Reverse transcriptase with improved performance

Abstract

The present invention relates to a reverse transcriptase comprising a modified Moloney murine leukemia virus reverse transcriptase amino acid sequence wherein the amino acid sequence comprises a mutation relative to the wild type Moloney murine leukemia virus reverse transcriptase and the enzyme has enhanced inhibitor resistance compared to the wild type Moloney murine leukemia virus reverse transcriptase. The invention also relates to a composition, a reaction mixture and a kit comprising the reverse transcriptase. The reverse transcriptase is used for synthesizing cDNA from RNA.

Description

Background Technology

[0001] This invention relates to reverse transcriptases with improved performance. Reverse transcriptases are RNA-dependent DNA polymerases that use RNA as a template to synthesize DNA. The synthesis of DNA using RNA as a template is typically followed by polymerase chain reaction (PCR) to rapidly detect and quantify the obtained complementary DNA (cDNA). Therefore, reverse transcriptases are essential reagents used in molecular biology applications.

[0002] Commonly used RT-PCR inhibitors are derived from Moloney murine leukemia virus (MMLV) and avian myeloblastoma virus (AMV). While these derivatives offer certain advantages, they are often insufficiently resistant to inhibitors that may be inherent in some samples, sometimes even after sample purification. Improved inhibitor resistance is particularly important in one-step RT-quantitative PCR (qPCR) and one-step RT-PCR, where cDNA synthesis and qPCR are performed in the same reaction vessel. Another important area requiring improved inhibitor resistance is direct RT-PCR. Therefore, freshly synthesized cDNA is susceptible to contamination from inhibitors from its source, such as blood or soil.

[0003] Furthermore, one-step RT-qPCR is preferred for high-throughput applications and diagnostics because it reduces sample handling, thereby lowering the risk of cross-contamination and experimental errors. Therefore, further reductions in benchtop time are desirable, and thus, the time efficiency of one-step RT-qPCR can be improved by providing reverse transcriptase that eliminates the need for additional repeat experiments and further purification steps.

[0004] In some cases, the disclosed reverse transcriptase contains additional desired properties, such as increased thermal activity, increased thermal stability, improved sustained synthesis capacity, and / or increased cDNA synthesis rate compared to wild-type MMLV reverse transcriptase.

[0005] Therefore, the present invention provides an improved reverse transcriptase based on MMLV reverse transcriptase and at least conferring improved inhibitor resistance. Summary of the Invention

[0006] In one aspect, the present invention provides a reverse transcriptase comprising a modified Moloney mouse leukemia virus (MMLV) reverse transcriptase amino acid sequence. The amino acid sequence contains mutations at positions I416 and / or K267 and / or N674 and / or T186 compared to the wild-type MMLV reverse transcriptase of SEQ ID NO: 1, and the enzyme exhibits enhanced inhibitor resistance compared to the wild-type MMLV reverse transcriptase.

[0007] In one embodiment, the reverse transcriptase has at least two mutations at a location selected from the group consisting of: I416, K267, N674, and T186.

[0008] In another embodiment, the reverse transcriptase has at least two mutations at positions I416 and K267.

[0009] In yet another embodiment, the reverse transcriptase has at least two mutations at positions N674 and K267.

[0010] In one embodiment, the reverse transcriptase contains at least one additional mutation at one or more of the following amino acid positions:

[0011] .

[0012] In one embodiment, the reverse transcriptase contains a mutation selected from the group consisting of I416Q, I416N, I416T, and I416S. In a preferred embodiment, the reverse transcriptase contains the I416T mutation.

[0013] In another embodiment, the reverse transcriptase contains a non-K267T mutation located at position K267. In a preferred embodiment, the reverse transcriptase contains a mutation selected from the group consisting of K267Q, K267N, and K267S. In a particularly preferred embodiment, the reverse transcriptase contains a K267Q mutation.

[0014] In yet another embodiment, the reverse transcriptase contains a mutation selected from the group consisting of N674D or N674E. In a preferred embodiment, the reverse transcriptase contains the N674D mutation.

[0015] In another embodiment, the reverse transcriptase comprises a non-T186C or T186D mutation located at position T186. In a preferred embodiment, the reverse transcriptase comprises a mutation selected from the group consisting of T186A, T186V, T186I, T186L, and T186M. In a particularly preferred embodiment, the reverse transcriptase comprises the T186A mutation.

[0016] In one embodiment, the reverse transcriptase comprises an I416T mutation, a K267Q mutation, an N674D mutation, and / or a T186A mutation. In a preferred embodiment, the reverse transcriptase comprises an I416T mutation, a K267Q mutation, and / or an N674D mutation. In another preferred embodiment, the reverse transcriptase comprises an I416T mutation and a K267Q mutation.

[0017] In another embodiment, reverse transcriptase confers enhanced resistance to heme chloride, tannic acid, humic acid, and guanidine isothiocyanate.

[0018] In another aspect, the present invention provides a composition comprising reverse transcriptase according to any of the foregoing embodiments.

[0019] In another aspect, the present invention provides a reaction mixture comprising the reverse transcriptase according to any of the foregoing embodiments.

[0020] Another aspect of the present invention is the use of the reverse transcriptase according to any of the foregoing embodiments for the reverse transcription of RNA.

[0021] In another aspect, the present invention provides a kit comprising the reverse transcriptase of any of the foregoing embodiments.

[0022] In another aspect, the present invention provides a polynucleotide encoding a reverse transcriptase of any of the foregoing embodiments. In one embodiment, the polynucleotide is codon-optimized for expression in a target host.

[0023] In one aspect, the present invention provides a plasmid comprising the polynucleotides according to the foregoing aspects and embodiments.

[0024] In another aspect, the present invention provides a cell comprising a reverse transcriptase according to any of the foregoing embodiments or a polynucleotide according to the foregoing aspects and related embodiments.

[0025] In another aspect, the present invention provides a method for reverse transcription of RNA. In one embodiment, a reverse transcriptase according to any of the foregoing embodiments is used to synthesize cDNA from RNA in a sample. In another embodiment, the sample is selected from the group consisting of biological samples, environmental samples, and food samples. In a preferred embodiment, the biological sample, environmental sample, or food sample is a treated or purified sample. In equally preferred embodiments, the biological sample, environmental sample, or food sample is an untreated or unpurified sample.

[0026] definition

[0027] "cDNA synthesis rate" refers to the ability of reverse transcriptase to synthesize a specific amount of cDNA within a specific time period under appropriate reaction conditions. An increased rate may be related to increased affinity for the template, improved sustained synthesis ability, and increased nucleotide incorporation rate.

[0028] "Inhibitor resistance" refers to the ability of reverse transcriptase to reverse transcribe in the presence of compounds, chemicals, proteins, buffers, etc., that typically inhibit reverse transcriptase (prevent or suppress reverse transcriptase activity).

[0029] Exemplary compounds or chemicals include, for example, detergents, anticoagulants, or polysaccharides. In particular, the reverse transcriptase according to the invention confers enhanced resistance to heme chloride, tannic acid, humic acid, and guanidine isothiocyanate. More specifically, the reverse transcriptase according to the invention exhibits enhanced resistance compared to wild-type MMLV reverse transcriptase (SEQ ID NO: 1), enabling it to perform reverse transcription in the presence of up to 15 µM heme chloride, up to 7.5 ng / µL tannic acid, up to 10 ng / µL humic acid, and up to 80 mM guanidine isothiocyanate. The reverse transcriptase according to the invention also confers resistance to heparin, xylan, isopropanol, and ELUGENT. TM Enhanced resistance to detergents.

[0030] The "continuous synthesis capacity" of reverse transcriptase refers to the number of nucleotides incorporated by the enzyme in a single binding event. Therefore, reverse transcriptases with high continuous synthesis capacity can synthesize longer cDNA chains in a shorter reaction time. The continuous synthesis capacity of an enzyme is also related to its affinity for the template.

[0031] "Sample" refers to a sample or specimen from a biological, environmental, or food source. Biological, environmental, and food samples contain target nucleic acids that can be detected and quantified. Samples can be fresh, frozen, or preserved, such as those preserved in formalin. Samples according to the invention may contain additional compounds, such as drug metabolites, antibiotics, anticoagulants, chemicals (such as preservatives, fixatives, or buffers), nutrients, fertilizers, etc. In some cases, the sample originates from a contaminated source.

[0032] A biological sample is a sample from a subject, which may be an animal, such as a mammal or a human. The sample or specimen may be solid, liquid, tissue, or cells. Biological samples also refer to cultures, such as cell cultures or microbial cultures. This includes all biological fluids and excretions.

[0033] Environmental samples include both samples from nature (such as soil or water) and the surrounding environment, including non-natural environments or surrounding environments such as the interior of buildings, such as walls, floors, surfaces, ventilation equipment, drains, conveyor belts and containers.

[0034] A food sample is a sample from any stage of a food or beverage source. The sample can be raw material, material being processed, an "in-process sample," or a sample from the final food product.

[0035] A processed sample is a sample that has undergone a defined process or treatment. This defined process or treatment may include multiple steps. In some cases, a purified sample (see definition below) undergoes a defined process or treatment to become a processed sample.

[0036] A purified sample is one from which any potentially harmful, unclean, or substandard substances have been removed. In other words, substances considered contaminants are removed from a purified sample. In some cases, a purified sample is a treated sample.

[0037] "Target host" refers to any suitable organism or its cellular subunit capable of carrying a plasmid or polynucleotide containing the reverse transcriptase according to the invention. Therefore, a suitable organism can be, for example, a yeast cell, prokaryotic cell, or eukaryotic cell or cell line. In some cases, the target host can express and thus produce the reverse transcriptase according to the invention.

[0038] "Thermoactive" or "thermally active" usually refers to the ability of reverse transcriptase to exhibit enzyme activity at elevated temperatures.

[0039] "Thermal stability" or "thermal-resistant" usually refers to the ability to withstand exposure to elevated temperatures, but does not necessarily mean that the substance is active at such elevated temperatures.

[0040] "Composition" generally refers to a product containing various components. Here, the composition contains the reverse transcriptase of the present invention. The composition may also contain salts. In a particular example, the composition contains Tris-HCl, KCl, and MgCl2. The composition may further contain actinomycin D, ribonucleotides, and / or dithiothreitol.

