reverse transcriptase, for example, EDTA, detergents / DDT, alcohol, salts, heparin, hematin, phenol:chloroform, proteases, nucleases, agarose, metal ions, and aldehydes all inhibit reverse transcriptase activity. In addition, some artificial sample preparation products are introduced into the sample for the very purpose of removing unwanted natural products in the sample. For example, ethanol, proteinase, and phenol:chloroform are typically used to wash away excess proteins, salts, and other natural products from the sample, and are nevertheless themselves transcriptase inhibitors. Other sample preparation products are necessary for the analysis of the sample type. For example, detergents are frequently introduced into samples containing cells to cause cell lysis and release the nucleic acids to be analyzed. Detergents are also frequently used in single-cell sequencing methods (e.g., droplet sequencing) to separate and compartmentalize individual cells for sequencing.

[0103] As a result, samples are typically washed multiple times during sample preparation to remove unwanted products or reagents that are no longer needed, but each wash results in the loss of at least a portion of the original sample and risks the introduction of contaminants.

[0104] Sample preservation steps can also result in the introduction of reverse transcriptase inhibitors. For example, formalin-fixed paraffin-embedded (FFPE) tissue preservation is a form of preservation and preparation for biopsy specimens. The tissue sample is first preserved by fixing it in formaldehyde to preserve proteins and important structures within the tissue. It is then embedded in a paraffin wax block, which allows for easy cutting of sections of the size required for mounting on a microscope slide for examination. However, FFPE sample preparation also results in the inhibition of reverse transcriptase activity.

[0105] The reverse transcriptase of the present invention exhibits improved reverse transcriptase activity in the presence of a reverse transcriptase inhibitor and a preserved sample (e.g., a FFPE sample) compared to either the wild-type MMLV enzyme or the reverse transcriptase shown in SEQ ID NO: 2. As a result, the reverse transcriptase of the present invention can be added directly to a sample containing an inhibitor without additional washing steps that result in loss of the original sample or introduction of contaminants. Furthermore, the reverse transcriptase of the present invention can be added directly to a preserved sample (e.g., a fixed sample), again without loss of reverse transcriptase activity.

[0106] Specifically, the reverse transcriptase of the present invention exhibits improved activity in the presence of a reverse transcription inhibitor relative to the reverse transcriptase shown in SEQ ID NO: 2, and is selected from the group consisting of M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q ; Y344H; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

[0107] In another embodiment, the activity of the reverse transcriptase is improved relative to the reverse transcriptase of SEQ ID NO:2 in formalin fixed samples, and is selected from the group consisting of M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L33 3Q; Y344H; A502V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

[0108] In yet other embodiments, the reverse transcriptase of the present invention exhibits increased thermostability compared to the reverse transcriptase of SEQ ID NO:2, and the reverse transcriptase of the present invention may contain at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

[0109] The activity of the reverse transcriptase of the present invention is also improved in the presence of a reverse transcriptase inhibitor compared to the reverse transcriptase of SEQ ID NO: 2, and the activity of the reverse transcriptase of the present invention may include at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R. Similarly, the activity of the reverse transcriptase of the present invention is also improved for formalin-fixed samples compared to the reverse transcriptase of SEQ ID NO: 2, and the activity of the reverse transcriptase of the present invention may include at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

[0110] A preferred reverse transcriptase of the present invention contains at least about 95% sequence identity to the reverse transcriptase of SEQ ID NO:2.

[0111] An embodiment of the present invention includes a method of reverse transcribing cDNA from RNA. A preferred method includes the steps of providing a sample containing RNA with a modified reverse transcriptase of the present invention that includes at least one mutation in at least one domain of the reverse transcriptase of SEQ ID NO:2, where the modified reverse transcriptase exhibits increased thermostability compared to the reverse transcriptase of SEQ ID NO:2, such that the activity of the reverse transcriptase at a temperature of about 50°C or higher is at least about 90% of the activity of the reverse transcriptase of SEQ ID NO:2 at 37°C; providing a reagent for reverse transcription to the sample, and performing a reverse transcription reaction between the reverse transcriptase and the RNA in the sample. The reverse transcription reaction is carried out at a temperature of 50°C or higher to form cDNA.

