WO2007117331A2

WO2007117331A2 - Novel dna polymerase from thermoanaerobacter tengcongenesis

Info

Publication number: WO2007117331A2
Application number: PCT/US2006/062527
Authority: WO
Inventors: Shanmuuga Sundaram; Scott Hamilton; Carl W. Fuller
Original assignee: GE Healthcare Bio Sciences Corp
Current assignee: Global Life Sciences Solutions USA LLC
Priority date: 2005-12-22
Filing date: 2006-12-22
Publication date: 2007-10-18
Anticipated expiration: 2008-06-22
Also published as: WO2007117331A3

Abstract

Disclosed is a DNA polymerase from Thermoanaerobacter tengcongenesis. This enzyme is useful for procedures requiring strand-displacing DNA synthesis such as SDA and for reverse transcription. Included within the scope of the present invention are various mutants (deletion and substitution) that retain the ability to replicate DNA with substantially the same efficiency as the native Thermoanaerobacter tengcongenesis polymerase.

Description

NOVEL DNA POLYMERASE FROM THERMOANAEROBACTER TENGCONGENESIS

Cross-Reference to Related Applications This application claims priority to United States provisional patent application number 60/753,075 filed December 22, 2005; the entire disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention The present invention relates to novel DNA polymerases obtainable from the thermophilic organism Therrnoanaerobacter tengcongenesis, to certain deletions and mutants of this enzyme, to genes and vectors encoding the wild type and mutant polymerases and their use in strand displacement activity and as reverse transcriptases.

Background of the Invention

DNA polymerases are a family of enzymes involved in DNA repair and replication. DNA polymerases have been isolated from E. coli (e.g. E. coli DNA polymerase I and the Klenow fragment thereof) and bacteriophage T4 DNA polymerase and more recently thermostable DNA polymerases have been isolated (e.g. from T. aquaticus, US Patent 4,889,818, and from T. litoralis). Thermostable DNA polymerases have been suggested (US Patent 4,683,195) for use in amplifying existing nucleic acid sequences in amounts that are large compared to that originally present. The polymerase chain reaction (PCR; US Patent 4,683,202) and strand displacement amplification (SDA) are two methods of amplifying nucleic acid sequences. PCR is based on the hybridization of oligonucleotide primers to specific sequences on opposite strands of the target DNA molecule, and subsequent extension of these primers with a DNA polymerase to generate two new strands of DNA which themselves can serve as a template for a further round of hybridization and extension. In PCR amplification the product of one cycle serves as the template for the next cycle such that at each repeat of the cycle the amount of specific sequence present in the reaction can double leading to an exponential amplification process.

In reverse transcription/polymerase chain reaction (RT/PCR), a DNA primer is hybridized to a strand of the target RNA molecule, and subsequent extension of this primer with a reverse transcriptase generates a new strand of DNA, which can serve as a template for PCR. Preparation of the DNA template is preferably carried out at an elevated temperature to avoid early termination of the reverse transcriptase reaction caused by RNA secondary structure. Since most of the known, efficient reverse transcriptases come from animal viruses, there is a lack of efficient reverse transcriptases that act at elevated temperatures, e.g. above 50⁰C.

SDA differs from PCR in being an isothermal amplification process, i.e. all reactions occur at the same temperature without the need for elevated temperature to melt DNA strands. This is made possible by adoption of a reaction scheme which uses the ability of certain DNA polymerases when extending along a DNA template strand to displace any DNA molecules already hybridized to the template. In SDA this strand displacement is used to separate the double stranded DNA produced earlier in the reaction process and hence to maintain continuous amplification of the target DNA sequence (Walker, G.T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Natl. Acad. Sci. USA 89:392-396). SDA is therefore in principle more suited to use with large numbers of samples than PCR as the isothermal process, which is performed at temperatures of 37⁰C to 6O⁰C, does not require stringent precautions to be taken to avoid evaporation and can be performed with simple temperature control equipment, for example in a standard laboratory incubator.