[0041] A “reaction mixture” generally refers to a mixture containing two or more components that cause a reaction of the original two or more components. For example, a reaction mixture may be an RT-qPCR or RT-PCR reaction mixture. Such reaction mixtures typically contain target RNA, reverse transcriptase, reverse transcriptase buffer, RNase inhibitors, ribonucleotides, one or more primers, and nuclease-free water. The reaction mixture may further contain additives known to promote or enhance reverse transcription, amplification, or a combination of both reactions (e.g., reagents that promote or enhance RT-PCR). Suitable additives are listed in detail below. The reaction mixture may also further contain one or more surfactants and / or detergents to support stability during storage; these surfactants and / or detergents are also listed in detail below. Additionally, the reaction mixture may further contain one or more dyes or fluorescent groups, which are also listed in detail below.

[0042] In the context of the composition, "stable" and "stability" mean that the composition (such as an enzyme composition) retains at least 70%, preferably at least 80%, and most preferably at least 90% of the original enzyme activity (in units) after the enzyme or the enzyme-containing composition is stored at a temperature of about 4°C for about one week, at a temperature of about -20°C for about two to six months, and at a temperature of about -80°C for about six months or longer.

[0043] "Working concentration" refers to the enzyme concentration that is at or near the optimal concentration for performing a specific function in solution, such as reverse transcription of nucleic acids. Attached Figure Description

[0044] Figure 1. Synthesis of a 1.3 kb cDNA fragment from GAPDH RNA using wild-type MMLV reverse transcriptase or its variants containing a single amino acid substitution shown above the image. The reaction mixture contained an increased amount of heme chloride. The numbers above the image indicate the concentration of heme chloride (µM) in each reaction mixture. L-ZipRuler Express 1 DNALadder (ID 1373, Thermo Scientific).

[0045] Figure 2. Resistance to inhibitors in single-point mutants compared to wild-type MMLV reverse transcriptase. Colors indicate the amount of 1.3 kb cDNA fragment obtained during the reverse transcription reaction: white – same amount of product (1.3 kb cDNA fragment) as the negative control (reaction mixture without inhibitor); light gray – reduced product amount; dark gray – no detectable product.

[0046] Figure 3. 0.5–9 kb cDNA fragments synthesized from Millennium™ RNA markers (Invitrogen, #AM7150) using single-point mutants I416T (SEQ ID NO: 2), K267Q (SEQ ID NO: 3), N674D (SEQ ID NO: 4), and T186A (SEQ ID NO: 5) compared to wild-type MMLV reverse transcriptase (SEQ ID NO: 1). The reaction products were analyzed on a 1.4% alkaline agarose gel.

[0047] Figure 4. Thermostability and thermal activity of the single-point mutant I416T (SEQ ID NO: 2) compared to wild-type MMLV reverse transcriptase (SEQ ID NO: 1). 0.5–9 kb cDNA fragments from the Millennium™ RNA marker (Invitrogen, #AM7150) were synthesized at different temperatures shown above the image (42°C, 44°C, 46°C, 48°C, and 50°C, respectively). The reaction products were analyzed on a 1.0% alkaline agarose gel.

[0048] Detailed description

[0049] reverse transcriptase

[0050] In one aspect, the present invention provides a reverse transcriptase comprising a modified Moloney mouse leukemia virus (MMLV) reverse transcriptase amino acid sequence. Compared to wild-type MMLV reverse transcriptase (SEQ ID NO: 1), the amino acid sequence contains mutations at positions I416 and / or K267 and / or N674 and / or T186, wherein the enzyme exhibits enhanced inhibitor resistance compared to the wild-type MMLV reverse transcriptase.

[0051] In one embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to SEQ ID NO: 2, 3, 4, or 5. In another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to SEQ ID NO: 2. In yet another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to SEQ ID NO: 3. In yet another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to SEQ ID NO: 4. In one embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to SEQ ID NO: 5. In a preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO: 2, 3, 4, or 5. In a particularly preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO: 2, 3, 4, or 5.

[0052] In another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to the wild-type MMLV reverse transcriptase. In a preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO: 1. In a particularly preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO: 1.

[0053] In another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to the sequence comprising amino acids 1-671 of SEQ ID NO: 2, 3, or 5. In another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to the sequence comprising amino acids 1-671 of SEQ ID NO: 2. In yet another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to the sequence comprising amino acids 1-671 of SEQ ID NO: 3. In one embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence is substantially identical or substantially homologous to the sequence comprising amino acids 1-671 of SEQ ID NO: 5. In a preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the sequence comprising amino acids 1-671 of SEQ ID NO: 2, 3, or 5. In a particularly preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the sequence comprising amino acids 1-671 of SEQ ID NO: 2, 3, or 5.

[0054] In another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the sequence comprising amino acids 1-671 of SEQ ID NO: 1. In a particularly preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the sequence comprising amino acids 1-671 of SEQ ID NO: 1.

[0055] In yet another embodiment, the mutation at position I416 is any one of I416R, I416H, I416K, I416D, I416E, I416S, I416T, I416N, I416Q, I416C, I416G, I416P, I416A, I416V, I416I, I416L, I416M, I416F, I416Y, and I416W. In a preferred embodiment, the mutation at position I416 is any one of I416Q, I416N, I416T, or I416S. In a particularly preferred embodiment, the mutation at position I416 is I416T.

[0056] In another embodiment, the mutation at position K267 is any one of K267R, K267H, K267K, K267D, K267E, K267S, K267T, K267N, K267Q, K267C, K267G, K267P, K267A, K267V, K267I, K267L, K267M, K267F, K267Y, and K267W. In a preferred embodiment, the mutation at position K267 is any one of K267S, K267N, or K267Q. In another preferred embodiment, the mutation at position K267 is not K267T. In a more preferred embodiment, the mutation at position K267 is K267N or K267Q. In a particularly preferred embodiment, the mutation at position K267 is K267Q.

[0057] In one embodiment, the mutation at position N674 is any one of N674R, N674H, N674K, N674D, N674E, N674S, N674T, N674N, N674Q, N674C, N674G, N674P, N674A, N674V, N674I, N674L, N674M, N674F, N674Y, and N674W. In a preferred embodiment, the mutation at position N674 is any one of N674A, N674V, N674I, N674L, N674M, N674F, N674Y, and N674W. In a particularly preferred embodiment, the mutation at position N674 is either N674D or N674E. In another particularly preferred embodiment, the mutation at position N674 is N674D.

[0058] In one embodiment, the mutation at position T186 is any one of T186R, T186H, T186K, T186D, T186E, T186S, T186T, T186N, T186Q, T186C, T186G, T186P, T186A, T186V, T186I, T186L, T186M, T186F, T186Y, and T186W. In a preferred embodiment, the mutation at position T186 is any one of T186A, T186V, T186I, T186L, T186M, T186F, T186Y, and T186W. In another preferred embodiment, the mutation at position T186 is not T186C or T186D. In a more preferred embodiment, the mutation is selected from the group consisting of T186A, T186V, T186I, T186L, and T186M. In another preferred embodiment, the mutation at position T186 is T186A.

[0059] In one embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence has at least two mutations at positions selected from the group consisting of I416, K267, N674, and T186. In a preferred embodiment, the at least two mutations are located at positions I416 and K267. In another preferred embodiment, the at least two mutations are located at positions N674 and K267.

[0060] In one embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence contains at least one additional mutation at one or more of the following amino acid positions: P51, H204, N249, T306, F309, D524, E562, K571, D583, T330, N479, L603, M289, M66, L139, D200, T287, H594, E607, Q22. 1. I49, A502, D653, K658, P130, Q237, A307, Y344, Q430, D449, A644, N649, L671, E673 , M39, Q91, W388, I179, L333, R390, Q374, E5, E69, E302, W313, L435, N454, D124, E286. In a preferred embodiment, the reverse transcriptase comprises at least one of the following amino acid mutations: P51L, H204R, N249D, T306K, T306R, T306L, F309N, F309R, D524G, D524A, E562Q, K571R, D583N, T330P, N479D, L603W, L603M, M289V, M66L, L139P, D200N, D200A, D200G, T287A, H594R, H594K, H594Q, E607K, E607G, E607A, Q221R, I49V, I49T, A502V , D653G, D653A, D653H, D653V, K658R, K658Q, P130S, Q237R, A307V, Y344H, Q430R, D449G, D449A, A644V, A644T, N649S, L671P, E673G, E673K , M39V, M39L, Q91R, Q91L, W388R, I179T, I179V, L333Q, R390W, Q374R, E5K, E69K, E302R, E302K, W313F, L435G, L435R, N454K, D124R, E286R.

[0061] In another embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence contains the I416T mutation, K267Q mutation, N674D mutation, and / or T186A mutation. In another preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence contains the I416T mutation, K267Q mutation, and / or N674D mutation. In yet another preferred embodiment, the reverse transcriptase comprising the modified MMLV reverse transcriptase amino acid sequence contains both the I416T and K267Q mutations.