[0112] In certain embodiments, the mutations are at positions 27, 138, 200, 204, 212, 218, 231, 232, 234, 237, 242, 249, 264, 265, 267, and 272 in the palm domain of reverse transcriptase, positions 43, 46, 48, 54, 56, 72, 163, and 177 in the finger domain of reverse transcriptase, positions 280, 281, 290, 306, 335, 338, 339, 344, 345, 346, and 349 in the thumb domain of reverse transcriptase, positions 376, 413, 420, 424, 443, and 444 in the connection domain of reverse transcriptase, and positions 413, 420, 424, 443, and 444 in the RNAse domain of reverse transcriptase. The invention relates to a method for producing a mutated sRNA comprising the steps of: (a) administering a mutated sRNA to a mammalian subject; and (b) administering an mutated sRNA to a mammalian subject. The method relates to administering an mutated sRNA to a mammalian subject.

[0113] For example, amino acid substitutions include S at position 56 substituted with G, A, V, L, or I; L at position 72 substituted with Q or R; G at position 138 substituted with S, C, T, or M; H at position 204 substituted with K or R; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; T at position 306 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 524 substituted with G, AV, L, or I; E at position 562 substituted with D, N, or Q; D at position 583 substituted with E, N, or Q; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q may be selected from:

[0114] The modified reverse transcriptase may comprise substitutions at at least three amino acid positions and up to at least twelve amino acid positions, the amino acid positions being selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642. Specifically, the at least one amino acid substitution may be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615E. In certain embodiments, the modified reverse transcriptase comprises at least one additional amino acid substitution selected from G524D, N583D, and Q562E. The amino acid substitution is selected from L72Q, T163K, N249E, and H642D. Additionally and / or alternatively, the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D. This embodiment may further comprise at least one additional amino acid substitution selected from D524G, D583N, E562Q, H204R, and T306K.

[0115] Generally, the activity of the reverse transcriptase at a temperature of about 65°C or greater is at least about 50% of the reverse transcriptase activity of SEQ ID NO:2 at about 42°C.

[0116] Further, in the methods of the invention, the RNA-containing sample may include one or more reverse transcription inhibitors, and the reverse transcription reaction may be more efficient than a reverse transcription reaction performed in the presence of one or more of the inhibitors using the reverse transcriptase of SEQ ID NO: 2. The RNA-containing sample may be formalin fixed, and the reverse transcription reaction may be more efficient than a reverse transcription reaction performed on a formalin-fixed sample using the reverse transcriptase of SEQ ID NO: 2.

[0117] In a further embodiment, the performing step is carried out at a temperature of about 65°C or greater and the reverse transcription reaction is at least about 50% as active as a reverse transcription reaction carried out at about 42°C using the reverse transcriptase of SEQ ID NO:2.

[0118] The methods of the invention can further include any known reagents and methods for reverse transcription. For example, the providing step can include providing the sample with a capture oligo that captures a target RNA molecule. EXAMPLES

[0119] (Example) 1 shows data characterizing the reverse transcriptase (RT) enzyme of the present disclosure.

[0120] FIG. 1A shows qPCR results indicating that prod-ana reverse transcriptase (RT) has thermo-activity competitive with Maxima in RT-qPCR.

[0121] FIG. 1B shows the efficiency scores for the enzymes.

[0122] First strand synthesis (FSS) was performed at 50° C. and 60° C. using 10 ng, 1 ng, and 0.1 ng of liver total RNA as input with the reverse transcription (RT) enzyme of the present disclosure and Maxima, followed by qPCR. First strand synthesis (FSS) reactions were developed on ice by mixing 10 ng of liver total RNA, 1x reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2), 10 U Murine RNase Inhibitor, 5 μM oligo dT(20), 0.5 mM dNTPs, and 200 U of the specified reverse transcriptase. FSS was performed for 25 min at various temperatures (42°C to 65°C), after which qPCR was performed to assess cDNA yield. qPCR reactions were developed by mixing 1x SsoAdvanced Universal SYBR Green Supermix, 10% of the FSS reaction, and 0.6 μM of a primer that arylates to the 5' end of the β-actin gene.

[0123] The resulting cDNA values ​​were assessed by the Ct values ​​shown in Figure 1. The cDNA yield from prod-blh FSS was 100-fold less when FSS was incubated at 50°C compared to 60°C, while prod-ana and Maxima were able to convert similar amounts of RNA to cDNA at 50°C and 60°C. prod-ana had competitive or better yield and efficiency than Maxima RNaseH minus reverse transcriptase at 50°C and 60°C.

