Modified dNTPs as well as preparation method and application thereof

Abstract

The invention provides modified dNTPs (deoxyribonucleoside triphosphates) and a preparation method and application thereof, the provided modified dNTPs are subjected to 3'substitution, specifically, the 3 'position is substituted by thermally unstable ethers, and the modified dNTPs can be applied to polymerase chain reaction to improve the specificity of nucleic acid amplification reaction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a class of modified dNTPs and a preparation method and application thereof. Background Art

[0002] PCR (polymerase chain reaction) is a molecular biology technique commonly used in biological and medical research to amplify and detect DNA fragments. It is widely used in genomics, genetics, pathology, medical diagnosis and other fields. Its basic principle is that through multiple cycles of temperature changes, the double-stranded DNA target sequence denatures and dissociates into single strands at high temperature (usually 95°C) in vitro, and then anneals (usually 72°C) to introduce specific oligonucleotide primers. These forward and reverse primers synthesize complementary chains along the direction of phosphate to pentose (5'-3'-) through the intervention of DNA polymerase following the principle of base complementary pairing. Accurate copies of the target DNA are obtained in each cycle. This process allows the specific DNA fragments being sought to be amplified in the sample, facilitating further analysis and research.

[0003] The dNTPs used in traditional PCR are a mixture containing all four nucleotides (A, T, C, G), which are also present at the beginning of the PCR reaction. In this way, before the temperature rises to the high temperature required for the melting step, the nucleotides may have randomly combined with non-specific DNA sequences, resulting in non-specific amplification or false positive results.

[0004] To solve this problem, hot-start dNTPs were introduced into the PCR reaction. At the beginning of the PCR reaction, before the temperature rises to the high temperature required for the melting step, these 3'-specifically modified dNTPs will not be attached to the primers. When the DNA polymerase intervenes, the heat-labile protective group is destroyed by heat, thereby obtaining a naked nucleotide substrate that can normally participate in the PCR reaction. In this way, by using hot-start dNTPs, nonspecific amplification and false positive results can be effectively suppressed, and the specificity and accuracy of the PCR reaction can be improved. In summary, hot-start dNTPs are a strategy to improve PCR specificity by controlling nucleotide binding at the beginning of the PCR reaction. They can bind weakly to primers and template DNA at low temperatures, thereby reducing the risk of nonspecific amplification and false positive results.

[0005] Currently, there are many methods for chemically modifying the necessary substrate dNTPs for PCR reactions, but the common disadvantage is that the cost is too high. The cost of modifying the substrate far exceeds the cost of the PCR reaction itself. Summary of the invention

[0006] The present invention aims to provide a type of chemically modified PCR reaction substrate, in which 3'-substitution is performed on dNTPs. Such modified dNTPs are applied to hot-start nucleic acid amplification in different scenarios, and the preparation process of the modified dNTPs is simple and the price is low.

[0007] In one aspect, the present invention provides a class of modified dNTPs, the structure of which is as follows:

[0008]

[0009] wherein B is selected from any "universal base" or "degenerate base" of a substituted or unsubstituted purine or pyrimidine, any aza or deaza derivative thereof, or any NTP analog;

[0010] R is selected from the following groups

[0011]

[0012] In some embodiments, the R group is

[0013] In some embodiments, the modified dNTP is 3'-substituted dATP, 3'-substituted dTTP, 3'-substituted dGTP, 3'-substituted dCTP, or 3'-substituted dUTP.

[0014] In another aspect, the present invention provides a method for replicating a nucleic acid, the method comprising:

[0015] Incubating a replication reaction mixture at 37° C. to 100° C., the mixture comprising the nucleic acid, at least one modified dNTP, and a replication reaction buffer without a chemical reagent for removing a 3′-substituent of the modified dNTP; and

[0016] replicating the nucleic acid in the replication reaction mixture,

[0017] Wherein the at least one modified dNTP is a modified dNTP described in the present invention.

[0018] In some embodiments, the modified dNTP impairs or prevents nucleic acid extension by a polymerase.

