Amplification of nucleic acid

Abstract

To provide alternative methods and reagents for amplifying target nucleic acids.SOLUTION: A method of amplifying a target nucleic acid in a DNA sample, comprises: (a) contacting a DNA sample containing the target nucleic acid with a DNA polymerase, at least two oligonucleotide primers designed to flank the target nucleic acid, a mixture of dATP, dGTP, dCTP, and dTTP, and a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer; (b) heating an output of the step (a) to a temperature at which the conjugate precipitates and thereby the DNA polymerase is no longer inhibited; and (c) amplifying the target nucleic acid by performing PCR steps of denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers, wherein the step (c) is repeated at least two times. Also disclosed are a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, a kit comprising the conjugate, and an aqueous composition comprising the conjugate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (introduction) The present invention relates to a conjugate for use in nucleic acid amplification, a composition comprising a DNA polymerase bound to the conjugate, a kit comprising a DNA polymerase and the conjugate, and a method for amplifying a target nucleic acid in a DNA sample using the conjugate. [Background technology]

[0002] BACKGROUND OF THE INVENTION Smart polymers, also known as phase-change polymers or stimuli-responsive polymers, are polymers that respond to their environment by changing their physical and / or chemical properties. Smart polymers respond to various environmental factors, including temperature, humidity, pH, chemical compounds, wavelength or intensity of light, and electric or magnetic fields, and can respond in various ways, such as by changing their shape, color, or solubility state. At key points, smart polymers can exhibit discontinuous responses to small changes in their environment, such that small changes in the environment are sufficient to cause large changes in the physical and / or chemical properties of the smart polymer.

[0003] In recent years, smart polymers have attracted great interest due to their use in a variety of applications, such as smart biocatalysts conjugated to enzymes and biopharmaceuticals for specifically targeted drug delivery.

[0004] Another application of smart polymers is in the field of polymerase chain reaction (PCR). In September 2015, Chen et al. reported in an ACS publication ("Improvement in the Thermal Stability of Pyrophosphatase by Conjugation to Poly(N-isopropylacrylamide): Application to the Polymerase Chain Reaction") that they conjugated the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) to pyrophosphatase (PPase) in an attempt to improve the enzyme's thermostability and thereby maintain its activity at high temperatures, such as those used in the extension step of PCR reactions. The optimum temperature of the PPase-PNIPAM conjugate was shown to increase from 45°C to 60°C compared to the unconjugated free enzyme. Furthermore, after 3 hours of incubation at 60°C, 77% of the enzyme activity was retained by the PPase-PNIPAM conjugate, a 6.8-fold increase compared to the unconjugated free enzyme. When used in PCR, the PPase-PNIPAM conjugate produced a yield 1.5-fold higher than using the unconjugated free enzyme alone.

[0005] However, the heat sensitivity of PPase is not the only challenge in PCR. Additional challenges arise from the ability of DNA polymerase enzymes to catalyze (1) the extension of partially annealed primers to nonspecific sites on DNA at room temperature (e.g., when PCR reaction components are combined and the reaction is set up) and (2) the extension of primer-dimers. The efficiency of thermostable DNA polymerases commonly used in PCR reactions is lower at room temperature than at higher temperatures; for example, those used in the extension step of PCR reactions still exhibit a small amount of activity at room temperature. Nonspecific amplification is not a major problem when the desired target nucleic acid is present at high concentrations. However, when the target nucleic acid is present at much lower concentrations, nonspecific amplification can become a much greater problem, as it can significantly impair detection of the desired target nucleic acid.

[0006] Several methods have been developed to reduce nonspecific amplification. A general term for such methods is hot-start PCR. The principle behind hot-start PCR is to keep the DNA polymerase in an inactive state until the reaction temperature exceeds the primer annealing temperature, which can be achieved in a variety of ways.

[0007] The first method is primitive and suggests keeping all PCR reaction vessels on ice and then transferring them to a thermocycler already set at 95°C. A slight modification of the method omits DNA polymerase from the reaction setup and adds it only after the PCR reaction vessels are placed in a thermocycler set at 95°C. However, this is difficult to implement and risks contamination / loss of reaction components when the PCR reaction vessels are opened.

[0008] More sophisticated methods include physically isolating the DNA polymerase from the other PCR reaction components, for example, by freezing the PCR reaction components except for the DNA polymerase and then adding the DNA polymerase in a liquid state. When the frozen layer melts, the DNA polymerase mixes with the other PCR reaction components. Other means of separating the DNA polymerase from the other PCR reaction components include using wax beads or petroleum jelly.

