N-terminal fragment of DNA polymerase, DNA polymerase, method for producing the same, and use thereof for isothermal DNA amplification at low temperatures

Abstract

The invention relates to an isolated N -terminal fragment of DNA polymerase responsible for maintaining enzymatic activity at low temperatures, as well as to a nucleotide sequence encoding the N-terminal fragment of DNA polymerase, and to an expression vector pET28-Efa enabling the expression of said N-terminal fragment of DNA polymerase. In another aspect, the invention also relates to the Efa DNA polymerase encoded by bacteriophage vB_EfaS-271, as well as to the nucleic acid sequence encoding said enzyme and to an expression vector containing a nucleotide sequence encoding a fusion DNA polymerase. The disclosure additionally concerns a method for producing the Efa DNA polymerase or its N-terminal domain, as well as the use of thereof for efficient isothermal DNA amplification at low temperatures. The invention finds application in scientific research (genotyping, mutation detection, DNA sequencing), diagnostics (mutation detection, diagnostic tests based on identification of pathogen genetic material), and in the biotechnology industry (DNA cloning and amplification).

Description

[0001] N-terminal fragment of DNA polymerase, DNA polymerase, method for producing the same, and use thereof for isothermal DNA amplification at low temperatures

[0002] The invention relates to an isolated N-terminal fragment of the Efa DNA polymerase, which is responsible for maintaining enzymatic activity at low temperatures only when constituting an integral part of the enzyme. The invention further relates to a nucleotide sequence encoding the N-terminal fragment of the Efa DNA polymerase and to the expression vector pET28-Efa enabling the expression of said N-terminal fragment of DNA polymerase. In another aspect, the invention also relates to the Efa DNA polymerase encoded by bacteriophage vB_EfaS-271, as well as to the nucleic acid sequence encoding said enzyme and to an expression vector containing a nucleotide sequence encoding a fusion DNA polymerase. The disclosure additionally concerns a method for producing the Efa DNA polymerase or its N-terminal domain, as well as the use of thereof for efficient isothermal DNA amplification at low temperatures. The invention finds application in scientific research (genotyping, mutation detection, DNA sequencing), diagnostics (mutation detection, diagnostic tests based on identification of pathogen genetic material), and in the biotechnology industry (DNA cloning and amplification).

[0003] Replication, that is, the duplication of DNA - the carrier of genetic information - is one of the most fundamental biological processes. It is essential for the propagation of bacteria and viruses, and it also constitutes a key process preceding mitosis and meiosis in eukaryotic cells [1,2], DNA replication occurs through the activity of enzymes known as DNA polymerases, which are capable of attaching nucleotides to the 3’ end of a DNA strand [3] . Although replication of genetic material is now one of the basic laboratory techniques, it was the discovery of the polymerase chain reaction (PCR) by Kary Mullis in 1985 that introduced this process into routine laboratory practice, leading to the rapid development of biological and medical sciences [4], The polymerase chain reaction described by Mullis allows quick and highly efficient amplification of small amounts of DNA, which has made it widely applicable in molecular biology, biotechnology, and medicine.

[0004] At present, PCR remains the gold standard for DNA amplification [5], It is used in the production of pharmaceuticals, the development of vaccines and the generation of genetically modified animal models. Moreover, it constitutes a primary diagnostic tool for the detection of viral, bacterial, and genetic diseases. This technique has also opened new avenues for progress in fields beyond medicine and molecular biology, such as agriculture and animal breeding, enabling the efficient production of high-quality meat, milk, and crops.

[0005] Although versatile, the PCR technique has its limitations. It relies on cyclic, rapid temperature changes that successively enable template denaturation, primer annealing, and extension. Therefore, its implementation requires specialized equipment - a thermocycler - and a thermostable enzyme capable of retaining activity at high temperatures. Consequently, DNA amplification by PCR is difficult to perform outside the laboratory. New approaches that allow DNA replication under isothermal conditions have been developed in response to this limitation [6].

[0006] Isothermal nucleic acid amplification (IA) utilizes knowledge of DNA replication in cells by recreating similar conditions in a test tube. A common feature of isothermal amplification methods is that the reaction is carried out at a constant temperature, although the exact one varies depending on the method applied and the polymerase used. It usually ranges from 30°C to 65°C. According to the literature, IA techniques can generate longer amplicons (fragments of the amplified DNA). They also exhibit higher amplification efficiency as well as lower sensitivity to inhibitors present in samples, and require fewer sample preparation steps compared to PCR-based methods [5], Nevertheless, since double -stranded DNA unwinding and primer annealing do not occur here in temperature -dependent manner, the IA reaction requires the participation of additional proteins or a polymerase with strand displacement activity (SDA) [7,8] . In the helicasedependent amplification (HDA) method, the unwinding of duplex DNA is carried out by a helicase. In turn, recombinase polymerase amplification (RPA) method enables replication of genetic material through the action of a recombinase, which catalyzes the hybridization of the primer with the homologous sequence of the template. Both techniques additionally require the presence of single -stranded DNA binding and stabilizing proteins [6], The simplicity of these techniques has led to the development of commercially available tests for pathogen detection in various environments, including water, food, and clinical samples. Such tests are produced, among others, by BioHelix Corporation (HDA) and TwistDx, Cambridge, United Kingdom (RPA) [9-11], The advantages of IA methods have also been successfully combined with nanotechnology to create devices such as microfluidic chips. This approach increases the sensitivity, reproducibility, speed, and cost-effectiveness of the method, resulting in more accurate outcomes. Miniaturization positively affects the sample quality by reducing the risk of contamination, thereby improving detection efficiency. Devices based in isothermal techniques, contrary to PCR-based ones, can be designed as simple and portable microsystems.

