Mutant reverse transcriptase with increased thermal stability as well as products, methods and uses involving the same

Abstract

The present invention relates to a mutant reverse transcriptase (RT) with increased thermal stability relative to the wildtype, a nucleic acid encoding the mutant RT, a cell comprising the mutant RT or the nucleic acid, a kit comprising the mutant RT, the use of the mutant RT for cDNA synthesis, method for reverse transcription of RNA comprising synthesizing cDNA with the use of the mutant RT and a method for detecting an RNA marker in a sample with the use of the mutant RT.

Claims

1. A mutant reverse transcriptase (RT) with an increased thermal stability relative to a wildtype RT consisting of SEQ ID NO:1, the mutant RT consisting of an amino acid sequence that has only six amino acid substitutions of SEQ ID NO:1, wherein Ala at position 32 is substituted with Val (A32V); Leu at position 72 is substituted with Arg (L72R); Glu at position 286 is substituted with Arg (E286R); Glu at position 302 is substituted with Lys (E302K); Trp at position 388 is substituted with Arg (W388R); and Leu at position 435 is substituted with Arg (L435R), wherein the mutant RT exhibits a reverse transcriptase activity, and wherein the mutant RT also has an increased thermal stability relative to a mutant MM3, wherein the mutant MM3 has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:1 and only has three amino acid substitutions of SEQ ID NO:1, wherein Glu at position 286 is substituted with Arg (E286R), Glu at position 302 is substituted with Lys (E302K) and Leu at position 435 is substituted with Arg (L435R).

2. The mutant RT of claim 1, wherein the increased thermal stability is determined by measuring the reverse transcriptase activity of the mutant RT after a thermal treatment.

3. The mutant RT of claim 1, wherein the reverse transcriptase activity of the mutant RT is at least 50% of the reverse transcriptase activity of the wildtype RT.

4. The mutant RT of claim 1 consisting of an amino acid sequence of SEQ ID NO:2.

5. A cell comprising the mutant RT of claim 1.

6. A kit for performing a reverse transcription reaction, comprising the mutant RT of claim 1.

Description

FIGURES

(1) FIG. 1: Nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 2) of mutant RT MM3.14. The six obligatory mutations (substitutions A32V, L72R, E286R, E302K, W388R and L435R) with respect to the wildtype are indicated.

(2) FIG. 2: Expression plasmids for MMLV-RT. The asterisk indicates the termination codon.

(3) FIG. 3: Activity and stability of single variants. (A) Specific activity. The dTTP incorporation reaction was carried out at 37° C. One unit is defined as the amount which incorporates 1 nmol of dTTP into poly(rA)-p(dT).sub.15 in 10 min. The relative specific activity is defined as the ratio of the specific activity of variant to that of WT. (B, C) Thermal stability. RT at 100 nM was incubated at 46° C. (B) or 49° C. (C) in the presence of poly(rA)-p(dT).sub.15 (28 μM) for 10 min. Then, the dTTP incorporation reaction was carried out at 37° C. The relative activity is defined as the ratio of the initial reaction rate of RT with the 10-min incubation at 46 or 49° C. to that without the incubation.

(4) FIG. 4: Activity and stability of multiple variants. (A, B) Specific activity. (A) The dTTP incorporation reaction was carried out at 5 nM RT at 37° C. One unit is defined as the amount which incorporates 1 nmol of dTTP into poly(rA)-p(dT).sub.15 in 10 min. The relative specific activity is defined as the ratio of the specific activity of variant to that of WT. (B) The PicoGreen incorporation reaction was carried out at 5 nM RT at 37° C. The initial reaction rates (ΔFI/min) were calculated and normalized with that of WT as 1.0. (C-F) Thermal stability. RT at 100 nM was incubated at 49 or 51° C. in the presence of poly(rA)-p(dT).sub.15 (28 μM) for 10 min. Then, the dTTP incorporation reaction (C, E) or the PicoGreen incorporation reaction (D, F) was carried out at 10 nM RT at 37° C. The relative activity is defined as the ratio of the initial reaction rate of RT with the 10-min thermal treatment to that without it.