[0124] FIG. 1C shows RT-qPCR yields at high temperature using the reverse transcription (RT) enzyme of the present disclosure. First strand synthesis (FSS) reactions were developed on ice by mixing 10 ng of liver total RNA, 1× reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2), 10 U Murine RNase Inhibitor, 5 μM oligo dT(20), 0.5 mM dNTPs, and 200 U of the specified reverse transcriptase. FSS was performed at various temperatures (42° C. to 65° C.) for 25 minutes, after which qPCR was performed to evaluate cDNA yields. qPCR reactions were developed by mixing 1× SsoAdvanced Universal SYBR Green Supermix, 10% of the FSS reaction, and 0.6 μM of a primer that allelizes to the 5′ end of the β-actin gene. The resulting cDNA values ​​were evaluated by Ct values. The resulting cDNA values ​​are shown in FIG. 1C.

[0125] Prod-blh had comparable cDNA yields at 42° C. and 50° C. Prod-ana was the most thermostable variant, with comparable yields at all temperatures tested.

[0126] Figure 2A shows that RT prod-ana has enhanced thermo-activity. In the RNA ladder assay, 10 polyA RNA transcripts of 0.5 kb to 9 kb are used as RNA inputs to first strand synthesis (FSS). In detail, FSS reactions were generated on ice by mixing 500 ng of Millennium RNA ladder (Millennium™ RNA Markers), 1× reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2), 10 U Murine RNase Inhibitor, 5 μM oligo dT(20), 0.5 mM dNTPs, and 200 U of a specific reverse transcriptase. FSS was performed at various temperatures (42°C-65°C) and the thermostability and processivity of the reverse transcriptase (RT) variants were analyzed with electrophoretic tools (e.g., gDNA TAPESTATION instrument, Agilent). For example, prod-blh, prod-ana, Thermo SSIII, and Thermo Maxima RNaseH minus were run in the RNA ladder assay, in which FSS was used at 50°C, 55°C, 60°C, or 65°C for 30 min. The processivity of the reverse transcriptase was quantitatively assessed by the conversion or lack thereof of higher molecular weight (MW) RNA transcripts to cDNA.

[0127] The gDNA tapestation and resulting cDNA products from the RNA ladder assay are shown in FIG. 2A. The length of the transcripts at various temperatures is consistent with the thermostability of the enzyme. prod-blh converts the entire RNA ladder to cDNA when performed at FSS temperatures of 42° C. or 50° C. The entire RNA ladder is not converted to cDNA when the FSS temperature is increased to 60° C. or 65° C. prod-blh has highly competitive cDNA conversion with Thermo SSIII reverse transcriptase across all temperatures. prod-ana converts the entire RNA ladder to cDNA when performed at FSS temperatures of 42° C., 50° C., or 60° C. The higher molecular weight (MW) RNA transcripts are not converted to cDNA when the FSS temperature is increased to 65° C. prod-ana has highly competitive cDNA conversion with Thermo Maxima RNaseH minus reverse transcriptase across all temperatures. RT prod-ana has enhanced thermostability and processivity compared to SSIII and competitive thermostability and processivity compared to Maxima RNaseH minus. Reverse transcriptase (RT) variants of the present disclosure were run in triplicate in thermal shift assays to assess enzyme thermostability.

[0128] FIG. 2B shows the processivity of the reverse transcription (RT) enzyme of an embodiment of the present disclosure at high temperature.

[0129] FSS reactions were generated on ice by mixing 500 ng Millennium RNA ladder (Millennium™ RNA Markers), 1× reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2), 10 U Murine RNase Inhibitor, 5 μM oligo dT(20), 0.5 mM dNTPs, and 200 U reverse transcriptase. FSS was performed at various temperatures (42°C to 65°C) and the thermostability and processivity of the reverse transcriptase (RT) variants were analyzed by gDNA tapestation. The processivity of the reverse transcriptase was quantitatively assessed by the conversion or lack thereof of higher molecular weight (MW) RNA transcripts to cDNA.