[0009] The most recent approach involves the inhibition of DNA polymerase enzymes. To date, two types of inhibitors are known: antibodies and oligonucleotides.

[0010] US 5,338,671 describes a method for amplifying a target nucleic acid, comprising contacting a sample suspected of containing the target nucleic acid with PCR reagents containing a temperature-sensitive inhibitor of DNA polymerase, the inhibitor being an antibody specific to the DNA polymerase that inhibits the DNA polymerase at a temperature T1 below about 85°C and irreversibly inactivates the DNA polymerase at a temperature T2 above T1 and above about 40°C. Thus, the antibody functions by inhibiting the DNA polymerase until the PCR temperature reaches a point at which the antibody is irreversibly denatured. At that point, the DNA polymerase is no longer inhibited. However, although this method is useful, there are still drawbacks to this approach. First, the high cost associated with the use of antibodies is high. Second, a separate antibody is required for each different enzyme. Third, the specific temperature at which the polymerase is activated cannot be controlled because the antibody-DNA polymerase is destroyed only slowly by increasing temperature. A further drawback is that, because the antibody is irreversibly denatured, the DNA polymerase is inhibited only at the beginning of the PCR reaction, not during the PCR reaction.

[0011] US 5,693,502 describes a method for performing PCR, including (1) mixing a sample containing a target nucleic acid with a single-stranded nucleic acid ligand capable of inhibiting DNA polymerase in a temperature-dependent manner, (2) heating the mixture to a temperature at which the nucleic acid no longer inhibits the DNA polymerase, and (3) performing PCR. Although nucleic acid ligands are cheaper than antibodies, they still have drawbacks. Namely, the nucleic acid-DNA polymerase is destroyed only slowly by increasing temperature, so the specific temperature at which the polymerase is activated cannot be controlled. Also, inhibition is only effective at room temperature or slightly above. Therefore, this technique does not provide a perfect hot start. Summary of the Invention [Problem to be solved by the invention]

[0012] Accordingly, one object of the present invention is to provide alternative methods and reagents for amplifying target nucleic acids. A further object is to provide improved methods and reagents for amplifying target nucleic acids. A related object is to provide conjugates for use in such methods, as well as aqueous solutions and kits containing the conjugates. [Means for solving the problem]

[0013] (Summary of the Invention) Therefore, the present invention provides (a) A DNA sample containing a target nucleic acid: (i) a DNA polymerase; (ii) at least two oligonucleotide primers designed to flank the target nucleic acid; (iii) a mixture of dATP, dGTP, dCTP, and dTTP, and (iv) a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer; contacting with (b) heating the product of step (a) to a temperature at which the conjugate precipitates and thereby the DNA polymerase is no longer inhibited; (c) amplifying the target nucleic acid, for example, by performing a PCR step of denaturing the target nucleic acid, annealing primers to the target nucleic acid, and extending the primers; 1. A method for amplifying a target nucleic acid in a DNA sample, comprising: Step (c) is repeated at least twice. to provide.

[0014] Also provided is a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer.

[0015] moreover, (a) a DNA polymerase, and (b) Conjugates containing a DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer. A kit is provided comprising:

[0016] Further provided is an aqueous composition comprising (i) a DNA polymerase bound to a conjugate comprising (ii) a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer.

[0017] In use according to the invention, for example, prior to DNA amplification by PCR at room temperature (20°C) in the presence of reagents, the conjugate is in solution and the inhibitor moiety binds to and inhibits the activity of the DNA polymerase. Upon raising the temperature, the conjugate remains in solution until it precipitates, removing the inhibitor from solution and liberating the DNA polymerase.

[0018] Suitably, the conjugate precipitates at a temperature between 35°C and 65°C, preferably between 40°C and 60°C, more preferably between 45°C and 55°C. [Brief explanation of the drawings]

[0019] [Figure 1-1] Not specified [Figure 1-2] Not specified DETAILED DESCRIPTION OF THE INVENTION

[0020] (Details of the invention) The present invention provides (a) A DNA sample containing a target nucleic acid: (i) a DNA polymerase; (ii) at least two oligonucleotide primers designed to flank the target nucleic acid; (iii) a mixture of dATP, dGTP, dCTP, and dTTP, and (iv) a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer contacting with (b) heating the product of step (a) to a temperature at which the conjugate precipitates and thereby the DNA polymerase is no longer inhibited; (c) amplifying the target nucleic acid, for example, by performing a PCR step of denaturing the target nucleic acid, annealing primers to the target nucleic acid, and extending the primers; wherein step (c) is repeated at least twice.