(5) FIG. 5: Temperature dependence on cDNA synthesis by WT, MM3, or MM3.14. cDNA synthesis reaction was carried out at 50 (A), 55 (B, C), 60 (D), or 65° C. (E) for 10 min using RT that had received thermal incubation at 55° C. for 5 min (B) or RT without the thermal incubation (A, C-E). Then, PCR was carried out. Fluorescence of real-time PCR using cDNA synthesis products was shown. The crossing points (CP) were 28.01, 25.22, and 25.67 min for WT, MM3, and MM3.14, respectively (A), 24.95 and 26.74 min for MM3 and MM3.14, respectively (B), 30.52 min for MM3.14 (C), 28.48 and 29.14 min for MM3 and MM3.14, respectively (D), and 32.51 min for MM3.14 (E).

(6) FIG. 6: Stability of WT, MM3, or MM3.14 as assessed by cDNA synthesis. RT at 100 nM was incubated at 48, 54, 57, 60, or 63° C. in the presence of poly(rA)-p(dT).sub.15 (28 μM) for 10 min. Then, the cDNA synthesis was carried out with 16 pg cesA RNA, 0.5 μM RV-R26 primer at 45° C. for 30 min. PCR was carried out with a primer combination of RV and F5. Amplified products were applied to 1% agarose gel followed by staining with ethidium bromide (1 μg/ml).

EXAMPLES

Methods

RT Concentration and Standard RNA

(7) RT concentration was determined as according to the method of Bradford (Bradford, 1976) using Protein Assay CBB Solution (Nacalai Tesque, Kyoto, Japan) with bovine serum albumin (Nacalai Tesque) as a standard. Standard RNA, which was an RNA of 1014-nucleotides corresponding to DNA sequence 8353-9366 of the cesA gene of Bacillus cereus (GenBank accession number DQ360825), was prepared by in vitro transcription.

Construction of Plasmids

(8) Expression plasmids of MMLV RT variants were constructed by site-directed mutagenesis using the expression plasmid for the wild-type MMLV RT, pET-MRTHis (FIG. 2), or the thermostable variant MM3, pET-MM3, as a template and an E. coli BL21(DE3) [F.sup.−, ompT, hsdS.sub.B (r.sub.B.sup.− m.sub.B.sup.−) gal dcm (DE3)] as a host. The nucleotide sequences of mutated MMLV RT genes were verified.

Expression and Purification of Single MMLV RT Variants

(9) Three ml of L broth containing 50 μg/ml ampicillin was inoculated with the glycerol stock of the transformed BL21(DE3) and incubated for 16 h with shaking at 30° C. The expression of the RT gene was induced by the autoinduction system (Novagen, Darmstadt, Germany). MMLV RT was purified from culture medium using HisLink Spin Protein Purification System (Promega, Madison, Wis.). Briefly, the bacterial cells were disrupted by FastBreak Cell Lysis Reagent, followed by addition of HisLink Protein Purification Resin to the culture. The samples were then transferred to HisLink Spin Column where unbound protein was washed away. MMLV RT was recovered by the elution with 0.2 ml of 100 mM HEPES-NaOH buffer (pH 7.5), 500 mM imidazole. The active fraction was desalted using pre-packed PD-10 gel filtration columns (GE Healthcare) equilibrated with 50 mM Tris-HCl buffer (pH 8.3), 200 mM KCl, 50% glycerol and stored at −80° C.