[0062] In another embodiment, the reverse transcriptase has at least one of the following mutation groups 1) to 61):

[0063] 1) I416;

[0064] 2) K267;

[0065] 3) N674;

[0066] 4) T186;

[0067] 5) I416, K267;

[0068] 6) K267, N674;

[0069] 7) I416, M289;

[0070] 8) I416, H204, T306;

[0071] 9) I416, D524, D583;

[0072] 10) I416, H204, T306, F309;

[0073] 11)I416, H204, T306, F309, M289;

[0074] 12)I416, H204, T306, F309, D524, D583, M289;

[0075] 13)I416, H204, T306, F309, D524, E562, D583, M289;

[0076] 14)I416, P51, H204, N249, D524, E562, K571, D583;

[0077] 15)I416, P51, H204, N249, D524, E562, K571, D583, M289;

[0078] 16)I416, P51, H204, N249, T306, F309, D524, E562, K571, D583;

[0079] 17)I416, P51, H204, N249, T306, F309, D524, E562, K571, D583, M289;

[0080] 18)K267, M289;

[0081] 19)K267, H204, T306;

[0082] 20)K267, D524, D583;

[0083] 21)K267, H204, T306, F309;

[0084] 22)K267, H204, T306, F309, M289;

[0085] 23)K267, H204, T306, F309, D524, D583, M289;

[0086] 24)K267, H204, T306, F309, D524, E562, D583, M289;

[0087] 25)K267, P51, H204, N249, D524, E562, K571, D583;

[0088] 26)K267, P51, H204, N249, D524, E562, K571, D583, M289;

[0089] 27)K267, P51, H204, N249, T306, F309, D524, E562, K571, D583;

[0090] 28)K267, P51, H204, N249, T306, F309, D524, E562, K571, D583, M289;

[0091] 29) I416, K267, M289;

[0092] 30) I416, K267, H204, T306;

[0093] 31) I416, K267, D524, D583;

[0094] 32) I416, K267, H204, T306, F309;

[0095] 33) I416, K267, H204, T306, F309, M289;

[0096] 34) I416, K267, H204, T306, F309, D524, D583, M289;

[0097] 35) I416, K267, H204, T306, F309, D524, E562, D583, M289;

[0098] 36) I416, K267, P51, H204, N249, D524, E562, K571, D583;

[0099] 37) I416, K267, P51, H204, N249, D524, E562, K571, D583, M289;

[0100] 38) I416, K267, P51, H204, N249, T306, F309, D524, E562, K571, D583;

[0101] 39) I416, K267, P51, H204, N249, T306, F309, D524, E562, K571, D583, M289;

[0102] 40)N674, M289;

[0103] 41)N674, H204, T306;

[0104] 42)N674, D524, D583;

[0105] 43)N674, H204, T306, F309;

[0106] 44)N674, H204, T306, F309, M289;

[0107] 45)N674, H204, T306, F309, D524, D583, M289;

[0108] 46)N674, H204, T306, F309, D524, E562, D583, M289;

[0109] 47) N674, P51, H204, N249, D524, E562, K571, D583;

[0110] 48) N674, P51, H204, N249, D524, E562, K571, D583, M289;

[0111] 49) N674, P51, H204, N249, T306, F309, D524, E562, K571, D583;

[0112] 50)N674, P51, H204, N249, T306, F309, D524, E562, K571, D583, M289;

[0113] 51)T186, M289;

[0114] 52)T186, H204, T306;

[0115] 53)T186, D524, D583;

[0116] 54)T186, H204, T306, F309;

[0117] 55)T186, H204, T306, F309, M289;

[0118] 56)T186, H204, T306, F309, D524, D583, M289;

[0119] 57)T186, H204, T306, F309, D524, E562, D583, M289;

[0120] 58)T186, P51, H204, N249, D524, E562, K571, D583;

[0121] 59)T186, P51, H204, N249, D524, E562, K571, D583, M289;

[0122] 60)T186, P51, H204, N249, T306, F309, D524, E562, K571, D583;

[0123] 61) T186, P51, H204, N249, T306, F309, D524, E562, K571, D583, M289.

[0124] In another embodiment, the reverse transcriptase has at least one of the following mutation groups (62) to (186):

[0125] 62)I416, T330;

[0126] 63)I416, N479;

[0127] 64)I416, L603;

[0128] 65)I416, M66;

[0129] 66)I416, D200;

[0130] 67)I416, T287;

[0131] 68)I416, L139;

[0132] 69)I416, L139, H594;

[0133] 70)I416, L139, E607;

[0134] 71)I416, L139, D653;

[0135] 72)I416, T330, N479, L139;

[0136] 73)I416, T330, L603, L139;

[0137] 74)I416, N479, L603, L139;

[0138] 75)I416, L603, L139;

[0139] 76)I416, T330, L139;

[0140] 77)I416, D200, L139, E607;

[0141] 78)I416, T330, L139, D653;

[0142] 79)I416, T330, N479, L603, L139;

[0143] 80)I416, T330, L603, D200, L139;

[0144] 81)I416, L603, D200, L139, D653;

[0145] 82) I416, D200, L139, E607;

[0146] 83)I416, T330, L603, D200, L139, D653;

[0147] 84)I416, N479, L603, D200, L139, H594;

[0148] 85) I416, T330, L603, D200, L139, E607;

[0149] 86) I416, T330, L603, D200, L139, E607;

[0150] 87)K267, T330, L139;

[0151] 88)K267, N479, L139;

[0152] 89)K267, L603, L139;

[0153] 90)K267, M66, L139;

[0154] 91)K267, D200, L139;

[0155] 92)K267, T287, L139;

[0156] 93)K267, L139;

[0157] 94)K267, L139, H594;

[0158] 95)K267, L139, E607;

[0159] 96)K267, L139, D653;

[0160] 97)K267, T330, N479, L139;

[0161] 98)K267, T330, L603, L139;

[0162] 99)K267, N479, L603, L139;

[0163] 100)K267, L603, L139;

[0164] 101)K267, T330, L139;

[0165] 102)K267, D200, L139, E607;

[0166] 103)K267, T330, L139, D653;

[0167] 104)K267, T330, N479, L603, L139;

[0168] 105)K267, T330, L603, D200, L139;

[0169] 106)K267, L603, D200, L139, D653;

[0170] 107)K267, D200, L139, E607;

[0171] 108)K267, T330, L603, D200, L139, D653;

[0172] 109)K267, N479, L603, D200, L139, H594;

[0173] 110)K267, T330, L603, D200, L139, E607;

[0174] 111)K267, T330, L603, D200, L139, E607;

[0175] 112)I416, K267, T330, L139;

[0176] 113)I416, K267, N479, L139;

[0177] 114) I416, K267, L603, L139;

[0178] 115)I416, K267, M66, L139;

[0179] 116) I416, K267, D200, L139;

[0180] 117)I416, K267, T287, L139;

[0181] 118)I416, K267, L139;

[0182] 119)I416, K267, L139, H594;

[0183] 120)I416, K267, L139, E607;

[0184] 121)I416, K267, L139, D653;

[0185] 122) I416, K267, T330, N479, L139;

[0186] 123) I416, K267, T330, L603, L139;

[0187] 124)I416, K267, N479, L603, L139;

[0188] 125)I416, K267, L603, L139;

[0189] 126)I416, K267, T330, L139;

[0190] 127) I416, K267, D200, L139, E607;

[0191] 128) I416, K267, T330, L139, D653;

[0192] 129) I416, K267, T330, N479, L603, L139;

[0193] 130) I416, K267, T330, L603, D200, L139;

[0194] 131)I416, K267, L603, D200, L139, D653;

[0195] 132) I416, K267, D200, L139, E607;

[0196] 133) I416, K267, T330, L603, D200, L139, D653;

[0197] 134) I416, K267, N479, L603, D200, L139, H594;

[0198] 135) I416, K267, T330, L603, D200, L139, E607;

[0199] 136) I416, K267, T330, L603, D200, L139, E607;

[0200] 137)N674, T330, L139;

[0201] 138)N674, N479, L139;

[0202] 139)N674, L603, L139;

[0203] 140)N674, M66, L139;

[0204] 141)N674, D200, L139;

[0205] 142)N674, T287, L139;

[0206] 143)N674, L139;

[0207] 144)N674, L139, H594;

[0208] 145)N674, L139, E607;

[0209] 146)N674, L139, D653;

[0210] 147)N674, T330, N479, L139;

[0211] 148)N674, T330, L603, L139;

[0212] 149)N674, N479, L603, L139;

[0213] 150)N674, L603, L139;

[0214] 151)N674, T330, L139;

[0215] 152)N674, D200, L139, E607;

[0216] 153)N674, T330, L139, D653;

[0217] 154)N674, T330, N479, L603, L139;

[0218] 155)N674, T330, L603, D200, L139;

[0219] 156)N674, L603, D200, L139, D653;

[0220] 157)N674, D200, L139, E607;

[0221] 158)N674, T330, L603, D200, L139, D653;

[0222] 159)N674, N479, L603, D200, L139, H594;

[0223] 160)N674, T330, L603, D200, L139, E607;

[0224] 161)N674, T330, L603, D200, L139, E607;

[0225] 162)T186, T330, L139;

[0226] 163)T186, N479, L139;

[0227] 164)T186, L603, L139;

[0228] 165)T186, M66, L139;

[0229] 166)T186, D200, L139;

[0230] 167)T186, T287, L139;

[0231] 168)T186, L139;

[0232] 169)T186, L139, H594;

[0233] 170)T186, L139, E607;

[0234] 171)T186, L139, D653;

[0235] 172)T186, T330, N479, L139;

[0236] 173)T186, T330, L603, L139;

[0237] 174)T186, N479, L603, L139;

[0238] 175)T186, L603, L139;

[0239] 176)T186, T330, L139;

[0240] 177)T186, D200, L139, E607;

[0241] 178)T186, T330, L139, D653;

[0242] 179)T186, T330, N479, L603, L139;

[0243] 180)T186, T330, L603, D200, L139;

[0244] 181)T186, L603, D200, L139, D653;

[0245] 182)T186, D200, L139, E607;

[0246] 183)T186, T330, L603, D200, L139, D653;

[0247] 184)T186, N479, L603, D200, L139, H594;

[0248] 185)T186, T330, L603, D200, L139, E607;

[0249] 186)T186, T330, L603, D200, L139, E607.