[0021] Typically, amplification is carried out in an aqueous buffer. (a) A DNA sample containing a target nucleic acid: (i) a DNA polymerase; (ii) at least two oligonucleotide primers designed to flank the target nucleic acid; (iii) a mixture of dATP, dGTP, dCTP, and dTTP; (iv) an aqueous buffer solution, and (v) a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer; contacting with (b) heating the product of step (a) to a temperature at which the conjugate precipitates and thereby the DNA polymerase is no longer inhibited; (c) performing standard PCR steps of denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers; wherein step (c) may comprise amplifying the target nucleic acid in the DNA sample, the amplifying step being repeated at least twice.

[0022] In the examples carried out to illustrate the present invention, as described in detail below, DNA polymerase was reversibly inhibited using the conjugate of the present invention in an RTase reaction prior to DNA amplification of cDNA. As can be seen, the use of the present invention is in improved DNA amplification reactions. Although the annealing temperature is lower than the typical primer annealing temperature used in PCR reactions, the presence of inhibitors at room temperature and at high temperatures reduces the formation of unwanted products that can contaminate the PCR reaction products. For example, non-specific binding of primers at low temperatures can result in the amplification of non-target sequences if DNA polymerase is present and active. The conjugate of the present invention prevents this.

[0023] The methods of the present invention use a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer. Accordingly, the present invention also provides a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer.

[0024] DNA polymerase inhibitors can inhibit DNA- or RNA-directed DNA polymerases, which catalyze the synthesis of DNA molecules from DNA or RNA templates, respectively.

[0025] Examples of DNA polymerases suitable for use in the conjugates of the invention, and which in turn are targets for inhibitors used in the methods of the invention, include those isolated from Thermus aquaticus, Pyrococcus furiosus, Thermococcus kodakaraensis, Thermus thermophilus, Pyrococcus woesei, Thermus filiformis, Thermus flavus, Thermus ubiquitous, Thermus litoralis, Thermotoga maritima, or recombinant versions of any of the foregoing DNA polymerases.

[0026] Examples of inhibitors of DNA polymerase include vidarabine, lamivudine, rifamycin SV monosodium salt, neobaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, tenofovir, and thiolutin.

[0027] In a preferred embodiment of the invention, the inhibitor is zidovudine.

[0028] Although examples of DNA polymerase inhibitors are identified, the type of inhibitor used to inhibit DNA polymerase and its mechanism of action are not actually critical. Inhibitors can inhibit DNA polymerase in a competitive or non-competitive manner. PCR is a technique well known to those skilled in the art. To test whether a given candidate inhibitor inhibits DNA polymerase, PCR reactions can be performed under standard conditions for at least 25 cycles using known target DNA, primers for the target, and standard reagents, with the candidate inhibitor and a known inhibitor (positive control) and without the inhibitor (negative control). The presence of product (relative to the negative control reaction) confirms that the DNA polymerase is active, so the absence of product from the candidate inhibitor indicates that the candidate substance is indeed an inhibitor of DNA polymerase. The absence of product in this context refers to the formation of 25% or less, preferably 10% or less, of product compared to the negative control.

[0029] The conjugates of the invention used in the methods of the invention include negative temperature sensitive polymers, a particular type of thermoresponsive polymer.

[0030] Thermoresponsive polymers, also known as temperature-responsive polymers, are polymers that exhibit dramatic and discontinuous changes in physical or chemical properties in response to changes in temperature.

[0031] In the conjugates of the invention used in the methods of the invention, it is the solubility of the thermoresponsive polymer, and thus the conjugate, that is sensitive to temperature.

[0032] Thermoresponsive polymers, which change their solubility state in response to temperature changes, can be divided into two classes: the first class includes thermoresponsive polymers that become insoluble above a critical temperature known as the lower critical solution temperature (LCST), and the second class includes thermoresponsive polymers that precipitate and undergo a phase change below a critical temperature known as the upper critical solution temperature (UCST).

[0033] When polymers exhibiting LCST are dissolved in aqueous systems, they are generally completely miscible below the LCST, but their solubility in aqueous solutions decreases with increasing temperature. Above a critical value, i.e., the LCST, they exhibit phase separation, and the polymer chains undergo a coil-to-globule-to-aggregate transition. Polymers exhibiting LCST are also known as "negative temperature-sensitive polymers." Examples of LCST polymers include poly(N-isopropylacrylamide) (PNIPAAm), polyvinylcaprolactam (PNVCL), poly(N,N-diethylacrylamide) (PDEAM), poly(N-ethylmethacrylamide) (PNEMAM), poly(methyl vinyl ether) (PMVE), and poly(2-ethoxyethyl vinyl ether) (PEOVE).

