JPWO2016153053A1 - Modified polymerase - Google Patents

Abstract

Disclosed is a modified polymerase comprising an amino acid sequence having a predetermined mutation in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. According to the present invention, a polymerase having LNA chain elongation activity and a polymerase having reverse transcription activity are provided.

Description

  The present invention relates to a modified polymerase.

  Nucleic acid aptamers are selected from nucleic acid libraries containing various sequences by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) method. A nucleic acid aptamer refers to a nucleic acid molecule that can specifically bind to a target molecule by its three-dimensional structure. Nucleic acid aptamers are expected as molecular target drugs because they bind specifically to target molecules and thus have few side effects; they can be chemically synthesized in large quantities;

  Nucleic acid aptamers can be synthesized using natural nucleic acids such as DNA and RNA, but such natural nucleic acids can be easily degraded in vivo by nucleolytic enzymes. Therefore, it is necessary to protect these natural nucleic acids from degradation by replacing them with artificial nucleic acids with chemical modifications. However, when they are replaced with artificial nucleic acids, a decrease in activity due to aptamer structural changes frequently occurs. .

  Xeno Nucleic Acid (XNA) is known as an artificial nucleic acid that can replace the natural nucleic acid. It is expected to obtain an aptamer having resistance to degrading enzymes by performing SELEX using a library containing artificial nucleic acid XNA in advance. In this case, an XNA library is prepared through transcription from a DNA library to XNA (DNA → XNA), and then the sequence obtained by selection is reverse-transcribed (XNA → DNA) from the XNA in the sequence to DNA. Amplify. In transcription, an XNA chain is synthesized using a DNA chain as a template, and in reverse transcription, a DNA chain is synthesized using an XNA chain as a template. These synthesis reactions are also called chain extension reactions, and are usually carried out using enzymes as catalysts.

  In the polymerase chain reaction (PCR) for amplifying the DNA strand (that is, extending the DNA strand using the DNA strand as a template), a heat-resistant DNA-dependent DNA polymerase is used. However, it is difficult for a wild-type DNA polymerase to elongate an artificial nucleic acid chain having a degradation enzyme resistance such as LNA.

  Non-Patent Document 1 discloses six types of XNA, namely 1,5-anhydrohexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), 2′-O, 4′-C-methylene-β-D. Ribonucleic acid (LNA: also referred to as “2,4-BNA (bridged nucleic acid) / LNA (locked nucleic acid)”), arabino nucleic acid (ANA) and 2′-fluoro-arabino nucleic acid (FANA), α-L— Studies on strand synthesis reactions using threofuranosyl nucleic acid (TNA) have been reported.

  In Non-patent Document 1, an attempt was made to produce a modified polymerase based on a DNA polymerase mutant (TgoT) derived from Thermococcus gorgonarius, a hyperthermophilic archaeon. Of the five modified polymerases, Pol6G12, PolC7 and PolD4K catalyze DNA → XNA, Pol6G12 is synthesized as HNA chain, PolC7 is synthesized as CeNA chain and LNA chain, and PolD4K is synthesized as ANA chain and FANA chain. It was. The remaining two, RT521 and RT521K, were reported to catalyze XNA → DNA, RT521 was reverse transcribed into HNA, TNA, ANA and FANA chains, and RT521K was reverse transcribed into CeNA and LNA chains.

  Non-Patent Document 2 reports the creation of a random library using LNA. Here, LNA-containing oligonucleotides are generated using LNA ATP or LNA TTP, that is, among A, C, G and T bases, only A or T is LNA, and LNA-containing oligonucleotide synthesis In this case, commercially available KOD XL polymerase (Novagen) is used.

  However, with respect to the XNA chain constituting the XNA library for use in SELEX, it is required to generate a stable chain from the template DNA. Thus, there is a need for a polymerase that allows such stable chain extension.

Science 2012, 336, 341-344 Molecules 2012, 17, 13087-13097

  An object of the present invention is to provide a modified polymerase capable of extending a chain containing an artificial nucleic acid. It is another object of the present invention to provide a modified polymerase capable of reverse transcription from a strand containing an artificial nucleic acid to DNA.

  The present invention provides modified polymerases as described in (i) to (iii) below.

The modified polymerase (i) of the present invention comprises an amino acid sequence having the following mutation (1) or (2) in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing:
(1) Asparagine at position 210 is aspartic acid, tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, aspartic acid or glutamic acid, alanine at position 485 is a neutral amino acid, and position 664 Glutamic acid is substituted with lysine, glutamine or arginine respectively; or (2) asparagine at position 210 is aspartic acid and tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, asparagine Acid or glutamic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with asparagine, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine. There.

The further modified polymerase (ii) of the present invention comprises an amino acid sequence having the following mutation (1) or (2) in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing:
(1) Asparagine at position 210 is substituted with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine; or (2) asparagine at position 210 Is substituted with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with asparagine, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine.

Still further modified polymerase (iii) of the present invention consists of an amino acid sequence having the following mutation in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing:
Asparagine at position 210 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine.

  The present invention also provides a method for synthesizing an LNA-containing oligonucleotide chain from a DNA-containing oligonucleotide. The method includes a step of reacting a template comprising the DNA-containing oligonucleotide, a primer, LNA-NTP, and modified polymerase (i) or (ii).

  In one embodiment, the primer is an LNA-containing primer.

  In one embodiment, the template includes a random sequence.

  The present invention further provides a method of synthesizing a DNA-containing oligonucleotide chain from an LNA-containing oligonucleotide. This method includes a step of reacting a template, a primer, dNTP comprising the LNA-containing oligonucleotide and the modified polymerase (ii) or (iii) of the present invention.

In one embodiment, in the method for synthesizing an LNA-containing oligonucleotide chain from the DNA-containing oligonucleotide and the method for synthesizing the DNA-containing oligonucleotide chain from the LNA-containing oligonucleotide, the reacting step comprises 120 mM Tris- Performed in a buffer containing HCl (pH 7-9), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , and 10 μg / mL bovine serum albumin (BSA).

  The present invention also relates to a modified polymerase having LNA chain elongation activity, a mutation that attenuates exonuclease activity, a mutation that contributes to binding of triphosphates, and a mutation that enhances binding to DNA / LNA duplex A modified polymerase is provided that is a polymerase modified to include

  In one embodiment, the modified polymerase further comprises a mutation that mitigates recognition of the triphosphate sugar structure.

  The present invention also relates to a modified polymerase having reverse transcription activity, including a mutation that attenuates exonuclease activity and a mutation that enhances binding to DNA / LNA duplex, or a mutation that attenuates exonuclease activity And a mutation modified to include a mutation that enhances the binding to the DNA / LNA duplex and a mutation that contributes to the binding of the triphosphate.

  According to the present invention, a modified polymerase capable of extending a chain containing an artificial nucleic acid LNA is provided. Furthermore, according to the present invention, a modified polymerase capable of reverse transcription from a strand containing LNA to DNA is also provided. This allows for stable extension of the LNA chain from DNA. Also, reverse transcription reaction from LNA chain to DNA is possible.

It is an electrophoresis photograph which shows the result of the LNA chain extension activity by N10 random arrangement | sequence template about various KOD polymerase mutants. It is an electrophoresis photograph which shows the result of the LNA chain extension activity by a N10 random sequence template about KOD polymerase variant A2, A34, A4, A13, A26, A48, and A53. It is an electrophoretogram showing the results of preparation of 30mer (left side) and 50mer (right side) LNA libraries for variant 1 and variant 2. It is an electrophoresis photograph which shows the result examined in various reaction time regarding preparation of a 30mer LNA library about the variant 2. Mutant 2 does not contain LNA-NTP (condition 1), lacks LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP (conditions 2-5), and lacks LNA-NTP. It is an electrophoresis photograph which shows the result of having performed LNA chain | strand extension reaction on the conditions (condition 6) which are not. It is an electrophoresis photograph which shows the comparison result of a DNA primer and a LNA primer about the LNA chain extension reaction of the variant 2. It is an electrophoresis photograph which shows the result of reverse transcription activity about various KOD polymerase mutants. It is an electrophoresis photograph which shows the result of the LNA chain | strand extension activity by the N10 random sequence template performed using the buffer solution for KOD-Plus-Ver.2 for the reaction liquid about the KOD polymerase variant which substituted variously for Y409. It is an electrophoresis photograph which shows the result of the LNA chain | strand extension activity by the N10 random sequence template performed using the buffer solution 1 for KOD DNA polymerase for the reaction liquid about the KOD polymerase variant which substituted Y409 variously. It is an electrophoresis photograph which shows the result of the LNA chain | strand extension activity by the N10 random sequence template performed using the buffer solution 2 for KOD DNA polymerase about the KOD polymerase variant which substituted variously for Y409. For A53, using the KOD DNA polymerase buffer 2 in the reaction solution, the conditions without LNA-NTP (Condition 1), the conditions lacking LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP ( It is an electrophoresis photograph which shows the result of having performed LNA chain | strand elongation reaction on the conditions (condition 6) which does not lack conditions 2-5) and LNA-NTP. For A53, using the buffer for KOD-Plus-Ver.2 in the reaction solution, the conditions not containing LNA-NTP (Condition 1), LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP are missing. 2 is an electrophoretic photograph showing the results of the LNA chain extension reaction performed under the conditions (conditions 2 to 5), and the conditions that do not lack LNA-NTP (condition 6). For A65, using the KOD DNA polymerase buffer 2 in the reaction solution, the conditions without LNA-NTP (Condition 1), the conditions lacking LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP ( It is an electrophoresis photograph which shows the result of having performed LNA chain | strand elongation reaction on the conditions (condition 6) which does not lack conditions 2-5) and LNA-NTP.