[0130] The gDNA tapestation and resulting cDNA products from the RNA ladder assay are shown in Figure 2B. The length of the transcripts at different temperatures is consistent with the thermostability of the enzyme: 1. In the absence of any reverse transcriptase (RT), there is no cDNA conversion (data not shown). 2. All reverse transcriptases are capable of converting RNA transcripts of all molecular weights (MW) into cDNA at 42°C and 50°C. 3. At 55° C., prod-dnd does not completely convert all transcripts of its RNA input into cDNA. prod-blh, prod-ana, and prod-cnc are able to completely convert all transcripts at 55° C. and 60° C. At 4.65° C., prod-ana converts 9 of its 10 polyA transcripts into cDNA.

[0131] Figure 3A shows the thermal stability of reverse transcriptase (RT) variants. The WMG reverse transcriptase (RT) variants were tested in triplicate in a thermal shift assay to evaluate enzyme thermal stability. The reverse transcriptase (RT) variants of the present disclosure were tested in triplicate in a thermal shift assay (GloMelt; Biotium) to evaluate enzyme thermal stability. A thermal ramp gradient was used according to the Biotium GloMelt manufacturing protocol. When the enzyme unfolds, the fluorescence signal increases, and the inflection point of the curve indicates the melting temperature for that enzyme.

[0132] Figure 3B shows melting curves, which indicate that the tested reverse transcriptase (RT) has various degrees of thermal stability: RTII < prod - alg = RTIII < prod - bmh < prod - ana. Here, prod - ana shows a substantial increase in thermal stability compared to RTII and RTIII. As shown, prod - aic (RTII) shows a melting curve at 63°C, prod - alg shows a melting curve at 67°C, prod - blh (RTIII) shows a melting curve at 67°C, prod - bmh shows a melting curve at 68°C, and prod - ana shows a melting curve at 71°C. The melting curves indicate that the tested reverse transcriptase (RT) has various degrees of thermal stability.

[0133] Figures 3C - 3E show the results of further evaluation of the aggregation temperature and thermal stability of the reverse transcriptase of the present disclosure.

[0134] FIG. 3C shows thermal ramp assay results for an embodiment of the disclosed reverse transcriptase (RT) enzyme. Reverse transcriptase (RT) variants were run in triplicate on an Unchained Labs Uncle instrument in a thermal ramp assay using static light scattering (SLS) to assess the onset of aggregation. A 266 nm laser was used to monitor the amount of scattered light while a thermal ramp from 15° C. to 95° C. was applied to the samples. All reverse transcriptase (RT) variants were diluted to 0.25 mg / mL in the following buffer system: 60 mM Tris-HCl (pH=8.0), 75 mM KCl, 50 mM NaCl, 25% (v / v) glycerol, 10 mM DTT, 3 mM MgCl2. The increase in the amount of scattered light directly measures the level of aggregation onset. Aggregation onset almost always occurs after a melting event occurs within the protein.

[0135] FIG. 3D shows a table of results for the thermal ramp assay of FIG. 3C for the reverse transcriptase (RT) variants tested. Results: 1.prod-dnd exhibits the lowest thermal stability and has the lowest aggregation onset temperature of 60.7°C. 2. Prod-ana exhibits the highest thermal stability and has the highest aggregation onset temperature of 68.2℃. 3. prod-blh exhibits intermediate thermal stability and has an aggregation onset temperature of 63.1° C. which is between the aggregation onset temperatures of prod-dnd and prod-ana.

[0136] FIG. 3E shows an isothermal reaction incubation for an embodiment of the reverse transcriptase (RT) enzyme of the present disclosure. Reverse transcriptase (RT) variants were run in triplicate on an Unchained Labs Uncle instrument in an isothermal assay using static light scattering (SLS) to assess the onset of aggregation. A 266 nm laser was used to monitor the amount of scattered light while the samples were kept at 50° C. for approximately 20 hours. All reverse transcriptase (RT) variants were diluted to 0.25 mg / mL in the following buffer system: 60 mM Tris-HCl (pH=8.0), 75 mM KCl, 50 mM NaCl, 25% (v / v) glycerol, 10 mM DTT, 3 mM MgCl2. The increase in the amount of scattered light directly measures the level of aggregation occurrence. The rate of aggregation can be determined by the increase in SLS counts over time. 1.prod-dnd exhibits the lowest thermal stability and has the steepest aggregation rate. 2. Prod-ana exhibits the highest thermal stability and has the most level aggregation rate. 3. prod-blh exhibits intermediate thermal stability and has an aggregation rate between that of prod-dnd and that of prod-ana.

