Reverse transcriptase variants

Abstract

The present invention provides an MMLV reverse transcriptase having improved thermal stability as compared to wild-type MMLV and AMV reverse transcriptase. The increased thermal stability results in the reverse transcription of RNA to cDNA at temperatures above 37 DEG C, thereby reducing the error rate introduced during cDNA synthesis. Therefore, the reverse transcriptase according to the present invention improves the accuracy of measuring the transcriptome in a living organism.

Description

[0001] Sequence Listing

[0002] A “Sequence Listing XML” is submitted herewith, which is in XML file format and (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. Field of the Invention

[0003] The present invention relates to reverse transcriptase. Background Art

[0004] The transcriptome refers to the RNA transcripts of a cell or organism at a given time. Knowledge of the transcriptome reveals active cellular processes and can provide information about cellular regulation, growth, and dysfunction.

[0005] Transcriptome analysis typically involves microarray technology, and more generally, next generation sequencing technology (e.g., RNA-Seq). The general goal of RNA-Seq is to detect all the various transcripts present, including mRNA and non-coding RNA, as well as to detect splice variants, mutations, mobile genetic elements, and expression levels during various developmental stages or under various conditions. Transcriptomics and RNA-Seq have been applied to diagnostics, disease analysis, pathogen detection, evolutionary biology, and other research areas. For example, RNA-Seq can potentially identify genes involved in resistance to environmental stresses (such as drought resistance in crops). In another example, transcriptomic analysis can provide information about drug resistance mechanisms, potentially revealing strategies to combat hospital-acquired antibiotic-resistant infections.

[0006] 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 a typical enzyme used to synthesize cDNA from RNA. Unlike DNA polymerase, reverse transcriptase does not have the ability to correct, so reverse transcription reactions usually require higher reaction temperatures. This is because higher temperatures reduce off-target primer binding. In addition, higher temperatures reduce secondary structures in RNA, which may cause steric hindrance to RT, thereby truncating transcripts. However, common commercially available reverse transcriptases, such as Moloneymurine leukemia virus (MMLV), Superscript II, and avian myeloblastosis virus (AMV), begin to lose effectiveness above 37°C. Therefore, the high error rate causes mutations to accumulate at an accelerated rate compared to the synthesis of the corrected form. For example, AMV reverse transcriptase typically has an error rate of 1 / 17,000 bases, and MMLV typically has an error rate of 1 / 30,000 bases. Therefore, commercially available reverse transcriptases hinder the accurate determination of the transcriptome. Summary of the invention

[0007] The present invention provides a modified reverse transcriptase having improved thermal stability, improved solubility, and reduced qPCR inhibition compared to conventional commercially available reverse transcriptases (e.g., Superscript II reverse transcriptase (shown herein as prod-dnd, SEQ ID NO: 2)). The improved thermal stability enables reverse transcription of RNA into cDNA at temperatures above 37°C, thereby reducing the error rate introduced during cDNA synthesis. Therefore, the reverse transcriptase of the present invention can achieve improved accuracy in transcriptomics.

[0008] The reverse transcriptase of the present invention may comprise a polypeptide comprising an amino acid sequence 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.

[0009] At least one amino acid substitution may be selected from:

[0010] S at position 56 is substituted with G, A, V, L or I;

[0011] G at position 138 substituted with S, C, T or M;

[0012] S at position 232 is substituted with C, T or M;

[0013] L at position 234 is substituted with D, E, N, Q;

[0014] G at position 290 is substituted with H, K or R;

[0015] Y at position 344 substituted with F or M;

[0016] T at position 420 is substituted with G, A, V, L or I;

[0017] G at position 424 substituted with D, E, N, Q;

[0018] V at position 444 is substituted with G, A, L or I; and

[0019] At position 615, D is substituted with E, N or Q.

[0020] For example, at least one amino acid substitution may be S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I and D615E. In addition, any of the following is an example of a mutation according to 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, V43 3T, V444I, D518E, G524D, L528F, A554P, Q562E, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D and K658E.

[0021] The reverse transcriptase of the present 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:

[0022] D at position 524 substituted with G, A, V, L or I;

[0023] D at position 583 is substituted with E, N or Q; and

[0024] At position 562, E is substituted with D, N or Q.

[0025] For example, the at least one additional amino acid substitution may be selected from D524G, D583N, and E562Q.

[0026] In some embodiments, the reverse transcriptase comprises any number of substitutions selected 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 3 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 5 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.

[0027] In various aspects of the invention, the reverse transcriptase of the invention comprises a polypeptide comprising an amino acid sequence having at least about 95% sequence identity with 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.

[0028] At least one amino acid substitution is selected from:

[0029] S at position 56 is substituted with G, A, V, L or I;

[0030] G at position 138 substituted with S, C, T or M;

[0031] S at position 232 is substituted with C, T or M;

[0032] L at position 234 is substituted with D, E, N or Q;

[0033] G at position 290 is substituted with H, K or R;

[0034] Y at position 344 substituted with F or M;

[0035] T at position 420 is substituted with G, A, V, L or I;

[0036] G at position 424 substituted with D, E, N or Q;

[0037] V at position 444 substituted with G, A, L or I;

[0038] D at position 615 substituted with E, N or Q; and

[0039] At position 642, H is substituted with D, E, N or Q.

[0040] For example, at least one amino acid substitution can be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E and H642D.

[0041] 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.

[0042] At least one additional amino acid substitution may be selected from:

[0043] D at position 524 substituted with G, A, V, L or I;

[0044] D at position 583 is substituted with E, N or Q;

[0045] E at position 562 is substituted with D, N or Q;

[0046] H at position 204 is substituted with K or R; and

[0047] At position 306, T is substituted with H, K or R.

[0048] For example, the additional amino acid substitution may be G524D, N583D, Q562E, R204H or K306T.

[0049] The reverse transcriptase of the present invention exhibits improved thermal stability relative to both wild-type reverse transcriptase and common commercially available reverse transcriptases such as Superscript II (SEQ ID NO: 2). For example, at 50°C, the reverse transcriptase of the present invention has at least about 50% of the reverse transcription activity of the wild-type reverse transcriptase or the reverse transcriptase of SEQ ID NO: 2 at 42°C. However, depending on the number and conditions of mutations, 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 the wild-type reverse transcriptase or the reverse transcriptase of SEQ ID NO: 2 at 42°C. For example, when there are 10 or more mutations, the reverse transcription activity exceeds 80% of the reverse transcription activity of the reverse transcriptase of SEQ ID NO: 2 at 42°C.

[0050] In addition, the activity of the reverse transcriptase of the present invention exceeds the physiological activity of the reverse transcriptase shown in, for example, SEQ ID NO: 2 at even higher temperatures (e.g., 65° C.). For example, the reverse transcriptase of the present invention exhibits at least 50% of the reverse transcription activity of the reverse transcriptase of SEQ ID NO: 2 at 42° C. at a temperature of about 65° C. or higher. In contrast, at a temperature above about 50° C., the reverse transcriptase activity of the wild-type transcriptase or the reverse transcriptase activity of the reverse transcriptase shown in SEQ ID NO: 2 will be significantly inhibited, and is almost inactive at a temperature close to about 65° C. However, similarly, depending on the number and conditions of mutations, at about 65° C. or higher, the reverse transcription activity of the reverse transcriptase of the present invention can even exceed 80% of the reverse transcription activity of the reverse transcriptase shown in SEQ ID NO: 2 or the wild-type reverse transcriptase at 42° C. For example, when there are 10 or more mutations, the reverse transcription activity can exceed 70% of the reverse transcription activity of the wild-type reverse transcriptase or the reverse transcriptase shown in SEQ ID NO: 2 at 42° C.

[0051] In the presence of inhibitors, the reverse transcriptase of the present invention has improved activity compared to conventional commercially available reverse transcriptases (including wild-type reverse transcriptases). Various inhibitors that reduce the efficiency of wild-type and commercially available reverse transcriptases have been found in a variety of samples. For example, plant samples typically contain polysaccharides, phenolic substances, and flavonoids that inhibit reverse transcription. Sample preparation steps also often introduce reverse transcription inhibitors, such as detergents, alcohols, salts, heparin, and hemoglobin.

[0052] The present invention further provides a method for reverse transcribing cDNA from RNA using the reverse transcriptase of the present invention.

[0053] A preferred method of the invention comprises the steps of providing a reverse transcriptase and reagents for reverse transcription to a sample comprising RNA, the reverse transcriptase comprising a polypeptide comprising an amino acid sequence 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 the group consisting of positions 56, 138, 234, 290, 344, 420, 424, 444, and 615. The method further comprises the step of performing a reverse transcription reaction between the reverse transcriptase and the RNA in the sample to form cDNA.

[0054] At least one amino acid substitution may be selected from:

[0055] S at position 56 is substituted with G, A, V, L or I;

[0056] G at position 138 substituted with S, C, T or M;

[0057] S at position 232 is substituted with C, T or M;

[0058] L at position 234 is substituted with D, E, N, Q;

[0059] G at position 290 is substituted with H, K or R;

[0060] Y at position 344 substituted with F or M;

[0061] T at position 420 is substituted with G, A, V, L or I;

[0062] G at position 424 substituted with D, E, N or Q;

[0063] V at position 444 is substituted with G, A, L or I; and

[0064] At position 615, D is substituted with E, N or Q.

[0065] For example, the at least one amino acid substitution can be S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, and D615.

[0066] 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, A, V, L, or I; D at position 583 substituted with E, N, or Q; and E at position 562 substituted with D, N, or Q.

[0067] In one example, the additional amino acid substitution is selected from G524D, N583d, and Q562E.

[0068] The reverse transcriptase may comprise any number of substitutions selected 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 3 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 5 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.

[0069] In various aspects of the invention, the reverse transcriptase of the invention comprises an amino acid sequence having at least about 95% sequence identity with 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.

[0070] At least one amino acid substitution is selected from:

[0071] S at position 56 is substituted with G, A, V, L or I;

[0072] G at position 138 substituted with S, C, T or M;

[0073] S at position 232 is substituted with C, T or M;

[0074] L at position 234 is substituted with D, E, N or Q;

[0075] G at position 290 is substituted with H, K or R;

[0076] Y at position 344 substituted with F or M;

[0077] T at position 420 is substituted with G, A, V, L or I;

[0078] G at position 424 substituted with D, E, N or Q;

[0079] V at position 444 substituted with G, A, L or I;

[0080] D at position 615 substituted with E, N or Q; and

[0081] At position 642, H is substituted with D, E, N or Q.

[0082] For example, the amino acid substitution can be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E and H642D.

[0083] 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.

[0084] Additional amino acid substitutions may also be selected from:

[0085] D at position 524 substituted with G, A, V, L or I;

[0086] D at position 583 is substituted with E, N or Q;

[0087] E at position 562 is substituted with D, N or Q;

[0088] H at position 204 is substituted with K or R; and

[0089] At position 306, T is substituted with H, K or R.

[0090] In one alternative, the additional amino acid substitution is selected from G524D, N583D, Q562E, R204H and K306T.

[0091] The use of the reverse transcriptase of the present invention enables the reaction to occur at 50°C or higher temperatures without losing enzyme activity like conventional reverse transcriptases. Therefore, the operating steps can be carried out at about 65°C or higher temperatures, and the activity is at least about 50% of the activity of the reverse transcriptase shown in SEQ ID NO:2 at 42°C. Due to the improved thermostability of the reverse transcriptase of the present invention, the improved reaction temperature achieves a high degree of reverse transcriptase activity in cDNA synthesis and reduced nucleotide base addition errors. In addition, the reverse transcription reaction can reduce the incubation time, for example, compared with the typical method requiring 50 minutes, the incubation time can be about 5, 10, 15, 20 or 30 minutes.

[0092] Aspects of the invention provide modified reverse transcriptases comprising a mutation in at least one domain of at least one reverse transcriptase of SEQ ID NO: 2. Importantly, the modified reverse transcriptases of the invention exhibit improved thermal stability relative to 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.

[0093] 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. In addition, the present invention contemplates mutations at positions 43, 46, 48, 54, 56, 72, 163 and / or 177 in the finger domain of SEQ ID NO: 2; 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 linker 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.