Brief Description of the Invention

The present invention provides a DNA polymerase from Thermoanaerobacter tengcongenesis. This enzyme is useful for procedures requiring strand-displacing DNA synthesis such as SDA and for reverse transcription. Included within the scope of the present invention arc various mutants (deletion and substitution) that retain the ability to replicate DNA with substantially the same efficiency as the native Thermoanaerobacter tengcongenesis polymerase.

Brief Description of the Drawings

Figure 1 is the DNA sequence from Thermoanaerobacter tengcongenesis encoding a full length novel DNA polymerase (SEQ ID NO:1).

Figure 2 is a contiguous open reading frame capable of encoding the full length polymerase from Thermoanaerobacter tengcongenesis (SEQ ID NO:2). Translation is of the open reading frame spanning SEQ ID NO:1 as shown in Figure 1, encoding native polymerase.

Figure 3 is a DNA sequence encoding the DNA polymerase from Thermoanaerobacter tengcongenesis, containing F705Y and D719R mutations (SEQ ID NO:3).

Figure 4 is the amino acid sequence of the DNA polymerase from Thermoanaerobacter tengcongenesis, containing F705Y and D719R mutations (SEQ ID NO:4). Figure 5 is the polynucleotide sequence encoding the DNA polymerase from Thermoanaerobacter tengcongenesis, containing Y72C, F705Y and D719R mutations (SEQ ID NO:5).

Figure 6 is the amino acid sequence of the DNA polymerase from Thermoanaerobacter tengcongenesis, containing Y72C, F705Y and D719R mutations (SEQ ID NO:6). The Y72C mutation decreased exonuclease activity.

Figure 7 is DNA sequence encoding a truncated version of a DNA polymerase from Thermoanaerobacter tengcongenesis, containing FY and DR mutations (SEQ ID

NO:7).

Figure 8 is the amino acid sequence of the truncated version of DNA polymerase from Thermoanaerobacter tengcongenesis, containing FY and DR mutations (SEQ ID NO:8).

Figure 9 is an alignment of several DNA polymerase protein sequences (SEQ ID NO:11 - SEQ ID NO:17).

Detailed Description of the Invention

In a first aspect, the present invention provides a purified DNA polymerase or fragment thereof having the DNA polymerase activity of Thermoanaerobacter tengcongenesis and having at least 80% amino acid homology, preferably at least 90% homology, to at least a contiguous 40 amino acid sequence shown in Figure 2 (SEQ ID NO:2). Figure 2 represents the translation of the open reading frame of DNA sequence encoding a DNA polymerase from Thermo anaerobacter tengcongenesis (Figure 1) (SEQ ID NO:1).

When used herein, the term amino acid homology means amino acid identity or conservative amino acid changes thereto. The DNA polymerase can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained. The amino acid sequence will be substantially similar to the sequence shown in Figure 2, or fragments thereof. A sequence that is substantially similar will preferably have at least 80% homology (more preferably at least 90% and most preferably 98-100%) to the sequence of Figure 2. By "identity" is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical or homologous residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.

The purified enzyme of the present invention has a molecular weight of approximately 101,000 daltons when measured on SDS-PAGE. It possesses a 5 -3' exonuclease activity. The temperature optimum of DNA synthesis is near 75⁰C under assay conditions. The optimum magnesium ion and manganese ion concentrations for DNA synthesis are approximately 1 mM and 0.5 mM respectively.

When used herein, the term a DNA polymerase or fragment thereof having the DNA polymerase activity of Thermoanaerobacter tengcongenesis means a DNA polymerase or fragment thereof (as hereinafter defined) which has the ability to replicate DNA with substantially the same efficiency as the enzyme encoded by the SEQ ID NO:1. By substantially the same efficiency is meant at least 80% and preferably at least 90% of the efficiency of the enzyme encoded by SEQ ID NO:1 to incorporate deoxynucleotides.