[0034] Thermoresponsive polymers that exhibit UCST are referred to as "positive temperature-sensitive polymers." These polymers remain miscible in solution above a certain temperature, but phase separation occurs when the solution temperature drops below a critical value, i.e., UCST. Examples of UCST polymers include poly(acrylic acid) (PAA), polyacrylamide (PAAm), and poly(acrylamide-co-butyl methacrylate).

[0035] The LCST or UCST is an inherent property of the thermoresponsive polymer and does not change simply by mixing the polymer with an inhibitor, such as a DNA polymerase inhibitor. However, if the polymer is chemically bound to an inhibitor, such as a DNA polymerase inhibitor, the LCST or UCST can change. The change in the LCST or UCST depends on the nature of the inhibitor, such as a DNA polymerase inhibitor.

[0036] For LCST polymers, if the inhibitor is hydrophilic, the LCST is usually increased, and therefore a higher temperature is required to precipitate the conjugate from the solution phase. Conversely, if the inhibitor is hydrophobic, the LCST is usually decreased.

[0037] For UCST polymers, if the inhibitor is hydrophilic, the UCST typically decreases, and therefore a lower temperature is required for the conjugate to go into solution. Conversely, if the inhibitor is hydrophobic, the UCST typically increases.

[0038] As mentioned above, a key feature of thermoresponsive polymers is that they exhibit dramatic and discontinuous changes in physical or chemical properties in response to changes in temperature.

[0039] When a thermoresponsive polymer is covalently attached to an inhibitor, such as an inhibitor of DNA polymerase, this property can be imparted substantially to the entire conjugate, i.e., the conjugate also exhibits dramatic and discontinuous changes in its physical or chemical properties in response to temperature changes.

[0040] In the present invention, it is advantageous to be able to precisely control when the DNA polymerase enzyme is activated and when it is inhibited.

[0041] References to the LCST herein refer to the LCST of the conjugate, not the isolated (non-conjugated polymer). Thus, in the conjugates of the present invention used in the methods of the present invention, the thermoresponsive polymer is a negative temperature-sensitive polymer, and 10% or less of the conjugate is in solution at 5°C above the LCST (i.e., precipitation temperature), and 90% or more of the conjugate is in solution at 5°C below the LCST (i.e., precipitation temperature). More preferably, 10% or less of the conjugate is in solution at 2°C above the LCST (i.e., precipitation temperature), and 90% or more of the conjugate is in solution at 2°C below the LCST (i.e., precipitation temperature). Even more preferably, 10% or less of the conjugate is in solution at 1°C above the LCST (i.e., precipitation temperature), and 90% or more of the conjugate is in solution at 1°C below the LCST (i.e., precipitation temperature).

[0042] In a preferred embodiment of the present invention, the negative temperature-sensitive polymer is or comprises a polymer selected from poly(N-substituted acrylamide) and its derivatives, poly(methyl vinyl ether), poly(N-vinyl caprolactam), poly(2-substituted 2-oxazoline) and its derivatives, poly(2-substituted 2-oxazine) and its derivatives, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethylhydroxyethyl cellulose, hydroxyethyl cellulose, poly(asparagine) peptide and its derivatives. Most preferably, the negative temperature-sensitive polymer is poly(N-isopropyl methacrylamide), also known as PNiPMAM.

[0043] The size, i.e., molecular weight, of the negative temperature sensitive polymer may range up to 200 KDa, or may range from 200 KDa to 10 KDa. Nevertheless, suitably, the negative temperature sensitive polymer is less than 200 The negative temperature sensitive polymer component of the conjugates of the invention used in the methods of the invention has a molecular weight of less than 100 KDa, more suitably less than 50 KDa, or even less than 20 KDa. In some preferred embodiments, the negative temperature sensitive polymer component of the conjugates of the invention used in the methods of the invention is selected to be relatively small / short to minimize the amount of material used without compromising the sensitivity of the polymer. For some polymers, e.g., acrylamide derivatives, the LCST does not vary significantly with molecular weight or polymer concentration, so a variety of sizes / weights can be used.