[0250] In another embodiment, the reverse transcriptase has at least one of the following mutation groups (187) to (256):

[0251] 187)I416, E69;

[0252] 188)I416, E302;

[0253] 189)I416, W313;

[0254] 190)I416, L435;

[0255] 191)I416, N454;

[0256] 192)I416, D124;

[0257] 193)I416, E286;

[0258] 194)I416, E69, E302;

[0259] 195)I416, E302, W313;

[0260] 196)I416, E302, W313, L435;

[0261] 197) I416, E69, E302, W313, L435;

[0262] 198)I416, E302, W313, L435, N454;

[0263] 199)I416, E69, E302, W313, L435, N454;

[0264] 200)I416, E69, E302, W313, L435, N454, D124, E286;

[0265] 201)K267, E69;

[0266] 202)K267, E302;

[0267] 203)K267, W313;

[0268] 204)K267, L435;

[0269] 205)K267, N454;

[0270] 206)K267, D124;

[0271] 207)K267, E286;

[0272] 208)K267, E69, E302;

[0273] 209)K267, E302, W313;

[0274] 210)K267, E302, W313, L435;

[0275] 211)K267, E69, E302, W313, L435;

[0276] 212)K267, E302, W313, L435, N454;

[0277] 213)K267, E69, E302, W313, L435, N454;

[0278] 214)K267, E69, E302, W313, L435, N454, D124, E286;

[0279] 215)I416, K267, E69;

[0280] 216)I416, K267, E302;

[0281] 217)I416, K267, W313;

[0282] 218)I416, K267, L435;

[0283] 219)I416, K267, N454;

[0284] 220)I416, K267, D124;

[0285] 221)I416, K267, E286;

[0286] 222)I416, K267, E69, E302;

[0287] 223)I416, K267, E302, W313;

[0288] 224)I416, K267, E302, W313, L435;

[0289] 225)I416, K267, E69, E302, W313, L435;

[0290] 226)I416, K267, E302, W313, L435, N454;

[0291] 227) I416, K267, E69, E302, W313, L435, N454;

[0292] 228)I416, K267, E69, E302, W313, L435, N454, D124, E286;

[0293] 229)N674, E69;

[0294] 230)N674, E302;

[0295] 231)N674, W313;

[0296] 232)N674, L435;

[0297] 233)N674, N454;

[0298] 234)N674, D124;

[0299] 235)N674, E286;

[0300] 236)N674, E69, E302;

[0301] 237)N674, E302, W313;

[0302] 238)N674, E302, W313, L435;

[0303] 239)N674, E69, E302, W313, L435;

[0304] 240)N674, E302, W313, L435, N454;

[0305] 241)N674, E69, E302, W313, L435, N454;

[0306] 242)N674, E69, E302, W313, L435, N454, D124, E286;

[0307] 243)T186, E69;

[0308] 244)T186, E302;

[0309] 245)T186, W313;

[0310] 246)T186, L435;

[0311] 247)T186, N454;

[0312] 248)T186, D124;

[0313] 249)T186, E286;

[0314] 250)T186, E69, E302;

[0315] 251)T186, E302, W313;

[0316] 252)T186, E302, W313, L435;

[0317] 253)T186, E69, E302, W313, L435;

[0318] 254)T186, E302, W313, L435, N454;

[0319] 255)T186, E69, E302, W313, L435, N454;

[0320] 256)T186, E69, E302, W313, L435, N454, D124, E286;

[0321] In another embodiment, the reverse transcriptase has at least one of the following mutation groups 257) to 311):

[0322] 257) 7 and one of 62–186; 258) 8 and one of 62–186; 259) 9 and one of 62–186; 260) 10 and one of 62–186; 261) 11 and one of 62–186; 262) 12 and one of 62–186; 263) 13 and one of 62–186; 264) 14 and one of 62–186; 265) 15 and one of 62–186; 266) 16 and one of 62–186; 267) 17 and one of 62–186; 268) 18 and one of 62–186; 269) 19 and one of 62–186; 270) 20 and 62–186 One of; 271) 21 and one of 62–186; 272) 22 and one of 62–186; 273) 23 and one of 62–186; 274) 24 and one of 62–186; 275) 25 and one of 62–186; 276) 26 and one of 62–186; 277) 27 and one of 62–186; 278) 28 and one of 62–186; 279) 29 and one of 62–186; 280) 30 and one of 62–186; 281) 31 and one of 62–186; 282) 32 and one of 62–186; 283) 33 and one of 62–186; 284) 34 285) 35 and one of 62–186; 286) 36 and one of 62–186; 287) 37 and one of 62–186; 288) 38 and one of 62–186; 289) 39 and one of 62–186; 290) 40 and one of 62–186; 291) 41 and one of 62–186; 292) 42 and one of 62–186; 293) 43 and one of 62–186; 294) 44 and one of 62–186; 295) 45 and one of 62–186; 296) 46 and one of 62–186; 297) 47 and 62–186 One of; 298) 48 and one of 62–186; 299) 49 and one of 62–186; 300) 50 and one of 62–186; 301) 51 and one of 62–186; 302) 52 and one of 62–186;303) One of 53 and 62–186; 304) One of 54 and 62–186; 305) One of 55 and 62–186; 306) One of 56 and 62–186; 307) One of 57 and 62–186; 308) One of 58 and 62–186; 309) One of 59 and 62–186; 310) One of 60 and 62–186; 311) One of 61 and 62–186.

[0323] In another embodiment, the reverse transcriptase has at least one of the following mutation groups (312) to (366):

[0324] 312) 7 and one of 187–256; 313) 8 and one of 187–256; 314) 9 and one of 187–256; 315) 10 and one of 187–256; 316) 11 and one of 187–256; 317) 12 and one of 187–256; 318) 13 and one of 187–256; 319) 14 and one of 187–256; 320) 15 and one of 187–256; 321) 16 and one of 187–256; 322) 17 and one of 187–256; 323) 18 and one of 187–256; 324) 19 and one of 187–256 One of; 325) 20 and one of 187–256; 326) 21 and one of 187–256; 327) 22 and one of 187–256; 328) 23 and one of 187–256; 329) 24 and one of 187–256; 330) 25 and one of 187–256; 331) 26 and one of 187–256; 332) 27 and one of 187–256; 333) 28 and one of 187–256; 334) 29 and one of 187–256; 335) 30 and one of 187–256; 336) 31 and one of 187–256; 337) 32 and 187–256 One of; 338) 33 and one of 187–256; 339) 34 and one of 187–256; 340) 35 and one of 187–256; 341) 36 and one of 187–256; 342) 37 and one of 187–256; 343) 38 and one of 187–256; 344) 39 and one of 187–256; 345) 40 and one of 187–256; 346) 41 and one of 187–256; 347) 42 and one of 187–256; 348) 43 and one of 187–256; 349) 44 and one of 187–256; 350) 45 and 187–256 One of; 351) 46 and one of 187–256; 352) 47 and one of 187–256; 353) 48 and one of 187–256; 354) 49 and one of 187–256; 355) 50 and one of 187–256;356) One of 51 and 187–256; 357) One of 52 and 187–256; 358) One of 53 and 187–256; 359) One of 54 and 187–256; 360) One of 55 and 187–256; 361) One of 56 and 187–256; 362) One of 57 and 187–256; 363) One of 58 and 187–256; 364) One of 59 and 187–256; 365) One of 60 and 187–256; 366) One of 61 and 187–256.

[0325] In another embodiment, the reverse transcriptase has at least one of the following mutation groups (367) to (491):

[0326] 367) One of 62 and 187–256; 368) One of 63 and 187–256; 369) One of 64 and 187–256; 370) One of 65 and 187–256; 371) One of 66 and 187–256; 372) One of 67 and 187–256; 373) One of 68 and 187–256; 374) One of 69 and 187–256; 375) One of 70 and 187–256; 376) One of 71 and 187–256; 377) One of 72 and 187–256; 378) One of 73 and 187–256; 379) One of 74 and 187–256 One of; 380) 75 and one of 187–256; 381) 76 and one of 187–256; 382) 77 and one of 187–256; 383) 78 and one of 187–256; 384) 79 and one of 187–256; 385) 80 and one of 187–256; 386) 81 and one of 187–256; 387) 82 and one of 187–256; 388) 83 and one of 187–256; 389) 84 and one of 187–256; 390) 85 and one of 187–256; 391) 86 and one of 187–256; 392) 87 and 187–256 One of; 393) 88 and one of 187-256; 394) 89 and one of 187-256; 395) 90 and one of 187-256; 396) 91 and one of 187-256; 397) 92 and one of 187-256; 398) 93 and one of 187-256; 399) 94 and one of 187-256; 400) 95 and one of 187-256; 401) 96 and one of 187-256; 402) 97 and one of 187-256; 403) 98 and one of 187-256; 404) 99 and one of 187-256; 405) 100 and 187-256 One of; 406) 101 and 187-256; 407) 102 and 187-256; 408) 103 and 187-256; 409) 104 and 187-256;410) One of 105 and 187-256; 411) One of 106 and 187-256; 412) One of 107 and 187-256; 413) One of 108 and 187-256; 414) One of 109 and 187-256; 415) One of 110 and 187-256; 416) One of 111 and 187-256; 417) One of 112 and 187-256; 418) One of 113 and 187-256; 419) One of 114 and 187-256; 420) One of 115 and 187-256; 421) One of 116 and 187-256; 422) 117 and one of 187-256; 423) 118 and one of 187-256; 424) 119 and one of 187-256; 425) 120 and one of 187-256; 426) 121 and one of 187-256; 427) 122 and one of 187-256; 428) 123 and one of 187-256; 429) 124 and one of 187-256; 430) 125 and one of 187-256; 431) 126 and one of 187-256; 432) 127 and one of 187-256; 433) 128 and one of 187-256; 434) 129 One of 187-256; 435) 130 One of 187-256; 436) 131 One of 187-256; 437) 132 One of 187-256; 438) 133 One of 187-256; 439) 134 One of 187-256; 440) 135 One of 187-256; 441) 136 One of 187-256; 442) 137 One of 187-256; 443) 138 One of 187-256; 444) 139 One of 187-256; 445) 140 One of 187-256; 446) 141 One of 187–256; 447) 142 one of 187–256; 448) 143 one of 187–256; 449) 144 one of 187–256; 450) 145 one of 187–256; 451) 146 one of 187–256;452) One of 147 and 187–256; 453) One of 148 and 187–256; 454) One of 149 and 187–256; 455) One of 150 and 187–256; 456) One of 151 and 187–256; 457) One of 152 and 187–256; 458) One of 153 and 187–256; 459) One of 154 and 187–256; 460) One of 155 and 187–256; 461) One of 156 and 187–256; 462) One of 157 and 187–256; 463) One of 158 and 187–256; 464) One of 159 and 187–256; 465) One of 160 and 187–256; 466) One of 161 and 187–256; 467) One of 162 and 187–256; 468) One of 163 and 187–256; 469) One of 164 and 187–256; 470) One of 165 and 187–256; 471) One of 166 and 187–256; 472) One of 167 and 187–256; 473) One of 168 and 187–256; 474) One of 169 and 187–256; 475) One of 170 and 187–256; 476) 171 477) 172 and one of 187–256; 478) 173 and one of 187–256; 479) 174 and one of 187–256; 480) 175 and one of 187–256; 481) 176 and one of 187–256; 482) 177 and one of 187–256; 483) 178 and one of 187–256; 484) 179 and one of 187–256; 485) 180 and one of 187–256; 486) 181 and one of 187–256; 487) 182 and one of 187–256; 488) 183 One of 187–256; 489) 184 one of 187–256; 490) 185 one of 187–256; 491) 186 one of 187–256.