[0094] The following is a list of preferred substitution mutations for use in the present invention:

[0095] S at position 56 is substituted with G, A, V, L or I;

[0096] L at position 72 is substituted with Q or R;

[0097] G at position 138 substituted with S, C, T or M;

[0098] H at position 204 is substituted with K or R;

[0099] S at position 232 is substituted with C, T or M;

[0100] L at position 234 is substituted with D, E, N, Q;

[0101] G at position 290 is substituted with H, K or R;

[0102] T at position 306 is substituted with H, K or R;

[0103] Y at position 344 substituted with F or M;

[0104] T at position 420 is substituted with G, A, V, L or I;

[0105] G at position 424 substituted with D, E, N or Q;

[0106] V at position 444 substituted with G, A, L or I;

[0107] D at position 524 substituted with G, A, V, L or I;

[0108] E at position 562 is substituted with D, N or Q;

[0109] D at position 583 is substituted with E, N or Q;

[0110] D at position 615 substituted with E, N or Q; and

[0111] At position 642, H is substituted with D, E, N or Q.

[0112] 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.

[0113] In addition, preferably amino acid substitutions include S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I and / or D615E. In addition to the foregoing, additional amino acid substitutions selected from G524d, N583D and Q562E may be added. In other embodiments, amino acid substitutions are selected from L72Q, T163K, N249E and H642D. In other embodiments, 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.

[0114] In a specific embodiment, the amino acid substitution is selected from 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, P3 38E, D339E, Y344F, Q345D, E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444I, D5 18E, G524D, L528F, A554P, E562Q, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D and K658E.

[0115] In a preferred embodiment, the modified reverse transcriptase of the present invention exhibits an enzyme activity at a temperature of about 65°C or higher that is equivalent to at least about 50% of the reverse transcription activity of the reverse transcriptase shown in SEQ ID NO: 2 at about 42°C. The activity of the reverse transcriptase of the present invention is increased in the presence of an inhibitor. In addition, the modified reverse transcriptase of the present invention exhibits increased thermal stability relative to the reverse transcriptase of SEQ ID NO: 2, and may comprise at least one amino acid substitution 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; 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.

[0116] Specifically, in the presence of an inhibitor, the activity of the reverse transcriptase of the present invention is increased relative to the reverse transcriptase shown in SEQ ID NO: 2, and may comprise at least one amino acid substitution 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; T330 P; 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.

[0117] In other embodiments, the reverse transcriptase of the invention exhibits increased activity relative to the reverse transcriptase of SEQ ID NO: 2 in formalin-fixed samples and may comprise at least one amino acid substitution 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; T3 30P; 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.

[0118] In other embodiments, the reverse transcriptase of the invention exhibits improved thermostability relative to the reverse transcriptase of SEQ ID NO: 2, and may comprise at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R and L435R.

[0119] In the presence of an inhibitor, the activity of the reverse transcriptase of the present invention is also improved relative to the reverse transcriptase of SEQ ID NO: 2, and may include at least one amino acid substitution selected from the group consisting of: A32V, E286R, E302K, W388R and L435R. Similarly, in formalin-fixed samples, the reverse transcription activity of the reverse transcriptase of the present invention is improved relative to the reverse transcriptase of SEQ ID NO: 2, and may include at least one amino acid substitution selected from the group consisting of: A32V, E286R, E302K, W388R and L435R.

[0120] In certain embodiments, the modified reverse transcriptase of the invention has at least about 95% sequence identity relative to the reverse transcriptase set forth in SEQ ID NO:2.

[0121] Aspects of the present invention include methods for reverse transcribing cDNA from RNA. The method includes utilizing a modified reverse transcriptase as provided herein. The modified enzyme exhibits improved thermal stability relative to existing reverse transcriptases, such that its activity at about 50°C or higher is at least about 90% of the reverse transcription activity of a conventional reverse transcriptase at 42°C, and a reverse transcription reagent is provided to the sample, and a reverse transcription reaction is performed between the reverse transcriptase and the RNA in the sample. The reverse transcription reaction is performed at 50°C or higher to form cDNA.

[0122] The modified enzyme of the present invention comprises at least one amino acid substitution at a position selected from the group consisting of 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 linker domain of SEQ ID NO: 2, and positions 447, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 501, 502 Positions 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642 and 658 in the RNase H domain of the reverse transcriptase shown in NO:2.

[0123] For example, amino acid substitutions may be selected from:

[0124] S at position 56 is substituted with G, A, V, L or I;

[0125] L at position 72 is substituted with Q or R;

[0126] G at position 138 substituted with S, C, T or M;

[0127] H at position 204 is substituted with K or R;

[0128] S at position 232 is substituted with C, T or M;

[0129] L at position 234 is substituted with D, E, N, Q;

[0130] G at position 290 is substituted with H, K or R;

[0131] T at position 306 is substituted with H, K or R;

[0132] Y at position 344 substituted with F or M;

[0133] T at position 420 is substituted with G, A, V, L or I;

[0134] G at position 424 substituted with D, E, N or Q;

[0135] V at position 444 substituted with G, A, L or I;

[0136] D at position 524 substituted with G, A, V, L or I;

[0137] E at position 562 is substituted with D, N or Q;

[0138] D at position 583 is substituted with E, N or Q;

[0139] D at position 615 substituted with E, N or Q; and

[0140] At position 642, H is substituted with D, E, N or Q.

[0141] The modified reverse transcriptase of the present invention may include substitutions at least 3 amino acid positions to at least 12 amino acid positions, wherein the amino acid positions are 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 may be selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I and D615E. In certain embodiments, the modified reverse transcriptase may include at least one additional amino acid substitution selected from G524D, N583D and Q562E. At least one amino acid substitution may be selected from L72Q, T163K, N249E and H642D. Additionally and / or alternatively, the amino acid substitution is selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I, D615E and H642D. This embodiment may further include an additional amino acid substitution selected from D524G, D583N, E562Q, H204R and T306K.

[0142] The methods of the present invention may further include any known reagents and methods for reverse transcription. For example, the providing step may include providing a capture oligonucleotide that captures a target RNA molecule to the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0143] Figure 1A Presented are qPCR results demonstrating the thermal activity of the RT enzymes of the present disclosure.

[0144] Figure 1B The efficiency of the enzyme is scored.

[0145] Figure 1C RT-qPCR yields at high temperatures using the RT enzymes of the present disclosure are demonstrated.

[0146] Figure 2A The enhanced thermal activity of the RT enzymes of the present disclosure is demonstrated.

[0147] Figure 2B The processivity of the RT enzymes of embodiments of the present disclosure at high temperatures is demonstrated.

[0148] Figure 3A The thermostability of the RT variant enzymes of the present disclosure is demonstrated.

[0149] Figure 3B Melt curves indicating thermal stability are shown.

[0150] Figure 3CThermal ramp assay results for embodiments of the disclosed RT enzymes are shown.

[0151] Figure 3D exhibit Figure 3C Results table of thermal ramp analysis.

[0152] Figure 3E Isothermal reaction incubations of embodiments of the disclosed RT enzymes are shown.

[0153] Figure 4A Embodiments of the disclosed RT enzymes are shown to be resistant to heparin inhibition.

[0154] Figure 4B Shown are the changes in Ct values ​​with increasing heparin concentrations for the RT enzymes of the present disclosure.

[0155] Figure 4C Embodiments of the disclosed RT enzymes are shown to be resistant to inhibition by guanidine thiocyanate.

[0156] Figure 4D Shown is the change in Ct value with increasing concentration of guanidine thiocyanate for the RT enzymes of the present disclosure.

[0157] Figure 4E Embodiments of the disclosed RT enzymes are shown to be tolerant to ethanol inhibition.

[0158] Figure 4F Shown are the changes in Ct values ​​with increasing ethanol concentrations for the RT enzymes of the present disclosure.

[0159] Figure 5 The cDNA production of the RT enzymes of the present disclosure is shown. DETAILED DESCRIPTION

[0160] The present invention provides a reverse transcriptase having improved thermal stability compared to conventional reverse transcriptases (such as Superscript II (SEQ ID NO: 2) or the wild-type reverse transcriptase shown in SEQ ID NO: 1). The improved thermal stability enables RNA to be reverse transcribed into cDNA at temperatures above about 42° C., thereby reducing the error rate introduced during cDNA synthesis. Therefore, the reverse transcriptase of the present invention can achieve an improvement in the accuracy of transcriptome determination of living organisms.

[0161] Specifically, disclosed are modified reverse transcriptases comprising at least one substitution at a 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 can be:

[0162] S at position 56 is substituted with G, A, V, L or I;

[0163] G at position 138 substituted with S, C, T or M;

[0164] S at position 232 is substituted with C, T or M;

[0165] L at position 234 is substituted with D, E, N or Q;

[0166] G at position 290 is substituted with H, K or R;

[0167] Y at position 344 substituted with F or M;

[0168] T at position 420 is substituted with G, A, V, L or I;

[0169] G at position 424 substituted with D, E, N or Q;

[0170] V at position 444 substituted with G, A, L or I;

[0171] D at position 615 substituted with E, N or Q;

[0172] At position 642, H is substituted with D, E, N or Q.

[0173] D at position 524 substituted with G, A, V, L or I;

[0174] D at position 583 is substituted with E, N or Q;

[0175] E at position 562 is substituted with D, N or Q;

[0176] H at position 204 is substituted with K or R; and

[0177] At position 306, T is substituted with H, K or R.

[0178] The present invention recognizes that an increase in temperature during the reverse transcription reaction reduces non-target primer binding, reduces error rates in cDNA synthesis, and reduces RNA secondary structure. Therefore, the improved thermal stability of the reverse transcriptase of the present invention enables RNA to be reversely transcribed into cDNA at temperatures above 37°C (the optimal temperature for wild-type and other conventional reverse transcriptases). By allowing the reaction temperature to increase, the reverse transcriptase of the present invention reduces the error rate in cDNA synthesis.

[0179] Furthermore, the reverse transcriptases of the present invention provide additional advantages over wild-type and other conventional reverse transcriptases, including improved efficiency in the presence of reverse transcriptase inhibitors and improved efficiency in formalin-fixed samples.

[0180] Reverse transcription

[0181] Reverse transcription is to synthesize DNA from RNA template and produce cDNA. Reverse transcriptase uses RNA template and short primer complementary to the 3' end of RNA to guide the synthesis of first-strand cDNA, which can be directly used as a template for polymerase chain reaction (PCR). The combination of this reverse transcription and PCR realizes the detection of RNA in the sample and produces corresponding cDNA. The RNase H activity from reverse transcriptase or exogenous supply can be used to separate RNA / cDNA heterocomplexes. Other components can also include buffer, one or more primers, dNTP mixture (a mixture of nucleotides dATP, dCTP, dGTP and dTTP), PCR, reagents required for the synthesis of the second DNA chain or amplification reagents.

[0182] Reverse transcription can be performed by a capture oligonucleotide with a free 3' polythymidylic acid (poly-T) region. The 3' portion of the capture oligonucleotide may contain a target-specific sequence or oligomer, such as a capture primer, for reverse transcribing an RNA molecule containing a capture sequence. The oligomer may be a random oligomer or a "not-so-random" (NSR) oligomer (NSRO), such as a random hexa-oligomer or an NSR hexa-oligomer. The oligonucleotide may contain one or more handles, such as a primer binding sequence homologous to a PCR primer used in the amplification step or a sequence of an NGS sequencing adapter. The reverse transcription primer may include a template exchange oligonucleotide (TSO), which may contain a polyguanylic acid (poly-G) sequence that hybridizes with and captures the polycytidylic acid (poly-C) segment added during reverse transcription.

[0183] Reverse transcription of RNA can include the use of capture sequences and capture primers or probes. Primer sequences can include binding sites, such as primer sequences that are expected to hybridize with complementary sequences in nucleic acid molecules. Primer sequences can also be "universal" primer sequences, i.e. sequences complementary to nucleotide sequences that are very common to nucleic acid fragments of a particular group. Primer sequences can be P5 and P7 primers provided by Illumina, Inc., San Diego, California. Primer sequences can also allow capture probes to bind to solid supports, such as template particles.

[0184] This process may include hybridizing a reverse transcription primer with a probe, followed by a reverse transcription reaction. When aligned, the complementarity of the nucleic acids need not be perfect; a stable duplex may contain mismatched base pairs or unmatched bases. One skilled in the art of nucleic acid technology may empirically determine duplex stability taking into account a variety of 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.

[0185] Reverse transcription can also be used to add barcodes, unique molecular identifiers (UMIs), sequencing adapters, or any additional nucleotide sequences to the final cDNA product. For example, a reverse transcription primer can include additional bases that add a barcode or UMI to the cDNA product.

[0186] A barcode is any nucleotide sequence that can be used to derive information about a nucleic acid product, such as 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 distinguish nucleic acids from different samples after sequencing the nucleic acids and the sample barcode.