[0137] (RT-qPCR yields in the presence of various inhibitors) First strand synthesis (FSS) reactions were developed on ice by mixing 10 ng of liver total RNA, 1x reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2), 10 U Murine RNase Inhibitor, 5 μM oligo dT(20), 0.5 mM dNTPs, and 200 U of the identified reverse transcriptase in the presence / absence of various inhibitors (see below). FSS was performed at the optimal temperature for the various reverse transcriptases (42°C for prod-dnd or 50°C for prod-blh) and incubated for 25 min, after which qPCR was performed to assess cDNA yield. qPCR reactions were developed by mixing 1x SsoAdvanced Universal SYBR Green Supermix, 10% of the FSS reaction, and 0.6 μM of a primer arylating to the 5' end of the β-actin gene.

[0138] Figures 4A and 4B show the resistance of the disclosed embodiment of the reverse transcription (RT) enzyme to inhibition by heparin. FSS was carried out as described above in the presence of 0ng / μL, 15ng / μL, 30ng / μL, 60ng / μL, 120ng / μL, 240ng / μL, or 600ng / μL heparin. The resulting cDNA values ​​were evaluated by the Ct values ​​shown in Figure 4A.

[0139] Figure 4B shows the change in Ct value with increasing heparin concentration for the reverse transcriptase (RT) enzyme of the present disclosure. The results are summarized below: No inhibition was observed for all reverse transcriptase (RT) variants at 1.15 ng / μL and 30 ng / μL heparin. 2.prod-dnd had the lowest yield (approximately 10-fold lower) in the presence of ≧60 ng / μL heparin, and no cDNA was produced at heparin concentrations >120 ng / μL. 3. In the presence of 240 ng / μL heparin, prod-blh had a cDNA yield that was approximately one-fourth to one-tenth that of prod-ana, and no cDNA was produced when 600 ng / μL heparin was added. 4. Prod-ana had the highest cDNA yield over a wide range of heparin concentrations, and cDNA was produced at every heparin concentration tested.

[0140] 4C and 4D show the resistance of embodiments of the reverse transcription (RT) enzyme of the present disclosure to inhibition by guanidine isothiocyanate.

[0141] FSS was carried out as above in the presence of 0 mM, 15 mM, 30 mM, 60 mM or 90 mM guanidine isothiocyanate. The resulting cDNA values ​​were assessed by the Ct values ​​shown in Figure 4C.

[0142] Figure 4D shows the change in Ct value with increasing guanidine isothiocyanate concentrations for the reverse transcriptase (RT) variants of the present disclosure. The results are summarized below: No or very minimal inhibition was observed for all reverse transcriptase (RT) variants at 1.30 mM and 60 mM guanidine isothiocyanate. 2. prod-dnd showed the greatest inhibition, being inhibited approximately 4-fold with 60 mM inhibitor and approximately 100-fold with 90 mM inhibitor. 3. prod-blh was inhibited approximately 4-fold by 60 mM inhibitor and less than 10-fold by 90 mM inhibitor. 4. Prod-ana had the highest cDNA yield over a wide range of guanidine isothiocyanate concentrations and was inhibited approximately 3-fold at 90 mM inhibitor.

[0143] FIG. 4E shows the resistance of embodiments of the reverse transcriptase (RT) enzyme of the present disclosure to inhibition by ethanol.

[0144] FSS was performed as above in the presence of 0%, 5%, 10% or 20% ethanol, and the resulting cDNA values ​​were assessed by the Ct values ​​shown in Figure 4E.

[0145] Figure 4F shows the change in Ct value with increasing ethanol concentration for the reverse transcription (RT) enzyme of the present disclosure. The results are summarized below: No or very minimal inhibition was observed for all reverse transcriptase (RT) variants at 1.5% and 10% ethanol. 2. prod-bmh and prod-dnd were inhibited by approximately 100-fold less in 20% ethanol. 3. prod-ana and prod-blh were inhibited by 20% ethanol by approximately 100-fold. Figure 5 shows that prod-bmh reverse transcriptase (RT) has cDNA yields competitive with Maxima from FFPE samples. First strand synthesis (FSS) reactions were developed on ice by mixing 10 ng, 1 ng, or 0.1 ng of FFPE RNA isolated from skin tissue with 1x reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2), 10 U Murine RNase Inhibitor, 5 μM oligo dT(20), 0.5 mM dNTPs, and 200 U of the specified reverse transcriptase. FSS was performed for 25 minutes at 42°C, after which qPCR was performed to assess cDNA yield. qPCR reactions were developed by mixing 1x SsoAdvanced Universal SYBR Green Supermix, 10% of the FSS reaction, and 0.6 μM of a primer that allylates to the 5' end of the β-actin gene.