[0044] Suitable methods for determining the size of thermoresponsive polymers, such as negative temperature sensitive polymers used in the conjugates of the invention and, in turn, in the methods of the invention, include polymer membrane osmometry, gel permeation chromatography, viscosity analysis, mass spectrometry, end group analysis by, e.g., NMR, and static light scattering.

[0045] One consideration that must be kept in mind when selecting a negative temperature sensitive polymer for use in the conjugates of the invention, and in turn in the methods of the invention, is the desired temperature at which the conjugate will exhibit a phase transition, as this is primarily determined by the negative temperature sensitive polymer component of the conjugate.

[0046] In a preferred embodiment of the invention, the conjugate precipitates at a temperature between 35°C and 65°C, preferably between 40°C and 60°C, more preferably between 45°C and 55°C, and even more preferably between 48°C and 52°C.

[0047] PCR is a technique used to generate multiple copies of a specific target DNA from a longer DNA sequence or a mixture of DNA molecules. In other words, PCR is a method for amplifying a target nucleic acid in a DNA sample. This technique requires two short sequences of nucleotides, known in the art as primers or oligonucleotide primers, which provide the starting point for DNA synthesis. Primers are short sequences of single-stranded DNA designed to flank the target DNA, typically about 20–25 nucleotides in length. That is, they are designed to be complementary to the opposite strand of the target DNA at the end of the region to be replicated. Therefore, one primer is called the forward primer, and the other is called the reverse primer. PCR reactions are performed by adding template DNA containing the specific target DNA to a reaction vessel along with two primers, deoxynucleotide triphosphates (dNTPs) such as dATP, dCTP, dGTP, and dTTP, a buffer (usually containing MgCl2), and a DNA polymerase. Once prepared, the reaction vessel is placed in a thermal cycler, which cycles the temperature up and down for a set period of time. First, the temperature is raised to approximately 90°C or higher to denature the template DNA, i.e., to separate the double-stranded DNA into single strands. This step is commonly referred to as the "denaturation phase." Next, the temperature is lowered to approximately 55°C to 65°C to anneal the primers to the single-stranded DNA. This step is commonly referred to as the "annealing phase." Next, the temperature is raised to approximately 72°C, allowing the DNA polymerase to extend the primers and synthesize new DNA strands. This step is commonly referred to as the "extension phase." After the extension phase is complete, the cycle of denaturation, annealing, and extension is typically repeated 25 to 35 times.

[0048] Reverse transcription PCR (RT-PCR) is a related technique that combines reverse transcription of RNA into complementary DNA (cDNA) and amplification of a target nucleic acid in a DNA sample. In RT-PCR, an RNA template is first converted to cDNA using the enzyme reverse transcriptase. The cDNA is then used as a template for conventional PCR, as described above.

[0049] In a preferred embodiment of the present invention, the DNA polymerase inhibitor is zidovudine and the negative temperature sensitive polymer is PNiPMAM.

[0050] The advantage of using a conjugate comprising a DNA polymerase inhibitor and a negative temperature-sensitive polymer in a PCR reaction is that the inhibitor maintains its effect even when the temperature increases. This is the opposite of, for example, when an antibody is used to inhibit DNA polymerase. When an antibody is used to inhibit DNA polymerase, an increase in temperature causes irreversible denaturation of the antibody. In contrast, the conjugate of the present invention used in the method of the present invention maintains its inhibitory effect throughout the PCR reaction, moving in and out of solution as needed.

[0051] The DNA polymerases used in certain methods, kits, and aqueous compositions of the invention are preferably thermostable / heat-stable DNA polymerases. Examples of species from which such polymerases can be isolated include Thermus aquaticus, Pyrococcus furiosus, Thermococcus kodakaraensis, Thermus thermophilus, Pyrococcus woesei, and the like. Examples of suitable DNA polymerases include Thermus woesei, Thermus filiformis, Thermus flavus, Thermus ubiquitous, Thermus litoralis, and Thermotoga maritima. The DNA polymerase can also be a recombinant version of any of the aforementioned naturally occurring DNA polymerases. In a preferred embodiment of the present invention, the DNA polymerase is Taq polymerase.

[0052] A key step in the method of the present invention is step (b), which involves heating the product of step (a) to a temperature at which the conjugate precipitates and the DNA polymerase is no longer inhibited. In a preferred embodiment of the present invention, the product of step (a) is heated to a temperature of at least 35°C, preferably at least 40°C, even more preferably at least 45°C, and most preferably at least 48°C. Thus, in a preferred embodiment of the present invention, the conjugate precipitates at a temperature of at least 35°C, preferably at least 40°C, even more preferably at least 45°C, and most preferably at least 48°C. Also preferably, the conjugate precipitates at a temperature of 60°C or less, suitably 55°C or less, or 52°C or less.