Expression and Purification of Multiple MMLV RT Variants

(10) The overnight culture of the transformants (5 mL) was added to 500 mL of L broth containing ampicillin (50 μg/ml) and incubated with shaking at 37° C. When OD.sub.660 reached 0.6-0.8, 0.15 mL of 0.5 M IPTG was added and growth was continued at 30° C. for 3 h. After centrifugation at 10,000×g for 5 min, the cells were harvested, suspended with 10 mL of 0.02 M potassium phosphate buffer (pH 7.2), 2.0 mM dithiothreitol (DTT), 10% glycerol (buffer A) containing 10 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5 and disrupted by sonication. After centrifugation at 20,000×g for 40 min, the supernatant was collected and applied to a column [25 mm (inner diameter)×120 mm] packed with Toyopearl DEAE-650M gel (Tosoh, Tokyo, Japan) equilibrated with buffer A. After the washing with buffer A containing 120 mM NaCl, the bound RT was eluted with buffer A containing 300 mM NaCl. Solid (NH.sub.4).sub.2SO.sub.4 was added to the eluate (30 mL) to a final concentration of 40% saturation. The solution was stirred for 5 min and left for 30 min on ice. After centrifugation at 20,000×g for 30 min at 4° C., the pellet was collected and dissolved in 10 mL of buffer A containing 100 mM NaCl. The solution was applied to the column packed with a Ni.sup.2+-sepharose (HisTrap HP 1 mL, GE Healthcare, Buckinghamshire, UK) equilibrated with 50 mM Tris-HCl buffer (pH 8.3), 200 mM KCl, 2 mM DTT, 10% glycerol (buffer B). After the washing with buffer B containing 50 mM imidazole, the bound RT was eluted with buffer B containing 500 mM imidazole. The active fraction was desalted using pre-packed PD-10 gel filtration columns equilibrated with 50 mM Tris-HCl buffer (pH 8.3), 200 mM KCl, 50% glycerol and stored at −80° C.

SDS-PAGE

(11) SDS-PAGE was performed in a 10% polyacrylamide gel under reducing conditions. Proteins were reduced by treatment with 2.5% of 2-mercaptoethanol at 100° C. for 10 min, and then applied onto the gel. A constant current of 40 mA was applied for 40 min. After electrophoresis, proteins were stained with Coomassie Brilliant Blue R-250. The molecular mass marker kit consisting of rabbit muscle phosphorylase B (97.2 kDa), bovine serum albumin (66.4 kDa), hen egg white ovalbumin (44.3 kDa), and bovine carbonic anhydrase (29.0 kDa) was a product of Takara Bio Inc (Otsu, Japan).

Reverse Transcription Assay Using [.SUP.3.H]-dTTP

(12) poly(rA)-p(dT).sub.15 was prepared by annealing (dT).sub.15 (Fasmac, Tokyo, Japan) and poly(rA) (GE Healthcare, Buckinghamshire, UK). The reaction was carried out in 25 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 2 mM DTT, 5 mM MgCl.sub.2, 25 μM poly(rA)-p(dT).sub.15 (this concentration is expressed based on p(dT).sub.15), 0.2 mM [.sup.3H]dTTP (1.85 Bq/pmol) (GE Healthcare), and 5 or 10 nM MMLV RT at 37° C. An aliquot (20 μl) was taken from the reaction mixture at 3 and 6 min and immediately spotted onto the glass filter. Unincorporated [.sup.3H]dTTP was removed by three washes of chilled 5% (w/v) trichloroacetic acid (TCA) for 10 min each, followed by one wash of chilled 95% ethanol. The radioactivity retained on the dried filters was counted in 2.5 ml of Ecoscint H (National Diagnostics, Yorkshire, UK). The initial reaction rate was estimated from the time-course for incorporation of [.sup.3H]dTTP.

Reverse Transcription Assay Using Fluorescent Dye PicoGreen

(13) EnzChek Reverse Transcriptase Assay Kit (Thermo Fisher Scientific, Waltham, Mass.) was used. 20×TE buffer (1 ml) was diluted by adding 19 ml of water to make 1×TE buffer. PicoGreen dsDNA quantification reagent (50 μl) was diluted by adding 17.5 ml of 1×TE buffer to make PicoGreen solution. poly(rA)-p(dT).sub.16 for use in thermal inactivation was prepared as follows: poly(rA) (3 μl of 1 mg/ml in 100 mM Tris-HCl buffer (pH 8.1), 0.5 mM EDTA; around 350 base) and p(dT).sub.16 (3 μl of 50 μg/ml in 100 mM Tris-HCl buffer (pH 8.1), 0.5 mM EDTA) were mixed and left for 1 h at room temperature followed by the dilution with 114 μl of PDGT (0.01 M potassium phosphate buffer (pH 7.6), 2 mM DTT, 10% glycerol, 0.2% Triton X-100). MMLV RT (8 μl of 500 nM), poly(rA)-p(dT).sub.16 (8 μl), and PDGT (64 μl) were mixed to make the MMLV RT concentration 50 nM. The resulting solution (40 μl out of 80 μl) was incubated at 49 or 51° C. for 10 min followed by the incubation on ice for 30 min.

