JP6826275B2 - Modified polymerase - Google Patents

Description

The present invention relates to a modified polymerase.

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

Nucleic acid aptamers can be synthesized using natural nucleic acids such as DNA and RNA, and such natural nucleic acids can be easily degraded in vivo by nucleic acid degrading enzymes. Therefore, these natural nucleic acids need to be protected from degradation by replacing them with chemically modified artificial nucleic acids, but when they are replaced with artificial nucleic acids, the activity is frequently reduced due to structural changes in aptamers. ..

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

In the polymerase chain reaction (PCR) for amplifying a 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 wild-type DNA polymerase to extend the strands of artificial nucleic acids having resistance to degrading enzymes such as LNA.

Non-Patent Document 1 describes six types of XNA, that is, 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 chain synthesis reactions using threofuranosyl nucleic acid (TNA) have been reported.

In Non-Patent Document 1, an attempt was made to create a modified polymerase based on a mutant (TgoT) of a DNA polymerase derived from Thermococcus gorgonarius, which is a hyperthermophilic archaea. Of the five modified polymerases, Pol6G12, PolC7 and PolD4K have been reported to catalyze DNA → XNA, Pol6G12 synthesizes HNA chains, PolC7 synthesizes CeNA and LNA chains, and PolD4K synthesizes ANA and FANA chains. It was. It was reported that the remaining two, RT521 and RT521K, catalyze XNA → DNA, RT521 reverse-transcribes the HNA, TNA, ANA and FANA chains, and RT521K reverse-transcribes the CeNA and LNA chains.

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

However, for the XNA strands that make up the XNA library for use in SELEX, stable strand generation from the template DNA is required. Therefore, there is a need for a polymerase that enables 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 also an object of the present invention to provide a modified polymerase capable of reverse transcribing DNA from a strand containing an artificial nucleic acid.

The present invention provides the following modified polymerases (i) to (iii).

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) Aspartic acid 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 replaced with lysine, glutamine, or arginine; or (2) aspartic acid at position 210 is aspartic acid and tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, asparagine. Alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine in acid or glutamic acid.

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) Aspartic acid at position 210 is replaced with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, and glutamic acid at position 664 is replaced with lysine, glutamic acid, or arginine; or (2) aspartic acid at position 210. Is replaced with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine.

A further modified polymerase (iii) of the present invention comprises an amino acid sequence having the following mutations in the amino acid sequence set forth in SEQ ID NO: 2 of 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, respectively.

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

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

In one embodiment, the template comprises a random sequence.

The present invention further provides a method for synthesizing a DNA-containing oligonucleotide chain from an LNA-containing oligonucleotide. The method comprises reacting a template, primer, dNTP consisting of the LNA-containing oligonucleotide with the modified polymerase (ii) or (iii) of the present invention.

In one embodiment, in the method of synthesizing an LNA-containing oligonucleotide chain from the above-mentioned DNA-containing oligonucleotide and the method of synthesizing a DNA-containing oligonucleotide chain from the above-mentioned LNA-containing oligonucleotide, the step of reacting is 120 mM Tris-. It is performed in 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 is also a modified polymerase having LNA chain elongation activity, which is a mutation that attenuates exonuclease activity, a mutation that contributes to triphosphate binding, and a mutation that strengthens binding to a DNA / LNA double strand. Provided is a modified polymerase, which is a polymerase modified to contain.

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

The present invention is also a modified polymerase having reverse transcription activity, which comprises a mutation that attenuates exonuclease activity and a mutation that enhances binding to a DNA / LNA duplex, or a mutation that attenuates exonuclease activity. To provide a polymerase modified to include a mutation that strengthens the binding to the DNA / LNA duplex and a mutation that contributes to the binding of the triphosphate.

According to the present invention, there is provided a modified polymerase capable of extending a chain containing LNA, which is an artificial nucleic acid. Furthermore, according to the present invention, there is also provided a modified polymerase capable of reverse transcribing DNA from a strand containing LNA. This enables stable extension of the LNA strand from DNA. In addition, a reverse transcription reaction from the LNA chain to DNA is also possible.

It is an electrophoretic photograph which shows the result of the LNA chain extension activity by the N10 random sequence template for various KOD polymerase mutants. FIG. 5 is an electrophoretic photograph showing the results of LNA chain extension activity by N10 random sequence template for KOD polymerase variants A2, A34, A4, A13, A26, A48, and A53. It is an electrophoretic photograph which shows the result of making the LNA library of 30 mer (left side) and 50 mer (right side) for mutant 1 and mutant 2. It is an electrophoretic photograph which shows the result of examining the mutant 2 with respect to the preparation of the LNA library of 30 mer at various reaction times. Variant 2 lacks LNA-NTP-free condition (Condition 1), LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP-deficient conditions (Conditions 2-5), and LNA-NTP-deficient. 6 is an electrophoretic photograph showing the result of performing an LNA chain extension reaction under the condition (condition 6). It is an electrophoretic photograph which shows the comparison result of the DNA primer and the LNA primer about the LNA chain extension reaction of mutant 2. It is an electrophoretic photograph which shows the result of the reverse transcription activity about various KOD polymerase mutants. It is an electrophoretic photograph which shows the result of the LNA chain extension activity by the N10 random sequence template performed by using the buffer solution for KOD-Plus-Ver.2 as the reaction solution for the KOD polymerase mutant which replaced Y409 variously. 6 is an electrophoretic photograph showing the results of LNA chain extension activity by N10 random sequence template performed using KOD DNA polymerase buffer 1 as a reaction solution for KOD polymerase mutants in which Y409 was variously substituted. 6 is an electrophoretic photograph showing the results of LNA chain extension activity by N10 random sequence template performed using KOD DNA polymerase buffer 2 as a reaction solution for KOD polymerase mutants in which Y409 was variously substituted. Regarding A53, the condition that KOD DNA polymerase buffer 2 was used as the reaction solution and that LNA-NTP was not contained (Condition 1) and that LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP were absent (Condition 1). It is an electrolysis photograph which shows the result of having performed the LNA chain extension reaction under the condition 2-5) and the condition (condition 6) which does not lack LNA-NTP. Regarding A53, KOD-Plus-Ver.2 buffer solution was used as the reaction solution, and the condition not containing LNA-NTP (condition 1), LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP were deficient, respectively. It is an electrophoretic photograph which shows the result of having performed the LNA chain extension reaction under the condition (condition 2-5) which was present, and the condition (condition 6) which did not lack LNA-NTP. Regarding A65, the condition that KOD DNA polymerase buffer 2 was used as the reaction solution and that LNA-NTP was not contained (Condition 1) and that LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP were absent (Condition 1). It is an electrolysis photograph which shows the result of having performed the LNA chain extension reaction under the condition 2-5) and the condition (condition 6) which does not lack LNA-NTP.