[0053] Step (c) of the PCR method involves performing standard PCR steps of denaturing the target nucleic acid, annealing primers to the target nucleic acid, and extending the primers, and then repeating the denaturation, annealing, and extension cycle at least twice once the extension step is complete. The number of times this step (c), i.e., the denaturation, annealing, and extension cycle, is repeated depends on the amount of DNA template input into the reaction and the desired yield of PCR product. The lower the input amount, the more times step (c) needs to be repeated. Similarly, the higher the desired yield of PCR product, the more times step (c) needs to be repeated. In a preferred embodiment of the present invention, step (c) is repeated at least 10 times, even more preferably at least 20 times, and most preferably at least 30 times.

[0054] The method used to prepare the conjugate of the present invention present in the kit and aqueous composition of the present invention and used in the method of the present invention is not critical, as long as the DNA polymerase inhibitor and the negative temperature-sensitive polymer are covalently conjugated.Methods for covalently conjugating molecules are well known to those skilled in the art.The conjugate can be direct, if the DNA polymerase inhibitor contains a functional group that allows it to be bound to the negative temperature-sensitive polymer, or indirect, if a linker can be present between the DNA polymerase inhibitor and the negative temperature-sensitive polymer.In the conjugate of Example 1, the DNA polymerase inhibitor is conjugated to the polymer via a linker.

[0055] In a preferred embodiment of the invention, the conjugate comprises a DNA polymerase inhibitor covalently attached to or near the terminus of the negative thermosensitive polymer, near in this context meaning within 20 monomers, preferably within 10 monomers, preferably within 5 monomers, more preferably within 2 monomers of the terminus of the negative thermosensitive polymer.

[0056] Even more preferably, the conjugate comprises a DNA polymerase inhibitor covalently attached to the terminus of the negative temperature-sensitive polymer. An advantage associated with this configuration is that the DNA polymerase inhibitor is more accessible to the DNA polymerase. To achieve this placement of the DNA polymerase inhibitor to the negative temperature-sensitive polymer, the monomers are combined and allowed to polymerize for a period of time before adding the inhibitor, after which the inhibitor is incorporated / attached to the polymer at or near its terminus. [Example]

[0057] (Example) The invention will now be further described by specific examples with reference to the accompanying drawings, in which Figure 1 shows the quantification of cDNA from RTase reactions carried out at various temperatures with RTase alone or conjugated to zidovudine-poly-N-isopropylmethacrylamide, and the RTase cDNA was subsequently amplified and quantified by qPCR.

[0058] Example 1 Preparation of thermoresponsive zidovudine-poly-N-isopropylmethacrylamide inhibitors Step 1: Preparation of Polymerizable Zidovudine by Reaction with Propargyl Acrylate Propargyl acrylate (413 μl, 2 equiv.) was added to ethanol (30 ml) in a sealable container and flushed with nitrogen for 5 minutes. Zidovudine (0.50 g), copper sulfate (30 mg, 1 mol%) in water, and sodium ascorbate (62 mg, 17 mol%) in water were then added. Copper sulfate and sodium ascorbate could be added as solids if enough water was added to the reaction to dissolve both. The container was sealed, heated to 30°C with gentle stirring, and left overnight. The solvent was then partially evaporated using nitrogen gas (approximately 4 hours per column was sufficient). Purification was then performed using flash silica chromatography (100% ethyl acetate), and the solvent was removed under reduced pressure at room temperature. 0.332 g of a white solid (0.88 mmol, 47% yield) was obtained.

[0059] (Step 2: Free radical polymerization and polymer purification) In a glass vial, 26 mg of polymerizable zidovudine, 50 mg of 1,1'-azobis(cyclohexanecarbonitrile), and 0.9 g of N-isopropylmethacrylamide were dissolved in 20 ml of ethanol. The mixture was then bubbled with nitrogen for 5 minutes, sealed with a screw cap, and placed in a 70°C oven for 24 hours. Polymer ("P") was prepared as above. Polymer ("P1X") was prepared as above, except that 26 mg of polymerizable zidovudine was added after 2 hours of polymerization. Polymer ("P2X") was prepared like polymer ("P1X"), except that 52 mg of polymerizable zidovudine was added instead of 26 mg. After polymerization, all polymers were purified using the same protocol. To this end, the polymerization mixture was added to approximately 150 ml of deionized water, and the initial precipitate was removed by filtration and discarded. The filter-through fraction was then heated to 60°C. The precipitate was removed by filtration using a glass fiber membrane with a nominal pore size of 1.2 μm. After recovery, the membrane was cooled to room temperature, and the temperature-responsive polymer was eluted with 50 / 50% ethanol / water (v / v). The resulting polymer had a lower critical solution temperature of approximately 52 °C, as observed by the formation of turbidity upon heating the polymer solution in water.