[0007] Despite the advantages mentioned above, a key challenge in optimizing and broadly applying methods based on isothermal amplification lies in selecting an appropriate DNA polymerase for a specific purpose. Crucial factors include the rate and fidelity of DNA amplification, as well as the temperature range within which given polymerase remains active [5,12], In rapid diagnostic and detection assays, the amplification rate is of primary importance, whereas in genotyping and sequencing, the amplicon fidelity constitutes the main priority. Although IA techniques do not require a thermocycler, the reaction temperature range (30°C - 65°C) still necessitates the use of equipment capable of maintaining a constant temperature [5,6], Moreover, isothermal methods do not address the problem of nucleases present in environmental or clinical samples, which remain active at temperatures similar to those of DNA polymerases and can adversely affect amplification results by degrading the input material. Therefore, there is a continuing need to search for new DNA-amplifying enzymes that - owing to their optimal conditions or unique properties - could overcome these limitations.

[0008] Enzymes can be classified into groups based on their optimal temperature of activity: psychrophilic (5-25°C), mesophilic (20-45°C), and thermophilic (60-80°C) [7], Thermophilic DNA polymerases are used in PCR, with Pfu DNA polymerase being among the most commonly employed. In contrast, mesophilic DNA polymerases exhibit broad applicability. For instance, the q>29 DNA polymerase, which possesses strand displacement activity, is utilized in isothermal PCR techniques [8], Nevertheless, mesophilic DNA polymerases are not suitable for DNA amplification at low temperatures, as their activity declines sharply with decreasing temperature. Psychrophilic DNA polymerases, capable of efficiently replicating DNA under such conditions, may bridge this gap and expand the temperature range applicable in diagnostic assays without the need for specialized equipment. Furthermore, such enzymes hold potential for supporting research and diagnostics in previously inaccessible environments, such as the Arctic or even outer space

[0013] , However, the production costs of psychrophilic enzymes are high. In general, they are also less thermostable than mesophilic or thermophilic proteins, therefore, their routine use in laboratories is more demanding and, in many cases, economically unfavorable for biotechnology companies. As a result, the application potential of such proteins remains largely underexploited [14-16], To date, only one psychrophilic DNA polymerase has been described in the literature, with its activity demonstrated by electrophoretic separation of extension products 4-5 nucleotides longer than the primer after seven hours of reaction

[0013] , Consequently, no DNA polymerase combining both high activity at low temperatures and high stability has yet been identified.

[0009] Bacteriophages (phages) - viruses that infect prokaryotic cells - represent a rich source of proteins with high biotechnological potential

[0017] , Given the fact that bacteriophages are the most abundant biological entities on Earth, with an estimated 1031virions (a number roughly ten times greater than that of bacterial cells)

[0018] , our understanding of the biology and biodiversity of phages infecting various bacterial species across different environments remains limited. Upon infection, a bacteriophage can rely on host enzymes, including DNA polymerase, to replicate its own genome. Nevertheless, numerous phages encode their own DNA polymerases, likely conferring an adaptive advantage

[0019] , To date, DNA polymerases isolated from phages have been shown to exhibit unusual structural features

[0020] and intriguing biochemical properties, such as the ability to initiate DNA replication without any primer

[0021] or to incorporate noncanonical or synthetic nucleotides [22, 23],

[0010] Extensive studies on the morphological, genetic, and functional diversity of bacteriophages isolated from municipal wastewater have led to the identification of several previously undescribed phages encoding their own DNA polymerases

[0024] , One such is vB_EfaS-271, characterized by a short latency period of approximately 8 minutes. This suggests that its DNA polymerase (hereinafter referred to as the Efa polymerase) is capable of replicating the 40197 base pair phage genome with high efficiency and speed

[0025] , The inventors’ research on the Efa polymerase resulted in sequencing of its gene, cloning into an expression vector, and subsequent overproduction and purification of the functional enzyme with high yield. The invention disclosed herein may provide a response to the ongoing need for novel DNA-amplifying enzymes efficiently functioning under isothermal conditions within the temperature range beyond previously known DNA polymerases.

[0011] The objective of the invention is to obtain an enzyme that remains active within a broad low- temperature range, including ambient or room temperature conditions and temperatures below 10°C. The proposed solution allows the enzymatic reaction to proceed independently of laboratory equipment maintaining a constant temperature, thereby permitting its use in situ, under natural environmental conditions.

[0012] The invention provides an N-terminal fragment of a DNA polymerase having the amino acid sequence set forth in SEQ ID NO: 7, or variants thereof having at least 90% sequence identity to said sequence.

[0013] In another aspect, the invention relates to a nucleic acid sequence set forth in SEQ ID NO: 8 encoding the N-terminal fragment of the DNA polymerase of SEQ ID NO: 7.

[0014] In a further aspect, the invention provides an expression vector pET28-Efa having the sequence set forth in SEQ ID NO: 9, which enables the expression and overproduction of the N- terminal fragment of the DNA polymerase of SEQ ID NO: 7.

[0015] The invention also relates to a DNA polymerase including N-terminal fragment of SEQ ID NO: 7 as an integral part of an enzyme, or variants thereof having at least 90% sequence identity to said sequence. Preferably, the DNA polymerase has the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least 90% sequence identity to said sequence. Equally preferably, the DNA polymerase has the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least 90% sequence identity to said sequence.

[0016] In another aspect, the invention relates to a nucleic acid sequence set forth in SEQ ID NO: 5 encoding the DNA polymerase of SEQ ID NO: 4, or a nucleic acid sequence set forth in SEQ ID NO: 2 encoding the DNA polymerase of SEQ ID NO: 1. In yet another aspect, the invention provides an expression vector set forth in SEQ ID NO: 6. comprising nucleic acid sequence encoding a fusion DNA polymerase with the amino acid sequence of SEQ ID NO: 4, or an expression vector set forth in SEQ ID NO: 3, comprising nucleic acid sequence encoding a fusion DNA polymerase with the amino acid sequence of SEQ ID NO: 1.