In the present specification, amino acid substitutions are expressed as follows. For example, "E664K" represents a variant in which glutamic acid at position 664 is replaced with lysine. When multiple parts are replaced, they are separated by "/" and each replacement is shown together. For example, in "N210D / Y409V / A485L / D614N / E664K", asparagine at 210th position is aspartic acid, tyrosine at 409th position is valine, alanine at 485th position is leucine, aspartic acid at 614th position is aspartic acid, and A variant in which glutamic acid at position 664 is replaced with lysine is shown. In addition, for example, the description "Y409" indicates that the 409th position 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) Aspartic acid 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 replaced with lysine, glutamine, or arginine; or (2) aspartic acid at position 210 is aspartic acid and tyrosine at position 409 is valine, glycine, alanine, serine, threonine, leucine, phenylalanine, asparagine. Alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine in acid or glutamic acid.

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) Aspartic acid at position 210 is replaced with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, and glutamic acid at position 664 is replaced with lysine, glutamic acid, or arginine; or (2) aspartic acid at position 210. Is replaced with aspartic acid, alanine at position 485 is replaced with a neutral amino acid, aspartic acid at position 614 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine.

A further modified polymerase (iii) of the present invention comprises an amino acid sequence having the following mutations in the amino acid sequence set forth in SEQ ID NO: 2 of 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, respectively.

The amino acid sequence of SEQ ID NO: 2 in the sequence listing corresponds to the amino acid sequence of the DNA-dependent DNA polymerase of Thermococcus Kodakaraensis KOD1 (also referred to as “KOD polymerase” in the present specification). The 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 among amino acids other than the predetermined substitutions are deleted, substituted or added, and is predetermined. Those having polymerase activity are also included. Preferably, one or several amino acids are deleted, substituted or added within a range that retains a given substitution and has 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, and the given polymerase activity. Those having the above are also included. As an example of the amino acid substitution in "one or several amino acids are deleted, substituted or added" here, any substitution may be used as long as each function or effect is substantially retained. For example, conservative replacement. Conservative substitutions specifically include substitutions within the following groups (ie, between the amino acids shown in parentheses): (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid). ), (Aspartic acid, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine).

As known herein, sequence "identity" is the relationship between two or more proteins or two or more polynucleotides, as is known in the art, determined by comparing sequences. Sequence "identity" is defined by the alignment between protein or polynucleotide sequences, or, in some cases, by the alignment between a series of partial sequences. It means the degree of sequence invariance. Also, "similarity" is between protein or polynucleotide sequences, as determined by the alignment between protein or polynucleotide sequences, or, in some cases, by the alignment between a series of partial sequences. It means the degree of correlation. More specifically, it is determined by sequence identity and conservativeness (substitutions that maintain physicochemical properties at a particular amino acid or sequence in the sequence). The similarity is also referred to as Similarity in the BLAST sequence homology search results described later. The method of determining identity and similarity is preferably the method designed for the longest alignment between contrasting sequences. Methods for determining identity and similarity are provided as publicly available programs. For example, a BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (For example, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; Altschyl et al., Nucleic Acids Res., 1997, 25: 3389-3402. ) Can be used for determination. 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 / D14. N210D / Y409S / A485L / D614N / E664K, N210D / Y409T / A485L / D614N / E664K, N210D / Y409L / A485L / D614N / E664K, N210D / Y409F / A485L / D614N / E664K, N210D / Y409F / A485L / D614N / E664K It has a mutation of N210D / Y409E / A485L / D614N / E664K.

The modified polymerase (ii) has LNA chain extension activity. 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 are 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 the present 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 one base selected from adenine (A), thymine (T), cytosine (C) and guanine (G)), and a nucleic acid chain having a predetermined chain length. Is. Further, in the present specification, "LNA chain extension activity" refers to a 3'-hydroxyl group of a DNA or LNA oligonucleotide (for example, a primer) annealed to a template DNA, and a 5'-triphosphate group of LNA. It refers to an activity that catalyzes a reaction that causes a template-dependent LNA-containing oligonucleotide synthesis of DNA, that is, an extension of an LNA chain 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, and as such a substrate, a methyl group is added to the 5-carbon atom of the pyrimidine ring of cytosine of LNA. ) Can also be used.

In the present specification, the reverse transcription activity from LNA to DNA (also simply referred to as “reverse transcription activity”) refers to an LNA-containing oligonucleotide as a template or an oligonucleotide of DNA annealed to a polynucleotide (for example, a primer). ) By covalently binding the α-phosphate of the 5'-triphosphate group of LNA to the 3'-hydroxyl group of), catalyzes the synthesis of template-dependent DNA-containing oligonucleotides, that is, the reaction that causes the elongation of DNA strands. The activity to do. 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.

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 in the N-terminal region (“N-ter”: 1-130). Positions and positions 327 to 368), exonuclease region ("Exo": positions 131 to 326), and polymerase active region (Polm region: positions 369 to 449 and 500 to 587, Fingers region: positions 450 to 499, and Thumb. -1, Thumb-2 region (in this specification, collectively referred to as "Thumb region"): 588 to 774 positions).