[0187] A UMI is a type of barcode that can be provided to a sample so that each nucleic acid molecule together with its barcode is 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, each nucleic acid molecule in the sample and its UMI will be unique or nearly unique.

[0188] UMIs are advantageous in that they can be used to correct errors generated during amplification, such as amplification bias or incorrect base pairing during amplification. For example, when UMIs are used, because each nucleic acid molecule in a sample is unique or nearly unique along with its one or more UMIs, molecules with the same sequence can be considered to refer to the same starting nucleic acid molecule after amplification and sequencing, thereby reducing amplification bias. Methods for using UMIs to correct errors are described in Karlsson et al., 2016, Counting Molecules in cell-free DNA and single cells RNA, Karolinska Institute, Stockholm Sweden, which is incorporated herein by reference.

[0189] For example, in the case 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 methods for RNA analysis first require reverse transcription of RNA molecules into cDNA copies, followed by amplification of the cDNA molecules. The cDNA molecules must then be amplified in order to produce enough nucleic acid for analysis. As a result, the absolute number of each original RNA molecule present in the sample is lost, and there are now multiple copies of each molecule in the prepared sample to be analyzed. In addition, information about the relative amount of each RNA molecule in the original sample is also lost due to amplification bias that causes some cDNA molecules to be amplified more unevenly than other cDNA molecules. By providing a UMI to each cDNA molecule before amplification, each cDNA molecule (corresponding to each RNA molecule in the sample) is made unique or nearly unique. After amplifying each cDNA molecule (along with its UMI), the amplified cDNA molecules can then be sequenced and sequence reads generated for each molecule. Sequence reads with the same base sequence and UMI can be collapsed into a single sequence read and regarded as reads 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 a different RNA molecule present in the original sample.

[0190] After reverse transcription, biotin-labeled capture bait or probe can also be used for targeted enrichment of specific cDNA molecules of interest. Biotin-labeled capture probes can include RNA, DNA, or a hybrid of RNA and DNA nucleotides. Biotin-labeled RNA capture probes can be added to the cDNA library, and are cultivated for sufficient time at a temperature sufficient to allow the biotin-labeled RNA capture probe to hybridize with its target cDNA molecule based on Watson-Crick base pairing. For example, a mixture containing cDNA and probes can be cultivated at 65 degrees Celsius for 24 hours. After hybridization, the biotin-labeled RNA capture probe hybridized with the target cDNA molecule can be captured and separated using streptavidin or antibodies. The target cDNA molecule can then be amplified and sequenced.

[0191] Any known sequencing technology can be used to analyze 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 use of fold-back PCR and anchor primers to amplify DNA on a solid surface. Genomic DNA is fragmented and attached to the surface of a flow cell channel. Four fluorophore-labeled reversibly terminating nucleotides are used to perform sequential sequencing. After the nucleotides are incorporated, a laser is used to excite the fluorophore, and an image is captured and the identity of the first base is recorded. Sequencing according to this technology is described in U.S. Publication 2011 / 0009278, U.S. Publication 2007 / 0114362, U.S. Publication 2006 / 0024681, U.S. Publication 2006 / 0292611, U.S. Patent 7,960,120, U.S. Patent 7,835,871, U.S. Patent 7,232,656, U.S. Patent 7,598,035, U.S. Patent 6,306,597, U.S. Patent 6,210,891, U.S. Patent 6,828,100, U.S. Patent 6,833,246, and U.S. Patent 6,911,345, each of which is incorporated by reference. For example, the Illumina Mi-Seq sequencer can be used to generate multiple sequence reads, which can be uploaded to a portal for analysis by, for example, the Genome Analysis Toolkit (GATK), and / or analyzed using the methods set forth in U.S. Patent No. 8,209,130, which is incorporated by reference.

[0192] Analyzing sequence reads can be performed using known software and following multi-step procedures known in the art. For example, first, the software FASTQC can be used to evaluate the quality of each sequence read, that is, the FASTQ sequence. Then, the reads can be pruned by, for example, Trimmomatic software. The pruned sequence reads can then be mapped to the human genome using HISAT2 software. The HISAT2 output is a file in SAM (sequence alignment / mapping format), which can be compressed into a binary sequence alignment / mapping file using a SAMtools version before the sequence reads are quantified. Then, feature Counts software can be used to count the mapped reads.

[0193] Reverse transcriptase

[0194] As discussed above, reverse transcriptase is an enzyme for producing cDNA (a process called reverse transcription) from an RNA template. It is mainly related to retroviruses. However, some non-retroviruses also use reverse transcriptase. The reverse transcription of retroviruses has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H and DNA-dependent DNA polymerase activity. Retroviruses use these activities to convert single-stranded genomic RNA into double-stranded cDNA. In the laboratory, RNA is converted into cDNA using the same reaction sequence for molecular cloning, RNA sequencing, polymerase chain reaction or genomic analysis.

[0195] Reverse transcriptase is a member of a specific enzyme family included in the DNA polymerase superfamily. Despite relatively low amino acid sequence homology and different sources, the enzymes of this superfamily share a similar 3D structure, in which the DNA polymerase consists of three different domains: the palm domain, the thumb domain, and the finger domain. In addition, reverse transcriptases with RNase H activity show a unique RNase H domain and a linker domain with corresponding active sites.

[0196] 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 finger, palm, thumb, link, and RNase H domains. The active site for the DNA polymerase reaction is present in the finger / palm / thumb domains, while the active site for the RNase H reaction is present in the RNase H domain.

[0197] The finger (41 to 124, 160 to 192 a.a.), palm (1 to 40, 125 to 159, 193 to 275 a.a.) and thumb (276 to 361) domains constitute the N-terminus of MMLV RT, while the linker domain (362 to 496 a.a.) and RNase H (497 to 671 a.a.) are located at the C-terminal portion. It has been proposed that the finger domain provides an intermediate binding site for the template-primer between the phosphate nucleotide transfer reactions. The thumb domain plays a key role in substrate binding and persistence. In the palm domain, regions I125-F155, L220-E233 and K257-E275 are located on the surface and interact with the template. The linker domain contains P360-K373, Y394-A436 and S453-A462, which interact with the template on the 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.

[0198] Superscript II reverse transcriptase (ThermoFisher) is a mutant MMLV reverse transcriptase with reduced RNase H activity and increased thermal stability compared to wild-type MMLV reverse transcriptase. Superscript II reverse transcriptase is a commonly used commercial enzyme form and is the primary basis for comparison of the reverse transcriptases of the present invention. The sequence of Superscript II reverse transcriptase is shown in SEQ ID NO: 2.

[0199] The reverse transcriptase of the present invention has at least one amino acid substitution compared to the reverse transcriptase of SEQ ID NO:2.

[0200] Modified reverse transcriptase of the present invention has improved thermostability relative to the wild-type form and Superscript II form of enzyme.The thermostability improvement of a kind of reverse transcriptase relative to another kind of reverse transcriptase means that the reverse transcriptase with improved thermostability is less prone to lose activity at high temperature (e.g., higher than 37°C).The stability improvement of enzyme can also include avoiding denaturation mechanism to realize its full potential as catalyst.In addition, because higher reaction temperature reduces RNA secondary structure and non-specific primer binding, for cDNA synthesis, it is expected that reaction temperature is higher.The stability between two enzymes can be determined by comparing the residual activity of two enzymes after being exposed to specific conditions (e.g., high temperature, in the presence of the inhibitor of the enzyme, or in fixed samples).Alternatively, stability can be determined by comparing the residual or residual acidity of enzyme after cultivation under given conditions (e.g., time or temperature) and the initial activity before cultivation.

[0201] An increase in the efficiency of one reverse transcriptase relative to another means that under a set of conditions, the more efficient enzyme will produce fewer base calling errors over time.

[0202] An increase in the activity of one enzyme relative to another means that the enzyme with the increased activity converts the substrate at a higher rate in reverse transcription, which means the rate at which RNA (substrate) is used to synthesize cDNA. The SI unit of enzyme activity is katal (1 katal = 1 mol s -1 A more practical and commonly used value is enzyme unit (U) = 1 μmol min -1 1 U corresponds to 16.67 nanokatal 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 (in μmol min -1 mg -1 express).

[0203] Reverse transcriptase activity can be determined in an assay that measures cDNA formation over time. For example, a reverse transcriptase can be incubated with an RNA template, primers, and a suitable dNTP mixture, and the production of cDNA or the consumption of dNTPs can be monitored.

[0204] Disclosed herein are reverse transcriptases comprising at least one amino acid substitution at an amino acid position selected from the group consisting of positions 56, 138, 234, 290, 344, 420, 424, 444, 615, 444, 615, 642, 524, 583, 562, 204, and 306 with respect to SEQ ID NO: 2. For example, the amino acid substitution can be:

[0205] S at position 56 is substituted with G, A, V, L or I;

[0206] G at position 138 substituted with S, C, T or M;

[0207] S at position 232 is substituted with C, T or M;

[0208] L at position 234 is substituted with D, E, N, Q;

[0209] G at position 290 is substituted with H, K or R;

[0210] Y at position 344 substituted with F or M;

[0211] T at position 420 is substituted with G, A, V, L or I;

[0212] G at position 424 substituted with D, E, N or Q;

[0213] V at position 444 substituted with G, A, L or I;

[0214] D at position 615 substituted with E, N or Q;

[0215] At position 642, H is substituted with D, E, N or Q.

[0216] D at position 524 substituted with G, A, V, L or I;

[0217] D at position 583 is substituted with E, N or Q;

[0218] E at position 562 is substituted with D, N or Q;

[0219] H at position 204 is substituted with K or R; and

[0220] At position 306, T is substituted with H, K or R.

[0221] It will be appreciated that the reverse transcriptase of the invention may comprise additional mutations that do not alter the function (ie, increased thermostability) of the reverse transcriptase.

[0222] For example, the reverse transcriptase may comprise additional conservative mutations. Conservative amino acid substitutions are substitutions of amino acids with different amino acids having similar properties, and therefore generally involve substitutions of amino acids in a polypeptide with amino acids within the same or similarly defined amino acid classes. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, such as alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain, such as serine and threonine; an amino acid with an aromatic side chain may be substituted with another amino acid with an aromatic side chain, such as phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain, such as lysine and arginine; an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain, such as aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid may be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0223] In various aspects of the invention, the reverse transcriptase does not comprise 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; A502 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; L671P, A32V, E286R, E302K, W388R and L435R. For example, the reverse transcriptase comprises a wild-type amino acid at the specified position, or comprises a conservative substitution at the wild-type amino acid position.

[0224] A reverse transcriptase having at least X% sequence identity means that the enzyme has an amino acid sequence characterized in that at least X amino acid residues are identical to the sequence of the corresponding sequence within a length of 100 amino acids. Sequence identity can be determined in a sequence comparison format by sequence alignment. Methods of sequence alignment are well known in the art and include various programs and alignment algorithms. The Basic Local Alignment Search Tool (BLAST) can also be used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0225] For example, because the wild-type MMLV is 671 amino acids in length, the enzyme can tolerate multiple substitutions, such as conservative substitutions, without losing the reverse transcription properties of the enzyme. However, it should be understood that mutations at certain positions relative to the wild-type will reduce the efficacy of the wild-type enzyme, while other less common substitutions can increase the efficacy of the wild-type enzyme. Thus, through the present invention, it has been found 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 can enhance the properties of the wild-type enzyme. It should be understood that an enzyme comprising substitutions at each of the 20 positions specified by the present invention will have 97.32% sequence identity with the wild-type enzyme. The enzyme of the present invention may comprise additional substitutions that do not change the properties of the present invention, such as additional 15 amino acid substitutions, such as conservative substitutions. Thus, the enzyme of the invention may have 95% sequence identity with the wild-type MMLV enzyme. The same principles apply to the SuperscriptII format.

[0226] Aspects of the invention provide modified reverse transcriptases comprising at least one mutation in at least one domain of the Moloney murine leukemia virus (MMLV) reverse transcriptase of SEQ ID NO: 2. Importantly, the modified reverse transcriptases of the invention exhibit increased thermal stability relative to wild-type reverse transcriptases, such that at temperatures of about 50°C or greater, their activity is at least about 90% of the activity of the wild-type reverse transcriptase at 42°C.