(14) Poly(rA)-p(dT).sub.16 for use in reverse transcription assay was prepared as follows: poly(rA) (5 μl) and p(dT).sub.16 (5 μl) were mixed and left for 1 h at room temperature followed by the dilution with 2 ml of polymerization buffer (60 mM Tris-HCl buffer (pH 8.1), 60 mM KCl, 8 mM MgCl.sub.2, 13 mM DTT, 100 μM dTTP). Poly(rA)-p(dT).sub.16 (96 μl) and 40 μl of 25 or 50 nM MMLV RT, either exposed to the thermal treatment or not, were incubated at 37° C. for 10 min. The reaction was initiated by adding the pre-incubated MMLV RT solution (24 μl) to the pre-incubated poly(rA)-p(dT).sub.16 solution (96 μl). An aliquot (25 μl) was taken from the reaction mixture at 2.5, 5.0, 7.5, and 10 min, to which 2 μl of 200 mM EDTA was immediately added, followed by the incubation on ice for 30 min or more. Blank solution was prepared by mixing poly(rA)-p(dT).sub.16 solution (20 μl) and 200 mM EDTA (2 μl) followed by the addition of MMLV RT solution (5 μl). To each solution (27 μl), PicoGreen solution (173 μl) was added. The tubes were wrapped with aluminum foil and left at room temperature for 10 min. The fluorescence at 523 nm was measured with EnSight (Perkin Elmer) with the excitation wavelength of 502 nm.

cDNA Synthesis

(15) Standard RNA, which is an RNA of 1,014-nucleotides corresponding to DNA sequence 8,353-9,366 of the cesA gene of Bacillus cereus (GenBank accession number DQ360825), was prepared by in vitro transcription. The reaction mixture for cDNA synthesis (20 μl) was prepared by mixing water (12 μl), 10×RT buffer (250 mM Tris-HCl buffer (pH 8.3), 500 mM KCl, 20 mM DTT, 50 mM MgCl.sub.2) (2 μl), 2.0 mM dNTP (1 μl), 160 pg/μl cesA RNA (2 μl), 10 μM RV-R26 primer 5′-TGTGGAATTGTGAGCGGTGTCGCAATCACCGTAACACGACGTAG-3′ (SEQ ID NO: 4) (1 μl) and 10 nM MMLV RT WT (2 μl). The reaction was run at 45° C. for 30 min and 65° C. for 5 min. The reaction mixture for PCR (25 μl) was prepared by mixing the reaction product of cDNA synthesis (2 μl), water (17.7 μl), 10×PCR buffer (2.5 μl), 2 mM dNTP (1.5 μl), 10 μM F5 primer 5′-TGCGCGCAAAATGGGTATCAC-3′ (SEQ ID NO: 5) (0.5 μl) and 10 μM RV primer 5′-TGTGGAATTGTGAGCGG-3′ (SEQ ID NO: 6) (0.5 μl), and Taq polymerase (0.3 μl). The reaction was run under 30 cycles of 30 s at 95° C., 30 s at 55° C., and 60 s at 72° C. The amplified products were separated on 1.0% w/v agarose gels and stained with ethidium bromide (1 μg/ml).

Example 1: Design of Mutations and Characterization of Single Variants

(16) We previously generated a thermostable triple MMLV RT variant MM3 (E286R/E302K/L435R) by introducing positive charges at positions that have been implicated in the interaction with a template-primer (Yasukawa et al., 2010). In order to further stabilize MMLV RT, we designed 29 mutations (Table 1). They are 8 mutations aimed to stabilize hydrophobic core, 8 mutations aimed to introduce salt bridge, 10 mutations aimed to introduce surface charge, and three mutations aimed to avoid disulfide bond.