N210D (replacement 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 triphosphates. Alanine at position 485 can be replaced with a neutral amino acid (eg, leucine, isoleucine, phenylalanine, methionine). At position 485, the bulky alanine can be replaced with a neutral amino acid that can also be a bulky hydrophobic amino acid (eg, leucine, isoleucine, phenylalanine, methionine).

D614N (replacement of aspartic acid at position 614 with asparagine) and E664K (replacement of glutamic acid at position 664 with lysine) can enhance binding to DNA / LNA duplexes. Negatively charged amino acids (aspartic acid at position D614: 614, glutamic acid at position E664: 664) are present in the Thumb region that binds to the duplex, but these amino acids are uncharged (eg, D614N) or positive. It is considered that by changing to an electric charge (for example, E664K), 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. Substitution mutations at positions 664 only, or substitution mutations at positions 614 and 664 may be used.

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

Modified polymerases with LNA chain elongation activity have been modified to include mutations that attenuate exonuclease activity, mutations that contribute to triphosphate binding, and mutations that strengthen 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 sugar structure of the triphosphate.

Structural features of the modified polymerase having reverse transcription activity include: The modified polymerase having activity is considered to have the following characteristics.

N210D (replacement 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, the polymerase needs to bind to the DNA / LNA duplex and properly position the 3'end of the primer strand at the reaction site. For example, reverse transcription activity was observed when the negatively charged amino acids in the Thumb region were changed to positive charges (eg, E664K or E664R), which was the result of enhanced binding of the polymerase to the DNA / LNA duplex. Is considered to be. Involved in binding to triphosphate, along with a mutation (E664Q) in which the negatively charged amino acids in the Thumb region are neutralized to positively charged (which can slightly increase the DNA / LNA double-strand binding capacity of the polymerase). Reverse transcription activity is also observed when A485L is introduced. In addition, the mutant having E664K also has improved reverse transcription activity in combination with A485L.

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

Modified polymerases with reverse transcriptional activity include mutations that attenuate exonuclease activity and mutations that strengthen binding to the DNA / LNA duplex, or mutations that attenuate exonuclease activity, and DNA / LNA duplex. It can be designed to be a polymerase that has been modified to include mutations that strengthen binding to the chain and mutations that contribute to the binding of triphosphates.

As a method for producing the modified polymerase, a conventionally known method can be used. For example, there is a method of introducing a mutation into a gene encoding a wild-type DNA polymerase to produce a mutant (modified) DNA polymerase having a new function by a protein engineering technique (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 is a base sequence available from a gene registry such as NCBI as a gene for the DNA-dependent DNA polymerase of Thermococcus kodakaraensis KOD1, or a base sequence for which codons have been optimized (for example, in the sequence listing). It can be obtained by a method usually used by those skilled in the art based on sequence information such as (base sequence of SEQ ID NO: 1).

Any known method may be used to introduce the mutation into the gene. For example, protein engineering techniques such as PCR and site-specific mutations can be used.

The modified polymerase gene is inserted into an expression vector as needed, transformed using, for example, Escherichia coli as a host, and then applied to an agar medium containing a selective drug such as kanamycin to form colonies. The colonies are inoculated into a nutrient medium, for example, LB medium, and appropriately cultured (for example, grown at 37 ° C. and then cultured at 30 ° C. in the presence of an expression inducer such as IPTG), and then the cells are collected and collected. Crush and extract the crude enzyme solution. The vector is preferably, but is not limited to, a pET vector. The medium composition, pH, temperature, cell density during culturing and culturing time can be appropriately set. Any known method may be used for crushing the cells, and for example, sonication, 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 heat-treated, for example, treated at 80 ° C. for 10 minutes, and then centrifuged, membrane filtration, or the like to obtain a crude enzyme. After this operation, it can be separated and purified by heparin sepharose column chromatography. Further, a step of purifying or concentrating the product can be carried out. These steps and the means required for them are appropriately selected by those skilled in the art.

Modified polymerases (i) and (ii) are used for synthesizing LNA-containing oligonucleotide chains from DNA-containing oligonucleotides (also referred to as "LNA chain extension method" in the present specification), and modified polymerases (also referred to as "LNA chain extension method"). 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 a "reverse transcription method" in the present specification).

The LNA chain extension method of the present invention includes a step of reacting a template consisting of a DNA-containing oligonucleotide, a primer, LNA-NTP and a modified polymerase (i) or (ii). Using the modified polymerase of the present invention, four types of LNA-nucleoside triphosphates using a DNA-containing oligonucleotide as a template and a primer, LNA-NTP (that is, each of A, T, C and G as a base) By reacting a mixture of LNA-ATP, LNA-TTP, LNA-CTP and LNA-GTP), the primer annealed to the template is extended while incorporating LNA, and the LNA chain (LNA-containing oligonucleotide) is used as the primer extension. Can be synthesized. The LNA chain extension method of the present invention may further include an annealing step prior to the reaction step using the 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 by the action of the modified polymerase of the present invention starting from the primer. DNA-LNA strands can be formed.

Here, the "oligonucleotide" may have 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 annealing and extension can be the same as that for ordinary PCR. For example, after heating at 95 ° C. for 1 minute, the temperature is lowered to room temperature for 1 hour to perform annealing, and then the polymerase is added. It can be added and stretched at 74 ° C. The composition of the reaction solution is shown in Table 2 below as an example, but is not limited thereto. The concentration of the enzyme added may depend on the length of the extended chain, the reaction time, and the like, but is not limited to, for example, 20 ng / μL to 300 ng / μL, preferably 200 ng / μL to 300 ng / μL. The reaction time may depend on the length of the extended chain, the concentration of the enzyme added, and the like, but 1.5 hours to 13.5 hours can be adopted, but the reaction time 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 the rate-determining step in the middle of the primer extension reaction, a primer containing at least a part of LNA (preferably a primer in which all are LNA) is preferable. The length of the primer can be appropriately determined, and examples thereof include those having a base number of 11 to 28, preferably 12 to 25. Primers can be appropriately designed and prepared by those skilled in the art based on the sequence information of the template.