  In this specification, amino acid substitutions are expressed as follows. For example, “E664K” represents a mutant in which glutamic acid at position 664 is replaced with lysine. When a plurality of places are replaced, each replacement is shown by separating them with “/”. For example, “N210D / Y409V / A485L / D614N / E664K” describes asparagine at position 210 as aspartic acid, tyrosine at position 409 as valine, alanine at position 485 as leucine, aspartic acid at position 614 as asparagine, and A mutant in which glutamic acid at position 664 is replaced with lysine is shown. For example, the description “Y409” indicates that position 409 is tyrosine.

The modified polymerase (i) of the present invention comprises an amino acid sequence having the following mutation (1) or (2) in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing:
(1) Asparagine at position 210 is aspartic acid, tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, aspartic acid or glutamic acid, alanine at position 485 is a neutral amino acid, and position 664 Glutamic acid is substituted with lysine, glutamine or arginine respectively; or (2) asparagine at position 210 is aspartic acid and tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, asparagine Acid or glutamic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with asparagine, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine. There.

The further modified polymerase (ii) of the present invention comprises an amino acid sequence having the following mutation (1) or (2) in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing:
(1) Asparagine at position 210 is substituted with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine; or (2) asparagine at position 210 Is substituted with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with asparagine, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine.

Still further modified polymerase (iii) of the present invention consists of an amino acid sequence having the following mutation in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing:
Asparagine at position 210 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine.

  The amino acid sequence of SEQ ID NO: 2 in the sequence listing corresponds to the amino acid sequence of Thermococcus Kodakaraensis KOD1 DNA-dependent DNA polymerase (also referred to herein as “KOD polymerase”). Its origin does not matter.

  Here, in the present invention, the amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids other than the predetermined substitution are deleted, substituted or added, Including those having polymerase activity. Preferably, one or several amino acids are deleted, substituted or added within a range in which the predetermined substitution is retained and the amino acid sequence of SEQ ID NO: 2 has a sequence identity of 95% or more, and the predetermined polymerase activity The thing including what has this is mentioned. Examples of amino acid substitution in “one or several amino acids are deleted, substituted or added” herein may be any substitution as long as each function or effect is substantially retained. For example, conservative substitution can be mentioned. Specific examples of the conservative substitution include substitution within the following groups (ie, between amino acids shown in parentheses): (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid) ), (Asparagine, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine).

  As used herein, “identity” of a sequence is a relationship between two or more proteins or two or more polynucleotides, as is known in the art, determined by comparing the sequences. Sequence “identity” means between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of sequence invariance. Also, “similarity” refers to a protein or polynucleotide sequence as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. It means the degree of correlation. More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is also referred to as Similarity in the BLAST sequence homology search result described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; Altschyl et al., Nucleic Acids Res., 1997, 25: 3389-3402). ) Can be determined. The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  The modified polymerase (i) has LNA chain extension activity. The modified polymerase (i) is preferably N210D / Y409V / A485L / E664K; or N210D / Y409V / A485L / D614N / E664K, N210D / Y409G / A485L / D614N / E664K, N210D / Y409A / A485L / D614, N210D / Y409S / A485L / D614N / E664K, N210D / Y409T / A485L / D614N / E664K, N210D / Y409L / A485L / D614N / E664K, N210D / Y409F / A485L / D614N / E664D / N614 / D664N / E664D It has a mutation of N210D / Y409E / A485L / D614N / E664K.

  The modified polymerase (ii) has LNA chain extension activity. The modified polymerase (ii) has reverse transcription activity from LNA to DNA. The modified polymerase (ii) preferably has a mutation of N210D / A485L / E664K or N210D / A485L / D614N / E664K. N210D / A485L / E664Q is also preferred.

  The modified polymerase (iii) has reverse transcription activity from LNA to DNA (“reverse transcription activity”). The modified polymerase (iii) preferably has a mutation of N210D / E664K or N210D / E664R.

  In this specification, “LNA” means 2′-O, 4′-C-methylene-β-D-ribonucleic acid (“2,4-BNA (bridged nucleic acid) / LNA (locked nucleic acid) ) "). The “LNA chain” is an oligonucleotide containing LNA, for example, the following formula:

(Here, “Base” is a base selected from adenine (A), thymine (T), cytosine (C) and guanine (G)), and a nucleic acid chain having a predetermined chain length It is. Furthermore, in this specification, “LNA chain elongation activity” refers to the 3′-hydroxyl group of DNA annealed to template DNA or the oligonucleotide (eg, primer) of LNA and the 5′-triphosphate group of LNA. This refers to the activity of catalyzing the reaction that causes the template-dependent synthesis of LNA-containing oligonucleotides of DNA, that is, the elongation of LNA chains, by covalently binding α-phosphate. The LNA chain extension activity can be determined by a method commonly used by those skilled in the art, for example, using the procedure described in Reference Example 3. LNA is used as a substrate for LNA chain extension by the modified polymerase of the present invention. As such a substrate, methyl C-type LNA (a methyl group is added to the 5-position carbon atom of the pyrimidine ring of cytosine of LNA). ) Can also be used.

  In the present specification, reverse transcription activity from LNA to DNA (also simply referred to as “reverse transcription activity”) refers to a template LNA-containing oligonucleotide or DNA oligonucleotide annealed to a polynucleotide (eg, primer ) Of 3) -hydroxyl group of LNA) is covalently bound to α-phosphate of 5′-triphosphate group of LNA to catalyze a reaction that causes synthesis of a template-dependent DNA-containing oligonucleotide, ie, DNA strand elongation. Activity. The reverse transcription activity can be determined by a method commonly used by those skilled in the art, for example, using the procedure described in Example 8.

  The structural features of the modified polymerase having LNA chain extension activity include the following. Here, the three-dimensional structure of KOD polymerase is shown, for example, in Kai et al., J Mol Biol. 2001, 306, 469-477, and the functional site is largely composed of an N-terminal region (“N-ter”: 1-130). And 327-368), exonuclease region ("Exo": 131-326), and polymerase active region (Palm region: 369-449 and 500-587, Fingers region: 450-499, and Thumb -1, Thumb-2 region (herein referred to as “Thumb region”): 588 to 774).

  N210D (substitution of asparagine at position 210 with aspartic acid) is located in the exonuclease region and attenuates exonuclease activity. This N210D is commonly present in modified polymerases having LNA chain extension activity.

  A485 (alanine at position 485) is located in the Fingers region and is thought to contribute to the binding of the triphosphate. The alanine at position 485 can be replaced with a neutral amino acid (eg, leucine, isoleucine, phenylalanine, methionine). At position 485, a small bulk alanine can be replaced with a neutral amino acid (eg, leucine, isoleucine, phenylalanine, methionine) that can also be a bulky hydrophobic amino acid.

  D614N (substitution of aspartic acid at position 614 to asparagine) and E664K (substitution of glutamic acid at position 664 to lysine) can enhance binding to the DNA / LNA duplex. There is a negatively charged amino acid (D614: aspartic acid at position 614, glutamic acid at position E664: 664) in the Thumb region that binds to the duplex, but this amino acid is uncharged (eg, D614N) or positive By changing the charge (for example, E664K), it is considered that the bond between the polymerase and the DNA / LNA duplex is strengthened, and the 3 ′ end of the primer strand can be correctly positioned at the reaction site. Here, aspartic acid at position 614 can be replaced with asparagine, and glutamic acid at position 664 can be replaced with lysine, glutamine, or arginine. It may be a substitution mutation only at position 664, or a substitution mutation at positions 614 and 664.