[0146] Results: The resulting cDNA values ​​were assessed by Ct values ​​and are shown in Figure 5. Note that the asterisk indicates that a non-specific melting curve was generated and no specific product was generated. 1.prod-dnd had the lowest yield (approximately 4-fold lower than prod-alg and prod-bmh) at 10 ng FFPE RNA input and generated only a specific cDNA product. 2. prod-alg had a cDNA yield equivalent to prod-bmh with 10 ng FFPE RNA input, but was unable to generate a specific cDNA product with lower RNA input. 3. prod-bmh generated specific products with 10 ng and 1 ng of FFPE RNA input, and the cDNA yield was competitive with that of Maxima RNaseH minus' reverse transcriptase. 4. WMG prod-ana has yield and efficiency scores that are competitive with Maxima in RT-qPCR at 50°C and 60°C.

[0147] Additional mutations that may be included as needed include S27T, V43K, A46P, L48I, A54P, S56A, S60W, Q68K, L72Q, L72R, E123Q, Y133C, G138S, T163K, M177R, D200H, I212T, I218T, T231E, S232T, L234E, Q237E, A242D, N249E, Q265E, K264Q, K267T, L272I T281S, G290K, N335Q P338E, D339E, Y344F, Q345D E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444I, D518E, G524D, L528F, A554P, Q562E, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D, and K658E.

[0148] (array) The following sequences may be referenced or used in the embodiments of the present disclosure. In wtMMLV-RT-aa (SEQ ID NO: 1), position 1 is homologous to position 34 in each of the following: prod-dnd-aa (SEQ ID NO: 2); prod-ana-aa (SEQ ID NO: 4); prod-amg-aa (SEQ ID NO: 5); prod-asb-aa (SEQ ID NO: 6); prod-bmh-aa (SEQ ID NO: 7); prod-cnc-aa (SEQ ID NO: 8); prod-cmi-aa (SEQ ID NO: 9); prod-bnb-aa (SEQ ID NO: 10); prod-bsc-aa (SEQ ID NO: 11); prod-csd-aa (SEQ ID NO: 12); and prod-dsf-aa (SEQ ID NO: 13). That is, position X in SEQ ID NO: 1 is homologous to position X+33 in any of SEQ ID NOs: 2 and 4 to 14. >wtMMLV-RT-aa (SEQ ID NO: 1) [ka] >prod-dnd-aa (SEQ ID NO:2) [ka] [ka] >prod-dnd-nt (SEQ ID NO:3) [ka] [ka] >prod-ana-aa (SEQ ID NO:4) [ka] [ka] >prod-amg-aa (SEQ ID NO:5) [ka] >prod-asb-aa (SEQ ID NO:6) [ka] [ka] >prod-bmh-aa (SEQ ID NO:7) [ka] >prod-cnc-aa (SEQ ID NO:8) [ka] >prod-cmi-aa (SEQ ID NO:9) [ka] [ka] >prod-bnb-aa (SEQ ID NO:10) [ka] >prod-bsc-aa (SEQ ID NO:11) [ka] [ka] >prod-csd-aa (SEQ ID NO:12) [ka] >prod-dsf-aa (SEQ ID NO:13) [ka] [ka] >prod-blh (SEQ ID NO:14) [ka] >prod-ajw (SEQ ID NO:15) [ka] [ka]

[0149] (Incorporated by reference) References and citations to other documents (e.g., patents, patent applications, patent publications, journals, books, articles, web content) have been made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0150] (Equivalent) Various modifications of the present invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this specification, including the references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. A modified reverse transcriptase comprising at least one modification in the reverse transcriptase sequence shown in SEQ ID NO:2, wherein said modified reverse transcriptase exhibits increased thermostability compared to the reverse transcriptase shown in SEQ ID NO:2, such that the activity of said reverse transcriptase at a temperature of about 50°C or higher is at least about 90% of the reverse transcriptase activity of the reverse transcriptase shown in SEQ ID NO:2 at 42°C.