The invention also encompasses a stable enzyme composition which comprises a purified DNA polymerase from Thermoanaerohacter tengcongenesis in a buffer.

The DNA polymerases of the present invention are preferably in a purified form. By purified is meant that the DNA polymerase is isolated from a majority of host cell proteins normally associated with it; preferably the polymerase is at least 10% (w/w), e.g. at least 50% (w/w) , of the protein of a preparation, even more preferably it is provided as a homogeneous preparation, e.g. homogeneous solution. Preferably the DNA polymerase is a single polypeptide on an SDS polyacrylamide gel. Buffers around neutral pH (5-9) such as 5-100 mM TrisHCl, HEPES or MES are suitable for use in the current invention.

The present invention also provides a gene encoding a polymerase of the present invention. Figure 1 represents nucleotide sequence of a cloned gene encoding the polymerase of the present invention (SEQ ID NO:1).

It has been found that the entire amino acid sequence of the polymerase is not required for enzymatic activity. Thus, for example, the exonuclease domain of the enzyme has been deleted to give a truncated enzyme which retains enzyme activity and also has reverse transcriptase activity making it useful for making cDNA. Thus, the present invention also provides fragments of the polymerase which retain the DNA polymerase activity of Thermoanaerobacter tengcongenesis but have one or more amino acids deleted, preferably from the amino-terminus, while still having at least 80% amino acid homology to at least a 40 contiguous amino acid sequence shown in Figure 2 (SEQ ID NO:2) . In a further aspect, the present invention provides a DNA polymerase which corresponds to the DNA polymerase from Thermoanaerobacter tengcongenesis in which up to one third of the amino acid sequence at the amino-terminus has been deleted. In particular, fragments of Thermoanaerobacter tengcongenesis having N-terminal deletions (See Figures 7 and 8) (SEQ ID NO:7 and SEQ ID NO:8) have been found to retain enzyme activity.

It is preferred that the 5'-3' exonuclease activity of the DNA polymerase is removed or reduced. This may be achieved by deleting the amino acid region of the enzyme responsible for this activity, e.g. by deleting up to one third of the amino acid sequence at the amino terminus, or by appropriate amino acid changes (Y72C, See Figures 5 and 6) (SEQ ID NO:5 and SEQ ID NO:6).

In addition to the N-terminal deletions and amino acid changes to remove the exonuclease activity, the enzyme may have conservative amino acid changes compared with the native enzyme which do not significantly influence enzyme activity. Such changes include substitution of like charged amino acids for one another or amino acids with small side chains for other small side chains, e. g. ala for val. More drastic changes may be introduced at non-critical regions where little or no effect on polymerase activity is observed by such a change.

Joyce and Steitz, Annu. Rev. Biochem, 63:777-822, 1994, discuss various functions of DNA polymerases including the catalytic center, the binding site for the 3 ' terminus of the primer, and the dNTP binding site. In particular, it mentions mutations that affect the binding of dNTP in the ternary complex. European patent application 0655506A discloses that the presence of a polar, hydroxyl containing amino acid residue at a position near the binding site for the dNTP substrate is important for the polymerase being able to efficiently incorporate a dideoxynucleotide. Applicant has discovered that the modification of the dNTP binding site for the dNTP substrate in DNA polymerase obtainable from Thermoanaerobacter tengcongenesis by the inclusion of a polar, hydroxyl containing amino acid residue at position 705 near the binding site increases the efficiency of the polymerase to incorporate dideoxynucleotides. Preferably the polar, hydroxyl containing amino acid is tyrosine. It has also been found that replacing the phenylalanine at the position corresponding to 705 of the native enzyme with tyrosine improves the incorporation of dideoxynucleotides when the enzyme is used for sequencing. In particular, a polymerase from Thermoanaerobacter tengcongenesis in which the exonuclease activity has been reduced e.g. by point mutation or deletion and which has the phenylalanine at the position corresponding to 705 of the native enzyme replaced by an amino acid (e.g. tyrosine) which increases the efficiency of the enzyme to incorporate dideoxynucleotides at least 20 fold compared to the wild type enzyme is a particularly preferred enzyme for use in sequencing. Several modified DNA polymerase sequences are provided, containing the FY mutation and other, preferable point mutations and truncations (See Figures 3 through 8) (SEQ ID NO:3 through SEQ ID NO:8).