[0017] Another aspect of the invention comprises a method for producing the DNA polymerase having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 1, or the N-terminal fragment thereof having the amino acid sequence of SEQ ID NO: 7, characterized by the following steps: a) E. coli Rosetta (pLysS) cells are transformed with the expression vector pET28-Efa, specifically of SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9; b) expression of the Efa polymerase gene is induced in a bacterial culture at the late logarithmic growth phase (OD6oo~0.6-0.8) with 0.5 mM isopropyl-P-d-1- thiogalactopiranoside (IPTG); c) protein overproduction is carried out at 16°C for 16-20 hours, followed by centrifugation of the bacterial culture and freezing of the pellet in liquid nitrogen; d) the thawed bacterial pellet is resuspended in buffer A supplemented with a protease inhibitor cocktail consisting of aprotinin, bestatin, E-64 epoxide, leupeptin, 4-(2- aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), and pepstatin A, followed by sonication, lysate centrifugation, and collection of the soluble fraction; e) the fusion Efa polymerase with an N-terminal His-SUMO tag is isolated from the soluble fraction (supernatant) by metal affinity chromatography on a nickel -charged Ni-NTA resin equilibrated with buffer A, contaminants are washed out with buffer B, and the polymerase is eluted from the resin using buffer C; f) proteolysis of the His-SUMO tag is carried out using Ulp 1 protease, occurring in parallel with dialysis of the Efa polymerase into buffer C for 18 hours at 4°C; g) the His-SUMO tag and Ulp 1 protease are separated from the Efa polymerase by a second round of metal affinity chromatography on the Ni-NTA resin equilibrated with buffer A, followed by washing with buffer B, and elution using buffer C; h) further assessment of the presence and purity of the Efa polymerase is performed using size exclusion chromatography (gel filtration) on a column with a cross-linked agarose resin with a particle size of 20-40 um and a fractionation range of 1-300 kDa equilibrated with buffer C to remove insoluble high-molecular weight aggregates and low-molecular weight impurities; i) the presence of purified Efa polymerase in the individual fractions is confirmed, and the final preparation is dialysed into buffer D, frozen in liquid nitrogen and stored at -80°C, wherein obtained Efa polymerase retains polymerization activity enabling isothermal amplification of DNA at temperatures from -10°C to +20°C.

[0018] Preferably, the method according to the invention employs buffer A comprising 50 mM Tris- HC1 (pH 7.5), 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 15 mM imidazole; buffer B comprising 50 mM Tris-HCl (pH 7.5), 1 M NaCl, 5% glycerol, 0.5 mM TCEP, and 15 mM imidazole; buffer C comprising 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 500 mM imidazole; and buffer D comprising 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 40% glycerol, 0.5 mM TCEP, 1 mM EDTA.

[0019] In another aspect, the invention also encompasses the use of Efa polymerase for isothermal DNA amplification at temperatures ranging from -10°C to +20°C, preferably from -10°C to +10°C, and more preferably from -10°C to 0°C.

[0020] In a further aspect, the invention provides the use of the N-terminal fragment of a DNA polymerase having the amino acid sequence of SEQ ID NO: 7, or variants thereof having at least 90% sequence identity, in the generation of hybrid DNA polymerases through translational fusion of said fragment.

[0021] Comparative analysis of Efa polymerase sequence with other available DNA polymerases revealed only less than 20% identity at the amino acid level, as described in further detail in the Examples. Efa DNA polymerase exhibits 5'-3' polymerase and 3'-5' exonuclease activities (so- called proofreading activity) in a wide range of reaction buffers containing divalent Mg2+ions. It is characterized by the ability to efficiently amplify DNA while maintaining enzyme stability in the temperature range of -10°C to 50°C. Its high activity at room temperature enables DNA amplification without the use of equipment maintaining a constant reaction temperature (the characteristics of Efa polymerase are described in more detail in the Examples and in the accompanying figures).

[0022] The invention also includes the nucleotide sequence of the pET28-Efa expression vector of SEQ ID NO: 3 containing the Efa DNA polymerase gene, as well as the nucleotide sequence of the pET28-Efa R79A expression vector (SEQ ID NO: 6) containing the Efa DNA polymerase mutant gene, and the nucleotide sequence of the pET28-Nter expression vector (SEQ ID NO: 9) containing the sequence of the N-terminal domain of the Efa DNA polymerase, and enabling their expression in the T7 system and overproduction of the respective proteins in E. coli cells. The expression vectors are characterized in that the polymerase gene or its variant is N-terminally fused with the His-SUMO-encoding sequence; furthermore, the vector sequences comprise the T7 bacteriophage promoter and terminator, the lad gene, the pBR322 and fl origins of replication, and the kanamycin resistance gene. Importantly, only the use of this specific construct enabled obtaining Efa polymerase in a soluble, enzymatically active form and in amounts sufficient for its purification and practical use, thereby demonstrating the inventive character of the solution.

[0023] The Efa DNA polymerase, its R79A mutant variant, and the isolated N-terminal domain are stable, and their production process is highly efficient, eliminating the need to maintain low temperatures during purification.

[0024] A characteristic feature of Efa polymerase and its mutant is the ability to extend DNA strands in the 5'-3' direction in linear primer-template DNA hybrids. This activity occurs within the temperature range examined to date, from -10 to 50°C. Efa polymerase and its mutant efficiently amplify products of approximately 40 base pairs in length within about 5 minutes (as described in more detail in Example 3). Furthermore, the Efa DNA polymerase and its mutant also possess 3 '-5' exonuclease activity, which becomes apparent in the absence of deoxyribonucleotides (DNA building blocks) in the reaction, as shown in Example 3. The 5'-3' polymerase activity of Efa DNA polymerase occurs efficiently even at -10°C (Example 4), whereas other commercially available DNA polymerases as well as the Efa polymerase variant lacking the N-terminal domain (Efa AN) show no such activity or exhibit it to a negligible extent, as demonstrated in Example 5.