[0227] Preferred amino acid substitutions occur 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, positions 43, 46, 48, 54, 56, 72, 163 and / or 177 in the finger domain of MMLV reverse transcriptase, position 280 in the thumb domain of MMLV reverse transcriptase. , 281, 290, 306, 335, 338, 339, 344, 345, 346 and / or 349, positions 376, 413, 420, 424, 443 and 444 in the linker domain of MMLV reverse transcriptase, and positions 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642 and / or 658 in the RNase H domain of MMLV reverse transcriptase.

[0228] Preferred substitutions are selected from:

[0229] S at position 56 is substituted with G, A, V, L or I;

[0230] L at position 72 is substituted with Q or R;

[0231] G at position 138 substituted with S, C, T or M;

[0232] H at position 204 is substituted with K or R;

[0233] S at position 232 is substituted with C, T or M;

[0234] L at position 234 is substituted with D, E, N, Q;

[0235] G at position 290 is substituted with H, K or R;

[0236] T at position 306 is substituted with H, K or R;

[0237] Y at position 344 substituted with F or M;

[0238] T at position 420 is substituted with G, A, V, L or I;

[0239] G at position 424 substituted with D, E, N or Q;

[0240] V at position 444 substituted with G, A, L or I;

[0241] D at position 524 substituted with G, A, V, L or I;

[0242] E at position 562 is substituted with D, N or Q;

[0243] D at position 583 is substituted with E, N or Q;

[0244] D at position 615 substituted with E, N or Q; and

[0245] At position 642, H is substituted with D, E, N or Q.

[0246] In addition, the modified reverse transcriptase can have substitutions at at least 1 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.

[0247] In some embodiments, the amino acid substitution is selected from S56A, G138S, S232T, L234E, G290K, Y344F, T420V, G424D, V444I and D615E. The modified enzyme may include an additional substitution selected from G524D, N583D and Q562E. In other embodiments, the amino acid substitution is selected from L72Q, T163K, N249E and H642D. In other embodiments, the amino acid substitution is 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.

[0248] In a specific embodiment, the amino acid substitution can be selected from 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, N 335Q, P338E, D339E, Y344F, Q345D, E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433T, V444 I, D518E, G524D, L528F, A554P, E562Q, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D and K658E.

[0249] The modified reverse transcriptase of the present invention generally has an activity at a temperature of 65°C or higher that is at least about 50% of the reverse transcription activity of the reverse transcriptase of SEQ ID NO: 2 at about 42°C. Preferably, the activity of the reverse transcriptase is increased in the presence of a reverse transcription inhibitor compared to the reverse transcriptase of SEQ ID NO: 2. In addition, the modified reverse transcriptase of the present invention exhibits increased thermal stability relative to, for example, the reverse transcriptase of SEQ ID NO: 2, and may comprise at least one amino acid substitution 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; 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.

[0250] Samples and sample preparation

[0251] Any sample suspected of containing 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, such as water or soil. The sample can be from an object, such as an animal or a human, and it can be a fluid, a solid, a suspension, or a tissue.

[0252] Examples of samples include cell or tissue culture, blood, serum, plasma, needle aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excrement, tears, saliva, sweat, biopsy, ascites, cerebrospinal fluid, pleural fluid, amniotic fluid, peritoneal fluid, interstitial fluid, sputum, milk, lymph, bronchial and other lavage samples or tissue extract samples. The source of the sample can be a solid tissue from a fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; or a cell from any time during pregnancy or development of the subject. In a preferred embodiment of the method, the sample is selected from the group consisting of: body fluid, blood, plasma, serum, urine, bile, cerebrospinal fluid, swab, clinical specimen, organ sample and tissue sample, especially human, animal or plant, especially human.

[0253] The sample may contain compounds that are naturally intermixed with the sample in nature as well as compounds that are not naturally intermixed with the sample in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0254] It is noteworthy that many samples contain natural and non-natural inhibitors of reverse transcriptase. For example, humic acid / fulvic acid, polysaccharides, phenolic substances and flavonoids in plant samples inhibit reverse transcription 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 fecal samples, proteases 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 transcription activity.

[0255] Sample preparation steps also often introduce products that inhibit reverse transcriptase, such as EDTA, detergent / DDT, alcohol, salt, heparin, hemoglobin, phenol: chloroform, protease, nuclease, agarose, metal ions and aldehydes all inhibit reverse transcription activity. In addition, in order to remove the purpose of undesired natural products in the sample, some artificial sample preparation products are introduced into the sample. For example, ethanol, protease and phenol: chloroform are generally used to wash away excessive protein, salt and other natural products in the sample, and are transcriptase inhibitors themselves. For the analysis of sample types, other sample preparation products are necessary. For example, detergents are often introduced into samples comprising cells so that cells are dissolved and nucleic acids to be analyzed are released. Detergents are also often used in methods (such as small droplet sequencing) for single cell sequencing to separate and divide individual cells for sequencing.

[0256] Therefore, samples are often washed multiple times during sample preparation to remove undesired products or reagents that are no longer needed. However, each wash results in the loss of at least some of the original sample and risks the introduction of contaminants.

[0257] Sample preservation steps may also lead to the introduction of reverse transcriptase inhibitors. For example, formalin-fixed paraffin-embedded (FFPE) tissue preservation method is a form of preservation and preparation of biopsy samples. Tissue samples are first preserved by fixing them in formaldehyde to preserve proteins and important structures within the tissue. Next, they are embedded in paraffin blocks, which makes it easy to cut into slices of the desired size to be placed on microscope slides for inspection. However, FFPE sample preparation also leads to inhibition of reverse transcription activity.

[0258] Compared to the wild-type MMLV enzyme or the reverse transcriptase shown in SEQ ID NO: 2, 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). Therefore, the reverse transcriptase of the present invention can be added directly to a sample containing an inhibitor without the need for additional washing steps that result in loss of the original sample or introduction of contaminants. In addition, the reverse transcriptase of the present invention can be added directly to a preserved sample, such as a fixed sample, without loss of reverse transcriptase activity.

[0259] Specifically, in the presence of a reverse transcription inhibitor, the reverse transcriptase of the present invention exhibits improved activity relative to the reverse transcriptase of SEQ ID NO: 2, and may comprise at least one amino acid substitution 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; T33 0P; 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.

[0260] In other embodiments, the activity of the reverse transcriptase is increased relative to the reverse transcriptase of SEQ ID NO: 2 in formalin-fixed samples and may comprise at least one amino acid substitution 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.

[0261] In other embodiments, the reverse transcriptase of the invention exhibits improved thermostability relative to the reverse transcriptase of SEQ ID NO: 2 and may comprise at least one amino acid substitution selected from the group consisting of A32V, E286R, E302K, W388R and L435R.

[0262] In the presence of a reverse transcriptase inhibitor, the activity of the reverse transcriptase of the present invention is also improved relative to the reverse transcriptase of SEQ ID NO: 2, and may include at least one amino acid substitution selected from the group consisting of: A32V, E286R, E302K, W388R and L435R. Similarly, for formalin-fixed samples, the activity of the reverse transcriptase of the present invention is improved relative to the reverse transcriptase of SEQ ID NO: 2, and may include at least one amino acid substitution selected from the group consisting of: A32V, E286R, E302K, W388R and L435R.

[0263] Preferred reverse transcriptases of the invention comprise at least about 95% sequence identity relative to the reverse transcriptase of SEQ ID NO:2.

[0264] Aspects of the present invention include methods for reverse transcribing cDNA from RNA. A preferred method includes providing a sample comprising RNA with a modified reverse transcriptase of the present invention comprising at least one mutation in at least one domain of the reverse transcriptase of SEQ ID NO: 2, wherein the modified reverse transcriptase exhibits improved thermal stability relative to the reverse transcriptase of SEQ ID NO: 2, such that the activity of the reverse transcriptase at 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, and 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. Perform a reverse transcription reaction at 50°C or higher to form cDNA.

[0265] In certain embodiments, the mutation comprises an amino acid substitution at a position selected from the group consisting of 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, and positions 164, 165, 167, and 178 in the thumb domain of reverse transcriptase. domain, positions 280, 281, 290, 306, 335, 338, 339, 344, 345, 346 and 349 in the linker domain of reverse transcriptase, positions 376, 413, 420, 424, 443 and 444 in the connecting domain of reverse transcriptase, and positions 518, 524, 528, 550, 554, 562, 583, 615, 619, 625, 629, 642 and 658 in the RNase H domain of reverse transcriptase.

[0266] For example, amino acid substitutions may be selected from:

[0267] S at position 56 is substituted with G, A, V, L or I;

[0268] L at position 72 is substituted with Q or R;

[0269] G at position 138 substituted with S, C, T or M;

[0270] H at position 204 is substituted with K or R;

[0271] S at position 232 is substituted with C, T or M;

[0272] L at position 234 is substituted with D, E, N, Q;

[0273] G at position 290 is substituted with H, K or R;

[0274] T at position 306 is substituted with H, K or R;

[0275] Y at position 344 substituted with F or M;

[0276] T at position 420 is substituted with G, A, V, L or I;

[0277] G at position 424 substituted with D, E, N or Q;

[0278] V at position 444 substituted with G, A, L or I;

[0279] D at position 524 substituted with G, A, V, L or I;

[0280] E at position 562 is substituted with D, N or Q;

[0281] D at position 583 is substituted with E, N or Q;

[0282] D at position 615 substituted with E, N or Q; and

[0283] At position 642, H is substituted with D, E, N or Q.

[0284] The modified reverse transcriptase may include substitutions at least 3 amino acid positions to at least 12 amino acid positions, wherein the amino acid positions are selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615 and 642. Specifically, 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 includes 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 substitution is 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.

[0285] Generally, the reverse transcriptase has an activity at a temperature of about 65°C or higher that is at least about 50% of the reverse transcription activity of the reverse transcriptase of SEQ ID NO: 2 at about 42°C.

[0286] In addition, in the methods of the present invention, the sample containing RNA may contain one or more inhibitors of reverse transcription, and the reverse transcription reaction may be more efficient than a reverse transcription reaction performed in the presence of one or more inhibitors using the reverse transcriptase of SEQ ID NO: 2. The sample containing RNA 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.

[0287] In other embodiments, the operating step is performed at a temperature of about 65°C or greater, and wherein the activity of the reverse transcription reaction is at least about 50% of a reverse transcription reaction performed at about 42°C using the reverse transcriptase of SEQ ID NO:2.

[0288] The methods of the present invention may further include any known reagents and methods for reverse transcription. For example, the providing step may include providing a capture oligonucleotide that captures a target RNA molecule to the sample.

[0289] Example

[0290] Data characterizing the RT enzymes of the present disclosure are presented.

[0291] Figure 1A qPCR results are presented, demonstrating that prod-ana RT has competitive thermal activity relative to Maxima in RT-qPCR.

[0292] Figure 1B The efficiency of the enzyme is scored.

[0293] First strand synthesis (FSS) was performed at 50°C and 60°C, followed by qPCR, using 10, 1 and 0.1 ng of total liver RNA as input, with the disclosed RT enzyme and Maxima.

[0294] First strand synthesis (FSS) reactions were performed on ice by mixing 10 ng of total liver RNA, 1× reaction buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 U of murine RNase inhibitor, 5 μM oligo-dT(20), 0.5 mM dNTPs, and 200 U of the indicated reverse transcriptase. FSS was performed at various temperatures (42°C to 65°C) for 25 minutes followed by qPCR to assess cDNA yield. qPCR reactions were performed by mixing 1× SsoAdvanced Universal SYBR Green Supermix, 10% FSS reactant, and 0.6 μM primer annealed to the 5' end of the β-actin gene.

[0295] The resulting cDNA values ​​were evaluated by Ct values ​​as shown in Figure 1. When FSS was incubated at 50°C, the cDNA yield produced by prod-blhFSS was more than 100-fold lower than that at 60°C, while prod-ana and Maxima were able to convert similar amounts of RNA into cDNA at both 50°C and 60°C. At both 50°C and 60°C, prod-ana had competitive or better yields and efficiencies than Maxima RNase H minus reverse transcriptase.

[0296] Figure 1CRT-qPCR yields at high temperatures using the disclosed RT enzymes are shown. First strand synthesis (FSS) reactions were performed on ice by mixing 10 ng total liver 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, followed by qPCR to assess cDNA yield. qPCR reactions were performed by mixing 1× SsoAdvanced Universal SYBR Green Supermix, 10% FSS reactant, and 0.6 μM primer annealed to the 5' end of the β-actin gene.

[0297] The obtained cDNA values ​​were evaluated by Ct values ​​and are shown in Figure 1C prod-blh had equal cDNA yields at 42°C and 50°C. prod-ana was the most thermostable variant and had equal yields at all temperatures tested.