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

The reverse transcription method of the present invention includes a step of reacting a template consisting of an LNA-containing oligonucleotide, a primer, dNTP and a modified polymerase (ii) or (iii). Using the modified polymerase of the present invention, four kinds of deoxynucleoside triphosphates (dATP, dTTP) using LNA-containing oligonucleotides as templates and primers and dNTPs (that is, A, T, C and G as bases) are used. , 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. The reverse transcription method of the present invention may further include an annealing step prior to the reaction step using the polymerase. In the annealing step, the primer and the complementary region on the template are hybridized, and in the subsequent extension step, the 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. LNA-DNA strands can be formed.

The primer for the reverse transcription method can be a DNA primer (a primer composed of DNA). The length of the primer can be appropriately determined, and examples thereof include those having a base number of 11 to 28, preferably 12 to 25. Primers can be appropriately designed and prepared by those skilled in the art based on the sequence information of the template.

The template for the reverse transcription method may consist 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 strand transcribed based on the DNA template (for example, an LNA strand prepared in the LNA strand extension method of the present invention).

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

In LNA chain elongation methods and reverse transcription methods, in addition to the modified polymerase and the nucleic acid component of the present invention, for example, divalent ions such as magnesium ions (e.g., as MgSO ₄₎ it is preferable to coexist. In the reaction using the modified polymerase, a commercially available PCR buffer solution (for example, Buffer for KOD-Plus-Ver.2 (manufactured by Toyo Boseki Co., Ltd.)) as a buffer solution, and an arbitrary buffer prepared by a person skilled in the art A solution (eg, buffer 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 solution can vary depending on the buffer solution temperature and the extension reaction temperature. For example, in Tris-HCl, when the temperature of the buffer solution rises by 1 ° C., the pH drops by about 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). It is done in a buffer solution. This buffer further contains 0.1% Triton® X-100. In this embodiment, for example, when the reaction is carried out under the same conditions as normal PCR with the buffer temperature at 25 ° C. or the extension reaction is carried out at 74 ° C., the pH of the buffer solution is preferably pH 8 to 8. It is 8.8, and pH 8.8 is preferable to pH 8 in terms of the extension efficiency of the LNA chain.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

(Example 1: Creation of modified polymerase)
(1-1: Construction of plasmid)
For the KOD polymerase gene, the intein sequence was removed in advance by artificial gene synthesis, and a codon-optimized sequence (the base sequence of SEQ ID NO: 1; the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 2) was prepared. Gene synthesis was commissioned to operon biotechnology. The KOD polymerase gene was inserted between the NdeI site and the SalI site of the 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 point mutation introduction. By performing the nucleotide sequence analysis of the KOD polymerase mutant gene for the KOD polymerase mutant expression plasmid, it was confirmed that the target sequence was correctly inserted. Table 1 shows 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, sprinkled on an LB / Km (50 μg / mL) plate, and allowed to stand at 37 ° C. The growing colonies were placed on TB medium / 1% glucose / Km (50 μg / mL) and shaken at 37 ° C. When the 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 the addition of IPTG, the culture solution was centrifuged to remove the supernatant, and Escherichia coli was recovered. E. coli was stored at -80 ° C and thawed during purification. The thawed Escherichia 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 centrifuging the E. coli crushed solution (4 ° C., 8000 g, 5 minutes), the supernatant was collected and heated in a hot water bath at 80 ° C. for 10 minutes. The contaminant protein denatured by heating was 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 obtained crude solution was purified by AKTA pure (GE Healthcare) using HiTrap Heparin HP Columns (5 mL). For the KOD polymerase variant 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 make the KOD polymerase variant into the elution fraction. I got the peak of. The elution fraction is concentrated by ultrafiltration using Amicon Ultra 4 (30K) (Millipore), the buffer is replaced, and finally the storage buffer (Storage Buffer: 50 mM Tris-HCl (pH 8.0)), It was dissolved in 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 in Storage Buffer and used in 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 stir 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). A proton sponge (117 mg, 5.46 × 10 ^-4 mol, 1.46 eq) that had been dried under reduced pressure overnight was added, and the mixture was further dried under reduced pressure overnight. Argon was replaced, a septum cap in the desiccator was attached, and an argon balloon was attached. Trimethyl phosphate ^{(1.3mL, 1.29 × 10 -2 mol} , 36eq) was added and stirred for 30 minutes in an ice bath was suspended. POCl ₃ (55 μL, 5.90 × 10 ^-4 mol, 1.5 eq) was added and stirred under an ice bath for 45 minutes. Dry-tributylamine (350 μL, 1.46 × 10 ^-3 mol, 4.0 eq) and 0.5 M pyrophosphate DMF (3.7 mL, 1.87 × 10 ^-3 mol, 5.0 eq) under an ice bath. It was added and reacted at room temperature for 1 hour. The mixture was stirred under an ice bath for 5 minutes, 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 to dissolve the solution, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphate, which was the target product, was separated by an anion exchange column (DEAE Sephadex A-25). Further, excess pyrophosphoric acid was removed by a medium pressure column (C18), purified by reverse phase HPLC (C18), fractions were 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 (for [(MH) ^- ]) = 518.0.