(17) TABLE-US-00006 TABLE 1 Designed mutations Mutation Aim Ala32.fwdarw.Val Stabilize hydrophobic core Leu41.fwdarw.Asp Introduce salt bridge Ala42.fwdarw.Val Stabilize hydrophobic core Val43.fwdarw.Glu Increase surface charge Gln63.fwdarw.Glu Introduce salt bridge Leu72.fwdarw.Arg Increase surface charge Cys90.fwdarw.Ser Avoid disulfide bonds Val118.fwdarw.Ile Stabilize hydrophobic core Tyr146.fwdarw.Phe Stabilize hydrophobic core Ala154.fwdarw.Ile Stabilize hydrophobic core Met177.fwdarw.Arg Increase surface charge Ile179.fwdarw.Arg Increase surface charge Ile212.fwdarw.Arg Increase surface charge Leu234.fwdarw.Arg Increase surface charge Ile261.fwdarw.Phe Stabilize hydrophobic core Cys262.fwdarw.Ser Avoid disulfide bonds Leu272.fwdarw.Glu Introduce salt bridge Met289.fwdarw.Arg Increase surface charge Cys310.fwdarw.Leu Stabilize hydrophobic core Trp336.fwdarw.Arg Introduce salt bridge Ile347.fwdarw.Glu Introduce salt bridge Leu351.fwdarw.Glu Increase surface charge Leu357.fwdarw.Asp Introduce salt bridge Asp361.fwdarw.Leu Stabilize hydrophobic core Leu368.fwdarw.Arg Increase surface charge Val370.fwdarw.Glu Introduce salt bridge Trp388.fwdarw.Arg Introduce salt bridge Cys409.fwdarw.Arg Avoid disulfide bonds Leu410.fwdarw.Arg Increase surface charge

(18) The wild-type MMLV RT (WT), the 29 single variants, and one double variant Y146F/D361L were expressed in 3-ml culture and purified from the cells. A thermostable quadruple variant MM4 (E286R/E302K/L435R/D524A) (Yasukawa et al., 2010) was also prepared. MM4 lacks the RNase H activity because Asp524 is a catalytic residue for the RNase H activity. Following SDS-PAGE under reducing conditions, purified WT and variants yielded a single band with a molecular mass of 75 kDa.

(19) FIG. 3A shows the specific activities of the reverse transcription reaction for WT and the 30 variants. The specific activity of WT was 14,000 units/mg. All variants can be classified into three groups. Group 1 comprises V43E, A1541, I261F, L357D, L368R, and V370E whose specific activities were less than 10% of that of WT. Group 2 comprises L41D, Q63E, L72R, L272E W388R, and L410R whose specific activities were 60-140% of that of WT. Group 3 comprises the other 18 variants whose specific activities were 10-60% of that of WT.

(20) FIGS. 3B and C show the stabilities of WT, MM4, and the 24 variants which belong to Group 2 or 3 at 49 and 51° C., respectively. Relative activity was defined as the ratio of the initial reaction rate for a 10-min incubation at 49 or 51° C. in the presence of T/P to the rate without incubation. The relative activities of WT and D524A at 49° C. were 66 and 120%, respectively, and those at 51° C. were 18 and 100%, respectively. No variants exhibited higher relative activity than MM4 at 49 or 51° C. However, A32V, L72R, 1212R, L272E, W388R, and C409R exhibited higher relative activity than WT both at 49 and 51° C.

Example 2: Design of Mutational Combination and Characterization of Multiple Variants

(21) Based on the results presented in FIG. 3, four mutations (Ala32.fwdarw.Val, Leu72.fwdarw.Arg, Ile212.fwdarw.Arg, Leu272.fwdarw.Glu, and Trp388.fwdarw.Arg) were selected as the stabilizing mutations and one mutation (Leu41.fwdarw.Asp) was selected as the activating mutation. Ten variants (MM3.1-MM3.10) were designed by combing one, two, or three out of the six mutations with the MM3 mutations (Glu286.fwdarw.Arg, Glu302.fwdarw.Lys, and Leu435.fwdarw.Arg) (Table 2).