[0025] The ability to perform DNA amplification reactions without specialized equipment, combined with the highly efficient and straightforward process of obtaining Efa DNA polymerase and its mutant, are factors that reduce industrial energy consumption and overall production costs.

[0026] The subject matter of the invention is further illustrated in the examples of embodiment and in the accompanying figures: fig. 1 shows maps of pET28-Efa (WT), pET28-Efa R79A (mutant), and pET28-Nter (N- terminal domain) plasmid vectors, indicating the respective gene sequences (Efa, Efa R79A, or N-terminal domain), the His-SUMO tag sequence, and the His-SUMO-protein gene fusion sequences; fig. 2 shows purification of Efa (WT) DNA polymerase (SEQ ID NO: 1), Efa R79A mutant (SEQ ID NO: 4), and Efa N-terminal domain (SEQ ID NO: 7. (A) Analytical size exclusion chromatography profiles (Superdex200 10 / 300 GL) obtained for the individual variants overlaid with molecular weight standards. (B) Coomassie blue-stained 10% SDS-PAGE analysis of 1 pg protein samples after purification of each variant. The corresponding molecular weight (kDa) standards are indicated with the letter M; fig. 3 shows the circular dichroism (CD) spectrum (A) from 197 nm to 260 nm and (B) the thermal CD stability of the native Efa DNA polymerase variant (SEQ ID NO: 1) at 220 nm; fig. 4 shows biochemical activity assays of Efa polymerase variants at 30°C. (A) Representative 5 '-3' polymerase (left) and 3 '-5' exonuclease (right) activity profiles of the native Efa polymerase variant (SEQ ID NO: 1). 50 nM primertemplate (13-mer: 40-mer) DNA substrate was incubated in the presence of 10 mM Mg2+with increasing concentrations of Efa polymerase (0.39; 0.78; 1.56; 3.125; 6.25; 12.5; 25; 50 or 100 nM) in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP and 5% glycerol, in the presence of 1 mM dNTPs (left) and without dNTPs (right). No enzyme was added to controls (K). The reactions were stopped after 5 min and analyzed after electrophoresis in a 20% polyacrylamide gel under denaturing conditions with 8 M urea. The corresponding molecular weight (nt) standards are marked with the letter M; (B) Densitometric analysis of substrate consumption as a function of protein concentration for Efa polymerase variants: native (SEQ ID NO: 1), Efa R79A (SEQ ID NO: 4), and the N-terminal domain-deficient variant (control). The graph shows mean values with standard deviations (error bars) from three independent experiments; fig. 5 shows the temperature -dependent 5'-3' polymerase activity of Efa polymerase. (A) Representative profile of the temperature -dependent 5'-3' polymerase activity of the native Efa polymerase variant (SEQ ID NO: 1). 50 nM primertemplate DNA substrate (13-mer:40-mer) was incubated in the presence of 10 mM of 50 nM Efa DNA polymerase in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP, and 5% glycerol in the presence of 1 mM dNTPs. No enzyme was added to the control sample (K). Reactions were performed at temperatures of -5, 0, 5, 10, 15, 20, 25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 65, 70°C, stopped after 5 minutes, and analyzed after electrophoretic separation of the reaction products in a 20% polyacrylamide gel under denaturing conditions with 8 M urea. The corresponding molecular weight (nt) standards are marked with the letter M; (B) Densitometric analysis of substrate consumption in (A). The graph shows the mean values with standard deviations (error bars) from three independent experiments; fig. 6 shows biochemical activity assays of Efa polymerase variants at -10°C. (A) Representative 5'— 3' polymerase activity profile of a native Efa polymerase variant (SEQ ID NO: 1). 50 nM primertemplate DNA substrate (13-mer:40-mer) was incubated in the presence of 10 mM Mg2+with increasing concentrations of Efa polymerase (0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 nM) in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP, and 40% glycerol, in the presence of 1 mM dNTPs. No enzyme was added to the control (K). The reaction was stopped after 30 minutes and analyzed after electrophoretic separation of the reaction products in a 20% polyacrylamide gel under denaturing conditions with 8 M urea. The corresponding molecular weight (nt) standards are marked with the letter M; (B) Densitometric analysis of substrate consumption as a function of protein concentration for Efa polymerase variants: native (SEQ ID NO: 1), Efa R79A (SEQ ID NO: 4), and the N-terminal domaindeficient variant (control). The graph shows mean values with standard deviations (error bars) from three independent experiments; fig. 7 shows comparative biochemical time-course assays of the activities of various DNA polymerases at 30°C; (A) 5'— 3' polymerase activity assay. 50 nM primertemplate (13- mer: 40-mer) DNA substrate was incubated in the presence of 10 mM Mg2+with 1 nM of the following polymerases: Efa (WT; SEQ ID NO: 1), Efa R79A (SEQ ID NO: 4), Efa AN (SEQ ID NO: 12), T4, T7, cp29 or Pol I in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP and 40% glycerol, in the presence of 1 mM dNTPs. No enzyme was added to controls (K). Reactions were stopped at the indicated time points (1, 5, 10, 15 min) and analyzed after electrophoretic separation of the reaction products in a 20% polyacrylamide gel under denaturing conditions with 8 M urea. The corresponding molecular weight (nt) standards are marked with the letter M; (B) Densitometric analysis of the final product growth in (A). The graph shows mean values with standard deviations (error bars) from three independent experiments; fig. 8 shows comparative biochemical time-course assays of the activity of various DNA polymerases at -10°C; (A) 5'— 3' polymerase activity assay. 50 nM primertemplate (13- mer: 40-mer) DNA substrate was incubated in the presence of 10 mM Mg2+with 1 nM of the following polymerases: Efa (WT; SEQ ID NO: 1), Efa R79A (SEQ ID NO: 4), Efa AN (SEQ ID NO: 12), T4, T7, <p29 or Pol I in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP and 40% glycerol, in the presence of 1 mM dNTPs. No enzyme was added to controls (K). Reactions were stopped at the indicated time points (1, 5, 10, 15 min) and analyzed after electrophoretic separation of the reaction products in a 20% polyacrylamide gel under denaturing conditions with 8 M urea. The corresponding molecular weight (nt) standards are marked with the letter M; (B) Densitometric analysis of the growth of the final product in (A). The graph shows mean values with standard deviations (error bars) from three independent experiments;

[0027] Example 1. Efa DNA polymerase gene sequence, amino acid sequence analysis, and expression vector construction.