[0060] Example 2 Reversible Inhibition Experiment of Reverse Transcriptase (RTase) Using the Thermoresponsive Zidovudine-Poly-N-Isopropylmethacrylamide Inhibitor of Example 1 This example demonstrates the use of the thermoresponsive zidovudine-poly-N-isopropylmethacrylamide inhibitor from Example 1 for the reversible inhibition of RTase. Reactions were performed using UltraScript 2.0 reverse transcriptase from PCR Bio (UK) according to the manufacturer's instructions. Typical conditions are for a total reaction volume of 20 μl.

[0061] RNA = 5µg template and random hexamer primers in PCR Bio buffer mix PCR Bio RTase = 324 nM 150 ng zidovudine-poly-N-isopropylmethacrylamide

[0062] (RTase reaction) Reactions were performed over a range of temperatures below and above the LCST of zidovudine-poly-N-isopropylmethacrylamide to assess both the inhibition and thermal reversibility of the conjugate. Individual reaction vials were held at a set temperature for 30 minutes to allow the reaction to proceed. Temperatures used were 25°C, 43°C, 55°C, and 35°C, with the latter occurring after the 55°C step. After each incubation, 1–2 μl of cDNA product was collected for qPCR amplification and quantification. The results are shown in Figure 1. Inhibition was detected by observing the loss of the product peak.

[0063] It should be noted that the zidovudine-poly-N-isopropylmethacrylamide prepared in Example 1 has a lower critical solution temperature of about 52°C, and therefore, at temperatures above about 52°C, substantially all of the conjugate was out of solution.

[0064] This confirmed the temperature dependence of RTase activity (availability) when RTase was bound to a thermoresponsive polymer, and the inhibition was reversible.

Claims

1. (a) A DNA sample containing a target nucleic acid is: (i) a DNA polymerase, (ii) at least two oligonucleotide primers designed to flank the target nucleic acid; (iii) a mixture of dATP, dGTP, dCTP, and dTTP, and (iv) Conjugates containing a DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer contacting with (b) heating the product of step (a) to a temperature at which the conjugate precipitates and thereby the DNA polymerase is no longer inhibited; (c) amplifying the target nucleic acid by performing a PCR step of denaturing the target nucleic acid, annealing primers to the target nucleic acid, and extending the primers; 1. A method for amplifying a target nucleic acid in a DNA sample, comprising: The method wherein step (c) is repeated at least twice.

2. 2. The method of claim 1, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, zidovudine, neovaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, or tenofovir.

3. The negative temperature sensitive polymer is: (i) poly(N-substituted acrylamide); (ii) poly(methyl vinyl ether); (iii) poly(N-vinylcaprolactam); (iv) poly(2-substituted 2-oxazolines), (v) poly(2-substituted 2-oxazines), (vi) hydroxypropyl methylcellulose, (vii) ethylhydroxyethylcellulose, (viii) hydroxyethyl cellulose, (ix) poly(asparagine), and (x) PNiPMAM The method according to claim 1 or 2, wherein the compound is selected from the group consisting of:

4. 4. The method of claim 1, wherein the conjugate precipitates at a temperature between 35°C and 65°C.

5. 5. The method of claim 4, wherein the conjugate precipitates at a temperature between 48°C and 52°C.

6. 6. The method of claim 1, wherein no more than 10% of the conjugate is in solution at 2° C. above the precipitation temperature and more than 90% of the conjugate is in solution at 2° C. below the precipitation temperature.

7. 7. The method of claim 6, wherein no more than 10% of the conjugate is in solution at 1° C. above the precipitation temperature and more than 90% of the conjugate is in solution at 1° C. below the precipitation temperature.

8. The method of any one of claims 1 to 7, wherein the conjugate comprises a DNA polymerase inhibitor covalently attached at or near the end of the negative temperature sensitive polymer.

9. The method of any one of claims 1 to 8, wherein the DNA polymerase is a thermostable DNA polymerase.

10. The method of claim 9, wherein the DNA polymerase is Taq polymerase.

11. (a) a DNA polymerase, and (b) Conjugates containing DNA polymerase inhibitors covalently linked to negative temperature-sensitive polymers. Includes a kit.