The DNA polymerases of the present invention can be constructed using standard techniques familiar to those who practice the art. By way of example, in order to prepare a polymerase with the phenylalanine to tyrosine mutation, mutagenic PCR primers can be designed to incorporate the desired Phe to Tyr amino acid change (FY mutation in one primer). Deletion of the exonuclease function is carried out by PCR to remove the amino terminus, or standard techniques of site directed mutagenesis to generate point mutations. Improved expression of the DNA polymerases of the present invention can be achieved by introducing silent codon changes (i.e., the amino acid encoded is not changed). Such changes can be introduced by the use of mutagenic PCR primers. Silent codon changes such as the following increase protein production in E. coli: substitution of the codon GAG for GAA; substitution of the codon AGG, AGA, CGG or CGA for CGT or CGC; substitution of the codon CTT, CTC, CTA, TTG or TTA for CTG; substitution of the codon ATA for ATT or ATC; substitution of the codon GGG or GGA for GGT or GGC.

Genes encoding the DNA polymerase from Thermoanaerobacter tengcongenesis polymerases in which up to one third of the amino acid sequence at the amino terminus has been deleted and which have the exonuclease activity removed by point mutation and such polymerases which incorporate the phenylalanine to tyrosine modification are also provided by the present invention.

In a yet further aspect, the present invention provides a host cell comprising a vector containing the gene encoding the DNA polymerase activity of the present invention, e.g., encoding an amino acid sequence corresponding to native Thermoanaerobacter tengcongenesis or differentiated from this in that it lacks up to one third of the N-terminal amino acids and optionally has mutations as shown in SEQ ID NO: 3 through SEQ ID NO:8. The DNA polymerases of the present invention are suitably used in SDA, preferably in combination with a restriction enzyme. Accordingly, the present invention provides a composition which comprises a DNA polymerase of the present invention in combination with a restriction enzyme, for example BsoBI from Bacillus stearothermophilus. The invention also features a kit or solution for SDA comprising a DNA polymerase of the present invention in combination and a thermostable restriction enzyme.

The polymerases of the present invention are also useful in methods for generating and amplifying a nucleic acid fragment via a strand displacement amplification (SDA) mechanism. The method generally comprises: a) specifically hybridizing a first primer 5' to a target nucleic acid sequence, the first primer containing a restriction enzyme recognition sequence 5' to a target binding region; b) extending the 3' ends of the hybridized material using a DNA polymerase of the present invention, preferably one in which the exonuclease activity has been removed, in the presence of three dNTPs and one dNTPα S; c) nicking at the hemiphosphorothioate recognition site with a restriction enzyme, preferably; d) extending the 3' end at the nick using a DNA polymerase of the present invention, displacing the downstream complement of the target strand; and e) repeating steps (c) and (d).

This SDA method proceeds at a linear amplification rate if one primer is used as above. However, if two primers are used which hybridize to each strand of a double-stranded DNA fragment, then the method proceeds exponentially (Walker, G.T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Nail. Acad. ScL USA 89:392-396).

In a further aspect, the present invention provides a method for preparing complementary DNA by combining an oligonucleotide primer, a sample of RNA, a DNA polymerase of the present invention, and between one and four deoxyribonucleoside phosphates, under conditions favoring preparation of the complementary DNA.