(22) TABLE-US-00007 TABLE 2 Multiple variants Variant Mutations MM3 E286R/E302K/L435R MM3.1 E286R/E302K/W388R/L435R MM3.2 L272E/E286R/E302K/L435R MM3.3 A32V/E286R/E302K/L435R MM3.4 L72R/E286R/E302K/L435R MM3.5 I212R/E286R/E302K/L435R MM3.6 L41D/E286R/E302K/L435R MM3.7 I212R/E286R/E302K/W388R/L435R MM3.8 L72R/E286R/E302K/W388R/L435R MM3.9 L72R/I212R/E286K/E302R/L435R MM3.10 L72R/I212R/E286R/E302K/W388R/L435R MM3.11 A32V/L72R/E286R/E302K/L435R MM3.12 A32V/I212R/E286R/E302K/L435R MM3.13 A32V/I212R/E286R/E302K/W388R/L435R MM3.14 A32V/L72R/E286R/E302K/W388R/L435R MM3.15 A32V/L72R/I212R/E286R/E302K/W388R/L435R

(23) They were expressed in E. coli and purified. Upon SDS-PAGE under reducing conditions, purified variants yielded a single band with a molecular mass of 75 kDa. The yields of the purified enzymes from 500 ml of culture were in the range of 0.38-4.26 mg, which were comparable to that of the WT (2.27 mg) (Table 3).

(24) TABLE-US-00008 TABLE 3 Yield, activity and stability of MMLV RT variants by the assay using using [.sup.3H]-dTTP Yields from 500-ml Variant culture (mg) Specific activity.sup.a (units/mg) WT 2.27 139,000 (1.0).sup.b MM3 4.26 92,000 (0.66).sup.b MM3.1 0.38 39,000 (0.28).sup.b MM3.2 3.24 0 (0).sup.b MM3.3 2.97 53,000 (0.38).sup.b MM3.4 2.30 80,000 (0.58).sup.b MM3.5 2.45 112,000 (0.81).sup.b MM3.6 2.22 103,000 (0.74).sup.b MM3.7 1.64 147,000 (1.1).sup.b MM3.8 2.65 84,000 (0.60).sup.b MM3.9 2.99 80,000 (0.58).sup.b MM3.10 2.23 60,000 (0.43).sup.b .sup.aThe reaction was carried out in 5 nM RT, 25 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 2 mM DTT, 5 mM MgCl.sub.2, 25 μM poly(rA)-p(dT).sub.15 (this concentration is expressed based on p(dT).sub.15), and 0.2 mM [.sup.3H]dTTP at 37° C. One unit is defined as the amount which incorporates 1 nmol of dTTP into poly(rA)-p(dT).sub.15 in 10 min. .sup.bNumbers in parentheses indicate values relative to WT (wildtype).

(25) FIG. 4A and Table 3 show the activities by the reverse transcription assay using [.sup.3H]-dTTP. Stabilities were assessed by the assay using fluorescent dye PicoGreen. MM3.2 (L272E/E286R/E302K/L435R) lacked the activity, indicating that the mutation of Leu272.fwdarw.Glu was incompatible with the MM3 mutations. The specific activities of the other nine variants (MM3.1 and MM3.3-MM3.10) were 30-100% of that of WT.