[0298] Figure 2A The RT prod-ana was shown to have enhanced thermal activity. In the RNA ladder analysis, 10 polyadenylic acid (polyA) RNA transcripts ranging from 0.5 kb to 9 kb were used as the RNA input into the first strand synthesis (FSS). Specifically, 500 ng of Millennium RNA ladder (Millennium TM The FSS reaction was performed on ice 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 at various temperatures (42°C to 65°C) and the thermal stability and processivity of the RT variants were analyzed on an electrophoresis tool (e.g., gDNA TAPESTATION instrument, Agilent). For example, prod-blh, prod-ana, Thermo SSIII and Thermo Maxima RNase Hminus were run in an RNA ladder analysis where FSS was performed at 50°C, 55°C, 60°C or 65°C for 30 minutes. The processivity of the reverse transcriptase was qualitatively assessed based on whether the higher MW RNA transcripts could be converted into cDNA.

[0299] The gDNA tapestation and the resulting cDNA products from the RNA ladder analysis are shown in Figure 2A In. The transcript length at various temperatures is consistent with the thermostability of the enzyme. When FSS is performed at 42°C or 50°C, prod-blh converts the entire RNA ladder into cDNA. When the FSS temperature is increased to 60°C or 65°C, the entire RNA ladder cannot be converted into cDNA. Relative to Thermo SSIII reverse transcriptase, prod-blh has very competitive cDNA conversion at all temperatures. When FSS is performed at 42°C, 50°C or 60°C, prod-ana converts the entire RNA ladder into cDNA. When the FSS temperature is increased to 65°C, higher MW RNA transcripts cannot be converted into cDNA. Relative to Thermo Maxima RNase H minus reverse transcriptase, prod-ana has very competitive cDNA conversion at all temperatures. RT prod-ana has enhanced thermostability and persistence compared to SSIII, and has competitive thermostability and persistence compared to Maxima RNase H minus. The RT variants disclosed in the present invention were operated in triplicate in a thermal shift assay to evaluate enzyme thermostability.

[0300] Figure 2B The persistence of the RT enzymes of the embodiments of the present disclosure at high temperatures is demonstrated.

[0301] By mixing 500 ng Millenium RNA ladder (Millennium TM The FSS reaction was performed on ice with 1x reaction buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 U mouse 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 thermal stability and processivity of the RT variants were analyzed on a gDNA tapestation. The processivity of the reverse transcriptase was qualitatively assessed based on the ability to convert higher MW RNA transcripts into cDNA.

[0302] The gDNA tapestation and the resulting cDNA products from the RNA ladder analysis are shown in Figure 2B The transcript lengths at various temperatures are consistent with the thermostability of the enzyme:

[0303] 1. No cDNA conversion when RT is absent (data not shown)

[0304] 2. All reverse transcriptases were able to convert all MW RNA transcripts into cDNA at 42°C and 50°C.

[0305] 3. At 55 °C, Prod-dnd cannot completely convert all transcripts input with RNA into cDNA. At 55 °C and 60 °C, prod-blh, prod-ana, and prod-cnc can completely convert all transcripts.

[0306] 4. At 65 °C, prod-ana converts 9 out of 10 polyadenylated transcripts into cDNA.

[0307] Figure 3A Show the thermal stability of RT variants. The WMG RT variants were run in triplicate in a thermal shift assay to evaluate enzyme thermal stability. The RT variants of the present disclosure were run in triplicate in a thermal shift assay (GloMelt; Biotium) to evaluate enzyme thermal stability. The Biotium GloMelt manufacturing protocol was followed, and a thermal ramp gradient was used. As the enzyme unfolds, the fluorescence signal increases, and the inflection point of the curve indicates the melting temperature of the enzyme.

[0308] Figure 3B Show melting curves indicating that the tested RTs have different degrees of thermal stability: RTII < prod-alg = RTIII < prod-bmh < prod-ana. Here, prod-ana exhibits a significant improvement in thermal stability compared to RTII and RTIII. As shown, prod-aic (RTII) exhibits a melting curve at 63 °C, prod-alg exhibits a melting curve at 67 °C, prod-blh (RTIII) exhibits a melting curve at 67 °C, prod-bmh exhibits a melting curve at 68 °C, and prod-ana exhibits a melting curve at 71 °C. The melting curves indicate that the tested RTs have different degrees of thermal stability.

[0309] Figures 3C to 3E Show the results of further evaluating the aggregation temperature and thermal stability of the reverse transcriptases of the present disclosure.

[0310] Figure 3CThe results of thermal ramp analysis of embodiments of the RT enzymes disclosed herein are presented. In a thermal ramp analysis using static light scattering (SLS), RT variants were run in triplicate on an Unchained Labs Uncle instrument to assess the onset of aggregation. A 266 nm laser was used to monitor the amount of light scattered while a thermal ramp from 15°C to 95°C was applied to the sample. All 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 at which aggregation occurs. The onset of aggregation almost always occurs after a denaturation event occurs within the protein.

[0311] Figure 3D Displays the specificity of the RT variants tested Figure 3C Results table of thermal ramp analysis. Results:

[0312] 1. prod-dnd exhibited the lowest thermal stability and the lowest aggregation onset temperature of 60.7°C.

[0313] 2. prod-ana exhibited the highest thermal stability and the highest aggregation onset temperature of 68.2°C.

[0314] 3. Prod-blh exhibited moderate thermal stability and an aggregation onset temperature of 63.1°C between Prod-dnd and Prod-ana.

[0315] Figure 3E Isothermal reaction incubations showing embodiments of the RT enzymes disclosed herein. RT variants were run in triplicate on a non-chained laboratory Uncle instrument in an isothermal analysis using static light scattering (SLS) to assess the onset of aggregation. The amount of light scattered while the samples were kept at 50°C for approximately 20 hours was monitored using a 266nm laser. All RT variants were diluted to 0.25mg / mL in the following buffer system: 60mM Tris-HCl (pH=8.0), 75mM KCl, 50mM NaCl, 25% (v / v) glycerol, 10mM DTT, 3mM MgCl2. The increase in the amount of scattered light directly measures the level at which aggregation occurs. The aggregation rate can be determined by the increase in SLS counts over time.

[0316] 1. prod-dnd exhibited the lowest thermal stability and the steepest aggregation rate.

[0317] 2. prod-ana exhibits the highest thermal stability and the flattest aggregation rate.

[0318] 3. Prod-blh exhibited moderate thermal stability and aggregation rate between Prod-dnd and Prod-ana.

[0319] RT-qPCR yield in the presence of various inhibitors

[0320] First strand synthesis (FSS) reactions were performed on ice by mixing 10 ng total liver 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 indicated reverse transcriptase + / - various inhibitors (see below). FSS was performed at the optimal temperature for each reverse transcriptase, 42°C for prod-dnd, or 50°C for prod-blh, and incubated for 25 minutes, followed by qPCR to assess cDNA yield. qPCR reactions were performed by mixing 1× SsoAdvanced Universal SYBR Green Supermix, 10% FSS reactant, and 0.6 μM primer annealed to the 5' end of the β-actin gene.

[0321] Figure 4A and 4B The tolerance of embodiments of the disclosed RT enzymes to heparin inhibition was demonstrated. FSS was performed as described above and in the presence of 0, 15, 30, 60, 120, 240, or 600 ng / μL heparin. Figure 4A The Ct values ​​shown in Figure 3 were used to evaluate the obtained cDNA values.

[0322] Figure 4B The change of Ct value with increasing heparin concentration for RT enzyme of the present disclosure is shown. The results are summarized as follows:

[0323] 1. No inhibition was observed for all RT variants at 15 and 30 ng / μL heparin.

[0324] 2. prod-dnd had the lowest yield (about 10-fold or more lower yield) in the presence of >=60 ng / μL and produced no cDNA at concentrations >120 ng / μL heparin.

[0325] 3. The cDNA yield of prod-blh in the presence of 240 ng / μL was approximately 4 to 10 times lower than that of prod-ana, and no cDNA was produced when 600 ng / μL heparin was added.

[0326] 4. prod-ana had the highest cDNA yield over a wide range of heparin concentrations and produced cDNA at every heparin concentration tested.

[0327] Figure 4C and 4D Embodiments of the disclosed RT enzymes are shown to be resistant to inhibition by guanidine thiocyanate.

[0328] FSS was performed as described above in the presence of 0, 15, 30, 60 or 90 mM guanidine isothiocyanate. Figure 4C The Ct values ​​shown in Figure 3 were used to evaluate the obtained cDNA values.

[0329] Figure 4D The change of Ct value with increasing concentration of guanidine thiocyanate for RT variants disclosed in the present invention is shown. The results are summarized as follows:

[0330] 1. No or minimal inhibition was observed for all RT variants at 30 and 60 mM guanidine thiocyanate.

[0331] 2. prod-dnd showed maximal inhibition and was inhibited approximately 4-fold at 60 mM inhibitor and approximately 100-fold at 90 mM inhibitor.

[0332] 3. prod-blh was inhibited approximately 4-fold at 60 mM inhibitor and >10-fold at 90 mM inhibitor.

[0333] 4. prod-ana had the highest cDNA yield over a wide range of guanidine thiocyanate concentrations and was inhibited approximately 3-fold at 90 mM inhibitor.

[0334] Figure 4E Embodiments of the disclosed RT enzymes are shown to be tolerant to ethanol inhibition.

[0335] FSS was performed as described above in the presence of 0, 5, 10 or 20% ethanol. Figure 4E The Ct values ​​shown in Figure 3 were used to evaluate the obtained cDNA values.

[0336] Figure 4F The change of Ct value with increasing ethanol concentration for RT enzyme of the present disclosure is shown. The results are summarized as follows:

[0337] 1. No or minimal inhibition was observed for all RT variants at 5% and 10% ethanol.

[0338] 2. Prod-BMH and Prod-DND were inhibited > about 100-fold under 20% ethanol.

[0339] 3. Prod-Ana and Prod-Blh were inhibited <100-fold in 20% ethanol.

[0340] Figure 5Prod-bmh RT was shown to have competitive cDNA yield from FFPE samples relative to Maxima. First strand synthesis (FSS) reactions were performed on ice by mixing 10, 1 or 0.1 ng of FFPE RNA isolated from skin tissue, 1× reaction buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 U of murine RNase inhibitor, 5 μM oligo-dT (20), 0.5 mM dNTPs and 200 U of the indicated reverse transcriptase. FSS was performed at 42°C for 25 minutes followed by qPCR to assess cDNA yield. qPCR reactions were performed by mixing 1× SsoAdvanced Universal SYBR Green Supermix, 10% FSS reactant and 0.6 μM primer annealed to the 5' end of the β-actin gene.

[0341] Results: The obtained cDNA values ​​were evaluated by Ct values ​​and displayed in Figure 5 It should be noted that the asterisk indicates that a non-specific melting curve was generated and no specific product was made.

[0342] 1. prod-dnd had the lowest yield (about 4-fold lower than prod-alg and prod-bmh) and only produced a specific cDNA product at 10 ng FFPE RNA input.

[0343] 2. prod-alg has the same cDNA yield as prod-bmh at 10 ng FFPE RNA input, but is unable to produce specific cDNA products at lower RNA input amounts.

[0344] 3. prod-bmh produces specific products at 10 ng and 1 ng FFPE RNA input. cDNA yields are competitive with Maxima RNase H minus reverse transcriptase.

[0345] 4. In RT-qPCR, WMG prod-ana had competitive yield and efficiency scores compared to Maxima at 50°C and 60°C.

[0346] Other additional mutations that may optionally be included include one or any combination of the following: 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, L 272I, T281S, G290K, N335Q, P338E, D339E, Y344F, Q345D, E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V 433T, V444I, D518E, G524D, L528F, A554P, Q562E, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D and K658E.

[0347] sequence

[0348] The following sequences may be referenced or used within 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 one of SEQ ID NOs: 2 and 4-14.