[0327] In a preferred embodiment, the reverse transcriptase has at least one of mutation groups 1) to 491, wherein the reverse transcriptase comprises at least one of the following amino acid mutations: P51L, H204R, N249D, T306K, T306R, T306L, F309N, F309R, D524G, D524A, E562Q, K571R, D583N, T330P, N479D, L603W, L603M, M289V, M66L, L139P, D200N, D200A, D200G, T287A, H594R, H594K, H594Q, E607K, E607G, E607A, D653G, D653A, D6 53H, D653V, K658R, K658Q, E69K, E302R, E302K, W313F, L435G, L435R, N454K, D124R, E286R.

[0328] In another embodiment, the reverse transcriptase has at least one group of mutation groups 1) to 491), wherein the reverse transcriptase further comprises at least one mutation at an amino acid position selected from: Q221, I49, A502, K658, P130, Q237, A307, Y344, Q430, D449, A644, N649, L671, E673, M39, Q91, W388, I179, L333, R390, Q374, E5. In a preferred embodiment, the reverse transcriptase comprises at least one of the following amino acid mutations: Q221R, I49V, I49T, A502V, P130S, Q237R, A307V, Y344H, Q430R, D449G, D449A, A644V, A644T, N649S, L671P, E673G, E673K, M39V, M39L, Q91R, Q91L, W388R, I179T, I179V, L333Q, R390W, Q374R, E5K.

[0329] Inhibitor resistance

[0330] In some embodiments, the reverse transcriptase confers enhanced resistance to inhibitors. In one embodiment, the inhibitor is a PCR inhibitor, such as antiviral substances, hemoglobin, IgG, myoglobin, heme chloride, lactoferrin, hormones, heparin, (complex)polysaccharides, lipids, urate, bile salts, glycogen, (poly)phenols, pectin, xylan, proteases, metal ions, calcium ions, debris, fulvic acid, porphyrin, polyphenols, formalin, paraffin, humic substances, bone meal, coprolites, peat extracts, clay-rich soils, and denaturants. In a preferred embodiment, the reverse transcriptase confers enhanced resistance to porphyrins, polyphenols, humic substances, and denaturants. In a particularly preferred embodiment, the reverse transcriptase confers enhanced resistance to heme chloride, tannic acid, humic acid, and guanidine isothiocyanate. In another particularly preferred embodiment, the reverse transcriptase according to the invention confers enhanced resistance to heparin, xylan, isopropanol, and ELUGENT. TM Detergent-enhanced resistance. In one embodiment, the reverse transcriptase exhibits enhanced resistance compared to wild-type MMLV reverse transcriptase, enabling the reverse transcriptase according to the invention to perform reverse transcription in the presence of up to 15 µM heme chloride, up to 7.5 ng / µL tannic acid, up to 10 ng / µL humic acid, and up to 80 mM guanidine isothiocyanate.

[0331] Further improved features

[0332] In one embodiment, the reverse transcriptase according to the invention increases the rate of cDNA synthesis by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to wild-type MMLV reverse transcriptase, such as by using a methyl group labeled with 3 The incorporation of nucleotides into deoxythymidine triphosphate (dTTP) (also known as tritium-labeled dTTP) by reverse transcriptase is measured. Typically, nucleotide incorporation (used here to monitor cDNA synthesis rate) is measured in units / mg and observed at predetermined time intervals. Those skilled in the art know how to perform this assay.

[0333] In another embodiment, the reverse transcriptase according to the invention increases the rate of cDNA synthesis, which is measured by a Cq value and compared with the Cq value of wild-type MMLV reverse transcriptase. Therefore, in yet another embodiment, the reverse transcriptase according to the invention exhibits enhanced inhibitor resistance and increases the rate of cDNA synthesis compared to wild-type MMLV reverse transcriptase.

[0334] In another embodiment, the reverse transcriptase according to the invention exhibits improved sustained synthesis capacity compared to wild-type MMLV reverse transcriptase. In a particularly preferred embodiment, the reverse transcriptase according to the invention can add 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides in a single binding event. In another embodiment, the reverse transcriptase according to the invention can add 500, 1000, or 1500 nucleotides in a single binding event, corresponding to sustained synthesis capacity that is 22 times, 43 times, and 65 times that of wild-type MMLV reverse transcriptase, respectively.

[0335] In another embodiment, the reverse transcriptase according to the present invention exhibits a higher affinity for the target template compared to the wild-type MMLV reverse transcriptase. Affinity for the target template is typically determined by the equilibrium dissociation constant (K0). D To measure. In a preferred embodiment, the K of the reverse transcriptase according to the invention. D The value is less than the K of wild-type MMLV reverse transcriptase. D .

[0336] In another embodiment, the reverse transcriptase exhibits optimal activity at 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, and 60°C. In yet another embodiment, compared to wild-type MMLV reverse transcriptase, this reverse transcriptase exhibits higher activity at temperatures of 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, and 60°C. In one embodiment, compared to wild-type MMLV reverse transcriptase, this reverse transcriptase exhibits higher activity at 55°C. In a preferred embodiment, the reverse transcriptase exhibiting higher activity at 55°C compared to wild-type MMLV reverse transcriptase is a reverse transcriptase containing the I416T mutation, K267Q mutation, N674D mutation, or T186A mutation. In a particularly preferred embodiment, the reverse transcriptase exhibiting higher activity at 55°C compared to wild-type MMLV reverse transcriptase is a reverse transcriptase containing the I416T mutation. In another embodiment, thermostability is determined by measuring the activity of the reverse transcriptase after heat treatment. In one embodiment, the thermostability is increased by at least 10%, 20%, 30%, 40%, or 50% compared to wild-type MMLV reverse transcriptase.

[0337] Uses of reverse transcriptase

[0338] In another aspect of the invention, the reverse transcriptase according to any of the foregoing embodiments is used for reverse transcription of RNA. Reverse transcription is used in various laboratory applications. Thus, in one embodiment, the reverse transcriptase according to any of the foregoing embodiments is used in laboratory applications such as reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), cDNA cloning and library construction, rapid amplification of cDNA ends (RACE), gene expression microarrays, RNA sequencing (RNA-Seq), reverse transcription loop-mediated isothermal amplification (RT-LAMP), single-cell and next-generation sequencing, or dPCR.

[0339] Therefore, in a preferred embodiment, the reverse transcriptase of the present invention can be used to synthesize DNA using RNA as a template, thereby obtaining complementary DNA (cDNA) for RT-PCR or RT-qPCR.

[0340] In another preferred embodiment, the RT-qPCR is a one-step RT-qPCR or a two-step RT-qPCR. In a preferred embodiment, the RT-qPCR is a one-step RT-qPCR. In an equally preferred embodiment, the RT-PCR is a two-step RT-qPCR.

[0341] In another embodiment, the RT-PCR is a one-step RT-PCR or a two-step RT-PCR. In a preferred embodiment, the RT-PCR is a one-step RT-PCR. In an equally preferred embodiment, the RT-PCR is a two-step RT-PCR.

[0342] Compositions containing reverse transcriptase and reaction mixtures

[0343] This invention also provides compositions and reaction mixtures comprising multiple components in various combinations. In some embodiments of the invention, the composition is prepared by mixing one or more reverse transcriptases. In some other embodiments, the composition is prepared in a buffered salt solution. Optionally, one or more DNA polymerases and / or one or more nucleotides and / or one or more primers may be added to the compositions of the invention. Alternatively, the reaction mixture according to the invention may contain one or more DNA polymerases and / or one or more nucleotides and / or one or more primers. These compositions and / or reaction mixtures can be used in the invention for reverse transcription, followed by one-step PCR or one-step qPCR. Alternatively, the compositions and / or reaction mixtures can be used for reverse transcription, followed by two-step PCR or one-step qPCR.

[0344] In some embodiments, the reverse transcriptase according to the invention is provided in a stable buffered salt solution at a working concentration (e.g., 1×). Such compositions can also be formulated as concentrated stock solutions (e.g., 2×, 3×, 4×, 5×, 6×, 10×, etc.). In some embodiments, preparing the composition as a concentrated (e.g., 5×) stock solution allows for the addition of larger quantities of nucleic acid samples (e.g., when the composition is used for nucleic acid synthesis).

[0345] In addition to the enzyme component, the compositions according to the invention may also contain one or more buffers and cofactors required for the synthesis of nucleic acid molecules (such as cDNA molecules). In some embodiments, the buffer used to form the compositions of the invention is in the form of acetate, sulfate, hydrochloride, phosphate, or free acid of tri-(hydroxymethyl)aminomethane (TRIS™) or 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), but alternative buffers with similar ionic strength and pKa to TRIS™ or HEPES can be used with the same results. For example, possible buffers used with the described enzyme may include 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (diglycine), tris(hydroxymethyl)methylamine (Tris™), N-tris(hydroxymethyl)methylglycine (wheat flavonoid), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 4-2-hydroxyethyl-1-piperazine ethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES), and dimethylarsonic acid (dimethylarsylate).

[0346] In addition to buffer salts, cofactor salts are also included for use in the compositions of the present invention, such as potassium salts (preferably potassium chloride or potassium acetate) and magnesium salts (preferably magnesium chloride or magnesium acetate).

[0347] Adding one or more carbohydrates and / or sugars to the composition and / or reaction mixture may also be advantageous to support enhanced stability of the composition after storage and / or enhanced stability of the reaction mixture during synthesis. In some embodiments, the carbohydrates or sugars used to include in the composition and / or reaction mixture of the present invention include sucrose, trehalose, glycerol, etc. In some embodiments, trehalose is provided in concentrations ranging from 0.01 M to 5 M (e.g., 0.01 M, 0.05 M, 0.1 M, 0.5 M, 0.75 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, or 5.0 M). In some embodiments, glycerol is provided in concentrations ranging from 5% to 60% (e.g., 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%).