[0019] In some embodiments, the modified dNTP is a non-substrate NTP. In some embodiments, the modified dNTP is a termination NTP. In some embodiments, the modified dNTP is converted into an open 3'-hydroxyl group during nucleic acid replication. In some embodiments, the modified dNTP is converted into an open 3'-hydroxyl group at a temperature between 37°C-100°C, for example, between 50-100°C, between 70-100°C. In some embodiments, the modified dNTP is converted into an open 3'-hydroxyl group at a temperature of 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 90°C, 95°C.

[0020] In some embodiments, the modified dNTP is selected from one or more of 3'-substituted dATP, 3'-substituted dTTP, 3'-substituted dGTP, 3'-substituted dCTP and 3'-substituted dUTP.

[0021] In some embodiments, in the modified dNTP, R is B is selected from In some embodiments, the modified dNTP is selected from IA, IG, IC, IT.

[0022] On the other hand, the present invention provides a method for preparing the modified dNTP IA, comprising:

[0023]

[0024] On the other hand, the present invention provides a method for preparing the modified dNTP IG, comprising:

[0025]

[0026]

[0027] On the other hand, the present invention provides a method for preparing the modified dNTP IC, comprising:

[0028] On the other hand, the present invention provides a method for preparing the modified dNTP IT, comprising:

[0029]

[0030]

[0031] In some embodiments, the preparation method further comprises the preparation of compound 1:

[0032]

[0033] In another aspect, the present invention provides use of the modified dNTPs of the present invention in nucleic acid amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the H NMR spectrum of compound 1;

[0035] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of compound 4-A;

[0036] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound 4-G;

[0037] Figure 4 is the C NMR hydrogen spectrum of compound 4;

[0038] Figure 5 is the H NMR spectrum of compound 4-T;

[0039] Figure 6 is the high performance liquid chromatogram of compound IA;

[0040] Figure 7 is the HPLC chromatogram of compound IG;

[0041] Figure 8 is the HPLC chromatogram of compound IC;

[0042] Fig. 9 is the HPLC chromatogram of compound IT;

[0043] Fig.10 This is a curve of the 3'-substituent release rate of modified dNTP IC, II-C, III-C, IV-C, VC, VI-C, and VII-C in a water system at 95°C. DETAILED DESCRIPTION

[0044] The technical scheme of the present invention is further described below in conjunction with specific examples. However, the following examples are merely examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. In the following examples, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0045] Embodiment 1:

[0046] 1. Synthesis of (2-methyl-2-(prop-1-enyloxy)propyl)benzene (Compound 1)

[0047] Dissolve dimethylbenzyl alcohol (12g, 0.8mol) in anhydrous tetrahydrofuran (320mL) and add it to a single-necked flask. Add sodium hydride (5.76g, 0.24mol) and allyl bromide (29.1g, 0.24mol) to the flask under a nitrogen atmosphere. Transfer the flask to 72°C for reflux reaction for 10h. Cool the reaction to room temperature, add water (20mL) to the system to quench the excess sodium hydride; extract the reaction liquid with petroleum ether, and concentrate the organic phase to obtain a colorless liquid. Add tris-(triphenylphosphine) ruthenium dichloride (82mg, 0.086mmol) to the colorless liquid, and stir the reaction in a closed manner at 140°C for 10h. After the reaction is completed, the system is naturally cooled to room temperature, the system is distilled under reduced pressure, and the fractions at 77°C-95°C are collected to obtain 12.5g of colorless liquid with a yield of 82.2%; the specific reaction formula is:

[0048]

[0049] NMR characterization data: 1H NMR (400MHz, DMSO-d6) δ7.32–7.16 (m, 5H), 6.45 (dq, J = 12.0, 1.6 Hz, 1H), 4.82 (dq, J = 12.0, 6.8 Hz, 1H), 2.79 (s, 2H), 1.54 (dd, J = 6.8, 1.6 Hz, 3H), 1.13 (s, 6H).