(2-2: Synthesis of LNA-TTP)

LNA-T (103 mg, 3.81 × 10 ^-4 mol, 1.0 eq) and a stir 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). A proton sponge (119 mg, 5.55 × 10 ^-4 mol, 1.46 eq) that had been dried under reduced pressure overnight was added, and the mixture was further dried under reduced pressure overnight. Argon was replaced, a septum cap in the desiccator was attached, and an argon balloon was attached. Trimethyl phosphate ^{(1.4mL, 1.39 × 10 -2 mol} , 36eq) was added and completely dissolved by stirring for 30 minutes in an ice bath. POCl ₃ (60 μL, 6.44 × 10 ^-4 mol, 1.5 eq) was added and stirred under an ice bath for 45 minutes. Dry-tributylamine (400 μL, 1.67 × 10 ^-3 mol, 4.0 eq) and 0.5 M pyrophosphate DMF (4.0 mL, 2.02 × 10 ^-3 mol, 5.0 eq) under an ice bath. It was added and reacted at room temperature for 1 hour. The mixture was stirred under an ice bath for 5 minutes, 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 to dissolve the solution, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphate, which was the target product, was separated by an anion exchange column (DEAE Sephadex A-25). Further, excess pyrophosphoric acid was removed by a medium pressure column (C18), purified by reverse phase HPLC (C18), fractions were 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 [(MH) ^- ]) = 509.18.

(2-3: Synthesis of ^LNA-G i TP)

LNA-G (101 mg, 2.76 × 10 ^-4 mol, 1.0 eq) and a stir 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). A proton sponge (118 mg, 5.51 × 10 ^-4 mol, 1.5 eq) that had been dried under reduced pressure overnight was added, and the mixture was further dried under reduced pressure overnight. Argon was replaced, 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 under an ice bath for 45 minutes. Dry-tributylamine (260 μL, 1.09 × 10 ^-3 mol, 4.0 eq) and 0.5 M pyrophosphate DMF (2.8 mL, 1.41 × 10 ^-3 mol, 5.0 eq) under an ice bath. It was added and reacted at room temperature for 1 hour. The mixture was stirred under an ice bath for 5 minutes, 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 to dissolve the solution, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphate, which was the target product, was separated by an anion exchange column (DEAE Sephadex A-25). Further, to remove excess pyrophosphate in the medium-pressure column (C18), reverse phase purified by HPLC (C18), evaporated under reduced pressure fractions were lyophilized to give ^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 [(MH) ^- ]) = 604.03.

(2-4: Synthesis of LNA-GTP)

^LNA-G i TP entering the recovery flask 100mL (1.86mg, 20.03μmol) was added and stirred 28% dissolved in water 1mL concentrated aqueous ammonia (7.1 mL). Sampling was performed every 4 hours and a peak was 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 hours. Until the peak of the ^LNA-G i TP disappears with HPLC, and repeated. After 48 hours, the reaction solution was distilled off under reduced pressure, purified by reverse phase HPLC (C18), the fraction was distilled off under reduced pressure, and freeze-dried to obtain LNA-GTP. Yield: 12.74 μmol. Yield: 63%. ESI-MS (negative ion mode) m / z, measured value = 533.8, calculated value (for [(MH) ^- ]) = 533.99.

(2-5: Synthesis of LNA-CTP)

LNA-C (101 mg, 3.72 × 10 ^-4 mol, 1.0 eq) and a stir 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). A proton sponge (113 mg, 5.27 × 10 ^-4 mol, 1.46 eq) that had been dried under reduced pressure overnight was added, and the mixture was further dried under reduced pressure overnight. Argon was substituted, and a septum cap in a desiccator was attached to perform argon substitution. Trimethyl phosphate ^{(4.5mL, 4.47 × 10 -2 mol} , 120eq) was added and stirred for 30 minutes in an ice bath was suspended. POCl ₃ (60 μL, 6.44 × 10 ^-4 mol, 1.5 eq) was added and stirred under an ice bath for 45 minutes. Dry-tributylamine (400 μL, 1.67 × 10 ^-3 mol, 4.0 eq) and 0.5 M pyrophosphate DMF (3.8 mL, 1.92 × 10 ^-3 mol, 5.0 eq) under an ice bath. It was added and reacted at room temperature for 1 hour. The mixture was stirred under an ice bath for 5 minutes, 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 to dissolve the solution, and the mixture was separated twice with diethyl ether. The aqueous phase was distilled off under reduced pressure, and triphosphate, which was the target product, was separated by an anion exchange column (DEAE Sephadex A-25). Further, excess pyrophosphoric acid was removed by a medium pressure column (C18), purified by reverse phase HPLC (C18), fractions were 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 [(MH) ^- ]) = 493.98.

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

(Reference Example 1: Modified Polyacrylamide Gel Electrophoresis (PAGE))
Electrophoresis buffer: 1 × TBE buffer, modified polyacrylamide gel: 1 × TBE buffer, 7M urea, 20% polyacrylamide. 40 mM EDTA, 0.1% bromophenol blue: 2 μL, and 10 M urea 12 μL were added per 10 μL of the reaction solution to prepare an electrophoresis sample. After performing a preliminary run at 55 ° C. and a constant voltage of 200 V for 30 minutes, the sample was applied to a gel and run under the same conditions.

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

As agarose, one for low molecular weight nucleic acid separation (Agarose 21 (Nippon Gene Co., Ltd.)) was used. The composition of the gel, the running buffer, and the loading die is as follows. Gel: 50 mM NaCl, 2 mM EDTA, 5% agarose, migration buffer: 5 mM NaOH, 2 mM EDTA, 6 x loading die: 300 mM NaOH, 12 mM EDTA, 30% glycerol, 0.1% bromophenol blue. Prior to electrophoresis, the gel was immersed in electrophoresis buffer for 1 hour for equilibration. The electrophoresis was performed under the conditions of room temperature and a constant voltage of 3 V / cm. For the detection of single-strand LNA, a fluorescent dye labeled with a primer was used, and analysis was performed by PharosFX (manufactured by Bio-Rad).

(Reference Example 3: LNA chain extension 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 the KOD polymerase mutant has not been added at 95 ° C. for 1 minute and then lowering it to room temperature for 1 hour for annealing, the KOD polymerase mutant is added and the LNA chain extension reaction is carried out at 74 ° C. I made it progress. Table 3 shows the sequences of the templates and primers used in the LNA chain extension reaction.