[0028] The gene encoding a potential family B DNA polymerase (SEQ ID NO: 2) was identified during functional annotation of the bacteriophage vB_EfaS-271 genome

[0025] , It consists of 2292 base pairs and encodes a 762-amino acid protein (SEQ ID NO: 1) with a theoretical molecular mass of 87.4 kDa and an isoelectric point of 5.92.

[0029] The inventors cloned the gene into the pET28 plasmid vector, enabling expression of the gene in the bacterial T7 system and overproduction of a fusion protein with an N-terminal fusion of 6xHis tag and SUMO, the sequence of which is recognized by a specific protease. The choice of plasmid vector was based on its easy replication in E. coli cells, easy selection of plasmid-bearing cells thanks to the kanamycin resistance marker, the simple and efficient overexpression of the target gene (here, the gene encoding Efa polymerase) in the T7 system, and the possibility to obtain native protein after tag removal during purification.

[0030] An analogous strategy was used to construct expression vectors containing: (i) the gene encoding the R79A variant of the Efa polymerase mutant (SEQ ID NO: 5) - vector pET28-Efa R79A (SEQ ID NO: 6), and (ii) the sequence encoding the N-terminal domain - vector pET28- Nter (SEQ ID NO: 9). Both constructs were designed to produce the protein in an N-terminal fusion with the His-SUMO tag, which ensured solubility and enabled efficient protein purification. Cloning was performed using a restriction enzyme -independent method based on two consecutive PCR reactions

[0026] . In the first reaction, the insert was amplified using modular primers containing 5'-terminal overhangs complementary to the insertion site in the pET28 vector. The primer sequences are presented in the sequence list and are designated as follows:

[0031] Efa RF F: SEQ ID NO: 10

[0032] Efa RF R: SEQ ID NO: 11

[0033] The product of the first PCR reaction was used in the second reaction as a so-called megaprimer to amplify the entire target construct. The template plasmid DNA was degraded using the Dpnl enzyme, and the reaction product (target vector) was transformed into E. coli DH5a cells. After clone selection and isolation of the target vectors pET28-Efa (SEQ ID NO: 3), pET28- Efa R79A (SEQ ID NO: 6), and pET28-Nter (SEQ ID NO: 9), their corectness was verified by Sanger sequencing. Maps (schematic representations) of the obtained constructs are shown in Fig. 1.

[0034] Example 2, Method for obtaining Efa polymerase and thermal stability of the enzyme

[0035] The first step in obtaining Efa DNA polymerase (SEQ ID NO: 1), its mutant Efa R79A (SEQ ID NO: 4), and the N-terminal domain (Efa_N) (SEQ ID NO: 7) involves overproduction of the protein in the bacterial T7 system

[0027] , E. coli Rosetta(DE3) pLysS cells were used for overproduction - this is an expression strain containing the T7 phage RNA polymerase gene (the enzyme that transcribes the target gene) under the control of a lactose promoter. The inducer of T7 RNA polymerase gene expression, and consequently of target gene expression and overproduction of the tested protein, is the lactose analog isopropyl [3-d- 1 -thiogalactopyranoside (IPTG). E. coli Rosetta(DE3) pLysS cells harboring the plasmid vector pET28-Efa (SEQ ID NO: 3), pET28-Efa R79A (SEQ ID NO: 6) or pET28-Nter (SEQ ID NO: 9) were grown in LB medium supplemented with kanamycin (50 pg / ml) and chloramphenicol (34 pg / ml) to ODeoo ~ 0.6-0.8, after which IPTG was added to a final concentration of 0.5 mM. Protein overproduction was carried out at 16°C for approximately 16-20 h. Bacterial cells were then centrifuged (4000 rpm, 10 min, 4°C), and the pellet was frozen in liquid nitrogen. The thawed bacterial cell pellet was resuspended in buffer A (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, 15 mM imidazole) supplemented with protease inhibitors cocktail (Thermo Scientific, cat. no. A32965; chemical composition: aprotinin, bestatin, epoxide E-64, leupeptin, 4-benzenesulfonyl fluoride hydrochloride (AEBSF), pepstatin A), using 10 ml of buffer for every 5 g of cell pellet. Cells were lysed by sonication, and the lysate was centrifuged (18,000 rpm, 45 min, 4°C) to separate the insoluble fraction. The soluble fraction (supernatant) containing the His-SUMO-Efa WT, His-SUMO-Efa R79A, or His-SUMO-Efa_N fusion proteins was applied to a 5 ml Ni-NTA column equilibrated with buffer A, and the column was washed with 5 volumes (25 ml) of buffer B (50 mM Tris-HCl pH 7.5, 1 M NaCl, 5% glycerol, 0.5 mM TCEP, 15 mM imidazole). The protein was eluted from the resin with 3-4 volumes (15-20 ml) of buffer C (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, 500 mM imidazole). The presence of Efa DNA polymerase variants in individual fractions was assessed by SDS-PAGE electrophoresis. Proteincontaining fractions were pooled, and Ulpl protease was added to a final concentration of 0.05 mg / ml. The mixture was transferred to a dialysis bag, which was immersed in 1-2 liters of buffer C (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, 500 mM imidazole) and incubated for 18 h at 4°C. After the dialysis step, the Efa, Efa R79A, or Efa_N DNA polymerase preparation was reapplied to a 5 ml Ni-NTA column equilibrated in buffer A to separate the cleaved tag and Ulpl protease from native Efa, EfaR79A, or Efa_N polymerase. The flowthrough fractions containing Efa variants were collected, the column washed, and eluted as before. The presence of Efa DNA polymerase variants in individual fractions was again assessed by SDS- PAGE electrophoresis. Protein-containing fractions were pooled and concentrated to approximately 1 ml using centrifugal concentrators. The concentrated protein was applied to a Superose 12 10 / 300 GL size exclusion chromatography column equilibrated with buffer C and eluted with 1.2 column volumes of buffer C at 0.25 ml / min (AKTA Purifier system), collecting 0.5 ml fractions. Protein presence in individual fractions was assessed by chromatogram analysis (absorbance at 280 nm). Protein -containing fractions were combined and dialyzed into buffer D (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 40% glycerol, 0.5 mM TCEP, 1 mM EDTA). The final preparation of Efa, Efa R79A or Efa_N DNA polymerase was divided into 10 pl aliquots, frozen in liquid nitrogen and stored at -80°C. Protein size and purity analysis of the preparation was performed using analytical size exclusion chromatography on a Superdex200 10 / 300 GL column in SEC buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, 1 mM EDTA). Furthermore, the homogeneity of the preparations was confirmed by SDS-PAGE electrophoresis (Fig . 2) . Proper folding and thermal stability of the enzyme were determined using circular dichroism (Fig. 3). The melting point of the native variant of Efa DNA polymerase was shown to be 40.76°C. Because the N-terminal domain of Efa lacks catalytic activity when separated from the polymerase core, a control Efa variant lacking the N-terminal domain, consisting only of the enzyme core, was constructed (hereinafter designated Efa AN, SEQ ID NO: 12). This variant was purified in a similar manner. It will allow for the assessment of the effect of the N-terminal domain on the enzyme's catalytic activity under various conditions.