(26) TABLE-US-00009 TABLE 4 Stability of MMLV RT variants by the assay using [.sup.3H]-dTTP. Initial reaction rate (nM/s) After heat After heat Before heat treatment Before heat treatment treatment.sup.a (49° C.).sup.b treatment (51° C.).sup.b WT 70.8 5.8 (0.08).sup.c 70.5 0.7 (0.0).sup.c MM3 92.4 48.8 (0.53).sup.c 78.8 20.9 (0.27).sup.c MM3.1 33.1 10.7 (0.32).sup.c NT.sup.d NT.sup.d MM3.2 NT.sup.d NT.sup.d NT.sup.d NT.sup.d MM3.3 47.8 36.4 (0.76).sup.c 46.2 17.6 (0.38).sup.c MM3.4 96.3 36.5 (0.38).sup.c NT.sup.d NT.sup.d MM3.5 64.9 40.7 (0.63).sup.c 120.7  49.4 (0.41).sup.c MM3.6 60.0 8.9 (0.15).sup.c NT.sup.d NT.sup.d MM3.7 87.3 63.7 (0.73).sup.c 71.9 55.3 (0.77).sup.c MM3.8 73.7 65.9 (0.89).sup.c 60.8 38.3 (0.63).sup.c MM3.9 83.0 58.9 (0.71).sup.c 47.1 14.0 (0.30).sup.c MM3.10 70.3 67.9 (0.97).sup.c 41.2 18.7 (0.45).sup.c .sup.aThe reaction was carried out in 10 nM RT, 25 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 2 mM DTT, 5 mM MgCl.sub.2, 25 μM poly(rA)-p(dT).sub.15 (this concentration is expressed based on p(dT).sub.15), and 0.2 mM [.sup.3H]dTTP at 37° C. One unit is defined as the amount which incorporates 1 nmol of dTTP into poly(rA)-p(dT).sub.15 in 10 min. .sup.bRT at 100 nM was incubated at 49 or 51° C. in the absence or presence of poly(rA)-p(dT).sub.15 (28 μM) for 10 min. Then, the dTTP incorporation reaction was carried out at 37° C. .sup.cNumbers in parentheses indicate the relative activity, which is defined as the ratio of the initial reaction rate with incubation to that without incubation. .sup.dNot tested

(27) FIG. 4C and Table 4 show the stabilities as assessed by the assay using [.sup.3H]-dTTP, and FIG. 4D shows the stabilities as assessed by the assay using PicoGreen. The relative activities of MM3.3 MM3.5, MM3.7, MM3.8, MM3.9, and MM3.10 were comparable to that of MM3 while those of MM3.1, MM3.4, and MM3.6 were lower than MM3. The relative activity of MM3.6 (L41D/E286R/E302K/L435R) was almost the same to that of WT, indicating that the mutation of Leu41.fwdarw.Asp was incompatible with the MM3 mutations.

(28) Additionally, five variants (MM3.11-MM3.15; see Table 2) were designed by combing two or more of the four mutations (Ala32.fwdarw.Val, Leu72.fwdarw.Arg, Ile212.fwdarw.Arg, and Trp388.fwdarw.Arg). MM3.11 was not expressed, but the other four variants (MM3.12.fwdarw.MM3.15) were expressed in E. coli and purified. FIGS. 4E and 4F show their stabilities as assessed by the assay using [.sup.3H]-dTTP and PicoGreen, respectively. The relative activity of MM3.14 was superior to that of MM3 while those of MM3.12, MM3.13, and MM3.15 were lower than MM3.

(29) MM3.14 was further evaluated. FIG. 5 shows the temperature dependence on cDNA synthesis by WT, MM3, or MM3.14. After the cDNA synthesis reaction, real-time PCR was carried out. When cDNA synthesis reaction was conducted with MM3.14 at 55, 60, or 65° C. for 10 min, the fluorescence increased in the PCR. On the other hand, when the cDNA synthesis reaction was conducted with WT or MM3, it was not. This indicates that MM3.14 was more thermostable and more suitable for use in cDNA synthesis than MM3.

(30) FIG. 6 shows the comparison of the thermostabilities of WT, MM3, and MM3.14. The cDNA synthesis reaction was carried out at 45° C. for 30 min with WT, MM3, or MM3.14 exposed to 48-63° C. for 10 min. The reaction product was subjected to PCR, followed by agarose gel electrophoresis. The highest temperatures at which cDNA was synthesized were 60° C. for MM3.

(31) In a further experiment it was proven that cDNA was synthesized at 60 and 65° C. in the reaction with MM3.14 (A32V/L72R/E286R/E302K/W388R/L435R), while it was little synthesized at 60° C. and not synthesized at 65° C. in the reaction with MM3 (E286R/E302K/L435R), indicating that MM3.14 is more thermostable than MM3 in the reaction.

(32) In summary, it could be proven that MM3.14 is more thermostable than MM3.

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