[0349] >wtMMLV-RT-aa (SEQ ID NO: 1)

[0350] TLNIEDEHRL HETSKEPDVS LGSTWLSDFP QAWAETGGMG LAVRQAPLII

[0351] <h2 style=";text-align:left;direction:ltr">PLKATSTPVS IKQYPMSQEA RLGIKPHIQR LLDQGILVPC QSPWNTPLLP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0352] <h2 style=";text-align:left;direction:ltr"> VKKPGTNDYR PVQDLREVNK RVEDIHPTVP NPYNLLS<h2 style=";text-align:left;direction:ltr"> G <h2 style=";text-align:left;direction:ltr"> LP PSHQWYTVLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0353] <h2 style=";text-align:left;direction:ltr"> LKDAFFCLRL HPTSQPLFAF EWRPEMGIS GQLTWTRLPQ GFKNSPTLFD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0354] <h2 style=";text-align:left;direction:ltr"> EALHRDLADF RIQHPDLILL QYVDDLLLAA T<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> E<h2 style=";text-align:left;direction:ltr"> L <h2 style=";text-align:left;direction:ltr"> DCQQGT RALLQTLGNL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0355] <h2 style=";text-align:left;direction:ltr"> GYRASAKKAQ ICQKQVKYLG YLLKEGQRWL TEARKETVM<h2 style=";text-align:left;direction:ltr"> G <h2 style=";text-align:left;direction:ltr"> QPTPKTPRQL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0356] <h2 style=";text-align:left;direction:ltr"> REFLGTAGFC RLWIPGFAEM AAPLYPLTKT GTLFNWGPDQ QKA<h2 style=";text-align:left;direction:ltr"> Y <h2 style=";text-align:left;direction:ltr"> QEIKQA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0357] <h2 style=";text-align:left;direction:ltr"> LLTAPALGLP DLTKPFELFV DEKQGYAKGV LTQKLGPWRR PVAYLSKKLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0358] <h2 style=";text-align:left;direction:ltr"> PVAAGWPPCL RMVAAIAVL<h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> KDA<h2 style=";text-align:left;direction:ltr"> G <h2 style=";text-align:left;direction:ltr"> KLTMGQ PLVILAPHAV EAL<h2 style=";text-align:left;direction:ltr"> V <h2 style=";text-align:left;direction:ltr"> KQPPDR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0359] <h2 style=";text-align:left;direction:ltr"> WLSNARMTHY QALLLDTDRV QFGPVVALNP ATLLLPPEEG LQHNCLDILA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0360] <h2 style=";text-align:left;direction:ltr"> EAHGTRPDLT DQPLPDADHT WYTDGSSLLQ EGQRKAGAAV TTETEVIWAK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0361] <h2 style=";text-align:left;direction:ltr"> ALPAGTSAQR AELIALTQAL KMAEGKKLNV YTDSRYAFAT AHIHGEIYRR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0362] <h2 style=";text-align:left;direction:ltr"> GRILLTSEGKE IKNK<h2 style=";text-align:left;direction:ltr"> D <h2 style=";text-align:left;direction:ltr"> EILAL LKALFLPKRL SIIHCPGHQK GHSAEARGNR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0363] <h2 style=";text-align:left;direction:ltr"> MADQAARKAA ITETPDTSTL L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0364] <h2 style=";text-align:left;direction:ltr"> >prod-dnd-aa(SEQ-ID NO 2)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0365] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0366] <h2 style=";text-align:left;direction:ltr"> DVSLGSTWLSDFPQAWAETG GMGLAVRQAP LIIPLKATST PVSIKQYPMS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0367] <h2 style=";text-align:left;direction:ltr"> QEARLGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN DYRPVQDLRE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0368] <h2 style=";text-align:left;direction:ltr"> VNKRVEDIHP TVPNPYNLLS<h2 style=";text-align:left;direction:ltr"> G <h2 style=";text-align:left;direction:ltr"> LPPSHQWYT VLDLKDAFFC LRLHPTSQPL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0369] <h2 style=";text-align:left;direction:ltr"> FAFEWRDPEM GISGQLTWTR LPQGFKNSPT LFDEALHRDL ADFRIQHPDL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0370] <h2 style=";text-align:left;direction:ltr"> ILLQYVDDLL LAAT<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> E<h2 style=";text-align:left;direction:ltr"> L <h2 style=";text-align:left;direction:ltr"> DCQ QGTRALLQTL GNLGYRASAK KAQICQKQVK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0371] <h2 style=";text-align:left;direction:ltr"> YLGYLLKEGQ RWLTEARKET VM<h2 style=";text-align:left;direction:ltr"> G <h2 style=";text-align:left;direction:ltr"> QPTPKTP RQLREFLGTA GFCRLWIPGF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0372] <h2 style=";text-align:left;direction:ltr"> AEMAAPLYPL TKTGTLFNWG PDQQKA<h2 style=";text-align:left;direction:ltr"> Y <h2 style=";text-align:left;direction:ltr"> QEI KQALLTAPAL GLPDLTKPFE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0373] <h2 style=";text-align:left;direction:ltr"> LFVDEKQGYA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMVAAIA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0374] <h2 style=";text-align:left;direction:ltr"> VL<h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> KDA<h2 style=";text-align:left;direction:ltr"> GKLT MGQPLVILAP HAVEAL V KQP PDRWLSNARM THYQALLLDT

[0375] DRVQFGPVVA LNPATLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA

[0376] DHTWYTGGSS LLQEGQRKAG AAVTTTETEVI WAKALPAGTS AQRAQLIALT

[0377] QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRGLLTSE GKEIKNK D EI

[0378] LALLKALFLP KRLSIIHCPG HQKGHSAEAR GNRMADQAAR KAAITETPDT STLL

[0379] >prod-dnd-nt(SEQ-ID NO 3)

[0380] ATGGGGGGTT CTCATCATCA TCATCATCAT GGTATGGCTA GCATGACTGG

[0381] TGGACAGCAA ATGGGTCGGG ATCTGTACGA CGATGACGAT AAGCATATGA

[0382] CCCTAATAT AGAAGATGAG CACCGGCTAC ATGAGACCTC AAAAGAGCCA

[0383] GATGTTTCTC TAGGGTCCAC ATGGTTATCG GATTTTCCCGC AAGCTTGGGC

[0384] TGAAACGGGG GGTATGGGGC TTGCTGTACG CCAGGCGCCC CTTATTATTC

[0385] CTCTTAAGGC AACAAGCACC CCCGTCAGCA TCAAACAATA CCCCATGTCA

[0386] CAAGAAGCGC GTTTGGGTAT TAAACCGCAC ATTCAACGCT TGCTGGACCA

[0387] AGGGATCCTT GTCCCCTGTC AGTCTCCATG GAACACGCCG CTGTTACCGG

[0388] TCAAGAAGCC TGGTACAAAC GACTACCGTC CTGTACAGGA TTTGCGCGAG

[0389] GTAAATAAGC GTGTAGAGGA TATCCACCCCC ACCGTGCCCA ACCCATACAA

[0390] CTTGTTGTCT GGTTTACCGC CTTCACATCA GTGGTATACG GTCCTTGATT

[0391] TGAAAGATGC ATTTTTCTGC CTGCGCTTGC ACCCTACGTC TCAGCCACTG

[0392] TTCGCATTCG AGTGGCGCGA CCCTGAAATG GGTATCAGTG GTCAGTTAAC

[0393] CTGGACACGC TTACCGCAAG GTTTCAAGAA TAGCCCGACC TTATTCGATG

[0394] AAGCACTTCA CCGCGATCTT GCCGATTTCC GCATCCAGCA CCCGGATCTT

[0395] ATTTTGTTGC AGTACGTAGA CGACCTGTTG TTGGCAGCCA CGTCGGAATT

[0396] GGATTGTCAG CAAGGAACAC GCGCGTTACT GCAAACTCTG GGTAATTTGG

[0397] GCTACCGTGC CTCCGCAAAG AAAGCGAGA TCTGCCAAAA GCAAGTAAAA

[0398] TACTTAGGAT ATTTATTGAA GGAGGGCCAG CGTTGGCTTA CAGAAGCTCG

[0399] CAAAGAAACT GTGATGGGAC AACCTACGCC TAAAACTCCT CGTCAGCTTC

[0400] GCGAATTCCT GGGAACAGCG GGGTTCTGTC GCCTGTGGAT TCCTGGTTTT

[0401] GCAGAGATGG CGGCGCCCCT TTACCCACTG ACCAAAACAG GGACATTATT

[0402] TAACTGGGGT CCCGATCAGC AGAAGGCCTA TCAGGAGATC AAGCAGGCTT

[0403] TACTTACAGC GCCAGCCTTA GGGTTACCGG ACCTTACGAA GCCCTTTGAG

[0404] TTATTCGTCG ACGAGAAACA GGGCTATGCA AAGGGTGTAC TGACCCAGAA

[0405] GTTGGGGCCG TGGCGTCGCC CGGTCGCCTA TTTGTCGAAA AAACTGGATC

[0406] CCGTGGCGGC CGGGTGGCCA CCTTGCCTGC GTATGGTTGC AGCTATTGCT

[0407] GTATTAACAA AAGACGCAGG AAAATTAACG ATGGGACAAC CGTTAGTCAT

[0408] TCTTGCGCCG CACGCAGTAG AAGCACTGGT AAAGCAACCG CCCGATCGCT

[0409] GGTTATCGAA CGCGCGCATG ACGCACTATC AGGCGTTGTT ACTTGATACA

[0410] GATCGTGTAC AGTTTGGCCC TGTCGTTGCA TTGAACCCAG CCACGCTGTT

[0411] ACCTCTTCCG GAAGAAGGAT TACAACACAA CTGTCTGGAC ATCCTGGCGG

[0412] AAGCTCATGG AACCCGCCCT GACTTGACAG ACCAACCGTT ACCCGACGCT

[0413] GATCATACCT GGTATACCGG GGGTTCGTCG CTTTTGCAGG AAGGGCAACG

[0414] TAAGGCAGGT GCAGCCGTAA CGACCGAGAC CGAAGTCATC TGGGCCAAGG

[0415] CCTTGCCAGC GGGGACTAGT GCCCAGCGTG CCCAATTAAT TGCCCTTACG

[0416] CAGGCATTAA AGATGGCTGA AGGAAAAAAG CTGAATGTAT ACACTAACAG

[0417] CCGCTACGCG TTCGCGACAG CGCATATTCA TGGAGAGATC TACCGTCGCC

[0418] GCGGACTTCT TACCTCCGAG GGTAAGGAGA TTAAGAACAA AGATGAGATT

[0419] CTGGCTTTGT TGAAGGCGTT GTTTTTGCCC AAACGTTTGT CAATCATTCA

[0420] CTGTCCCGGG CATCAAAAAAG GGCACAGCGC CGAGGCCCGT GGTAACCGTA

[0421] TGGCAGACCA GGCCGCACGT AAAGCAGCCA TTACGGAGAC ACCTGATACG

[0422] AGTACGTTAC TTTAA

[0423] >prod-ana-aa(SEQ ID NO:4)

[0424] MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKHMTLNIEDEHRLHETSKEPDVSLGS

[0425] TWLSDFPQAWAETGGMGLAVRQAPLIIPLKATATPVSIKQYPMSQEARQGIKPHIQRLLD

[0426] QGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSSLPPSH

[0427] QWYTVLDLKDAFFCLRLHPKSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEA

[0428] LRRDLADFRIQHPDLILLQYVDDLLLAATTEEDCQQGTRALLQTLGELGYRASAKKAQI

[0429] CQKQVKYLGYLLKEGQRWLTEARKETVMKQPTPKTPRQLREFLGKAGFCRLWIPGFAE

[0430] MAAPLYPLTKTGTLFNWGPDQQKAFQEIKQALLTAPALGLPDLTKPFELFVDEKQGYA

[0431] KGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLVKDADKLTMGQPLVI

[0432] LAPHAVEALIKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ

[0433] HNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTETEVIWAK

[0434] ALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHGEIYRRRGLLTSEGK

[0435] <h2 style=";text-align:left;direction:ltr">EIKNKEEILALLKALFLPKRLSIIHCPGHQKGDSAEARGNRMADQAARKAAITETPDTST<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0436] <h2 style=";text-align:left;direction:ltr"> LLIENSSPNSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0437] <h2 style=";text-align:left;direction:ltr"> >prod-amg-aa(SEQ ID NO:5)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0438] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0439] <h2 style=";text-align:left;direction:ltr"> DVSLGSMTWL SDFPQAWAET GGMGLAVRQA PLIIPLKATA TPVSIKQYPM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0440] <h2 style=";text-align:left;direction:ltr"> SQEARLGIKP HIQRLLDQGI LVPCQSPWNT PLLPVKKPGT NDYRPVQDLR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0441] <h2 style=";text-align:left;direction:ltr"> EVNKRVEDIH PTVPNPYNLL SSLPPSHQWY TVLDLKDAFF CLRLHPTSQP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0442] <h2 style=";text-align:left;direction:ltr"> LFAFEWRDPE MGISGQLTWT RLPQGFKNSP TLFDEALHRD LADFRIQHPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0443] <h2 style=";text-align:left;direction:ltr"> LILLQYVDDL LLAATTEEDC QQGTRALLQT LGNLGYRASA KKAQICQKQV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0444] <h2 style=";text-align:left;direction:ltr"> KYLGYLLKEG QRWLTEARKE TVMKQPTPKT PRQLREFLGT AGFCRLWIPG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0445] <h2 style=";text-align:left;direction:ltr"> FAEMAAPLYP LTKTGTLFNW GPDQQKAFQE IKQALLTAPA LGLPDLTKPF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0446] <h2 style=";text-align:left;direction:ltr"> ELFVDEKQGY AKGVLTQKLG PWRRPVAYLS KKLDPVAAGW PPCLRMVAAI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0447] <h2 style=";text-align:left;direction:ltr"> AVLVKDADKL TMGQPLVILA PHAVEALIKQ PPDRWLSNAR MTHYQALLLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0448] <h2 style=";text-align:left;direction:ltr">TDRVQFGPVV ALNPATLLPL PEEGLQHNCL DILAEAHGTR PDLTDQPLPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0449] <h2 style=";text-align:left;direction:ltr"> ADHTWYTGGS SLLQEGQRKA GAAVTTETEV IWAKALPAGT SAQRAQLIAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0450] <h2 style=";text-align:left;direction:ltr"> TQALKMAEGK KLNVYTNSRY AFATAHIHGE IYRRRGLLTS EGKEIKNKEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0451] <h2 style=";text-align:left;direction:ltr"> ILALLKALFL PKRLSIIHCP GHQKGHSAEA RGNRMADQAA RKAAITETPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0452] <h2 style=";text-align:left;direction:ltr"> TSTLLIENSS PNSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0453] <h2 style=";text-align:left;direction:ltr"> >prod-asb-aa(SEQ ID NO:6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0454] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0455] <h2 style=";text-align:left;direction:ltr"> DVSLGSTWLT DFPQAWAETG GMGLAKRQPP IIIPLKPTAT PVWIKQYPMS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0456] <h2 style=";text-align:left;direction:ltr"> KEARQGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN DYRPVQDLRE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0457] <h2 style=";text-align:left;direction:ltr"> VNKRVQDIHP TVPNPCNLLS SLPPSHQWYT VLDLKDAFFC LRLHPKSQPL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0458] <h2 style=";text-align:left;direction:ltr"> FAFEWRDPER GISGQLTWTR LPQGFKNSPT LFHEALHRDL ADFRTQHPDL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0459] <h2 style=";text-align:left;direction:ltr"> TLLQYVDDLL LAAETEEDCE QGTRDLLQTL GELGYRASAK KAQICQQEVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0460] <h2 style=";text-align:left;direction:ltr"> YLGYILKEGQ RWLSEARKET VMKQPTPKTP RQLREFLGTA GFCRLWIPGF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0461] AEMAAPLYPL TKTGTLFQWG EEQQKAFDDI KRALLTAPAL GLPDLTKPFE

[0462] LFVDEKQGFA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMIAAIA

[0463] VLVKDADKLT MGQPLTILAP HAVESLIKQP PDRWLSNARM THYQALLLDT

[0464] DRVQFGPVVA LNPATLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA

[0465] EHTWYTGGSS FLQEGQRKAG AAVTTTETEVI WASALPPGTS AQRAQLIALT

[0466] QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRRGLLTSE GKEIKNKEEI

[0467] LELLKALWLP KKLAIIHCPG HQKGDSAEAR GNRMADQAAR EAAITETPDT

[0468] STLLIENSSP NSRLIN

[0469] >prod-bmh-aa(SEQ ID NO:7)

[0470] MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP

[0471] DVSLGSMTWL SDFPQAWAET GGMGLAVRQA PLIIPLKATA TPVSIKQYPM

[0472] SQEARLGIKP HIQRLLDQGI LVPCQSPWNT PLLPVKKPGT NDYRPVQDLR

[0473] EVNKRVEDIH PTVPNPYNLL SSLPPSHQWY TVLDLKDAFF CLRLHPTSQP

[0474] <h2 style=";text-align:left;direction:ltr">LFAFEWRDPE MGISGQLTWT RLPQGFKNSP TLFDEALRRD LADFRIQHPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0475] <h2 style=";text-align:left;direction:ltr"> LILLQYVDDL LLAATTEEDC QQGTRALLQT LGNLGYRASA KKAQICQKQV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0476] <h2 style=";text-align:left;direction:ltr"> KYLGYLLKEG QRWLTEARKE TVMKQPTPKT PRQLREFLGK AGFCRLWIPG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0477] <h2 style=";text-align:left;direction:ltr"> FAEMAAPLYP LTKTGTLFNW GPDQQKAFQE IKQALLTAPA LGLPDLTKPF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0478] <h2 style=";text-align:left;direction:ltr"> ELFVDEKQGY AKGVLTQKLG PWRRPVAYLS KKLDPVAAGW PPCLRMVAAI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0479] <h2 style=";text-align:left;direction:ltr"> AVLVKDADKL TMGQPLVILA PHAVEALIKQ PPDRWLSNAR MTHYQALLLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0480] <h2 style=";text-align:left;direction:ltr"> TDRVQFGPVV ALNPATLLPL PEEGLQHNCL DILAEAHGTR PDLTDQPLPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0481] <h2 style=";text-align:left;direction:ltr"> ADHTWYTGGS SLLQEGQRKA GAAVTTETEV IWAKALPAGT SAQRAQLIAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0482] <h2 style=";text-align:left;direction:ltr"> TQALKMAEGK KLNVYTNSRY AFATAHIHGE IYRRRGLLTS EGKEIKNKEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0483] <h2 style=";text-align:left;direction:ltr"> ILALLKALFL PKRLSIIHCP GHQKGHSAEA RGNRMADQAA RKAAITETPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0484] <h2 style=";text-align:left;direction:ltr"> TSTLLIENSS PNSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0485] <h2 style=";text-align:left;direction:ltr"> >prod-cnc-aa(SEQ ID NO:8)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0486] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0487] DVSLGSTWLS DFPQAWAETG GMGLAKRQAP LIIPLKATAT PVSIKQYPMS

[0488] QEARQGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN ANIMALS

[0489] VNKRVEDIHP TVPNPYNLLS SLPPSHQWYT VLDLKDAFFC LRLHPKSQPL

[0490] FAFEWRDPEM GISGQLTWTR LPQGFKNSPT LFDEALRRDL ADFRIQHPDL

[0491] ILLQYVDDLL LAATTEEDCE QGTRALLQTL GELGYRASAK KAQICQKEVK

[0492] YLYLLKEGQ RWLTEARKET VMKQPTPKTP RQLREFLGKA GFCRLWIPGF

[0493] AEMAAPLYPL TKTGTLFNWG PDQQKAFQEI KQALLTAPAL GLPDLTKPFE

[0494] LFVDEKQGVA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMVAAIA

[0495] VLVKDADKLT MGQPLVILAP GARDENALIKQP PDRWLSNARM THYQALLLDT

[0496] DRVQFGPVVA LNPATLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA

[0497] DHTWYTGGSS FLQEGQRKAG AAVTTETEVI WAKALPAGTS AQRAQLIALT

[0498] QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRRGLLTSE GKEIKNKEEI

[0499] <h2 style=";text-align:left;direction:ltr">LALLKALFLP KRLSIIHCPG HQKGDSAEAR GNRMADQAAR KAAITETPDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0500] <h2 style=";text-align:left;direction:ltr"> STLLIENSSP NSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0501] <h2 style=";text-align:left;direction:ltr"> >prod-cmi-aa(SEQ ID NO:9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0502] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0503] <h2 style=";text-align:left;direction:ltr"> DVSLGSMTWL SDFPQAWAET GGMGLAVRQA PLIIPLKATA TPVSIKQYPM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0504] <h2 style=";text-align:left;direction:ltr"> SQEARLGIKP HIQRLLDQGI LVPCQSPWNT PLLPVKKPGT NDYRPVQDLR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0505] <h2 style=";text-align:left;direction:ltr"> EVNKRVEDIH PTVPNPYNLL SSLPPSHQWY TVLDLKDAFF CLRLHPTSQP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0506] <h2 style=";text-align:left;direction:ltr"> LFAFEWRDPE MGISGQLTWT RLPQGFKNSP TLFDEALRRD LADFRIQHPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0507] <h2 style=";text-align:left;direction:ltr"> LILLQYVDDL LLAATTEEDC QQGTRALLQT LGNLGYRASA KKAQICQKQV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0508] <h2 style=";text-align:left;direction:ltr"> KYLGYLLKEG QRWLTEARKE TVMKQPTPKT PRQLREFLGK AGNCRLWIPG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0509] <h2 style=";text-align:left;direction:ltr"> FAEMAAPLYP LTKTGTLFNW GPDQQKAFQE IKQALLTAPA LGLPDLTKPF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0510] <h2 style=";text-align:left;direction:ltr"> ELFVDEKQGY AKGVLTQKLG PWRRPVAYLS KKLDPVAAGW PPCLRMVAAI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0511] <h2 style=";text-align:left;direction:ltr"> AVLVKDADKL TMGQPLVILA PHAVEALIKQ PPDRWLSNAR MTHYQALLLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0512] <h2 style=";text-align:left;direction:ltr">TDRVQFGPVV ALNPATLLPL PEEGLQHNCL DILAEAHGTR PDLTDQPLPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0513] <h2 style=";text-align:left;direction:ltr"> ADHTWYTGGS SLLQEGQRKA GAAVTTETEV IWAKALPAGT SAQRAQLIAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0514] <h2 style=";text-align:left;direction:ltr"> TQALKMAEGK KLNVYTNSRY AFATAHIHGE IYRRRGLLTS EGKEIKNKEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0515] <h2 style=";text-align:left;direction:ltr"> ILALLKALFL PKRLSIIHCP GHQKGHSAEA RGNRMADQAA RKAAITETPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0516] <h2 style=";text-align:left;direction:ltr"> TSTLLIENSS PNSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0517] <h2 style=";text-align:left;direction:ltr"> >prod-bnb-aa(SEQ ID NO:10)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0518] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0519] <h2 style=";text-align:left;direction:ltr"> DVSLGSTWLS DFPQAWAETG GMGLAVRQAP LIIPLKATAT PVSIKQYPMS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0520] <h2 style=";text-align:left;direction:ltr"> QEARQGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN DYRPVQDLRE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0521] <h2 style=";text-align:left;direction:ltr"> VNKRVEDIHP TVPNPYNLLS SLPPSHQWYT VLDLKDAFFC LRLHPKSQPL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0522] <h2 style=";text-align:left;direction:ltr"> FAFEWRDPEM GISGQLTWTR LPQGFKNSPT LFDEALRRDL ADFRIQHPDL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0523] <h2 style=";text-align:left;direction:ltr"> ILLQYVDDLL LAATTEEDCQ QGTRALLQTL GELGYRASAK KAQICQKQVK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0524] <h2 style=";text-align:left;direction:ltr"> YLGYLLKEGQ RWLTEARKET VMKQPTPKTP RQLREFLGKA GNCRLWIPGF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0525] <h2 style=";text-align:left;direction:ltr">AEMAAPLYPL TKTGTLFNWG PDQQKAFQEI KQALLTAPAL GLPDLTKPFE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0526] <h2 style=";text-align:left;direction:ltr"> LFVDEKQGYA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMVAAIA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0527] <h2 style=";text-align:left;direction:ltr"> VLVKDADKLT MGQPLVILAP HAVEALIKQP PDRWLSNARM THYQALLLDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0528] <h2 style=";text-align:left;direction:ltr"> DRVQFGPVVA LNPATLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0529] <h2 style=";text-align:left;direction:ltr"> DHTWYTGGSS LLQEGQRKAG AAVTTETEVI WAKALPAGTS AQRAQLIALT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0530] <h2 style=";text-align:left;direction:ltr"> QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRRRGLLTSE GKEIKNKEEI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0531] <h2 style=";text-align:left;direction:ltr"> LALLKALFLP KRLSIIHCPG HQKGDSAEAR GNRMADQAAR KAAITETPDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0532] <h2 style=";text-align:left;direction:ltr"> STLLIENSSP NSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0533] <h2 style=";text-align:left;direction:ltr"> >prod-bsc-aa(SEQ ID NO:11)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0534] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0535] <h2 style=";text-align:left;direction:ltr"> DVSLGSTWLT DFPQAWAETG GMGLAKRQPP IIIPLKPTAT PVWIKQYPMS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0536] <h2 style=";text-align:left;direction:ltr"> KEARQGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN DYRPVQDLRE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0537] <h2 style=";text-align:left;direction:ltr"> VNKRVQDIHP TVPNPCNLLS SLPPSHQWYT VLDLKDAFFC LRLHPKSQPL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0538] FAFEWRDPER GISGQLTWTRLPQGFKNSPT LFHEALRRDL ADFRTQHPDL