The DNA polymerases of the present invention which act as reverse transcriptases lack appreciable, and preferably have no RNaseH activity and, as such, are useful in RT/PCR, the generation of hybridization probes and RNA sequencing.

In a yet further aspect, the present invention provides a purified reverse transcriptase having activity of greater than 1000 units per milligram. Preferably, the reverse transcriptase lacks RNaseH activity; the reverse transcriptase is from Thermoanaerobac tengcongenesis; the reverse transcriptase has an N-terminal deletion or amino acid changes that remove the exonuclease function. In a further aspect, the invention features a method for reverse transcription/polymerase chain reaction (RT/PCR) which utilizes a DNA polymerase of the present invention and a DNA polymerase suitable for PCR in the same reaction vessel. Preferably, the DNA polymerase of the present invention is from Thermoanaerobacter tengcongenesis, the polymerase has one or more amino acids deleted from the amino terminus or amino acid changes to remove the exonuclease activity, the DNA polymerase suitable for PCR is Taq DNA polymerase. In another aspect, the present invention features a kit or solution for RT/PCR comprising a DNA polymerase of the present invention and a DNA polymerase suitable for PCR. Preferably, the DNA polymerase of the present invention is from Thermoanaerobacter tengcongenesis, the polymerase has one or more amino acids deleted from the amino terminus or amino acid changes to remove the exonuclease function, and the DNA polymerase suitable for PCR is Taq DNA polymerase.

In a further preferred embodiment the invention features polymerases that have exonuclease activity removed by replacing an aspartic acid residue at position 8 with an alanine residue. This is a preferred mutation for it not only removes the exonuclease activity but removes an RNaseH activity, which may affect applications using RNA. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.

Examples

The following examples serve to illustrate the DNA polymerases of the present invention and are not intended to be limiting.

Example 1 : Cloning of native polymerase and generation of mutant en/ymes

The Thermoanaerobacter tengcongenesis bacterial strain (DSM No. 15242) obtained from DSMZ was dispensed in Medium 965 (DSM, Germany) and lysed with KOH, Tris, EDTA (blood GenomiPhi protocol, GE Healthcare) and the sample was heat treated at 95⁰C for 3 min and the GenomiPhi reaction started. The amplified genomic DNA was cleaned by passing through a G-50 column. The polymerase gene was then amplified by PCR and cloned into PKK vector at the EcoRI and Kpnl Site. The primers were designed using the known genome sequence, with the introduction of restriction enzyme sites. The primer at N-terminus (with EcoRI site) is 5'- CCG GAA TTC AGT GGT TTA ATG GCA AAG TTT CTG TTA ATT G - 3' (SEQ ID NO:9). The primer at the C-terminus (with Kpnl site) is 5' - CGG GGG TAC CAT TTC CAC CTC ACT TAG CCA AAA ACC AGT TA - 3' (SEQ ID NO: 10). The PCR product was generated using Pfu DNA polymerase and cloned into PKK vector.

The point mutants were generated by using the Quickchange site directed mutagenesis kit (Stratagene). The truncated version was generated by designing primers covering amino acids 294 till end of the polymerse gene with EcoRl and Kpnl sites and cloning into PKK vector. Example 2: Purification of Cloned Enzymes

The full length and truncated enzyme clones were fermented using 2X YT medium. Overnight cultures of the plasmid-containing strains were inoculated into 2X YT medium containing Kanamycin. The polymerase expression was induced at 1.0 OD by adding 1 mM IPTG. The cells were harvested after 2 hours of induction.

The cell pastes containing full length or truncated enzymes were lysed using the lysis buffer (50 mM Tris-Cl, pH 8.5; 50 mM NaCl; 1 mM EDTA; 0.2% NP40; 0.2% Tween 20) and heat treatment. Lysis was carried out at 74°C for 20 min. The suspension was centrifuged and supernatant loaded onto 5ml HiTrap Heparin Sepharose HP. The column was washed 10 column volumes with Buffer A (5OmM Tris pH 8.5; ImM EDTA; 5OmM NaCl). The column was further washed with gradient: 0 - 60% Buffer B (5OmM Tris pH 8.3; ImM EDTA; IM NaCl) in 10 column volumes. Elution was continued by stepping up to 100%B for 5 column volumes. 5ml fractions were collected.