[0036] Example 3, 5 '-3' polymerase and 3 '-5' exonuclease activity of Efa polymerase

[0037] To investigate the activity of Efa DNA polymerase (SEQ ID NO: 1), its R79A mutant (SEQ ID NO: 4), and a variant lacking the N-terminal domain (Efa AN; SEQ ID NO: 12), primertemplate DNA duplex extension reactions were performed using fluorescently labelled substrates. In this case, a 13-mer fluorescein-labeled primer was hybridized to a 40-mer template. Due to the 5 '-3' polymerase activity of the tested DNA polymerase, the primer is extended in the presence of divalent magnesium ions (Mg2+) and deoxyribonucleotides (dNTPs), which can be observed by electrophoresis of the obtained reaction products in a 20% polyacrylamide gel under denaturing conditions with 8M urea (UREA -PAGE) as a longer DNA band. Simultaneously, in the absence of dNTPs in the reaction, DNA polymerases characterized by 3'-5' exonuclease activity degrade the DNA duplex from the 3' end, which can also be observed by UREA -PAGE as shorter DNA bands.

[0038] Reactions for the Efa polymerase variants were performed under isothermal conditions, allowing DNA amplification at a constant temperature of 30°C. 50 nM DNA substrate was incubated with 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 nM Efa DNA polymerase in the presence of 10 mM Mg2+for 5 minutes, in two variants - with the addition of 1 mM dNTPs (favoring 5'-3' polymerase activity) and without dNTPs (favoring 3'-5' exonuclease activity). The reaction was carried out in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP, and 5% glycerol. After 5 minutes, the reaction was stopped by the addition of 1 volume of STOP buffer (86% formamide in IxTBE), followed by UREA -PAGE electrophoresis. Based on the results obtained from electrophoretic separation (Fig. 4A, left) as well as densitometric analysis of substrate consumption (Fig. 4B, left), it is noted that Efa WT polymerase, its R79A mutant, and the variant lacking the N-terminal domain (Efa AN) are characterized by efficient 5'- 3' polymerase activity - at an enzyme: substrate molar ratio of 1 :2, all substrate was converted into the final product within 5 minutes of reaction (at lower enzyme: substrate molar ratios, intermediate products are visible). This proves that Efa polymerase and its variants are capable of efficient DNA amplification in vitro at constant temperature. In the absence of dNTPs, 3'-5' exonuclease activity was observed in the reaction (Fig. 4A, right), with even greater efficiency - the entire substrate was consumed at an enzyme: substrate molar ratio of 1:4 (Fig. 4B, right).

[0039] Next, an experiment was performed to test the temperature dependence of the 5'-3' polymerase activity of the native Efa polymerase variant. 50 nM DNA substrate was incubated with 50 nM Efa DNA polymerase in the presence of 10 mM Mg2+for 5 minutes, with the addition of 1 mM dNTPs. The reaction was carried out in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP, and 5% glycerol. After 5 minutes, the reaction was stopped by adding 1 volume of STOP buffer (86% formamide in IxTBE), and UREA-PAGE electrophoresis was performed. Based on the results obtained from electrophoretic separation (Fig. 5A) and densitometric analysis of substrate consumption (Fig. 5B), a wide temperature range of Efa polymerase activity is observed, ranging from -5 °C to 55 °C. No products are formed at temperatures above 55 °C. Therefore, the Efa polymerase efficiently performs isothermal DNA amplification even at low temperatures.

[0040] Example 4, 5 '-3' polymerase activity of Efa polymerase at -10°C

[0041] Due to the high efficiency of Efa polymerase in primer extension at -5 °C, an experiment was performed to test the 5 '-3' polymerase activity of the enzyme at -10°C for three variants: WT Efa polymerase (SEQ ID NO: 1), its R79A mutant (SEQ ID NO: 4), and a variant lacking the N- terminal domain (AN; SEQ ID NO: 12). 50 nM DNA substrate was incubated with 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 nM Efa in the presence of 10 mM Mg2+for 30 minutes with the addition of 1 mM dNTPs . The reaction was carried out in a buffer consisting of 20 mM HEPES pH 7.5, 140 mM KC1, 1 mM TCEP, and 40% glycerol. After 30 min, the reaction was stopped by adding 1 volume of STOP buffer (86% formamide in IxTBE), followed by UREA-PAGE electrophoresis. Based on the obtained results (Fig. 6A) and densitometric analysis (Fig. 6B), it was determined that the Efa WT polymerase and the R79A mutant exhibit polymerase activity at -10 °C, with the R79A mutant having a higher reaction efficiency than the wild-type enzyme. The variant lacking the N-terminal domain (AN) did not show significant activity at this temperature, indicating that the N-terminal domain plays a key role in the adaptation of the polymerase to extremely low temperatures.