[0348] Similarly, it may be advantageous to add one or more surfactants and / or detergents to the composition and / or reaction mixture to support enhanced stability of the composition and / or reaction mixture after storage. Preferred detergents for inclusion in the compositions and / or reaction mixtures of the present invention include Tween™ 20, Nonidet™ P 40 (NP-40), Brij™ 58, Triton™ X-100, Ecosurf™ SA, Ecosurf™ EH-9, Tergitol™, CHAPS, etc. In some embodiments, the addition of one or more detergents from Tween™ 20, Nonidet™ P40 (NP-40), Brij™ 58, Triton™ X-100, Ecosurf™ SA-9, Ecosurf™ EH-9, Tergitol™, and CHAPS can enhance enzyme activity in the reaction mixture.

[0349] In some embodiments, component degradation can be reduced by storing the composition and / or reaction mixture at a temperature of about -80°C (for up to two years) or at a temperature of about -20°C (for up to one year).

[0350] In some embodiments, the compositions and / or reaction mixtures of the present invention may be packaged in suitable containers or vessels capable of preserving the compositions and not significantly interacting with the components of the compositions. These containers or vessels may be designed to allow for easy dispensing of dosage forms, either individually or via a liquid-operated instrument. Such containers or vessels of compositions and / or reaction mixtures may be further packaged in multi-packaging units.

[0351] In another aspect, the compositions and reverse transcriptases of the present invention can be prepared and stored in a dry form (e.g., lyophilized) in the presence of one or more carbohydrates, sugars, or synthetic polymers. Preferred carbohydrates, sugars, or polymers for preparing the dried compositions or reverse transcriptases include sucrose, trehalose, and polyvinylpyrrolidone (PVP) or combinations thereof (see, for example, U.S. Patent Nos. 5,098,893, 4,891,319, and 5,556,771). Such dried compositions and enzymes can be stored at various temperatures for extended periods without significant degradation of the components of the enzymes or compositions of the present invention. In some preferred embodiments, the dried reverse transcriptases or compositions are stored at about -20°C to about 25°C.

[0352] In another aspect, the compositions and reverse transcriptases of the present invention can be prepared as stable glycerol-free enzyme formulations. Such formulations may have the advantage of being lyophilizable compared to glycerol-containing formulations. In some embodiments, the glycerol-free solution contains no glycerol. In some embodiments, the glycerol-free solution contains negligible or undetectable glycerol. In some embodiments, the glycerol-free buffer contains no more than 2% glycerol, preferably no more than 1% glycerol, more preferably no more than 0.5% glycerol, or even more preferably no more than 0.1% glycerol. In some embodiments, the stable enzyme formulation contains a K+ and / or Na+-based salt having a stable effective concentration (see, for example, U.S. Patent No. 11,268,084). In some embodiments, the stable enzyme further comprises at least one of the following: a buffer salt, a reducing agent, a detergent, a cryoprotectant, and / or other stabilizers.

[0353] The present invention also includes compositions and / or reaction mixtures for reverse transcription of nucleic acid molecules, methods for reverse transcription using such compositions and / or reaction mixtures, and nucleic acid molecules produced using such methods. In some embodiments, the compositions of the present invention may comprise one or more of the following components: (1) one or more buffers (e.g., sodium phosphate, sodium acetate, 2-(N-morpholino)-ethanesulfonic acid (MES), tris-(hydroxymethyl)aminomethane (Tris), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPS), citrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), acetate, 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), etc.); (2) one or more monovalent cationic salts (e.g., NaCl, KCl, etc.); (3) one or more divalent cationic salts (e.g., MnCl2, MgCl2, MgSO4, CaCl2, etc.); (4) one or more reducing agents (e.g., dithiothreitol, β-mercaptoethanol, etc.); (5) one or more ionic or nonionic detergents (e.g., TRITON™ X-100, NONIDET™ P40 Ecosurf™). (6) nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP, dTTP, etc.); (7) RNA to be reverse transcribed and / or amplified; (8) one or more RNase inhibitors (e.g., RNASEOUT™, Ingenie, Carlsbad, CA, catalog number 10777-019, etc.); (9) reverse transcriptase (e.g., the reverse transcriptase of this invention); and / or (10) one or more diluents (e.g., water). Other components and / or ingredients (e.g., primers, DNA polymerases, etc.) may also be present in the composition and / or reaction mixture.

[0354] The concentration of the buffer in the compositions and / or reaction mixtures of the present invention will vary depending on the specific buffer used. Typically, the working concentration of the buffer will be from about 5 mM to about 500 mM.

[0355] The final pH of the solution of the present invention will generally be set and maintained by the buffer present in the composition and / or reaction mixture of the present invention. The pH of the composition of the present invention, and therefore the pH of the reaction mixture of the present invention, will vary depending on the specific application and the buffer present, but is typically from about pH 5.5 to about pH 9.0.

[0356] As described above, one or more monovalent cation salts (e.g., NaCl, KCl, etc.) may also be included in the compositions and / or reaction mixtures of the present invention. In many cases, the salts used in the compositions of the present invention will dissociate in solution to produce at least one monovalent substance (e.g., Na+). + K + (etc.). When these salts are added to the compositions of the present invention, they are often present alone or in combination, with a concentration ranging from about 0.5 mM to about 500 mM.

[0357] As described above, one or more divalent cation salts (e.g., MnCl2, MgCl2, MgSO4, CaCl2, etc.) may also be included in the compositions and / or reaction mixtures of the present invention. In many cases, the salts used in the compositions and / or reaction mixtures of the present invention will dissociate in solution to produce at least one monovalent substance (e.g., Mg). ++ Mn ++ Ca ++ (etc.). When these salts are added to the compositions and / or reaction mixtures of the present invention, they are often present alone or in combination, with concentrations ranging from about 0.5 mM to about 500 mM.

[0358] When reducing agents (e.g., dithiothreitol, β-mercaptoethanol, TCEP, etc.) are included in the compositions and / or reaction mixtures of the present invention, they are often present alone or in combination, with a concentration range of about 0.1 mM to about 50 mM.

[0359] Other additives, besides those disclosed herein, that can promote or enhance reverse transcription, amplification, or a combination of both reactions (e.g., reagents for promoting or enhancing RT-PCR) are also known in the art. According to the invention, one or more of these additives can be incorporated to optimize the generation and replication of nucleic acids from ribonucleic acid or deoxyribonucleic acid templates. The additives can be organic or inorganic compounds. Some additives suitable for use in this invention include peptides and non-peptide additives. Such additives may include, for example, RNase inhibitor proteins (RIPs), uracil DNA glycosidase (UDG), lectins, Escherichia coli single-strand binding (SSB) proteins, tRNA, rRNA, 7-dezo-2-deoxyguanosine (dC7GTP), sulfur-containing compounds, acetate-containing compounds, dimethyl sulfoxide (DMSO), ribonuclease inhibitors (e.g., RNase OUT™) formamide, betaine, tetramethylammonium chloride (TMAC), polyethylene glycol (PEG), tetrahydropyrimidine, sodium azide, kathon, and polyols, to name just a few. Those skilled in the art will be able to identify the additional additives used according to the present invention.

[0360] The compositions and / or reaction mixtures of the present invention may also contain one or more hot-start components. Hot-start is a common technique for reducing nonspecific amplification at room temperature at which nucleic acid synthesis reactants are assembled. In some embodiments, the reverse transcriptase is reversibly inactivated or physically isolated from one or more key components in the reactants. For example, magnesium may be isolated in wax beads that melt upon heating of the reactants, releasing the components only at higher temperatures (see, for example, Carothers et al. 1989; Krishnan et al. 1991; Clark, 1988). Reverse transcriptase may also remain inactive by binding to oligonucleotides, also known as aptamers (see, for example, Lin and Jayasena, 1997; Dang and Jayasena, 1996) or antibodies (see, for example, Scalice et al. 1994; Sharkey et al. 1994). This binding may then be interrupted at higher temperatures, releasing the functional reverse transcriptase.

[0361] In other embodiments, the reverse transcriptase may be chemically modified to maintain an inactive state (see, for example, Moretti, T. et al 1998). In some embodiments, the chemical modification is reversible. Thus, in some embodiments, the reverse transcriptase is chemically modified such that it is inactive at lower temperatures (e.g., less than about 55°C) and fully functional / active at higher temperatures (e.g., greater than about 55°C).

[0362] In some embodiments, nucleotides, such as dNTPs, such as dGTP, dATP, dCTP, dTTP, etc., will be present in the reaction mixture of the present invention.

[0363] In some embodiments, the composition and / or reaction mixture comprises at least one protein stabilizer. In some embodiments, the protein stabilizer is selected from BSA, inactive polymerase, and deferferrin.

[0364] In some embodiments, the reaction mixture comprises at least one dye or fluorescent group. Dyes that can be used in the compositions provided herein include xylenenitrile FF, tartrazine, phenol red, quinoline yellow, brilliant blue, lacquer blue, indigo carmine, acid red 1, m-cresol violet, cresol red, neutral red, bromocresol green, acid violet 5, bromophenol blue, and orange G (see, for example, US20190270975A1 and US Patent No. 8,663,925 B2). Additional exemplary dyes are described, for example, in US Patent No. 6,942,964. Those skilled in the art will understand that any dye that does not inhibit nucleic acid synthesis by polymerase may be used.

[0365] In some embodiments, the composition and / or reaction mixture contains at least one agent that increases the density of the composition. In some embodiments, the composition and / or reaction mixture contains at least one agent selected from PEG 4000 and / or sucrose.

[0366] The compositions and / or reaction mixtures of the present invention can be prepared as concentrated solutions (e.g., 5× solutions), which are then diluted to working concentrations for final use.

[0367] In some embodiments, the compositions and / or reaction mixtures of the present invention are provided in a sterile form. The individual components of the compositions and / or reaction mixtures may be sterilized prior to mixing or after their preparation. Sterilization of such solutions can be performed by any suitable means, including autoclaving or ultrafiltration.