Fractions with activity from the Heparin HP column were dialyzed overnight against Buffer A so that the conductivity is less than lOmS/cm. The resulting sample was loaded onto a 5ml HiTrap Q column, washed with 10 column volumes of Buffer A. Followed by gradient: 0 - 60% Buffer B in 10 column volumes. Elution was continued by stepping up to 100%B for 5 column volumes. Collect 5ml fractions. The fractions containing polymerase activity were pooled and dialyzed against the final buffer (50 mM Tris-Cl, pH 8.5, 25 mM KCL, 0.2 mM EDTA, 0.5% NP 40 and 0.5% Tween 20). Example 3: Characterization of Enzymes Reverse transcriptase activity

The cloned enzyme functioned as a reverse transcriptase in an assay using the EnzCheck RT assay kit (Molecular Probes- E 220664). The result shows that RT activity of the novel polymerase from Thermoanaerobacter tengcongenesis has similar specific activity to that of Thermoanaerobacter thermohydrosulfuricus (Tts).

Cy dye incorporation

Truncated Tten enzyme incorporate Cy dyes with similar specific activity as the Tts enzyme.

Strand Displacement Activity

The exonuclease deficient cloned enzyme described above (SEQ ID NO: 8) was used in a Strand Displacement Amplification (SDA) reaction (Walker et al, (1992) Proc. Natl. Acad. ScL USA 89 :392-396). The reaction mixtures contain:

40 mM tris-cl, pH 7.5

20 mM MgC12

1 mM DTT

80 ng M13 130 ng Ml 3 primer

200 uM dNTPs

The mixture was heated at 95°C for 5 min and then at 55°C for 10 min. The mixture was cooled and 1 ul of the truncated enzyme added. The reaction was incubated at 60⁰C for 5, 10 and 15 min, respectively. The reaction was stopped by adding 500 mM EDTA and loaded on 0.8% agars gels for detection. As a control, similar reactions were carried out with T4 and T7 DNA polymerases (negative and positive controls). The strand displacement activity was measured by the ability of the enzyme to produce a high molecular weight product. Truncated FYDR enzyme is able to catalyze strand displacement activity, with an efficiency similar to that of T7 DNA polymerase. One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, kits, solutions, and molecules, described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

Other embodiments are within the following claims.

Claims

What is claimed is:

1. A novel DNA polymerase protein comprising: a) a DNA polymerase from Thermoanaerobacter tengeongenesis having amino acid sequence of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or

SEQ ID NO:8; or b) a polypeptide having a sequence with 80% similarity to that of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

2. The DNA polymerase of claim 1 with strand displacement activity.

3. The DNA polymerase of claim 1 with reverse transcriptase activity.

4. A nucleic acid sequence encoding a DNA polymerase, comprising: a) a nucleotide having the sequence of SEQ ID NO: 1 , SEQ ID NO:3, SEQ

ID NO:5 or SEQ ID NO:7; or b) a polynucleotide having a sequence with 90% similarity to that of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

5. A vector containing a nucleic acid sequence encoding a protein of claim 1.

6. A transformed cell containing a vector of claim 5.

7. A vector containing a nucleic acid sequence of claim 4.

8. A transformed cell containing a vector of claim 7.

9. In a method for reverse transcription of a RNA strand to a DNA strand, the improvement comprises using the novel DNA polymerase of claim 1 as the reverse transcriptase.

10. In a strand displacement amplification method, the improvement comprises using the novel DNA polymerase of claim 1 as the amplification enzyme.