2. 2. The modified reverse transcriptase of claim 1, wherein the modification comprises an amino acid substitution at an amino acid position selected from positions 27, 138, 200, 204, 212, 218, 231, 232, 234, 237, 242, 249, 264, 265, 267, and 272 43, 46, 48, 54, 56, 72, 163, 177, 280, 281, 290, 306, 335, 338, 339, 344, 345, 346, 349, 376, 413, 420, 424, 443, 444, 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642, and 658 of SEQ ID NO:

2.

3. The amino acid substitution is S at position 56 substituted with G, A, V, L, or I; L at position 72 substituted with Q or R; G at position 138 substituted with S, C, T, or M; H at position 204 substituted with K or R; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; T at position 306 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 524 substituted with G, AV, L, or I; E at position 562 substituted with D, N, or Q; D at position 583 substituted with E, N, or Q; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q The modified reverse transcriptase of claim 2, selected from the group consisting of

4. 2. The modified reverse transcriptase of claim 1, comprising substitutions at at least 3 amino acid positions to at least 14 amino acid positions selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642.

5. 3. The modified reverse transcriptase of claim 2, wherein the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615E.

6. 3. The modified reverse transcriptase of claim 2, comprising at least one additional amino acid substitution selected from G524D, N583D, and Q562E.

7. 3. The modified reverse transcriptase of claim 2, wherein the amino acid substitutions are selected from L72Q, T163K, N249E, and H642D.

8. 3. The modified reverse transcriptase of claim 2, wherein the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D.

9. 9. The modified reverse transcriptase of claim 8, further comprising at least one additional amino acid substitution selected from G524D, N583D, Q562E, R204H, and K306T.

10. The amino acid substitutions are S27T, V43K, A46P, L48I, A54P, S56A, L72Q, G138S, T163K, M177R, D200H, I212T, I218T, T231E, S232T, L234E, Q237E, A242D, N249E, Q265E, K264Q, K267T, L272I T281S, G290K, N335Q P338E, D339E, Y344F, Q345D 3. The modified reverse transcriptase of claim 2, selected from E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444I, D518E, G524D, L528F, A554P, E562Q, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D, and K658E.

11. 2. The modified reverse transcriptase of claim 1, wherein the activity of the reverse transcriptase at a temperature of 65°C or higher is at least 50% of the reverse transcriptase activity of the reverse transcriptase set forth in SEQ ID NO:2 at 42°C.

12. 2. The modified reverse transcriptase of claim 1, wherein the activity of the reverse transcriptase is improved in the presence of a reverse transcription inhibitor, as compared to the reverse transcriptase set forth in SEQ ID NO:

2.

13. The reverse transcriptase exhibits increased thermostability relative to the reverse transcriptase of SEQ ID NO:2, and is selected from the group consisting of E5K; M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502V; D524A; L 2. The modified reverse transcriptase of claim 1, comprising at least one amino acid substitution selected from the group consisting of: 528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

14. The reverse transcriptase has improved reverse transcription activity in the presence of a reverse transcription inhibitor relative to the reverse transcriptase of SEQ ID NO: 2, and is selected from the group consisting of M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502V; 2. The modified reverse transcriptase of claim 1, comprising at least one amino acid substitution selected from the group consisting of D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

15. The reverse transcriptase has improved reverse transcription activity in formalin-fixed samples relative to the reverse transcriptase of SEQ ID NO: 2, and is selected from the group consisting of M39V or M39L; I49V or I49T; M66L; Q91R or Q91L; P130S; L139P; I179T or I179V; D200N, D200A or D200G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502 2. The modified reverse transcriptase of claim 1, comprising at least one amino acid substitution selected from the group consisting of: V; D524A; L528I; H594R, H594K or H594Q; L603W or L603M; E607K, E607G or E607A; H634Y; A644V or A644T; N649S; D653G, D653A, D653H or D653V; K658R or K658Q; and L671P.

16. 2. The modified reverse transcriptase of claim 1, wherein the reverse transcriptase exhibits increased thermostability relative to the reverse transcriptase of SEQ ID NO:2 and comprises at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

17. 2. The modified reverse transcriptase of claim 1, wherein the activity of the reverse transcriptase is improved relative to the reverse transcriptase of SEQ ID NO: 2 when compared in the presence of a reverse transcription inhibitor, and the modified reverse transcriptase comprises at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

18. The modified reverse transcriptase of claim 1, wherein the reverse transcriptase has improved reverse transcription activity relative to the reverse transcriptase of SEQ ID NO: 2 for formalin-fixed samples and comprises at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R, and L435R.