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

The analysis results are shown in FIG. 1 (FIGS. 1-1, 2, 3, 4). FIG. 2 shows the results of analysis by denatured PAGE by selecting a representative example from FIG. In these figures, the results of the unmodified wild type (“WT”) as a control are shown. Further, in order to clarify the degree of extension, only the primer (“Primer”) and the case where the template is extended corresponding to the extension (that is, 10 bases) (“Primer + 10mer”) are shown together (the same applies to the following figures). is there).

All variants carry the mutant N210D with attenuated exonuclease activity. Mutant A485L, which is 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. In addition, the mutant Y409V common to A34 and A53 is considered to alleviate 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, so that NTP uptake is restricted by recognition of the sugar portion of the nucleotide. However, this limitation causes a decrease in the uptake activity of LNA-NTP having a modified sugar portion. Mutant Y409V improves the uptake rate of LNA-NTP by relaxing sugar recognition.

Mutants A485L and Y409V that contribute to these triphosphates are not sufficient for stable extension of the LNA chain. Since the structure of the DNA / LNA double helix (type A helix) is different from the structure of the DNA / DNA double helix (type B helix), it is considered that ordinary polymerase cannot stably bind to the DNA / LNA strand. This is to be done. In fact, 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 double strand. Negatively charged amino acids (D614, E664) are present in the Thumb region bound to the duplex, but by changing these amino acids to uncharged (D614N) or positively charged (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. Although the LNA elongation activity is not so high only by strengthening the binding to the DNA / LNA duplex as in the mutant A4, mutations such as A13, A26, A48, and A53 that contribute to each of the nucleotide monomer and the nucleotide polymer are generated. By introducing them at the same time, stable elongation of the LNA chain is possible. Mutants A48 and A53, which have these mutations comprehensively, show particularly high LNA chain elongation activity. Hereinafter, A48 is also referred to as mutant 1 and A53 is also referred to as mutant 2.

(Example 4: Preparation of LNA library using KOD polymerase mutant)
The composition of the reaction solution used to prepare the LNA library is shown in Table 4 below. The reaction solution to which the KOD polymerase mutant is not added is heated at 95 ° C. for 1 minute, then annealed by lowering the reaction solution to room temperature over 1 hour, and then KOD polymerase mutant 1 or 2 is added to carry out the LNA chain extension reaction. I made it progress. 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.

Primer strands: 5'_6-FAM_labeled primer P2, template strands: Template DNA N30, Template DNA N50 were used to prepare 30 mer and 50 mer LNA libraries. The LNA chain extension proceeded at 74 ° C. for 9 hours. 1/6 volume of 6 × loading die was added to the obtained reaction solution, and 1 pmol of primer chain was applied to an alkaline agarose gel for electrophoresis. As a result, we succeeded in creating an LNA library comparable to the DNA library created using dNTP (Fig. 3).

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

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 a DNA library was prepared using dATP, dCTP, dGTP and dTTP as controls instead of LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP. .. The KOD polymerase mutant 2 was used for comparison under the conditions of reaction time: 1.5 hours, 3 hours, 6 hours, and 9 hours. It was shown that a 30 mer LNA extension reaction product began to form at an extension time of 1.5 hours, and the reaction time for preparing the LNA library could be shortened from 9 hours (Fig. 4).

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

Using primer strand: 5'_6-FAM_labeled primer P2, template strand: Template DNA L3-1 (Table 3), and 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 free of LNA-NTP, the primers were degraded by the weak exonuclease activity of the KOD polymerase mutant (FIG. 5, condition 1). Compared with the condition without LNA-NTP (Condition 6), the extension reaction is stopped halfway under the condition without LNA-ATP, LNA-CTP, LNA-GTP and LNA-TTP (Conditions 2 to 5). It was shown to do. This showed that the uptake of erroneous bases was restricted to some extent and had a certain degree of accuracy.

(Example 7: LNA chain extension reaction using LNA primer with KOD polymerase mutant)
When an LNA strand library is prepared using a DNA primer, there is a rate-determining step LNA strand length in which the progress of the extension reaction is slowed down in the middle. It is considered that this is because the structure of the double strand including the DNA-LNA junction region of the primer strand is irregular, which makes it difficult to bind the polymerase. Therefore, by using an LNA primer, the reaction under the condition that 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, reaction time 1.5 hours, The reaction proceeded in 3 hours, 4.5 hours and 6 hours. As a control, a DNA library using dNTP was prepared for the DNA primer. Each reaction solution is as shown in Table 4 except that the conditions in Table 6 below are followed. In the case of DNA library preparation, 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, the reaction conditions are shown in Table 6 below.

As a result, under the condition using the LNA primer, the rate-determining in the middle stage of the extension reaction did not occur unlike the condition using the DNA primer (Fig. 6). Although there are some primer chains in which the extension reaction has not progressed at all, no change with time is observed, which is considered to be a problem in the synthesis of LNA primers.

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

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

All mutants have introduced mutant N210D, which attenuates exonuclease activity. During reverse transcription, the polymerase needs to bind to the DNA / LNA duplex and properly position the 3'end of the primer strand at the reaction site. It is considered that the reverse transcription activity is observed in the mutants A4 (E664K) and A43 (E664R) in which the negatively charged amino acids in the Thumb region are changed to positive charges as a result of improving the DNA / LNA double-stranded binding ability. Be done. On the other hand, A485L alone, which is involved in binding to triphosphate, does not show reverse transcription activity (mutant A2). However, when A485L is introduced (mutant A51) together with a mutation (E664Q: negative charge → neutral) that slightly increases the DNA / LNA double-stranded binding ability, reverse transcription activity is observed. This is considered to indicate that the required DNA / LNA double-stranded binding ability may be low if the phosphate diester bond forming reaction proceeds rapidly. Further, even in the mutant having E664K with improved DNA / LNA double-strand binding ability, the reverse transcription activity was improved by A485L (mutant A13), and in addition to these mutations, the mutant having D614N was also reversed. Photoactivity 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, A48 (variant 1) and A53 (variant 2) were used for transcription (DNA → LNA), and A51 was used for reverse transcription (LNA → DNA) to obtain the results. The DNA strand was inserted into pT7Blue T-Vector (manufactured by Takara Bio Co., Ltd.) by TA cloning, and the base sequence was analyzed to evaluate the accuracy of polymerase. Since errors can occur in steps other than transcription and reverse transcription, a control experiment was conducted in which the reactions of all steps were advanced with DNA, and the error rates of transcription and reverse transcription were corrected. The procedure is described below.