  Y409 (tyrosine at position 409) can be further substituted. Y409 is located in the Palm region and touches the sugar part of LNA-NTP. Y409 can be substituted with valine, glycine, alanine, serine, threonine, leucine, phenylalanine, aspartic acid or glutamic acid. Y409 substitution mutations can be made with the substitution mutations described above, ie, substitution mutations at positions 210, 485 and 664, or positions 210, 485, 614 and 664. In one embodiment, substitution of Y409 can be made with substitution of N210D, A485L, D614N and E664K. Substitution of Y409 can alleviate the recognition of the sugar structure of the triphosphate and improve the uptake rate of LNA-NTP. By replacing Y409 with an amino acid having a small size (bulk) group (for example, glycine, alanine or serine), higher LNA chain elongation activity can be obtained, and Y409 is valine (Y409V) or threonine. By substituting (Y409T), LNA extension can be obtained with higher accuracy.

  Modified polymerases with LNA chain extension activity have been modified to include mutations that attenuate exonuclease activity, mutations that contribute to triphosphate binding, and mutations that enhance binding to DNA / LNA duplexes It can be designed to be a polymerase. Preferably, the modified polymerase further comprises a mutation that alleviates recognition of the triphosphate sugar structure.

  Structural features of the modified polymerase having reverse transcription activity include the following. It is considered that the modified polymerase having activity has the following characteristics.

  N210D (substitution of asparagine at position 210 with aspartic acid) attenuates exonuclease activity. This N210D is commonly present in modified polymerases having reverse transcription activity.

  During reverse transcription, it is necessary that the polymerase binds to the DNA / LNA duplex and that the 3 'end of the primer strand is correctly positioned at the reaction site. For example, reverse transcription activity is seen when the negatively charged amino acid in the Thumb region is changed to a positive charge (eg, E664K or E664R), which is a result of increased binding between the polymerase and the DNA / LNA duplex. It is thought that. Involved in binding to triphosphate with a mutation (E664Q) in which the negatively charged amino acid in the Thumb region is neutrally changed to a positive charge (which may slightly increase the DNA / LNA duplex binding capacity of the polymerase) Reverse transcription activity is also observed when A485L is introduced. A mutant having E664K also has improved reverse transcription activity in combination with A485L.

  When E614N is included together with E664K, reverse transcription activity is improved in combination with A485L. Here, the alanine at position 485 can be substituted with a neutral amino acid (eg, leucine, isoleucine, phenylalanine, methionine). The glutamic acid at position 664 can be replaced with lysine, glutamine, or arginine.

  The modified polymerase having reverse transcription activity includes a mutation that attenuates exonuclease activity and a mutation that enhances binding to DNA / LNA duplex, or a mutation that attenuates exonuclease activity and DNA / LNA duplex It can be designed to be a polymerase modified to include mutations that enhance binding to the strand and mutations that contribute to triphosphate binding.

  As a method for producing the modified polymerase, a conventionally known method can be used. For example, there is a method for producing a mutant (modified) DNA polymerase having a new function by introducing a mutation into a gene encoding a wild-type DNA polymerase (J. Biol. Chem., 264). (11), 6447-6458 (1989)). The gene encoding the DNA polymerase into which the mutation is introduced is not particularly limited as long as it is a gene encoding the amino acid sequence of SEQ ID NO: 2. For example, the gene may be a base sequence that can be obtained from a gene registration organization such as NCBI as a DNA-dependent DNA polymerase gene of Thermococcus kodakaraensis KOD1, or a base sequence that has been subjected to codon optimization (for example, in the sequence listing). Based on sequence information such as the base sequence of SEQ ID NO: 1, it can be obtained by methods commonly used by those skilled in the art.

  Any known method may be used for introducing a mutation into a gene. For example, protein engineering techniques such as PCR and site-specific mutation can be used.

  The modified polymerase gene is inserted into an expression vector as necessary, and transformed with the expression vector using, for example, Escherichia coli as a host, and then applied to an agar medium containing a selective agent such as kanamycin to form colonies. After inoculating the colony in a nutrient medium such as LB medium and appropriately culturing (eg, growing at 37 ° C. and then culturing at 30 ° C. in the presence of an expression inducer such as IPTG), the cells are recovered and Crush and extract the crude enzyme solution. The vector is preferably a pET vector, but is not limited thereto. The culture medium composition, pH, temperature, cell density during culture, and culture time can be appropriately set. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. The crude enzyme solution can be treated by heat treatment, for example, at 80 ° C. for 10 minutes, and then the crude purified enzyme can be obtained by centrifugation, membrane filtration or the like. After this operation, it can be separated and purified by heparin sepharose column chromatography. Furthermore, the process of refine | purifying or concentrating this can also be implemented. These steps and the means required for them are appropriately selected by those skilled in the art.

  The modified polymerases (i) and (ii) are synthesized by a method for synthesizing an LNA-containing oligonucleotide chain from a DNA-containing oligonucleotide (also referred to as “LNA chain extension method” in this specification), and a modified polymerase ( ii) and (iii) can be used in a method for synthesizing a DNA-containing oligonucleotide chain from an LNA-containing oligonucleotide (also referred to as “reverse transcription method” in the present specification).

  The LNA chain extension method of the present invention comprises a step of reacting a template comprising a DNA-containing oligonucleotide, a primer, LNA-NTP and a modified polymerase (i) or (ii). Using the modified polymerase of the present invention, a DNA-containing oligonucleotide as a template and a primer, LNA-NTP (that is, four types of LNA-nucleoside triphosphates each having A, T, C and G as bases) LNA-ATP, LNA-TTP, LNA-CTP, and LNA-GTP) reaction), the primer annealed to the template is extended while incorporating LNA, and the LNA chain (LNA-containing oligonucleotide) is used as a primer extension product. Can be synthesized. The LNA chain extension method of the present invention may further include an annealing step before the reaction step using a polymerase. In the annealing step, the primer and the complementary region on the template are hybridized, and in the subsequent extension step, the LNA strand complementary to the single-stranded DNA of the template is extended from the primer as a starting point by the action of the modified polymerase of the present invention. DNA-LNA strands can be formed.

  Here, the “oligonucleotide” may be any length as long as it is suitable for the template, and examples thereof include those having a base number of, for example, 30 to 100, preferably 35 to 75.

  The reaction temperature for the annealing and extension may be the same conditions as in normal PCR. For example, after annealing at 95 ° C. for 1 minute and then lowering to room temperature over 1 hour, the polymerase is used. Can be added and extended at 74 ° C. Examples of the composition of the reaction solution include those shown in Table 2 below, but are not limited thereto. The addition concentration of the enzyme may depend on the length of the extended chain, the reaction time, and the like, but is, for example, 20 ng / μL to 300 ng / μL, preferably 200 ng / μL to 300 ng / μL, but is not limited thereto. The reaction time may depend on the length of the extended chain, the concentration of the enzyme added, etc., but may be 1.5 hours to 13.5 hours, but is not limited thereto.

  The primer may be either a DNA primer (a primer composed of DNA) or an LNA primer (a primer composed of LNA). The primer may contain both DNA and LNA. From the viewpoint of avoiding a rate-limiting step in the middle of the primer extension reaction, a primer containing LNA at least in part (preferably a primer that is all LNA) is preferred. Although the length of a primer can be determined suitably, what has the base number is 11-28, for example, Preferably, it is 12-25. Primers can be appropriately designed and produced by those skilled in the art based on the sequence information of the template.

  The template may be composed of a specific sequence or may include a random sequence. The length of the random sequence is, for example, 10 to 50 bases. By using a template containing a random sequence, an LNA library can be created.

  The reverse transcription method of the present invention comprises a step of reacting a template comprising an LNA-containing oligonucleotide, a primer, dNTP, and a modified polymerase (ii) or (iii). Using the modified polymerase of the present invention, an LNA-containing oligonucleotide is used as a template, and a primer, dNTP (that is, four types of deoxynucleoside triphosphates (dATP, dTTP) each having A, T, C, and G as bases. , A mixture of dCTP and dGTP)), the primer annealed to the template is extended while incorporating DNA, and a DNA strand (DNA-containing oligonucleotide) can be synthesized as a primer extension product. The reverse transcription method of the present invention may further include an annealing step before the reaction step using a polymerase. In the annealing step, the primer and the complementary region on the template are hybridized, and in the subsequent extension step, a DNA strand complementary to the single-stranded LNA of the template is extended by the action of the modified polymerase of the present invention starting from the primer. And an LNA-DNA strand can be formed.

  The primer for the reverse transcription method can be a DNA primer (a primer composed of DNA). Although the length of a primer can be determined suitably, what has the base number is 11-28, for example, Preferably, it is 12-25. Primers can be appropriately designed and produced by those skilled in the art based on the sequence information of the template.

  The template for the reverse transcription method may be composed of a specific sequence or may include a random sequence. The length of the random sequence is, for example, 10 to 50 bases. The template for the reverse transcription method may be an LNA chain transcribed based on a DNA template (for example, an LNA chain produced by the LNA chain extension method of the present invention).