[0368] Reagent test kit

[0369] In another aspect, the present invention provides a kit comprising the reverse transcriptase of any of the foregoing embodiments. In one embodiment, the kit may, in addition to comprising the reverse transcriptase of the present invention, also comprise additional components suitable for PCR, such as additional reagents, such as DNA polymerase or primers, one or more buffers, nuclease-free water, salts (such as magnesium sulfate), one or more dyes / fluorescent groups, ribonucleotides, deoxynucleotides, etc.

[0370] In some embodiments, these additional components may be present in the premix. In a preferred embodiment, the additional components are present in the premix along with the reverse transcriptase of the present invention. Furthermore, the kit may also include reaction dishes, control samples, and instructions.

[0371] In one embodiment, the kit is a two-step RT-qPCR kit. In a preferred embodiment, the kit is a one-step RT-qPCR kit.

[0372] Polynucleotides

[0373] In another aspect, the present invention provides a polynucleotide encoding the reverse transcriptase of any of the foregoing embodiments.

[0374] In some embodiments, the polynucleotide may be DNA or RNA. In a preferred embodiment, the polynucleotide is DNA. In some embodiments, in addition to the sequence encoding the reverse transcriptase of the present invention, the polynucleotide may also comprise other nucleic acid sequences. In other embodiments, the polynucleotide consists of a sequence encoding the reverse transcriptase.

[0375] In another embodiment, the polynucleotide undergoes codon optimization for expression in the target host. Codon optimization is a method that improves gene expression and translation efficiency of the target gene, ultimately enhancing protein expression in the target host or host organism. A wide range of codon optimization techniques are well-known to those skilled in the art, such as: using the most commonly used codons for all instances of amino acids; adjusting codon usage based on the natural distribution in the target host or host organism; using codons corresponding to abundant tRNAs; replacing rate-restricting codons; avoiding codon pairs known to translate slowly; or methods that do not take these considerations into account, also known as hypothesis-free methods.

[0376] As specified in the definition section above, the target host is any suitable organism or its cellular subunit. Thus, a suitable organism may be, for example, yeast cells, prokaryotic cells, or mammalian cells or cell lines carrying plasmids or polynucleotides containing the reverse transcriptase according to the invention.

[0377] plasmid

[0378] In another aspect, the present invention provides a plasmid comprising a polynucleotide according to any of the foregoing embodiments. Suitable plasmids comprising the polynucleotides of the present invention are, for example, cloning and expression vectors, bacteriophages, phage particles, granules, or any other nucleic acid that can replicate independently in a suitable host cell, has at least one recognition sequence (such as a nuclease restriction site), and is capable of inserting another nucleic acid therein. Suitable cloning and expression vectors for expressing polynucleotides in a target host (such as prokaryotic cells, yeast cells, or mammalian cells or cell lines) are known to those skilled in the art.

[0379] cell

[0380] In another aspect, the present invention provides a cell comprising a reverse transcriptase, a polynucleotide, or a plasmid according to any of the foregoing embodiments.

[0381] In a preferred embodiment, the cell containing the reverse transcriptase is a host cell. The host cell can be any cell suitable for expressing one or more recombinant proteins. Examples of suitable host cells include prokaryotic and eukaryotic cells. In one embodiment, a prokaryotic cell is, for example, a species of *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, or *Pseudomonas*, *Streptomyces*, or *Staphylococcus*. Suitable eukaryotic cells are, for example, animal cells, vertebrate cells, yeast cells, plant cells, fungal cells, insect cells, mammalian cells, reptile cells, or algal cells. In a preferred embodiment, the prokaryotic cells are Escherichia coli, preferably Escherichia coli JM109, Escherichia coli JM101, Escherichia coli XL-1 Blue, Escherichia coli MV1 190, Escherichia coli HB101, Escherichia coli DH5α, Escherichia coli DH10B, and Escherichia coli BL21.

[0382] The maintenance of host cells according to the present invention is known to those skilled in the art. Similarly, the introduction of reverse transcriptase or polynucleotides or plasmids from any of the foregoing embodiments to produce the corresponding proteins is a conventional procedure known to those skilled in the art.

[0383] method

[0384] In another aspect, the present invention provides a method for reverse transcription of RNA. Reverse transcription refers to the conversion of RNA into cDNA. Therefore, in one embodiment, the reverse transcriptase according to any of the foregoing embodiments is used to synthesize cDNA from RNA. In a preferred embodiment, the reverse transcriptase according to any of the foregoing embodiments is used to synthesize cDNA from RNA in a sample.

[0385] In one embodiment, the sample is selected from the group consisting of biological samples, environmental samples, or food samples. In some embodiments, the biological, environmental, or food samples may be fresh, frozen, or preserved samples, such as those preserved in formalin and / or embedded in paraffin. In other embodiments, the biological, environmental, or food samples may contain additional compounds, such as drug metabolites, antibiotics, anticoagulants, chemicals (such as preservatives, fixatives, or buffers), nutrients, fertilizers, etc. In some embodiments, the biological, environmental, or food samples originate from a contaminated source.

[0386] In one embodiment, the biological sample is derived from a human, animal, or plant. In a preferred embodiment, the human is a patient. In another preferred embodiment, the animal is a mammal. In a preferred embodiment, the biological sample is a cell, cell culture, blood, urine, feces, saliva, cerebrospinal fluid, tissue fluid, ascites, peritoneal fluid, amniotic fluid, breast milk, mucus, semen, bile, skin swab, or a combination of the above.

[0387] In another preferred embodiment, the environmental sample is a soil sample, peat sample, sediment sample, or water sample. In some embodiments, the water sample may be a process water sample, surface water sample, drinking water sample, freshwater sample, or seawater sample. In yet another preferred embodiment, the environmental sample is a swab from a wall, floor, surface, ventilation equipment, drain pipe, conveyor belt, or container.

[0388] In another preferred embodiment, the food sample is a sample of food or beverage. In some embodiments, the food sample is a raw material, material being processed (i.e., a "process sample"), or a sample from the final food or beverage product. In one embodiment, the raw material is raw vegetables, raw fruit, raw meat, spices, nuts, drinking water, syrup, additives, preservatives, sugar, or artificial sweeteners. In another embodiment, the processed material is processed vegetables, processed fruit, processed meat, processed spices, and processed nuts. In yet another embodiment, the final food is a dairy product, such as cheese or butter. In still another embodiment, the final beverage product is milk, lemonade, vegetable juice, fruit juice, or drinking water.

[0389] In one embodiment, the biological sample, environmental sample, or food sample is a processed sample or a purified sample.

[0390] In one embodiment, the processed sample is a sample that has undergone a defined process or treatment. The defined process or treatment may include multiple steps. In some embodiments, the defined process or treatment may be, for example, RNA or DNA extraction, reverse transcription, qPCR, RT-qPCR, or PCR. In some embodiments, a purified sample undergoes a defined process or treatment to become a processed sample. Therefore, in some embodiments, a purified sample is a processed sample.

[0391] In another embodiment, a purified sample refers to a sample from which any potentially harmful, impure, or inferior substances have been removed. Therefore, in some embodiments, substances considered contaminants are removed from the purified sample. In another embodiment, the purified sample corresponds to a concentrated sample. In some embodiments, the purified sample is a processed sample. Various chemical and physical purification methods, such as filtration, drying, chromatography, distillation, extraction, ion exchange, etc., are well known to those skilled in the art.

[0392] In another embodiment, the biological sample, environmental sample, or food sample may be an untreated sample or an unpurified sample.

[0393] In one embodiment, an untreated sample is a sample that has not yet undergone a defined process or treatment. In another embodiment, an unpurified sample is a sample from which any potentially harmful, impure, or inferior substances have not been removed. Therefore, in some embodiments, substances considered contaminants are present in the unpurified sample. In another embodiment, an unpurified sample corresponds to an unconcentrated sample.

[0394] Example

[0395] Example 1: Single-point mutants of wild-type MMLV reverse transcriptase (SEQ ID NO: 1) I416T (SEQ ID NO: 2), K267Q (SEQ ID NO: 3), N674D (SEQ ID NO: 4) and T186A (SEQ ID NO: 5)

[0396] To generate reverse transcriptases with improved inhibitor resistance, four single-point mutants were designed: I416T (SEQ ID NO: 2), K267Q (SEQ ID NO: 3), N674D (SEQ ID NO: 4), and T186A (SEQ ID NO: 5).

[0397] Mutagenesis was introduced via site-directed mutagenesis using Invitrogen Platinum™ SuperFi™ II PCR Master Mix with mutagenic primers, following the recommendations in Application Note, Mutagenesis Protocol A: https: / / tools.thermofisher.com / content / sfs / brochures / site-directed-mutagenesis-platinum-superfi-app-note.pdf. In short, point mutations were generated by designing two partially complementary mutagenic primers with 3' overhangs. PCR amplification was performed using Platinum™ SuperFi™ PCR Master Mix, followed by direct transformation of the PCR product into *E. coli* (using the pET21 expression system) without any additional steps in the *E. coli* expression process, and purification. SDS-PAGE under reducing conditions was then performed, resulting in a single band of 78 kDa from the purified variant, achieving a purity exceeding 80%.

[0398] Example 2: Inhibitor resistance of single-point mutants I416T (SEQ ID NO: 2), K267Q (SEQ ID NO: 3), N674D (SEQ ID NO: 4) and T186A (SEQ ID NO: 5) to wild-type MMLV reverse transcriptase (SEQ ID NO: 1).

[0399] The reaction mixture was prepared as shown in Table 1 below:

[0400] Table 1: Reaction mixture

[0401] The 5x RT buffer (see the second row of Table 1 above) consists of the following components: 250 mM Tris-HCl (pH 8.3, 25°C), 250 mM KCl, 20 mM MgCl2, and 50 mM DTT.

[0402] The following inhibitors were added to the reaction mixture in increased amounts: heme chloride (10 µM, 15 µM, 17 µM), tannic acid (5 ng / µL, 7.5 ng / µL, 10 ng / µL), humic acid (6 ng / µL, 8 ng / µL, 10 ng / µL), and guanidine isothiocyanate (70 mM, 75 mM, 80 mM). The reaction mixture was incubated at 42°C for 60 min, followed by electrophoresis with 5 µL of basic electrophoretic dye (180 mM NaOH, 6 mM EDTA, 18% Ficoll). TM Mix 400 mg / mL of 0.05% bromocresol green and heat at 70°C for 10 minutes. Afterward, cool the sample on ice and load it onto a 1.4% agarose gel as described in Baranauskas et al. (2012), Protein Engineering Design & Selection. Prepare the gel in 30 mM NaCl, 2 mM EDTA (pH 7.5) buffer and equilibrate overnight at 4°C in electrophoresis buffer (30 mM NaOH, 2 mM EDTA). Electrophoresis was performed at 3 V / cm for 1 hour, the gel was neutralized for 30 minutes in 300 mL of 0.5 M Tris–HCl buffer (pH 7.5), stained for 30 minutes in 0.5 µg / mL ethidium bromide solution, and then visualized under UV light. All reactions were performed in triplicate.