<Transcription (DNA → LNA)>
Reaction solution composition (reaction solution total 50 μL):
1x
Buffer solution for KOD-Plus-Ver.2 1.5 mM sulfonyl ₄
0.2 mM LNA-NTP (control: dNTP)
0.5 μM primer (Primer hmP3)
0.6 μM 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.)).

The reaction solution (liquid volume: 40 μL) before the addition of the polymerase was annealed (slowly cooled to 25 ° C. over 1 hour at 94 ° C. for 30 seconds), and then 10 μL of 1500 ng / μL polymerase mutant A48 or A53 was added for the reaction. (Control: The reaction solution was used as it was for PCR).

Reaction conditions:
9 hours at 74 ° C. (Control: Step 1: 2 minutes at 95 ° C. (x1), Step 2: 30 seconds at 95 ° C., 30 seconds at 55 ° C., 1 minute at 68 ° C. (x3)) ).

50 μL of 100 mM NaOH was added to the LNA extended sample to denature the polymerase. After neutralizing by adding 250 μL of 50 mM Tris-HCl (pH 7.4), purification by ethanol precipitation is performed, and 1 × B / W Buffer (binding and washing buffer: 5 mM Tris-HCl (pH 7.4)), 0. 5 mM EDTA, 1M NaCl, 0.05% v / v Tween 20): 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).

50 μL of Dynabeads® M-280 Streptavidin (Thermo Fisher Scientific, Inc.) was washed 3 times with 1 mL of 1 × B / W Buffer, then 1 mL of the above-mentioned post-reaction sample 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 sterile water. Further, 50 μL of 100 mM NaOH was added and mixed for 5 minutes, and then the supernatant was collected. It can be contained in a trace amount by adding 1 mL of 100 mM Tris-HCl (pH 7.4) to neutralize it, then adding 50 μL of new Dynabeads® M-280 Streptavidin (cleaned) and mixing for 30 minutes. Sexual template strands were trapped on the beads and removed. The supernatant was collected, replaced with sterile water using Vivaspin 500 (5,000 MWCO) (Sartorius AG), and concentrated.

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

Reaction conditions:
Step 1: 95 ° C. for 2 minutes (x1), Step 2: 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 74 ° C. (control: 68 ° C.) for 1 minute (x20).

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

After washing 50 μL of Dynabeads (registered trademark) M-280 Streptavidin 3 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 wash operation removed the LNA extension strands as much as possible and trapped the reverse transfer strands in the beads. The beads were suspended in 50 μL of sterile water.

PCR (for TA cloning): 25 μL of reaction solution. 1 × Taq reaction buffer, 1.5 mM polymerase ₄ , 0.2 mM dNTP, 0.3 μM primers (Primer hP1, Primer hP5), bead suspension 2.5 μL, 0.1 U / μL rTaq trapped with reverse transcriptase Polymerase (manufactured by Toyo Boseki Co., Ltd.). Reaction conditions: Step 1: 94 ° C. for 2 minutes (x1), Step 2: 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 1 minute (x20). Purified using the 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 (manufactured by Toyobo Co., Ltd.) 2 μL are mixed, reacted at 16 ° C for 60 minutes, and then Escherichia coli. Introduced into DH5α. Each plasmid was extracted from the obtained single colony and base sequence analysis was performed.

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

Due to the convenience 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 error rate was investigated by proceeding with the reaction with DNA in all steps. Table 9 shows the results of nucleotide sequence analysis for evaluating the accuracy of LNA transcription / reverse transcription.

As a result, in the control experiment, 6 errors were included in the 1024 bases analyzed. Therefore, the error rate of the experimental system was determined to be 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 (transcription) and A51 (reverse transcription), the raw error rate = 17 errors / (31 samples x 32 bases) = 17.1 x 10 ^-3 without correction, and the control error rate was set. When pulling corrected, it was determined to correct the error rate ^{= 17.1 × 10 -3 -5.8 × 10} -3 = 11.3 × 10 -3 (1.1% error per base). In addition, under the conditions using A53 (transcription) and A51 (reverse transcription), the raw error rate = 9 errors / (26 samples x 32 bases) = 10.8 x 10 ^-3 without correction, and the control error rate is subtracted. determined and a correction error rate correcting ^{= 10.8 × 10 -3 -5.8 × 10} -3 = 5.0 × 10 -3 (0.5% error per nucleotide) Te. Therefore, it was shown that the LNA transcription of A53 maintained its accuracy from the base sequence analysis of the DNA strand obtained by the subsequent reverse transcription reaction. Further, in SELEX, A53 (transcription) and A51 (reverse transcription) can be used more preferably.

(Example 10: Creation of modified polymerase (KOD polymerase mutant) and examination of LNA chain extension activity)
The modified polymerase (KOD polymerase variant) shown in Table 10 was prepared, and the LNA chain extension activity using the DNA strand as a template was evaluated. These KOD polymerase variants are obtained by substituting Y409 with the following amino acids, respectively.

Annealing is performed by heating the reaction solution to which the KOD polymerase mutant is not added at 95 ° C. for 1 minute and then lowering it to 25 ° C. over 60 minutes, and then adding the KOD polymerase mutant and extending the LNA chain at 74 ° C. The reaction proceeded. 1/6 volume of 6 × loading die was added to the obtained reaction solution, and electrophoresis was performed on an alkaline agarose gel.

For various KOD polymerase mutants, the LNA chain extension reaction was carried out with the following three reaction solution compositions.