  Similarly, in the case of the reverse transcription method, the reaction temperature for annealing and extension can be the same as that for normal PCR. For example, after annealing at 95 ° C. for 1 minute, the temperature is lowered to room temperature over 1 hour for annealing. After performing, the polymerase can be added and extended at 74 ° C. Examples of the composition of the reaction solution include those shown in Example 8, but are not limited thereto. The concentration of the enzyme added may depend on the length of the extended chain, the reaction time, and the like, and is, for example, 10 ng / μL to 300 ng / μL, preferably 10 ng / μL to 100 ng / μL, but is not limited thereto. The reaction time may depend on the length of the extended chain, the concentration of the enzyme added, etc., but may be 30 minutes to 3 hours, but is not limited thereto.

In the LNA chain extension method and reverse transcription method, it is preferable that a divalent ion such as magnesium ion (for example, as MgSO ₄ ) coexists in addition to the modified polymerase of the present invention and the nucleic acid component. In the reaction using the modified polymerase, as a buffer solution, a commercially available PCR buffer solution (for example, KOD-Plus-Ver.2 Buffer (manufactured by Toyobo Co., Ltd.)) and any buffer prepared by those skilled in the art. Solutions (eg, buffers containing 120 mM Tris-HCl (pH 7-9), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , and 10 μg / mL bovine serum albumin (BSA)) can be used. Here, the pH of the buffer may vary depending on the buffer temperature and the extension reaction temperature. For example, in Tris-HCl, when the buffer temperature increases by 1 ° C., the pH decreases by 0.025, and the extension reaction temperature can also affect the working pH.

In one embodiment, the LNA chain extension reaction and reverse transcription method comprises 120 mM Tris-HCl (pH 7-9), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , and 10 μg / mL bovine serum albumin (BSA). In buffer. This buffer further contains 0.1% Triton® X-100. In this embodiment, for example, when the buffer temperature is 25 ° C. and the reaction is performed under the same conditions as in normal PCR or the extension reaction is performed at 74 ° C., the pH of the buffer is preferably pH 8-8. And pH 8.8 is more preferable than pH 8 in terms of the elongation efficiency of the LNA chain.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by these Examples.

(Example 1: Production of modified polymerase)
(1-1: Construction of plasmid)
The KOD polymerase gene was prepared by removing the intein sequence in advance by artificial gene synthesis and optimizing the codon (base sequence of SEQ ID NO: 1; the amino acid sequence encoded thereby is shown in SEQ ID NO: 2). Gene synthesis was commissioned to operon biotechnology. The KOD polymerase gene was inserted between the NdeI site and SalI site of pET49 vector (Novagen), and an expression plasmid was constructed so that the KOD polymerase protein did not contain extra sequences such as tags. The method of PrimeSTAR (registered trademark) Mutagenesis Basal Kit (TAKARA) was used for the point mutation introduction. The nucleotide sequence of the KOD polymerase mutant gene was analyzed for the KOD polymerase mutant expression plasmid to confirm that the target sequence was correctly inserted. Table 1 lists the combinations of mutations introduced.

(1-2: Expression and purification of KOD polymerase mutant)
The KOD polymerase mutant expression plasmid was introduced into T7 Express Competent E. coli (High Efficiency) (NEB) by the heat shock method, spread on an LB / Km (50 μg / mL) plate, and allowed to stand at 37 ° C. The grown colonies were shaken at 37 ° C. on TB medium / 1% glucose / Km (50 μg / mL). When OD ₆₀₀ reached 0.4 to 0.6, 1/1000 vol of 1M IPTG was added (final 1 mM), and the culture temperature was changed to 30 ° C. to induce expression. Six hours after adding IPTG, the culture was centrifuged to remove the supernatant, and E. coli was recovered. E. coli was stored at −80 ° C. and thawed during purification. The thawed E. coli was suspended in a buffer solution (10 mM sodium phosphate, 100 mM NaCl, 1 mM DTT, 10% glycerol), and the cells were disrupted by ultrasonic waves. After the E. coli disrupted solution was centrifuged (4 ° C., 8000 g, 5 minutes), the supernatant was recovered and heated in an 80 ° C. hot water bath for 10 minutes. Contaminating proteins denatured by heating were removed by centrifugation (4 ° C., 8000 g, 5 minutes), and the supernatant was passed through a 0.45 μm PVDF membrane filter to completely remove fine particles. The resulting crude purified solution was purified by AKTA pure (GE Healthcare) using HiTrap Heparin HP Columns (5 mL). For the KOD polymerase mutant adsorbed on the column, gradually increase the NaCl concentration of the buffer solution (10 mM sodium phosphate, 100 mM NaCl, 1 mM DTT, 10% glycerol) from 100 mM to 2 M to the elution fraction. The peak was obtained. The elution fraction was concentrated by ultrafiltration using Amicon Ultra 4 (30K) (Millipore), and then the buffer solution was replaced. Finally, a storage buffer solution (Storage Buffer: 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 50% glycerol, 0.1% Tween 20, 0.1% Nonidet P40) and stored at −20 ° C. The concentration of the KOD polymerase mutant was determined by the Bradford method, diluted to the desired concentration with Storage Buffer, and used for the reaction.

(Example 2: Synthesis of sugar-modified base)
(2-1: Synthesis of LNA-ATP)

LNA-A (102 mg, 3.65 × 10 ⁻⁴ mol, 1.0 eq) and a stirring bar were placed in a 100 mL eggplant flask, dried under reduced pressure overnight, and azeotroped twice with dry-DMF (6 mL). It was then azeotroped 3 times with dry-MeCN (6 mL). Proton sponge (117 mg, 5.46 × 10 ⁻⁴ mol, 1.46 eq), which had been dried under reduced pressure overnight, was added and further dried under reduced pressure overnight. The atmosphere was replaced with argon, a septum cap in the desiccator was attached, and an argon balloon was attached. Trimethyl phosphate (1.3 mL, 1.29 × 10 ⁻² mol, 36 eq) was added, suspended and allowed to stir in an ice bath for 30 minutes. POCl ₃ (55 μL, 5.90 × 10 ⁻⁴ mol, 1.5 eq) was added, and the mixture was stirred for 45 minutes in an ice bath. In an ice bath, dry-tributylamine (350 μL, 1.46 × 10 ⁻³ mol, 4.0 eq) and 0.5 M DMF pyrophosphate (3.7 mL, 1.87 × 10 ⁻³ mol, 5.0 eq) were added. The mixture was added and reacted at room temperature for 1 hour. The mixture was stirred for 5 minutes in an ice bath, about 6 mL of TEAB buffer was added to stop the reaction, and the mixture was distilled off under reduced pressure. A small amount of distilled water was added and dissolved, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphoric acid as the target product was fractionated with an anion exchange column (DEAE Sephadex A-25). Furthermore, excess pyrophosphoric acid was removed with a medium pressure column (C18) and purified by reverse phase HPLC (C18). The fraction was distilled off under reduced pressure and freeze-dried to obtain LNA-ATP. Yield: 6.16 μmol. Yield: 1.6%. ESI-MS (negative ion mode) m / z, measured value = 517.8, calculated value (relative to [(M−H) ⁻ ]) = 518.0.

  (2-2: Synthesis of LNA-TTP)

LNA-T (103 mg, 3.81 × 10 ⁻⁴ mol, 1.0 eq) and a stirring bar were placed in a 100 mL eggplant flask, dried under reduced pressure overnight, and azeotroped twice with dry-DMF (6 mL). It was then azeotroped 3 times with dry-MeCN (6 mL). Proton sponge (119 mg, 5.55 × 10 ⁻⁴ mol, 1.46 eq) that had been dried under reduced pressure overnight was added, and then further dried under reduced pressure overnight. The atmosphere was replaced with argon, a septum cap in the desiccator was attached, and an argon balloon was attached. Trimethyl phosphate (1.4 mL, 1.39 × 10 ⁻² mol, 36 eq) was added and dissolved completely and allowed to stir in an ice bath for 30 minutes. POCl ₃ (60 μL, 6.44 × 10 ⁻⁴ mol, 1.5 eq) was added and stirred for 45 minutes in an ice bath. Dry-tributylamine (400 μL, 1.67 × 10 ⁻³ mol, 4.0 eq) and 0.5 M DMF pyrophosphate (4.0 mL, 2.02 × 10 ⁻³ mol, 5.0 eq) in an ice bath The mixture was added and reacted at room temperature for 1 hour. The mixture was stirred for 5 minutes in an ice bath, about 6 mL of triethylammonium bicarbonate (TEAB) buffer was added to stop the reaction, and the mixture was distilled off under reduced pressure. A small amount of distilled water was added and dissolved, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphoric acid as the target product was fractionated with an anion exchange column (DEAE Sephadex A-25). Furthermore, excess pyrophosphoric acid was removed with a medium pressure column (C18) and purified by reverse phase HPLC (C18). The fraction was distilled off under reduced pressure and freeze-dried to obtain LNA-TTP. Yield: 115 μmol. Yield: 30.2%. ESI-MS (negative ion mode) m / z, measured value = 508.9, calculated value (for [(M−H) ⁻ ]) = 509.18.