[0403] The results showed that, compared with wild-type MMLV reverse transcriptase, mutants T186A, K267Q, I416T, and N674D were still able to synthesize 1.3 kb cDNA fragments in the presence of higher inhibitor concentrations, indicating that these mutants have enhanced resistance to inhibitors. The results of cDNA synthesis in the presence of heme chloride in the reaction mixture are shown in Figure 1. The results of cDNA synthesis in the presence of various inhibitors (i.e., heme chloride, tannic acid, humic acid, and guanidine isothiocyanate) are summarized and shown in Figure 2.

[0404] Example 3: Improved continuous synthesis capacity of single-point mutant reverse transcriptases I416T (SEQ ID NO: 2), K267Q (SEQ ID NO: 3), N674D (SEQ ID NO: 4), and T186A (SEQ ID NO: 5) compared to wild-type MMLV reverse transcriptase (SEQ ID NO: 1).

[0405] Synthesize 0.5–9 kb cDNA fragments from Millennium™ RNA markers (Invitrogen, #AM7150).

[0406] Except for the addition of 1 µg Millennium™ RNA marker to the mixture in place of GAPDH RNA, the composition of the reaction mixture is the same as that provided in Table 2.

[0407] The results showed that, compared with the wild-type MMLV reverse transcriptase (SEQ ID NO: 1), the single-point mutants I416T (SEQ ID NO: 2), K267Q (SEQ ID NO: 3), N674D (SEQ ID NO: 4), and T186A (SEQ ID NO: 5) had enhanced continuous synthesis capacity, especially the K267Q mutant (SEQ ID NO: 3). The results are shown in Figure 3.

[0408] Example 4. Enhanced thermostability and thermoactivity of the single-point mutant reverse transcriptase I416T (SEQ ID NO: 2) compared to the wild-type MMLV reverse transcriptase (SEQ ID NO: 1).

[0409] Except for the addition of 1 µg Millennium™ RNA marker to the mixture in place of GAPDH RNA, the composition of the reaction mixture is the same as that provided in Table 1.

[0410] cDNA synthesis reactions were performed at 42°C, 44°C, 46°C, 48°C, and 50°C for 60 minutes, followed by mixing with 5 µL of alkaline electrophoresis buffer and heating at 70°C for 10 minutes. Samples were analyzed on a 1.0% alkaline agarose gel as described in Example 2. The cDNA synthesis results at the temperature gradients are shown in Figure 4.

[0411] The results showed that, compared with the wild-type MMLV reverse transcriptase, the mutant RTI416T was able to synthesize longer cDNA fragments at 48°C and 50°C, indicating that the mutant has higher thermal stability and thermal activity.

[0412] Example 5. Enhanced thermostability of single-point mutant reverse transcriptase I416T (SEQ ID NO: 2) compared to wild-type MMLV reverse transcriptase (SEQ ID NO: 1).

[0413] The reaction mixture was prepared as shown in Table 2 below:

[0414] Table 2: Reaction mixture section 1

[0415] Then, incubate the mixture at 55°C for 30 minutes. After incubation, add the remaining components:

[0416] Table 3: Reaction mixture section 2

[0417] cDNA synthesis was performed at 50°C for 30 minutes and terminated by heating at 80°C for 10 minutes, followed by mixing with 6X TrackIt™ Cyan / Orange Loading Buffer. 12 µL of each mixture was analyzed on a 1% TAE agarose gel stained with 0.5 µg / ml ethidium bromide solution.

[0418] The single-point mutant reverse transcriptase I416T (SEQ ID NO: 2) still exhibited cDNA synthesis activity even after heat treatment, while the wild-type MMLV reverse transcriptase almost lost its activity after incubation at 55°C (data not shown).

[0419] sequence

[0420] Table 4: Sequences (SEQ ID NO: 1-16)

[0421] References

[0422] BARANAUSKAS, A.Generation and characterization of new highlythermostable and processive M-MuLV reverse transcriptase variants.ProteinEngineering, Design and Selection, October 2012, Vol. 25, No. 10, pages 657–668.

[0423] CAROTHERS, AM et al. Point mutation analysis in a mammalian gene:Rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencingby a novel method.Biotechniques, May 1989, Vol. 7, No. 5, pages 494-499.

[0424] KRISHNAN, BR et al. Linear amplification DNA sequencing directly fromsingle phage plaques and bacterial colonies.Nucleic Acid Research, March1991, Vol.19, No.5, page 1153.

[0425] CLARK, JM.Novel non-templated nucleotide addition reactions catalyzedby procaryotic and eucaryotic DNA polymerases.Nucleic Acid Research, October1988, Vol.16, No. 20, pages 9677– 9686.

[0426] LIN, Y AND JAYASENA, SD.Inhibition of multiple thermostable DNApolymerases by a heterodimeric aptamer.Journal of Molecular Biology, August1997, Vol. 264, Issue 1, pages 100-111.

[0427] DANG, C AND JAYESENA, SD.Oligonucleotide inhibitors of Taq DNApolymerase facilitate detection of low copy number targets by PCR.Journal ofMolecular Biology, November 1996, Vol. 271, Issue 2, pages 268-278.

[0428] SCALICE, ER et al. Monoclonal antibodies prepared against the DNApolymerase from Thermus aquaticus are potent inhibitors of enzymeactivity.Journal of Immunological Methods, June 1994, Vol. 172, Issue 2,pages 147-163.

[0429] SHARKEY, D et al. Antibodies as Thermolabile Switches:HighTemperature Triggering for the Polymerase Chain Reaction.NatureBiotechnology, Vol.12, pages 506–509.

[0430] MORETTI, T. et al. Enhancement of PCR Amplification Yield andSpecificity Using AmpliTaq Gold™ DNA Polymerase.BioTechniques, October 1998,Vol. 25, pages 716-722

Claims

1. A reverse transcriptase comprising a modified amino acid sequence of Moloney mouse leukemia virus reverse transcriptase, wherein the amino acid sequence contains mutations at positions K267 and / or I416 and / or N674 and / or T186 compared to the wild-type Moloney mouse leukemia virus reverse transcriptase of SEQ ID NO: 1, wherein the enzyme has enhanced inhibitor resistance compared to the wild-type Moloney mouse leukemia virus reverse transcriptase.

2. The reverse transcriptase according to claim 1, wherein the amino acid sequence has at least two mutations at positions selected from the group consisting of K267, I416, N674 and T186.

3. The reverse transcriptase according to claim 2, wherein the at least two mutations are located at positions K267 and I416.

4. The reverse transcriptase according to claim 2, wherein the at least two mutations are located at positions N674 and K267.

5. The reverse transcriptase according to any of the preceding claims, wherein the amino acid sequence contains at least one additional mutation at one or more of the following amino acid positions: 。 6. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase comprises a mutation selected from the group consisting of I416Q, I416N, I416T, and I416S.

7. The reverse transcriptase according to claim 6, wherein the reverse transcriptase contains the I416T mutation (SEQ ID NO:2).

8. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase comprises a mutation selected from the group consisting of K267Q, K267N, and K267S.

9. The reverse transcriptase according to claim 8, wherein the reverse transcriptase contains a K267Q mutation (SEQ ID NO:3).

10. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase comprises a mutation selected from the group consisting of N674D or N674E.

11. The reverse transcriptase according to claim 10, wherein the reverse transcriptase contains the N674D mutation (SEQ ID NO:4).

12. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase contains a non-K267T mutation at position K267.

13. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase contains a mutation at position 186 that is neither T186C nor T186D.

14. The reverse transcriptase according to claim 12, wherein the reverse transcriptase is selected from the group consisting of T186A, T186V, T186I, T186L, and T186M.

15. The reverse transcriptase according to claim 13, wherein the reverse transcriptase comprises the T186A mutation (SEQ ID NO:5).

16. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase comprises an I416T mutation, a K267Q mutation, an N674D mutation, and / or a T186A mutation.

17. The reverse transcriptase of claim 15, wherein the reverse transcriptase comprises an I416T mutation, a K267Q mutation, and / or an N674D mutation.

18. The reverse transcriptase according to claim 15, wherein the reverse transcriptase comprises the I416T mutation and the K267Q mutation (SEQ ID NO: 6).

19. The reverse transcriptase according to any of the preceding claims, wherein the reverse transcriptase confers enhanced resistance to heme chloride, tannic acid, humic acid and guanidine isothiocyanate.

20. A composition comprising the reverse transcriptase according to any of the preceding claims.

21. A reaction mixture comprising the reverse transcriptase according to any one of claims 1 to 19.

22. Use of the reverse transcriptase according to any one of claims 1 to 19 for reverse transcription of RNA.

23. A kit comprising the reverse transcriptase according to any one of claims 1 to 19.

24. A polynucleotide encoding a reverse transcriptase according to any one of claims 1 to 19.

25. The polynucleotide of claim 24, wherein the polynucleotide is codon-optimized for expression in the target host.

26. A plasmid comprising the polynucleotide according to claim 24 or 25.

27. A cell comprising a reverse transcriptase according to any one of claims 1 to 19, a polynucleotide according to claim 24 or 25, or a plasmid according to claim 26.

28. A method for reverse transcription of RNA, wherein cDNA is synthesized from RNA in a sample using a reverse transcriptase according to any one of claims 1 to 19.

29. The method of claim 28, wherein the sample is selected from the group consisting of biological samples, environmental samples, or food samples.

30. The method of claim 29, wherein the biological sample, environmental sample, or food sample is a processed sample or a purified sample.

31. The method of claim 30, wherein the biological sample, environmental sample, or food sample is an untreated sample or an unpurified sample.

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