[0042] Example 5 , 5 '-3 ' polymerase activity of Efa polymerase compared to other commercially available DNA polymerases

[0043] To evaluate the 5'-3' polymerase activity of Efa polymerase compared to other disclosed and commercially available DNA polymerases, comparative biochemical studies were conducted with Efa WT polymerase (SEQ ID NO: 1), the Efa R79A mutant (SEQ ID NO: 4), and the N-terminal domain-deficient (AN) variant of SEQ ID NO: 12, as well as commercially available polymerases: T4, T7, q>29, and Pol I. The primer extension reaction (50 nM DNA substrate) in the presence of 1 mM dNTPs was performed using 1 nM enzyme (molar ratio of enzyme to substrate 1:50) under the conditions described above. Isothermal DNA amplification was performed at 30°C (Fig. 7) and -10°C (Fig. 8). The reaction was stopped at four time points - after 1, 5, 10, and 15 minutes. At 30°C, the Efa WT polymerase and the R79A mutant were characterized by higher activity than three of the four commercial polymerases tested. The R79A mutant showed the highest efficiency in this range, while the AN variant was characterized by reduced polymerase activity. The cp 29 polymerase had the lowest activity, while the T4 polymerase had the highest activity (Fig. 7B). At -10°C, the Efa WT polymerase and the R79A mutant were able to convert the substrate to the final reaction product, with the R79A mutant showing significantly higher activity than the wildtype enzyme. The remaining commercial polymerases tested (T4, T7, q>29, Pol I) showed only residual or no activity at this temperature. The AN variant showed no measurable polymerase activity at -10°C, and therefore Efa polymerase replicates DNA at low temperatures only when the N-terminal domain is an integral part of it. This confirms that the N-terminal domain (SEQ ID NO: 7) is crucial for enzyme activity at extremely low temperatures (Fig. 8B).

[0044] The results indicate that translational fusion of the N-terminal domain with any DNA polymerase confers low -temperature activity to the fusion enzyme. The hybrid DNA polymerases constructed in this manner retain their intrinsic properties, while gaining expanded temperature range of activity due to the translational fusion with the Efa N-terminal domain (SEQ ID NO: 7).

[0045] Literature:

[0046] 1. Hillers, K.J. et al.. Meiosis. WormBook 2017, 1-20.

[0047] 2. Moreno-Andres, D.; Holl, K.; Antonin, W. The second half of mitosis and its implications in cancer biology. Semin. Cancer Biol. 2023, 88, 1-17.

[0048] 3. Garcia-Diaz, M.; Bebenek, K. Multiple functions of DNA polymerases. CRC. Crit. Rev. Plant Sci. 2007, 26, 105-122.

[0049] 4. Kubista, M. et al. The real-time polymerase chain reaction. Mol. Aspects Med. 2006, 27, 95-125.

[0050] 5. Oliveira, B.B. et al.. Isothermal Amplification of Nucleic Acids: The Race for the Next “Gold Standard.” Front. Sensors 2021, 2, 1-22.

[0051] 6. Zhao, Y. et al. Isothermal Amplification of Nucleic Acids. Chem. Rev. 2015, 115, 12491-12545.

[0052] 7. Salas, M.; Martin, G.; Bernard, A.; Garmendia, C.; Lazaro, J.M.; Zaballos, A.; Serrano, M.; Otero, M.J.; Gutierrez, J.; Pares, E.; etal. Protein-primed replication of bacteriophage phi29. 1988, 951, 419- 424.

[0053] 8. Kroneis, T.; El-Heliebi, A. Whole Genome Amplification by Isothermal Multiple Strand Displacement Using Phi29 DNA Polymerase. Methods Mol. Biol. 2015, 1347, 111-117,.

[0054] 9. Wang, M.; Liu, H.; Ren, J.; Huang, Y.; Deng, Y.; Liu, Y.; Chen, Z.; Chow, F.W.-N.; Leung, P.H.-M.; Li, S. Enzyme-Assisted Nucleic Acid Amplification in Molecular Diagnosis: A Review. Biosensors 2023, 13, 160, doi:10.3390 / biosl3020160. Tan, M. et al. Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications. Front. Cell. Infect. Microbiol. 2022, 12, 1-13, doi:10.3389 / fcimb.2022.1019071. Gavrilov, M. et al. Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics. Nat. Commun. 2022, 13, 1-14, doi:10.1038 / s41467 -022-34076-0. Boonbanjong, P et al. Isothermal Amplification Technology for Disease Diagnosis. Biosensors 2022, 12, doi:10.3390 / biosl2090677. Xue, Y. et al . Temperature effect on polymerase fidelity. J. Biol. Chem. 2021, 297 (5):101270. Margesin, R.; Feller, G. Biotechnological applications of psychrophiles. Environ. Technol. 2010, 31, 835-844. Saleem Farooq, R.N. and B.U. Psychrophiles: Their habitat and applications. J. Himal. Ecol. Sustain. Dev. 2016, 11, 27-36. Feller, G.; Gerday, C. Psychrophilic enzymes: Hot topics in cold adaptation. Nat. Rev. Microbiol. 2003, 1, 200-208. Santos SB et al. Exploiting Bacteriophage Proteomes: The Hidden Biotechnological Potential. Trends Biotechnol. 2018 Sep;36(9):966-984. Weitz JS et al. Phage-bacteria infection networks. Trends Microbiol. 2013 Feb;21(2):82-91. Morcinek-Orlowska J et al. Bacteriophage-Encoded DNA Polymerases-Beyond the Traditional View of Polymerase Activities. Int J Mol Sci. 2022 Jan 7;23(2):635. Guo H et al . Crystal structure and biochemical studies of the bifunctional DNA primase -polymerase from phage NrS-1. Biochem Biophys Res Commun. 2019 Mar 19;510(4):573-579. Zhu B, et al . Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers. Proc Natl Acad Sci U S A. 2017 Mar 21;114(12):E2310-E2318. Pezo V et al. Noncanonical DNA polymerization by aminoadenine -based siphoviruses. Science. 2021 Apr 30;372(6541):520-524. Pinheiro VB, Holliger P. Towards XNA nanotechnology: new materials from synthetic genetic polymers. Trends Biotechnol. 2014 Jun;32(6):321-8. Jurczak-Kurek A et al. Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage. Sci Rep. 2016 Oct 4;6:34338. Topka-Bielecka G et al. Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis. Int J Mol Sci. 2020 Sep 1;21(17):6345. van den Ent F, Lowe J. RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods. 2006 Apr 30;67(l):67-74. Studier FW. T7 Expression Systems for Inducible Production of Proteins from Cloned Genes in E. coli. CurrProtoc Mol Biol. 2018 Oct;124(l):e63.