(2-3: Synthesis of LNA-G ⁱ TP)

LNA-G (101 mg, 2.76 × 10 ⁻⁴ mol, 1.0 eq) and a stirring bar were placed in a 100 mL eggplant flask, dried under reduced pressure overnight, and azeotroped twice with dry-DMF (6 mL). It was then azeotroped 3 times with dry-MeCN (6 mL). Proton sponge (118 mg, 5.51 × 10 ⁻⁴ mol, 1.5 eq) that had been dried under reduced pressure overnight was added, and further dried under reduced pressure overnight. The atmosphere was replaced with argon, a septum cap in the desiccator was attached, and an argon balloon was attached. Trimethyl phosphate (1.0 mL, 9.94 × 10 ⁻³ mol, 36 eq) was added, suspended, and stirred in an ice bath for 30 minutes. POCl ₃ (40 μL, 4.29 × 10 ⁻⁴ mol, 1.5 eq) was added and stirred for 45 minutes in an ice bath. In an ice bath, dry-tributylamine (260 μL, 1.09 × 10 ⁻³ mol, 4.0 eq) and 0.5 M DMF pyrophosphate (2.8 mL, 1.41 × 10 ⁻³ mol, 5.0 eq) were added. The mixture was added and reacted at room temperature for 1 hour. The mixture was stirred for 5 minutes in an ice bath, about 6 mL of TEAB buffer was added to stop the reaction, and the mixture was distilled off under reduced pressure. A small amount of distilled water was added and dissolved, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphoric acid as the target product was fractionated with an anion exchange column (DEAE Sephadex A-25). Furthermore, excess pyrophosphoric acid was removed with a medium pressure column (C18) and purified by reverse phase HPLC (C18). The fraction was distilled off under reduced pressure and lyophilized to obtain LNA-G ⁱ TP. Yield: 32.3 μmol. Yield: 11.7%. ESI-MS (negative ion mode) m / z, measured value = 603.8, calculated value (for [(M−H) ⁻ ]) = 604.03.

  (2-4: Synthesis of LNA-GTP)

LNA-G ⁱ TP (1.86 mg, 20.03 μmol) in a 100 mL eggplant flask was dissolved in 1 mL of water, and 28% concentrated aqueous ammonia (7.1 mL) was added and stirred. Samples were taken every 4 hours and peaks were observed by HPLC. The reaction mixture was concentrated, 28% concentrated aqueous ammonia (7.1 mL) was added, and the mixture was further stirred for 4 hr. This was repeated until the LNA-G ⁱ TP peak disappeared on HPLC. After 48 hours, the reaction solution was distilled off under reduced pressure, purified by reverse phase HPLC (C18), and the fraction was distilled off under reduced pressure and lyophilized to obtain LNA-GTP. Yield: 12.74 μmol. Yield: 63%. ESI-MS (negative ion mode) m / z, found value = 533.8, calculated value (for [(M−H) ⁻ ]) = 533.99.

  (2-5: Synthesis of LNA-CTP)

LNA-C (101 mg, 3.72 × 10 ⁻⁴ mol, 1.0 eq) and a stirring bar were placed in a 100 mL eggplant flask, dried under reduced pressure overnight, and azeotroped twice with dry-DMF (6 mL). It was then azeotroped 3 times with dry-MeCN (6 mL). Proton sponge (113 mg, 5.27 × 10 ⁻⁴ mol, 1.46 eq) that had been dried under reduced pressure overnight was added, and further dried under reduced pressure overnight. After replacing with argon, a septum cap in a desiccator was attached and replaced with argon. Trimethyl phosphate (4.5 mL, 4.47 × 10 ⁻² mol, 120 eq) was added, suspended and allowed to stir in an ice bath for 30 minutes. POCl ₃ (60 μL, 6.44 × 10 ⁻⁴ mol, 1.5 eq) was added and stirred for 45 minutes in an ice bath. In an ice bath, dry-tributylamine (400 μL, 1.67 × 10 ⁻³ mol, 4.0 eq) and 0.5 M DMF pyrophosphate (3.8 mL, 1.92 × 10 ⁻³ mol, 5.0 eq) were added. The mixture was added and reacted at room temperature for 1 hour. The mixture was stirred for 5 minutes in an ice bath, about 6 mL of TEAB buffer was added to stop the reaction, and the mixture was distilled off under reduced pressure. A small amount of distilled water was added and dissolved, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphoric acid as the target product was fractionated with an anion exchange column (DEAE Sephadex A-25). Furthermore, excess pyrophosphoric acid was removed with a medium pressure column (C18) and purified by reverse phase HPLC (C18). The fraction was distilled off under reduced pressure and freeze-dried to obtain LNA-CTP. Yield: 18.28 μmol. Yield: 4.89%. ESI-MS (negative ion mode) m / z, measured value = 493.9, calculated value (for [(M−H) ⁻ ]) = 493.98.

(Reference example: Test method of LNA chain elongation activity of KOD polymerase mutant)
The methods used for examining the activity of KOD polymerase mutants are shown in Reference Examples 1 to 3. In this example, the methods of Reference Examples 1 to 3 below were used unless otherwise specified.

(Reference Example 1: Denaturing polyacrylamide gel electrophoresis (PAGE))
Running buffer: 1 × TBE buffer, denaturing polyacrylamide gel: 1 × TBE buffer, 7M urea, 20% polyacrylamide. To 10 μL of the reaction solution, 40 mM EDTA, 0.1% bromophenol blue: 2 μL, 12 μL of 10 M urea was added to prepare a migration sample. Preliminary electrophoresis was performed at 55 ° C. and a constant voltage of 200 V for 30 minutes, and then the sample was applied to a gel and migrated under the same conditions.

(Reference Example 2: Alkaline agarose gel electrophoresis)
For analysis of a single strand of DNA / RNA, denatured PAGE is usually used to dissociate the double strand. However, since the Tm value of DNA / LNA duplex is very high, it cannot be dissociated under the conditions of denaturing PAGE, and is not suitable for analysis of LNA single strand. Therefore, alkaline agarose gel electrophoresis was used to completely dissociate and analyze the DNA / LNA duplex. Even a DNA / LNA duplex is dissociated in a sodium hydroxide solution, so that LNA can be analyzed in a single-stranded state.

  The agarose used was for separating low molecular weight nucleic acids (Agarose 21 (Nippon Gene)). The composition of the gel, electrophoresis buffer, and loading dye are as follows. Gel: 50 mM NaCl, 2 mM EDTA, 5% agarose, running buffer: 5 mM NaOH, 2 mM EDTA, 6 × loading dye: 300 mM NaOH, 12 mM EDTA, 30% glycerol, 0.1% bromophenol blue. Before running, the gel was equilibrated by soaking in running buffer for 1 hour. Electrophoresis was performed under conditions of room temperature and 3 V / cm constant voltage. For detection of single-stranded LNA, analysis was performed by PharosFX (manufactured by Bio-Rad) using a fluorescent dye labeled with a primer.

(Reference Example 3: LNA chain elongation activity reaction of KOD polymerase mutant)
The composition of the reaction solution used for comparing the LNA chain extension activity of the KOD polymerase mutant is shown in Table 2 below. After heating the reaction solution to which KOD polymerase mutant has not been added at 95 ° C. for 1 minute and then lowering to room temperature over 1 hour, KOD polymerase mutant was added and LNA chain elongation reaction was performed at 74 ° C. Proceeded. Table 3 shows the template and primer sequences used in the LNA chain extension reaction.

(Example 3: Examination of LNA chain elongation activity of KOD polymerase mutant)
About the KOD polymerase variant produced in Example 1, LNA chain | strand extension activity was compared using primer strand: 5'_6-FAM_labeled primer P2 and template strand: Template DNA N10. For LNA chain extension, the reaction was allowed to proceed at 74 ° C. for 30 minutes. 40 mM EDTA, 0.1% bromophenol blue: 2 μL, 10 M urea 12 μL was added to the resulting reaction solution to prepare an electrophoresis sample. Analysis was performed by denaturing PAGE using 0.625 pmol of primer strand. If the LNA chain is a short chain, the DNA / LNA chain can be dissociated under the conditions of denaturing PAGE, so alkaline agarose gel electrophoresis was not used.