<Evaluation 1>
Reaction solution composition: 1 × buffer solution for KOD-Plus-Ver.2, KOD polymerase mutant: 300 ng / μL, 0.2 mM LNA-NTP, 1.5 mM sulfonyl 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, 35V, 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 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® X -100, 10 μg / mL BSA), KOD polymerase variant: 300 ng / μL, 0.2 mM LNA-NTP, 1.5 mM sulfonyl 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, 35V, 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 composition: 1 × KOD DNA polymerase buffer 2 (120 mM Tris-HCl (pH 8.8) (25 ° C), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton® X -100, 10 μg / mL BSA), KOD polymerase variant: 300 ng / μL, 0.2 mM LNA-NTP, 1.5 mM sulfonyl 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, 35V, 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, the KOD polymerase mutants A62, A63 and A64 showed particularly high LNA chain extension activity. These are variants in which Y409 is replaced with less bulky amino acids (glycine, alanine and serine, respectively). In FIG. 8, the LNA chain elongation activity was also observed for A65 (threonine), A53 (valine) and A68 (aspartic acid), and further A66 (leucine) and A67 (phenylalanine). It was found that efficient LNA chain extension is possible by changing the composition of the buffer solution of the reaction solution (FIGS. 8 to 10). The LNA chain extension efficiency is higher under the condition of using the 1 × KOD DNA polymerase buffer solution 1 (evaluation 2) in FIG. 9 than the condition using the 1 × KOD-Plus-Ver.2 buffer solution (evaluation 1). Further, under the condition (evaluation 3) using the 1 × KOD DNA polymerase buffer solution 2 of FIG. 10, the LNA chain extension efficiency was particularly high. According to FIGS. 8 to 10, gel band movement was also observed for A69 (glutamic acid), and it was evaluated that the gel had LNA chain elongation activity. Comparing the LNA chain extension activities of A53 and A62 to A69, higher LNA chain extension activity was observed when the size (bulkness) of the amino acid group after substitution with respect to Y409 was smaller.

(Example 11: Examination of accuracy of LNA chain synthesis using KOD polymerase mutant)
The modified polymerase produced was evaluated for accuracy of LNA uptake. The type of modified polymerase was evaluated using A53 in the case of two buffer solutions. As a method for evaluating accuracy, an extension reaction is performed under the condition that one of the four types of bases is missing (conditions 2 to 5 below: condition 1 when there is no base and condition 6 when all four types of bases are present). Was done.

The LNA chain extension reaction was carried out under the following reaction solution composition and conditions, and the LNA chain extension activity was evaluated.

Reaction composition: 1 × KOD DNA polymerase buffer 2 (120 mM Tris-HCl (pH 8.8) (25 ° C), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton® X -100, 10 μg / mL BSA), KOD polymerase variant A53: 300 ng / μL, 1.5 mM sulfonyl 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 Running conditions: 25 ° C, 35V, 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).

Further, the 1 × KOD DNA polymerase buffer 2 having the above reaction composition was replaced with the 1 × KOD-Plus-Ver.2 buffer, the reaction time was 15 hours, and the sample volume was 1.5 μL (0.625 pmol). The LNA chain extension reaction was carried out as a primer), and the LNA chain extension activity was evaluated.

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

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

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

According to the present invention, there is provided a modified polymerase capable of extending a chain containing LNA, which is an artificial nucleic acid. This makes it possible to stably extend the LNA strand from the DNA strand. Furthermore, an LNA library can be created by 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 a degrading enzyme can be obtained in advance, and thus the time required for optimizing the aptamer can be shortened. In addition, the artificial nucleic acid library allows the size of the library to be increased, expanding the scope of screening for candidate aptamers. The creation of an artificial nucleic acid library will be useful for screening sequences containing candidate artificial nucleic acids such as pharmaceuticals and for efficient development of nucleic acid pharmaceuticals containing artificial nucleic acids.

Claims (9)

A modified polymerase consisting of an amino acid sequence having the following mutation in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing:
Aspartic acid 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 aspartic acid at position 614. Asparagine and glutamic acid at position 664 have been replaced with lysine, glutamine, or alanine, respectively. In the amino acid sequence described in SEQ ID NO: 2 in Sequence Listing, the amino acid sequence having a mutation in the following, modified polymerase with LNA chain elongation activity and / or reverse transcription activity:
2 Aspartic acid at position 10 is replaced with aspartic acid, alanine at position 485 is replaced with neutral amino acid, asparagine at position 614 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine, glutamine, or arginine .
Here, position 521 of the amino acid sequence set forth in SEQ ID NO: 2 is isoleucine . A modified polymerase having reverse transcription activity consisting of an amino acid sequence having the following mutation in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing:
Asparagine at position 210 is replaced 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.
Here, position 521 of the amino acid sequence set forth in SEQ ID NO: 2 is isoleucine . In the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, a modified polymerase having reverse transcription activity consisting of an amino acid sequence having the following mutation:
Asparagine at position 210 is replaced with aspartic acid, and glutamic acid at position 664 is replaced with lysine or arginine , respectively .
Here, position 521 of the amino acid sequence set forth in SEQ ID NO: 2 is isoleucine . A method for synthesizing an LNA-containing oligonucleotide chain from a DNA-containing oligonucleotide.
A method comprising reacting a template consisting of the DNA-containing oligonucleotide, a primer, LNA-NTP, and the modified polymerase according to claim 1 or 2. The method according to claim 5 , wherein the primer is an LNA-containing primer. The method of claim 5 or 6 , wherein the DNA-containing oligonucleotide comprises a random sequence. A method for synthesizing a DNA-containing oligonucleotide from an LNA-containing oligonucleotide.
A method comprising reacting a template consisting of the LNA-containing oligonucleotide, a primer, dNTP, and the modified polymerase according to any one of claims 2 to 4 . The reaction is carried out in a buffer 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). The method according to any one of 5 to 8 .

Images