Claims

Patent claims1. N-terminal fragment of a DNA polymerase having the amino acid sequence of SEQ ID NO: 7, or variants thereof having at least 90% sequence identity to said sequence.

2. Nucleic acid sequence set forth in SEQ ID NO: 8 encoding the N-terminal fragment of the DNA polymerase according to claim 1.

3. Expression vector pET28-Efa having the sequence of SEQ ID NO: 9, which enables the expression and overproduction of the N-terminal fragment of the DNA polymerase according to claim 1.

4. A DNA polymerase including N-terminal fragment having the sequence as defined in claim 1.

5. The DNA polymerase according to claim 4, characterized in that it constitutes the SEQ ID NO: 4 or a variant having at least 90% sequence identity thereto.

6. The DNA polymerase according to claim 4, characterized in that it constitutes the SEQ ID NO: 1 or a variant having at least 90% sequence identity thereto.

7. Nucleic acid sequence of SEQ ID NO: 5 encoding the DNA polymerase as defined in claim 5.

8. Nucleic acid sequence of SEQ ID NO: 2 encoding the DNA polymerase as defined in claim 6.

9. An expression vector of SEQ ID NO: 6 comprising the nucleotide sequence encoding the fusion DNA polymerase as defined in claim 5.

10. An expression vector of SEQ ID NO: 3 comprising the nucleotide sequence encoding the fusion DNA polymerase as defined in claim 6.

11. A method for obtaining the DNA polymerase having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 1, or the N-terminal fragment thereof having the amino acid sequence of SEQ ID NO: 7, characterized in that it comprises the following steps: a) E. coli Rosetta (pLysS) cells are transformed with the expression vector pET28-Efa, specifically of SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9; b) expression of the Efa polymerase gene is induced in a bacterial culture at the late logarithmic growth phase (OD6oo~0.6-0.8) with 0.5 mM isopropyl-P-d-1- thiogalactopiranoside (IPTG); c) protein overproduction is carried out at 16°C for 16-20 hours, followed by centrifugation of the bacterial culture and freezing of the pellet in liquid nitrogen; d) the thawed bacterial pellet is resuspended in buffer A supplemented with a protease inhibitor cocktail consisting of aprotinin, bestatin, E-64 epoxide, leupeptin, 4-(2- aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), and pepstatin A, followed by sonication, lysate centrifugation, and collection of the soluble fraction;e) the fusion Efa polymerase with an N-terminal His-SUMO tag is isolated from the soluble fraction (supernatant) by metal affinity chromatography on a nickel -charged Ni-NTA resin equilibrated with buffer A, contaminants are washed out with buffer B, and the polymerase is eluted from the resin using buffer C; f) proteolysis of the His-SUMO tag is carried out using Ulp 1 protease, occurring in parallel with dialysis of the Efa polymerase into buffer C for 18 hours at 4°C; g) the His-SUMO tag and Ulp 1 protease are separated from the Efa polymerase by a second round of metal affinity chromatography on the Ni-NTA resin equilibrated with buffer A, followed by washing with buffer B, and elution using buffer C; h) further assessment of the presence and purity of the Efa polymerase is performed using size exclusion chromatography (gel filtration) on a column with a cross-linked agarose resin with a particle size of 20-40 um and a fractionation range of 1-300 kDa equilibrated with buffer C to remove insoluble high-molecular weight aggregates and low-molecular weight impurities; i) the presence of purified Efa polymerase in the individual fractions is confirmed, and the final preparation is dialysed into buffer D, frozen in liquid nitrogen and stored at -80°C, wherein obtained Efa polymerase retains polymerization activity enabling isothermal amplification of DNA at temperatures from -10°C to +20°C.

12. The method according to claim 11 characterized in that it employs buffer A comprising 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 15 mM imidazole; buffer B comprising 50 mM Tris-HCl (pH 7.5), 1 M NaCl, 5% glycerol, 0.5 mM TCEP, and 15 mM imidazole; buffer C comprising 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 500 mM imidazole, and buffer D comprising 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 40% glycerol, 0.5 mM TCEP, 1 mM EDTA.

13. Use of the DNA polymerase according to any of claims 4 to 6 for isothermal DNA amplification at temperatures from -10°C to +20°C, preferably from -10°C to +10°C, and more preferably from -10°C to 0°C.

14. Use of the N-terminal fragment of the DNA polymerase according to claim 1 for producing DNA polymerases by performing translational fusions with said sequence.

Images