  The analysis results are shown in FIG. 1 (FIGS. 1-1, 2, 3, and 4). The result of selecting a representative example from FIG. 1 and analyzing by denaturing PAGE is shown in FIG. In these figures, the result of the unmodified wild type (“WT”) is shown as a control. In addition, in order to clarify the extent of extension, only the primer (“Primer”) and the extension corresponding to the extension of the template (ie, 10 bases) (“Primer + 10mer”) are also shown (the same applies to the following figures). is there).

  All mutants have a mutation N210D that attenuates exonuclease activity. Mutation A485L common to mutants A2, A34, A13, A26, A48, and A53 is located in the Fingers region and is thought to contribute to the binding of triphosphates. Moreover, it is considered that the mutation Y409V common to A34 and A53 relaxes the recognition of the sugar structure of the triphosphate. Originally, in wild-type polymerases, the inclusion of RNA in the DNA strand is inconvenient for survival, and thus is restricted by nucleotide sugar recognition so that NTP incorporation does not occur. However, this restriction causes a decrease in the uptake activity of LNA-NTP with a modified sugar moiety. Mutation Y409V improves the uptake rate of LNA-NTP by relaxing sugar part recognition.

  The mutations A485L and Y409V that contribute to these triphosphates alone are insufficient for the stable extension of the LNA chain. Since the structure of DNA / LNA duplex (A-type helix) is different from the structure of DNA / DNA duplex (B-type helix), it is considered that ordinary polymerase cannot bind to DNA / LNA chain stably. Because it is. Actually, in A2 and A34, the extension of the LNA chain is stopped at an early stage. Therefore, D614N (A26, A53) and E664K (A4, A13, A26, A48, A53) are introduced in order to strengthen the binding with the DNA / LNA duplex. There is a negatively charged amino acid (D614, E664) in the Thumb region that binds to the double chain. By changing this amino acid to no charge (D614N) or positive charge (E664K), DNA / LNA It is considered that the bond with the double strand was strengthened and the 3 ′ end of the primer strand was correctly positioned at the reaction site. LNA elongation activity is not so high just by strengthening the binding with DNA / LNA duplex like mutant A4, but mutations contributing to nucleotide monomer and nucleotide polymer like A13, A26, A48 and A53, respectively. By introducing simultaneously, it is possible to stably extend the LNA chain. Mutants A48 and A53, which have these mutations comprehensively, show particularly high LNA chain elongation activity. Hereinafter, A48 is also referred to as variant 1 and A53 as variant 2.

(Example 4: Preparation of LNA library using KOD polymerase mutant)
The composition of the reaction solution used to create the LNA library is shown in Table 4 below. After heating the reaction solution to which KOD polymerase mutant has not been added at 95 ° C. for 1 minute and then lowering it to room temperature over 1 hour, KOD polymerase mutant 1 or 2 was added to perform the LNA chain extension reaction. Proceeded. As a control, a DNA library was prepared using the same amount of dATP, dCTP, dGTP and dTTP instead of LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP in Table 4.

  30-mer and 50-mer LNA libraries were prepared using primer strand: 5′_6-FAM_labeled primer P2 and template strands: Template DNA N30 and Template DNA N50. For LNA chain extension, the reaction was allowed to proceed at 74 ° C. for 9 hours. 1/6 volume of 6 × loading dye was added to the resulting reaction solution, and 1 pmol of primer strand was applied to an alkaline agarose gel for electrophoresis. As a result, an LNA library comparable to a DNA library prepared using dNTP was successfully created (FIG. 3).

(Example 5: Examination of LNA chain elongation reaction time using KOD polymerase mutant)
The LNA chain extension reaction time was examined.

  Primer strand: 5'_6-FAM_labeled primer P2, Template strand: Template DNA N30 was used to prepare a 30-mer LNA library. The composition of the reaction solution is as shown in Table 4, and as a control, a DNA library was prepared using dATP, dCTP, dGTP and dTTP instead of LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP. . Comparison was performed using KOD polymerase mutant 2 under the conditions of reaction time: 1.5 hours, 3 hours, 6 hours, and 9 hours. A 30-mer LNA extension reaction product began to be produced at an extension time of 1.5 hours, indicating that the reaction time for preparing an LNA library can be reduced from 9 hours (FIG. 4).

(Example 6: Accuracy of LNA chain synthesis using KOD polymerase mutant)
Does the extension reaction stop when an LNA chain extension reaction is performed using a single sequence template under the reaction conditions except one of LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP? Examined. It was found that the reaction proceeds to the end without stopping the extension reaction in the middle when the wrong base incorporation is allowed.

  Using the primer strand: 5′_6-FAM_labeled primer P2, the template strand: Template DNA L3-1 (Table 3), and the polymerase: mutant 2, the reaction was allowed to proceed in a large excess with a reaction time of 13.5 hours. Each reaction solution was as shown in Table 4 except that the conditions in Table 5 below were followed. The result is shown in FIG. Corresponding to FIG. 5, the reaction conditions are shown in Table 5 below.

  Under conditions that did not contain LNA-NTP, the primer was degraded by the weak exonuclease activity of the KOD polymerase mutant (FIG. 5, Condition 1). Compared with the condition that does not lack LNA-NTP (Condition 6), the extension reaction stopped halfway under the conditions (Condition 2 to 5) that lacked LNA-ATP, LNA-CTP, LNA-GTP, and LNA-TTP, respectively. Was shown to do. This has shown that incorporation of incorrect bases is limited to some extent and has a certain accuracy.

(Example 7: LNA chain extension reaction using LNA primer by KOD polymerase mutant)
When an LNA chain library is prepared using DNA primers, there is a rate-determining LNA chain length that slows the progress of the extension reaction in the middle. This is thought to be because the structure of the double strand containing the DNA-LNA junction region of the primer strand is irregular, making it difficult for the polymerase to bind. Thus, by using an LNA primer, the reaction under the condition in which the DNA-LNA junction region of the primer strand was deleted was examined.

  Primer strand: 5'_6-FAM_labeled primer P2 (DNA primer), 5'_6-FAM_labeled primer LP2 (LNA primer), template strand: Template DNA N30, polymerase: Mutant 2 with a reaction time of 1.5 hours, The reaction proceeded at 3 hours, 4.5 hours and 6 hours. As a control, a DNA library using dNTP was prepared for DNA primers. Each reaction solution is as shown in Table 4 except that the conditions in Table 6 below are followed. In the case of preparing a DNA library, dATP is used instead of LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP. , DCTP, dGTP and dTTP were used. The result is shown in FIG. Corresponding to FIG. 6, reaction conditions are shown in Table 6 below.

  As a result, under the condition using the LNA primer, the rate-determination did not occur in the middle of the extension reaction as in the condition using the DNA primer (FIG. 6). Although there are primer strands in which the extension reaction has not progressed at all, no change over time is observed, which is considered to be a problem of LNA primer synthesis.

(Example 8: Synthesis of DNA strand by reverse transcription from LNA oligonucleotide by various KOD polymerase mutants)
Using the prepared KOD polymerase mutant, the reverse transcription activity of LNA chain → DNA chain was compared. Table 7 shows the template and primer sequences used in the reverse transcription reaction. After heating the reaction solution to which KOD polymerase mutant has not been added at 95 ° C. for 1 minute and then lowering to room temperature over 1 hour, KOD polymerase mutant is added and reverse transcription proceeds at 74 ° C. I let you. In the reverse transcription reaction, the composition of the reaction solution was the same as that shown in Table 2 except that dATP, dCTP, dGTP and dTTP were used instead of LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP. A thing was used. The reverse transcription reaction was performed for 30 minutes. 1/6 vol of 6 × loading dye was added to the obtained reaction solution, and 1 pmol of primer chain was applied to an alkaline agarose gel for electrophoresis.

  Comparison of reverse transcription activity is shown in FIG. 7 (A) and (B) show the results of the examined KOD polymerase mutants, and FIG. 7 (c) shows those exhibiting characteristic activities.

  In all mutants, a mutation N210D that attenuates exonuclease activity is introduced. During reverse transcription, it is necessary that the polymerase binds to the DNA / LNA duplex and that the 3 'end of the primer strand is correctly positioned at the reaction site. In mutants A4 (E664K) and A43 (E664R) in which the negatively charged amino acid in the Thumb region is changed to a positive charge, the reverse transcription activity is considered to be a result of improved DNA / LNA duplex binding ability. It is done. On the other hand, only A485L involved in binding to the triphosphate does not show reverse transcription activity (mutant A2). However, reverse transcription activity is observed when A485L is introduced (mutant A51) together with a mutation (E664Q: negative charge → neutral) that slightly increases the DNA / LNA duplex binding ability. This is considered to indicate that if the phosphodiester bond formation reaction proceeds rapidly, the required DNA / LNA duplex binding ability may be low. In addition, even the mutants having E664K with improved DNA / LNA duplex binding ability improved reverse transcription activity by A485L (mutant A13), and even those mutants further having D614N in addition to these mutations were reversed. Copying activity was observed (mutant A26).