19. 2. The modified reverse transcriptase of claim 1, wherein the modified reverse transcriptase comprises a sequence of amino acids having at least 95% sequence identity with the wild-type MMLV reverse transcriptase of SEQ ID NO:

1.

20. 1. A method for reverse transcribing cDNA from RNA, comprising: providing to a sample comprising RNA a modified reverse transcriptase comprising at least one modification in at least one domain of the reverse transcriptase of SEQ ID NO:2 and reagents for reverse transcription, said modified reverse transcriptase exhibiting increased thermostability compared to the reverse transcriptase of SEQ ID NO:2 such that the activity of said reverse transcriptase at a temperature of 50° C. or greater is at least about 90% of the reverse transcriptase activity of the reverse transcriptase of SEQ ID NO:2 at 42° C.; and performing a reverse transcription reaction between the reverse transcriptase and the RNA in the sample, the reverse transcription reaction being carried out at a temperature of 50° C. or higher to form cDNA; A method comprising:

21. 21. The method of claim 20, wherein the modification comprises an amino acid substitution at an amino acid position selected from positions 27, 138, 200, 204, 212, 218, 231, 232, 234, 237, 242, 249, 264, 265, 267, 272, 43, 46, 48, 54, 56, 72, 163, 177, 280, 281, 290, 306, 335, 338, 339, 344, 345, 346, 349, 376, 413, 420, 424, 443, 444, 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642, and 658.

22. The amino acid substitution is S at position 56 substituted with G, A, V, L, or I; L at position 72 substituted with Q or R; G at position 138 substituted with S, C, T, or M; H at position 204 substituted with K or R; S at position 232 substituted with C, T, or M; L at position 234 substituted with D, E, N, Q; G at position 290 substituted with H, K, or R; T at position 306 substituted with H, K, or R; Y at position 344 substituted with F or M; T at position 420 substituted with G, A, V, L, or I; G at position 424 substituted with D, E, N, or Q; V at position 444 substituted with G, A, L, or I; D at position 524 substituted with G, A V, L, or I; E at position 562 substituted with D, N, or Q; D at position 583 substituted with E, N, or Q; D at position 615 substituted with E, N, or Q; and H at position 642 substituted with D, E, N, or Q 22. The method of claim 21 , wherein the

23. 22. The method of claim 21, wherein the modified reverse transcriptase comprises substitutions at at least 3 amino acid positions to at least 12 amino acid positions, the amino acid positions being selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642.

24. 22. The method of claim 21, wherein the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615E.

25. 22. The method of claim 21, wherein the modified reverse transcriptase comprises at least one additional amino acid substitution selected from G524D, N583D, and Q562E.

26. 22. The method of claim 21 , wherein the amino acid substitutions are at positions 72, 163, 249, and 642.

27. 27. The method of claim 26, wherein the amino acid substitution is selected from L72Q, T163K, N249E, and H642D.

28. 22. The method of claim 21, wherein the amino acid substitutions are selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E, and H642D.

29. 29. The method of claim 28, wherein the modified reverse transcriptase further comprises at least one additional amino acid substitution selected from G524D, N583D, and R204H Q562E.

30. 21. The method of claim 20, wherein the activity of the reverse transcriptase at a temperature of 65°C or higher is at least 50% of the reverse transcriptase activity of SEQ ID NO:3 at 42°C.

31. 21. The method of claim 20, wherein the sample contains one or more reverse transcription inhibitors, and the reverse transcription reaction is more efficient than a reverse transcription reaction performed with the reverse transcriptase of SEQ ID NO:2 in the presence of the one or more inhibitors.

32. 21. The method of claim 20, wherein the sample containing RNA is formalin fixed and the reverse transcription reaction is more efficient than a reverse transcription reaction carried out with the reverse transcriptase of SEQ ID NO: 2 on a formalin fixed sample.

33. 21. The method of claim 20, wherein the performing step is carried out at a temperature of 65° C. or greater, and the reverse transcription reaction is at least 50% as active as a reverse transcription reaction carried out at 42° C. using the reverse transcriptase of SEQ ID NO:2.

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