[0539] TLLQYVDDLL LAAETEEDCE QGTRDLLQTL GELGYRASAK KAQICQQEVT

[0540] YLGYILKEGQ RWLSEARKET VMKQPTPKTP RQLREFLGKA GFCRLWIPGF

[0541] AEMAAPLYPL TKTGTLFQWG EEQQKAFDDI KRALLTAPAL GLPDLTKPFE

[0542] LFVDEKQGFA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMIAAIA

[0543] VLVKDADKLT MGQPLTILAP HAVESLIKQP PDRWLSNARM THYQALLLDT

[0544] DRVQFGPVVA LNPATLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA

[0545] EHTWYTGGSS FLQEGQRKAG AAVTTTETEVI WASALPPGTS AQRAQLIALT

[0546] QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRRGLLTSE GKEIKNKEEI

[0547] LELLKALWLP KKLAIIHCPG HQKGDSAEAR GNRMADQAAR EAAITETPDT

[0548] STLLIENSSP NSRLIN

[0549] >prod-csd-aa(SEQ ID NO:12)

[0550] MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KHMTLNIEDE HRLHETSKEP

[0551] DVSLGSTWLT DFPQAWAETG GMGLAKRQPP IIIPLKPTAT PVWIKQYPMS

[0552] KEARQGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN DYRPVQDLRE

[0553] VNKRVQDIHP TVPNPCNLLS SLPPSHQWYT VLDLKDAFFC LRLHPKSQPL

[0554] FAFEWRDPER GISGQLTWTRLPQGFKNSPT LFHEALRRDL ADFRTQHPDL

[0555] TLLQYVDDLL LAAETEEEDCE QGTRDLLQTL GELGYRASAK KAQICQQEVT

[0556] YLGYILKEGQ RWLSEARKET VLKQPTPKTP RQLREFLGKA GNCRLWIPGF

[0557] AEMAAPLYPL TKTGTLFQWG EEQQKAFDDI KRALLTAPAL GLPDLTKPFE

[0558] LFVDEKQGFA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMIAAIA

[0559] VLVKDADKLT MGQPLTILAP HAVESLIKQP PDRWLSNARM THYQALLLDT

[0560] DRVQFGPVVA LNPATLLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA

[0561] EHTWYTGGSS FLQEGQRKAG AAVTTETEVI WASALPPGTS AQRAQLIALT

[0562] QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRRGLLTSE GKEIKNKEEI

[0563] LELLKALWLP KKLAIIHCPG HQKGDSAEAR GNRMADQAAR EAAITETPDT

[0564] STLLIENSSP NSRLIN

[0565] >prod-dsf-aa(SEQ ID NO:13)

[0566] MGGSHHHHHH GMASMTGGQQ MGRDSOUNDDDDD KHMTLNIED SAFETY SHIP

[0567] DVSLGSTWLS DFPQAWAETG GMGLAKRQAP LIIPLKPTAT PVWIKQYPMS

[0568] QEARQGIKPH IQRLLDQGIL VPCQSPWNTP LLPVKKPGTN ANIMALS

[0569] VNKRVEDIHP TVPNPYNLLS SLPPSHQWYT VLDLKDAFFC LRLHPKSQPL

[0570] FAFEWRDPEM GISGQLTWTR LPQGFKNSPT LFHEALHRDL ADFRIQHPDL

[0571] ILLQYVDDLL LAATTEEDCE QGTRALLQTL GELGYRASAK KAQICQKEVT

[0572] VALID RWLTEARKET VMKQPTPKTP RQLREFLGTA GFCRLWIPGF

[0573] AEMAAPLYPL TKTGTLFNWG PEQQKAFQEI KQALLTAPAL GLPDLTKPFE

[0574] LFVDEKQGFA KGVLTQKLGP WRRPVAYLSK KLDPVAAGWP PCLRMVAAIA

[0575] VLVKDADKLT MGQPLTILAP GARDENALIKQP PDRWLSNARM THYQALLLDT

[0576] <h2 style=";text-align:left;direction:ltr">DRVQFGPVVA LNPATLLPLP EEGLQHNCLD ILAEAHGTRP DLTDQPLPDA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0577] <h2 style=";text-align:left;direction:ltr"> DHTWYTGGSS FLQEGQRKAG AAVTTETEVI WAKALPPGTS AQRAQLIALT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0578] <h2 style=";text-align:left;direction:ltr"> QALKMAEGKK LNVYTNSRYA FATAHIHGEI YRRRRGLLTSE GKEIKNKEEI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0579] <h2 style=";text-align:left;direction:ltr"> LALLKALWLP KRLSIIHCPG HQKGDSAEAR GNRMADQAAR EAAITETPDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0580] <h2 style=";text-align:left;direction:ltr"> STLLIENSSP NSRLIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0581] <h2 style=";text-align:left;direction:ltr"> >prod-blh(SEQ ID NO:14)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0582] <h2 style=";text-align:left;direction:ltr"> MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKHMTLNIEDEHRLHETSKEPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0583] <h2 style=";text-align:left;direction:ltr"> VSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0584] <h2 style=";text-align:left;direction:ltr"> QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0585] <h2 style=";text-align:left;direction:ltr"> GLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0586] <h2 style=";text-align:left;direction:ltr"> TLFDEALRRRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRAS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0587] <h2 style=";text-align:left;direction:ltr"> AKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGNCRL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0588] <h2 style=";text-align:left;direction:ltr"> WIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0589] EKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLT

[0590] MGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLP

[0591] LPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE

[0592] TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHGEIYRRR

[0593] GLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAI

[0594] TETPDTSTLL

[0595] >prod-ajw(SEQ ID NO:15)

[0596] MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKHMTLNIEDEHRLHETSKEPD

[0597] VSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARQGIKPHI

[0598] QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLS

[0599] SLPPSHQWYTVLDLKDAFFCLRLHPKSQPLFAEWRDPEMGISGQLTWTRLPQGFKNSP

[0600] TLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATTEEDCQQGTRALLQTLGELGYRAS

[0601] AKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMKQPTPKTPRQLREFLGTAGFCRL

[0602] WIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAFQEIKQALLTAPALGLPDLTKPFELFVD

[0603] EKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLVKDADKLT

[0604] MGQPLVILAPHAVEALIKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLP

[0605] LPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE

[0606] TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHGEIYRRR

[0607] GLLTSEGKEIKNKEEILALLKALFLPKRLSIIHCPGHQKGDSAEARGNRMADQAARKAAI

[0608] TETPDTSTLLIENSSPNSRLIN

[0609] Incorporation by Reference

[0610] Other documents, such as patents, patent applications, patent publications, journals, books, papers, and web content, are referenced and cited throughout this disclosure. All of the aforementioned documents are hereby incorporated herein by reference in their entirety for all purposes.

[0611] Equivalent

[0612] Various modifications of the invention other than those shown and described herein and many other embodiments thereof will become apparent to those skilled in the art from the full content of this document, which includes references to the scientific and patent literature cited herein. The subject matter herein contains important information, examples, and guidance that can be adapted for implementing the invention in its various embodiments and their equivalents.

Claims

1. A modified reverse transcriptase comprising at least one modification in the reverse transcriptase sequence shown in SEQ ID NO:2, wherein the modified reverse transcriptase exhibits improved thermal stability relative to 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 reverse transcription activity of the reverse transcriptase shown in SEQ ID NO:2 at 42°C.

2. The modified reverse transcriptase of claim 1, wherein the modification comprises an amino acid substitution at an amino acid position selected from the group consisting of 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 modified reverse transcriptase according to claim 2, wherein the amino acid substitution is selected from: S at position 56 is substituted with G, A, V, L or I; L at position 72 is substituted with Q or R; G at position 138 substituted with S, C, T or M; H at position 204 is substituted with K or R; S at position 232 is substituted with C, T or M; L at position 234 is substituted with D, E, N, Q; G at position 290 is substituted with H, K or R; T at position 306 is substituted with H, K or R; Y at position 344 substituted with F or M; T at position 420 is 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 is substituted with D, N or Q; D at position 583 is substituted with E, N or Q; D at position 615 substituted with E, N or Q; and At position 642, H is substituted with D, E, N or Q.

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

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

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

7. The modified reverse transcriptase of claim 2, wherein the amino acid substitution is selected from the group consisting of L72Q, T163K, N249E and H642D.

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

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

10. The modified reverse transcriptase of claim 2, wherein the amino acid substitution is selected from 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, E346D, Q349R, Y376F, V413I, T420V, G424D, A442S, V433 T, V444I, D518E, G524D, L528F, A554P, E562Q, D583N, K550S, D615E, A619E, F625W / H, R629K, H642D and K658E.

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

12. The modified reverse transcriptase according to claim 1, wherein the activity of the reverse transcriptase is increased compared to the reverse transcriptase shown in SEQ ID NO: 2 in the presence of a reverse transcription inhibitor.

13. The modified reverse transcriptase of claim 1, wherein the reverse transcriptase exhibits improved thermal stability relative to the reverse transcriptase of SEQ ID NO: 2, the reverse transcriptase comprising at least one amino acid substitution 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; 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.

14. The modified reverse transcriptase of claim 1, wherein the reverse transcription activity of the reverse transcriptase is improved relative to the reverse transcriptase of SEQ ID NO: 2 in the presence of a reverse transcription inhibitor, the reverse transcriptase comprising at least one amino acid substitution 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; A30 7V; 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.

15. The modified reverse transcriptase of claim 1, wherein the reverse transcription activity of the reverse transcriptase is improved relative to the reverse transcriptase of SEQ ID NO: 2 in a formalin-fixed sample, the reverse transcriptase comprising at least one amino acid substitution 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; A30 7V; 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.

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

17. 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 in the presence of a reverse transcription inhibitor, the reverse transcriptase comprising 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 transcription activity of the reverse transcriptase is improved relative to the reverse transcriptase of SEQ ID NO: 2 for formalin-fixed samples, the reverse transcriptase comprising at least one amino acid substitution selected from the group consisting of: A32V, E286R, E302K, W388R and L435R.

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

1.

20. A method for reverse transcribing cDNA from RNA, the method comprising the following steps: For samples containing RNA: A modified reverse transcriptase comprising a modification in at least one domain of at least one reverse transcriptase of SEQ ID NO: 2, wherein the modified reverse transcriptase exhibits improved thermal stability relative to the reverse transcriptase of SEQ ID NO: 2, such that the activity of the reverse transcriptase at a temperature of 50° C. or higher is at least 90% of the reverse transcription activity of the reverse transcriptase of SEQ ID NO: 2 at 42° C., and Reagents for reverse transcription; and A reverse transcription reaction is performed between the reverse transcriptase and the RNA in the sample, wherein the reverse transcription reaction is performed at a temperature of 50° C. or higher to form cDNA.

21. The method of claim 20, wherein the modification comprises an amino acid substitution at an amino acid position selected from the group consisting of 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 method of claim 21, wherein the amino acid substitution is selected from the group consisting of: S at position 56 is substituted with G, A, V, L or I; L at position 72 is substituted with Q or R; G at position 138 substituted with S, C, T or M; H at position 204 is substituted with K or R; S at position 232 is substituted with C, T or M; L at position 234 is substituted with D, E, N, Q; G at position 290 is substituted with H, K or R; T at position 306 is substituted with H, K or R; Y at position 344 substituted with F or M; T at position 420 is 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 is substituted with D, N or Q; D at position 583 is substituted with E, N or Q; D at position 615 substituted with E, N or Q; and At position 642, H is substituted with D, E, N or Q.

23. 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, wherein the amino acid positions are selected from positions 56, 72, 138, 204, 232, 234, 290, 306, 344, 420, 424, 444, 524, 562, 583, 615, and 642.

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

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

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

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

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

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

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

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

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

33. The method of claim 20, wherein the operating step is performed at 65°C or higher, and wherein the activity of the reverse transcription reaction is at least 50% of the activity of a reverse transcription reaction performed at 42°C using the reverse transcriptase of SEQ ID NO: 2.

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