(Example 9: Examination of accuracy using KOD polymerase mutant for transcription (DNA → LNA) and reverse transcription (LNA → DNA) reactions)
Based on the DNA template, a reaction was performed using A48 (mutant 1) and A53 (mutant 2) for transcription (DNA → LNA) and A51 for reverse transcription (LNA → DNA). The DNA strand was inserted into pT7Blue T-Vector (Takara Bio Inc.) by TA cloning, the base sequence was analyzed, and the accuracy of the polymerase was evaluated. Since errors can occur in steps other than transcription / reverse transcription, a control experiment was conducted in which the reactions of all steps were carried out with DNA, and the error rates of transcription and reverse transcription were corrected. The procedure is described below.

<Transcription (DNA → LNA)>
Reaction solution composition (total reaction solution 50 μL):
1x
Buffer for KOD-Plus-Ver.2 1.5 mM MgSO ₄
0.2 mM LNA-NTP (control: dNTP)
0.5μM primer (Primer hmP3)
0.6μM template (3'-B-Template_FA2-1A)
300 ng / μL KOD-A48 or A53 (control: 0.02 U / μL KOD-Plus- (manufactured by Toyobo Co., Ltd.)).

  After annealing the reaction solution (volume: 40 μL) before addition of polymerase (slow cooling to 25 ° C. for 30 seconds at 94 ° C. for 1 hour), 10 μL of 1500 ng / μL polymerase mutant A48 or A53 was added, and the reaction was performed. (Control: The reaction solution was used for PCR as it was).

Reaction conditions:
9 hours at 74 ° C. (control: step 1: 95 ° C. for 2 minutes (× 1), step 2: 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute (× 3) ).

  50 μL of 100 mM NaOH was added to the LNA extension sample to denature the polymerase. After neutralization by adding 250 μL of 50 mM Tris-HCl (pH 7.4), purification by ethanol precipitation, 1 × B / W Buffer (binding and washing buffer: 5 mM Tris-HCl (pH 7.4), 0. 5 mM EDTA, 1 M NaCl, 0.05% v / v Tween 20) was dissolved in 1 mL (control: 50 μL of 2 × B / W Buffer and 900 μL of 1 × B / W Buffer were added to the sample after the reaction).

  After washing 50 μL of Dynabeads (registered trademark) M-280 Streptavidin (Thermo Fisher Scientific, Inc.) three times with 1 mL of 1 × B / W Buffer, 1 mL of the sample after the above reaction was added and mixed for 60 minutes. After washing 3 times with 1 mL of 1 × B / W Buffer, it was washed twice with 1 mL of sterilized water. Further, 50 μL of 100 mM NaOH was added and mixed for 5 minutes, and then the supernatant was recovered. After neutralization by adding 1 mL of 100 mM Tris-HCl (pH 7.4), 50 μL of fresh Dynabeads (registered trademark) M-280 Streptavidin (washed) is added and mixed for 30 minutes. Unique template strands were trapped on the beads and removed. The supernatant was collected and replaced with sterilized water and concentrated using Vivaspin 500 (5,000 MWCO) (Sartorius AG).

<Reverse transcription (LNA → DNA)>
Reaction solution composition (reaction solution total 50 μL):
Buffer solution for 1 × KOD-Plus-Ver.2 1.5 mM MgSO ₄
0.2 mM dNTP
0.5μM primer (5'-Primer hP5)
LNA template solution 2.5μL
2 ng / μL KOD_A51 (control: 0.02 U / μL KOD-Plus-).

Reaction conditions:
Step 1: 95 ° C. for 2 minutes (× 1), Step 2: 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 74 ° C. (control: 68 ° C.) for 1 minute (× 20).

  50 μL of 2 × B / W Buffer and 900 μL of 1 × B / W Buffer were added to the sample after the reaction.

  After washing 50 μL of Dynabeads (registered trademark) M-280 Streptavidin three times with 1 mL of 1 × B / W Buffer, 1 mL of the sample after the reverse transcription reaction described above was added and mixed for 60 minutes. It was washed 3 times with 1 mL of 1 × B / W Buffer, 4 times with 1 mL of 100 mM NaOH, once with 1 mL of 50 mM Tris-HCl (pH 7.4) and twice with 1 mL of sterile water. The LNA extension strand was removed as much as possible by washing operation, and the reverse transcription strand was trapped on the beads. The beads were suspended in 50 μL of sterile water.

PCR (for TA cloning): 25 μL of reaction mixture. 1 × Taq reaction buffer, 1.5 mM MgSO ₄ , 0.2 mM dNTP, 0.3 μM primer (Primer hP1, Primer hP5), 2.5 μL of bead suspension trapping reverse transcription strand, 0.1 U / μL rTaq Polymerase (Toyobo Co., Ltd.). Reaction conditions: Step 1: 94 ° C. for 2 minutes (× 1), Step 2: 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 1 minute (× 20). It was purified using QIAquick PCR Purification Kit (QIAGEN, Inc.) and used for TA cloning.

  TA cloning: PCR product 1.5 μL, 2.5 ng / μL pT7Blue T-Vector 0.5 μL, Ligation High Ver.2 (Toyobo Co., Ltd.) 2 μL were mixed, reacted at 16 ° C. for 60 minutes, then E. coli It was introduced into DH5α. Each plasmid was extracted from the obtained single colony and subjected to base sequence analysis.

  Table 8 shows the sequences of primers and templates used for the accuracy evaluation of LNA transcription / reverse transcription.

  Due to the circumstances of the experimental system, errors may occur during cloning other than transcription / reverse transcription. Therefore, as a control experiment for determining the background error rate, the reaction was progressed with DNA in all steps, and the error rate was examined. Table 9 shows the results of base sequence analysis for evaluating the accuracy of LNA transcription / reverse transcription.

As a result, the control experiment contained 6 errors in the analyzed 1024 bases. Therefore, the error rate of the experimental system was determined as error rate = 6 errors / (32 samples × 32 bases) = 5.8 × 10 ⁻³ (0.58% error per base). On the other hand, under the conditions using A48 (transfer) and A51 (reverse transfer), the raw error rate without correction was 17 errors / (31 samples × 32 bases) = 17.1 × 10 ⁻³ , and the control error rate was When subtracted and corrected, the correction error rate was calculated to be 17.1 × 10 ⁻³ −5.8 × 10 ⁻³ = 11.3 × 10 ⁻³ (1.1% error per base). Under the conditions using A53 (transfer) and A51 (reverse transfer), the raw error rate without correction is 9 errors / (26 samples × 32 bases) = 10.8 × 10 ⁻³ , and the control error rate is subtracted. The correction error rate was calculated as 10.8 × 10 ⁻³ −5.8 × 10 ⁻³ = 5.0 × 10 ⁻³ (0.5% error per base). Therefore, it was shown that the L53 transcription of A53 maintained accuracy from the base sequence analysis of the DNA strand obtained by the subsequent reverse transcription reaction. In SELEX, A53 (transfer) and A51 (reverse transfer) can be used more suitably.

(Example 10: Production of modified polymerase (KOD polymerase mutant) and examination of LNA chain elongation activity)
Modified polymerases (KOD polymerase mutants) shown in Table 10 were prepared and evaluated for LNA chain elongation activity using a DNA chain as a template. These KOD polymerase mutants are obtained by substituting Y409 with the following amino acids, respectively.

  The reaction solution to which the KOD polymerase mutant was not added was heated at 95 ° C. for 1 minute, and then annealed by lowering to 25 ° C. over 60 minutes. Then, the KOD polymerase mutant was added to extend the LNA chain at 74 ° C. The reaction was allowed to proceed. 1/6 volume of 6 × loading dye was added to the resulting reaction solution, and electrophoresis was performed on an alkaline agarose gel.

  The various KOD polymerase mutants were subjected to LNA chain extension reaction in the following three reaction solution compositions.

<Evaluation 1>
Reaction solution composition: 1 × KOD-Plus-Ver.2 buffer, KOD polymerase mutant: 300 ng / μL, 0.2 mM LNA-NTP, 1.5 mM MgSO ₄ , primer: 0.5 μM 5′_6-FAM_labeled primer LP2, template: 0.6 μM Template DNA N50
Reaction temperature: 74 ° C., reaction time: 60 minutes Running conditions: 25 ° C., 35 V, 2 hours Gel: 50 mM NaCl, 2 mM EDTA, 5% agar Running buffer: 50 mM NaOH, 2 mM EDTA
Sample: 1.2 μL (0.5 pmol primer).