12. 12. The kit of claim 11, wherein the conjugate precipitates at a temperature between 35°C and 65°C.

13. 13. The kit of claim 12, wherein the conjugate precipitates at a temperature between 48°C and 52°C.

14. 14. The kit of any of claims 11 to 13, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, neobaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, or tenofovir.

15. 14. The kit according to any one of claims 11 to 13, wherein the DNA polymerase inhibitor is zidovudine.

16. 16. The kit of any of claims 11-15, wherein no more than 10% of the conjugate is in solution at 2°C above the precipitation temperature and more than 90% of the conjugate is in solution at 2°C below the precipitation temperature.

17. 17. The kit of claim 16, wherein no more than 10% of the conjugate is in solution at 1°C above the precipitation temperature and more than 90% of the conjugate is in solution at 1°C below the precipitation temperature.

18. 18. The kit of any of claims 11 to 17, wherein the conjugate comprises a DNA polymerase inhibitor covalently attached at or near the end of the negative temperature sensitive polymer.

19. 19. The kit of any one of claims 11 to 18, wherein the DNA polymerase is a thermostable DNA polymerase.

20. 20. The kit of claim 19, wherein the DNA polymerase is Taq polymerase.

21. (ii) An aqueous composition comprising (i) a DNA polymerase associated with a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer.

22. 22. The aqueous composition of claim 21, wherein the conjugate precipitates at a temperature between 35°C and 65°C.

23. 23. The aqueous composition of claim 22, wherein the conjugate precipitates at a temperature between 48°C and 52°C.

24. 24. The aqueous composition of any of claims 21 to 23, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, neobaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, or tenofovir.

25. 24. The aqueous composition of any of claims 21 to 23, wherein the DNA polymerase inhibitor is zidovudine.

26. 26. The aqueous composition of any of claims 21-25, wherein no more than 10% of the conjugate is in solution at 2°C above the precipitation temperature and more than 90% of the conjugate is in solution at 2°C below the precipitation temperature.

27. 27. The water-soluble composition of claim 26, wherein no more than 10% of the conjugate is in solution at 1°C above the precipitation temperature and more than 90% of the conjugate is in solution at 1°C below the precipitation temperature.

28. 28. The aqueous composition of any of claims 21 to 27, wherein the conjugate comprises a DNA polymerase inhibitor covalently attached at or near the terminus of the negative temperature sensitive polymer.

29. 29. The aqueous composition of any one of claims 21 to 28, wherein the DNA polymerase is a thermostable DNA polymerase.

30. 30. The aqueous composition of claim 29, wherein the DNA polymerase is Taq polymerase.

31. 1. A conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, the DNA polymerase inhibitor is vidarabine, lamivudine, zidovudine, neovaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, or mithramycin A; The conjugate precipitates at temperatures between 35°C and 65°C, and A conjugate wherein no more than 10% of the conjugate is in solution at 2°C above the precipitation temperature and more than 90% of the conjugate is in solution at 2°C below the precipitation temperature.

32. 32. The conjugate of claim 31, wherein the conjugate precipitates at a temperature between 40°C and 60°C.

33. 33. The conjugate of claim 32, wherein the conjugate precipitates at a temperature between 48°C and 52°C.

34. 34. The conjugate of any one of claims 31 to 33, wherein no more than 10% of the conjugate is in solution at 1°C above the precipitation temperature and more than 90% of the conjugate is in solution at 1°C below the precipitation temperature.

35. 35. The conjugate of any one of claims 31 to 34, wherein the conjugate comprises a DNA polymerase inhibitor covalently attached at or near the terminus of the negative temperature sensitive polymer.

36. (a) An RNA sample containing a target nucleic acid is: (i) a DNA polymerase, wherein the DNA polymerase is an RNA-directed DNA polymerase; (ii) at least two oligonucleotide primers; (iii) a mixture of dATP, dGTP, dCTP, and dTTP, and (iv) Conjugates containing an RNA-directed DNA polymerase inhibitor covalently attached to a negative temperature-sensitive polymer contacting with (b) heating the product of step (a) to a temperature at which the conjugate precipitates and thereby the DNA polymerase is no longer inhibited; (c) amplifying the target nucleic acid from the cDNA; 1. A method for amplifying a target nucleic acid in an RNA sample, comprising: A method wherein step (c) is repeated at least twice.

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