<Evaluation 2>
Reaction solution composition: 1 × KOD DNA polymerase buffer 1 (120 mM Tris-HCl (pH 8.0) (25 ° C.), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton (registered trademark) X −100, 10 μg / mL BSA), KOD polymerase mutant: 300 ng / μL, 0.2 mM LNA-NTP, 1.5 mM MgSO ₄ , primer: 0.5 μM 5′_6-FAM_labeled primer LP2, template: 0.6 μM Template DNA N50
Reaction temperature: 74 ° C., reaction time: 60 minutes Running conditions: 25 ° C., 35 V, 2 hours Gel: 50 mM NaCl, 2 mM EDTA, 5% agar Running buffer: 50 mM NaOH, 2 mM EDTA
Sample: 1.2 μL (0.5 pmol primer).

<Evaluation 3>
Reaction solution composition: Buffer solution for 1 × KOD DNA polymerase 2 (120 mM Tris-HCl (pH 8.8) (25 ° C.), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton (registered trademark) X −100, 10 μg / mL BSA), KOD polymerase mutant: 300 ng / μL, 0.2 mM LNA-NTP, 1.5 mM MgSO ₄ , primer: 0.5 μM 5′_6-FAM_labeled primer LP2, template: 0.6 μM Template DNA N50
Reaction temperature: 74 ° C., reaction time: 60 minutes Running conditions: 25 ° C., 35 V, 2 hours Gel: 50 mM NaCl, 2 mM EDTA, 5% agar Running buffer: 50 mM NaOH, 2 mM EDTA
Sample: 1.2 μL (0.5 pmol primer).

  The results of evaluations 1, 2 and 3 are shown in FIGS. 8, 9 and 10, respectively. As shown in FIG. 8, KOD polymerase mutants A62, A63 and A64 showed particularly high LNA chain elongation activity. These are mutants in which Y409 is replaced with low-volume amino acids (glycine, alanine and serine, respectively). In FIG. 8, LNA chain elongation activity was also observed for A65 (threonine), A53 (valine) and A68 (aspartic acid), as well as A66 (leucine) and A67 (phenylalanine). It was found that by changing the composition of the reaction buffer, efficient LNA chain extension was possible (FIGS. 8 to 10). LNA chain extension efficiency is higher under the conditions (Evaluation 2) using the 1 × KOD DNA polymerase buffer 1 in FIG. 9 than under the conditions (Evaluation 1) using the 1 × KOD-Plus-Ver.2 buffer. Furthermore, under the conditions (Evaluation 3) using the 1 × KOD DNA polymerase buffer 2 in FIG. 10, the LNA chain elongation efficiency was particularly high. According to FIGS. 8-10, the movement of the gel band was seen also about A69 (glutamic acid), and it was evaluated that it has LNA chain extension activity. When comparing the LNA chain elongation activity between A53 and A62 to A69, a higher LNA chain elongation activity was observed when the size (bulk) of the amino acid group after substitution for Y409 was smaller.

(Example 11: Examination of accuracy of LNA chain synthesis using KOD polymerase mutant)
The produced modified polymerase was evaluated for the accuracy of LNA incorporation. The type of modified polymerase was evaluated using A53 for two types of buffer solutions. As an accuracy evaluation method, elongation reaction under the condition of lacking one of the four types of bases (Conditions 2 to 5 below: Condition 1 when no base is present and Condition 6 when four types of bases are aligned) I did it.

  The LNA chain elongation reaction was performed under the following reaction solution composition and conditions, and the LNA chain elongation activity was evaluated.

Reaction solution composition: Buffer solution for 1 × KOD DNA polymerase 2 (120 mM Tris-HCl (pH 8.8) (25 ° C.), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton (registered trademark) X −100, 10 μg / mL BSA), KOD polymerase mutant A53: 300 ng / μL, 1.5 mM MgSO ₄ ,
Condition 1: No LNA-NTP
Condition 2: 0.2 mM LNA- (C, G, T) TP (ie lacking LNA-ATP),
Condition 3: 0.2 mM LNA- (A, G, T) TP (ie lacking LNA-CTP),
Condition 4: 0.2 mM LNA- (A, C, T) TP (ie lacking LNA-GTP),
Condition 5: 0.2 mM LNA- (A, C, G) TP (ie lacking LNA-TTP),
Condition 6: 0.2 mM LNA-NTP,
Primer: 0.5 μM 5'_6-FAM_labeled primer P2,
Template: 0.6 μM Template DNA L3-2 (SEQ ID NO: 14)
Reaction temperature: 74 ° C., reaction time: 120 minutes Electrophoretic conditions: 25 ° C., 35 V, 2 hours Gel: 50 mM NaCl, 2 mM EDTA, 5% agar Electrophoresis buffer: 50 mM NaOH, 2 mM EDTA
Sample: 1.2 μL (0.5 pmol primer).

  Further, the buffer solution for 1 × KOD DNA polymerase 2 having the above reaction solution composition was replaced with a buffer solution for 1 × KOD-Plus-Ver.2, the reaction time was 15 hours, and the sample amount was 1.5 μL (0.625 pmol). As a primer), an LNA chain extension reaction was performed, and the LNA chain extension activity was evaluated.

  These results are shown in FIGS. When the KOD DNA polymerase buffer 2 (FIG. 11) was used, the elongation was similar to that when the KOD-Plus-Ver.2 buffer (FIG. 12) was used. It was shown that the reaction buffer greatly improved the elongation efficiency, but the accuracy did not change significantly.

(Example 12: Examination of accuracy of LNA chain synthesis using KOD polymerase mutant)
Except that the modified polymerase A53 of Example 11 was replaced with A65, the accuracy of LNA incorporation was evaluated in the same manner as in the case of using 1 × KOD DNA polymerase buffer 2. The result is shown in FIG.

  Also in A65, as in A53, excessive extension reaction was efficiently suppressed under the conditions (conditions 2 to 5) lacking one type of base (FIG. 13).

  According to the present invention, a modified polymerase capable of extending a chain containing an artificial nucleic acid LNA is provided. This makes it possible to stably extend the LNA chain from the DNA chain. Furthermore, an LNA library can be created using a DNA strand consisting of a random sequence as a template. By selecting an aptamer from an artificial nucleic acid library, an aptamer having resistance to degrading enzymes can be obtained in advance, and thus the time required for optimizing the aptamer can be shortened. In addition, artificial nucleic acid libraries can increase the size of the library and broaden the scope of candidate aptamer screening. Creation of an artificial nucleic acid library is useful for screening sequences containing artificial nucleic acids that are candidates for drugs and the like and for efficient development of nucleic acid drugs containing artificial nucleic acids.

Claims (11)

A modified polymerase comprising an amino acid sequence having the following mutation (1) or (2) in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing:
(1) Asparagine at position 210 is aspartic acid, tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, aspartic acid or glutamic acid, alanine at position 485 is a neutral amino acid, and position 664 Glutamic acid is substituted with lysine, glutamine or arginine respectively; or (2) asparagine at position 210 is aspartic acid and tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, asparagine Acid or glutamic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with asparagine, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine. There. A modified polymerase comprising an amino acid sequence having the following mutation (1) or (2) in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing:
(1) Asparagine at position 210 is substituted with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine; or (2) asparagine at position 210 Is substituted with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with asparagine, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine. A modified polymerase comprising an amino acid sequence having the following mutation in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing:
Asparagine at position 210 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine. A method of synthesizing an LNA-containing oligonucleotide chain from a DNA-containing oligonucleotide comprising:
A method comprising reacting a template comprising the DNA-containing oligonucleotide, a primer, LNA-NTP, and the modified polymerase according to claim 1 or 2.   The method of claim 4, wherein the primer is an LNA-containing primer.   6. The method according to claim 4 or 5, wherein the DNA-containing oligonucleotide comprises a random sequence. A method of synthesizing a DNA-containing oligonucleotide from an LNA-containing oligonucleotide comprising:
A method comprising reacting a template, a primer, dNTP comprising the LNA-containing oligonucleotide and the modified polymerase according to claim 2 or 3. The step of reacting is performed in a buffer containing 120 mM Tris-HCl (pH 7 to 9), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , and 10 μg / mL bovine serum albumin (BSA). The method according to any one of 4 to 7.   Modified polymerase with LNA chain elongation activity modified to include mutations that attenuate exonuclease activity, mutations that contribute to triphosphate binding, and mutations that enhance binding to DNA / LNA duplexes A modified polymerase, which is a modified polymerase.   The modified polymerase according to claim 9, wherein the modified polymerase further comprises a mutation that relaxes recognition of a sugar structure of a triphosphate.   A modified polymerase having reverse transcription activity, comprising a mutation that attenuates exonuclease activity and a mutation that enhances binding to DNA / LNA duplex, or a mutation that attenuates exonuclease activity; A modified polymerase, which is a polymerase that has been modified to include a mutation that enhances binding to an LNA duplex and a mutation that contributes to binding of a triphosphate.

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