[0050] 2. Synthesis of (2-methyl-2-(prop-1-enyloxy)propyl)benzene substituted deoxyribonucleoside 3'-hydroxyl hydrogen

[0051] 10 mmol of N6-benzoyl-2'-deoxyadenosine (Huanda, HD00827), N2-isobutyryl-2'-deoxyguanosine (Huanda, HD00826), N4-benzoyl-2'-deoxycytidine (Huanda, HD00828), and 2'-deoxythymidine were dissolved in tetrahydrofuran (50 mL), and Novozymes 435 enzyme (5 g) and oxime ester (5.2 g, 30 mmol) were added, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the 435 enzyme was filtered off, and the tetrahydrofuran was removed by vacuum distillation to obtain a colorless to light yellow viscous liquid. The thick liquid was redissolved with DCM (100 mL), and the system was washed three times with saturated sodium bicarbonate aqueous solution (100 mL). The organic phase was concentrated to a volume of about 10 mL, poured into methyl tert-butyl ether (200 mL) for slurry precipitation, and the precipitate was vacuum dried to obtain 2-A, 2-G, 2-C, and 2-T. The yields were: 2-A = 73.5%, 2-G = 66.2%, 2-C = 80.1%, and 2-T = 85.9%. The specific reaction formula is:

[0052]

[0053]

[0054] Anhydrous dichloromethane (23.3 mL) containing compound 2-A / G / C / T (2.33 mmol) was added to a single-necked flask, and (2-methyl-2-(prop-1-enyloxy)propyl)benzene (1.32 g, 6.99 mmol) and pyridinium p-toluenesulfonate (0.58 g, 2.33 mmol) were added; the reaction was carried out at room temperature for 8 h. After the reaction was completed, the reaction system was washed three times with saturated sodium bicarbonate aqueous solution (20 mL), and the organic phase was concentrated to obtain a colorless to light yellow viscous liquid, which was the crude product of compound 3-A / G / C / T;

[0055] Add methanol solution of ammonia (46.6 mL) to the crude compound 3-A / G / C / T respectively, and stir at room temperature for 12 h. The system was distilled under reduced pressure, and purified by silica gel column. The TLC points of Rf=0.4 (4-A), 0.4 (4-G), 0.25 (4-C), and 0.6 (4-T) were collected under the system of methanol / dichloromethane=5%. The effluent was distilled under reduced pressure to obtain a white powdery solid, which was compound 4-A / G / C / T. The yields 4-A=72.8%, 4-G=56.3%, 4-C=85.4%, and 4-T=81.1%; the specific reaction formula is:

[0056]

[0057]

[0058] 4-A:1H NMR(400MHz,DMSO-d6)δ8.32(s,1H),8.13(d,J=1.9Hz,1H),7.28(d,J=25.1Hz,3H),7.23(d,J=2.2Hz,2H),7.22-7.09(m,2H),6.26(ddd,J=10.8,7.9,6.0Hz,1H),5.31(ddd,J=8.9,6.6,4.8Hz,1H),4.87(t,J=5.3Hz,1H),4.49(dq,J=8.2,3.0Hz,1H),4.12(q,J=5.2Hz,1H),3.98(dd,J=22.0,2.7Hz,1H),3.65-3.57(m,1H),3.54-3.46(m,1H),3.17(d,J=5.2Hz,2H),2.77(d,J=4.4Hz,2H),2.62(dt,J=13.5,6.7Hz,1H),2.35-2.27(m,1H),2.17-1.87(m,2H),1.57(tq,J=13.6,6.4Hz,2H),1.19(d,J=4.1Hz,3H),1.12(d,J=3.0Hz,3H),0.84(td,J=7.4,2.5Hz,3H).

[0059] 4-G:1H NMR(400MHz,DMSO-d6)δ10.62(s,1H),7.92(d,J=2.8Hz,1H),7.30-7.12(m,5H),6.46(s,2H),6.04(ddd,J=13.4,8.0,5.9Hz,1H),5.07-4.95(m,1H),4.85(td,J=5.3,2.3Hz,1H),4.40(ddt,J=11.2,5.6,2.7Hz,1H),3.90(dtd,J=21.7,4.5,2.5Hz,1H),3.60-3.44(m,2H),2.82-2.70(m,2H),2.47-2.17(m,2H),1.62-1.46(m,2H),1.18(d,J=4.2Hz,3H),1.11(d,J=4.2Hz,3H),0.83(td,J=7.4,1.5Hz,3H).

[0060] 4-C:1H NMR (400MHz, DMSO-d6) δ7.79(d,J=7.4Hz,1H),7.32-7.14(m,7H),6.10(td,J=7.4,5.8Hz,1H),5.74(dd,J=7.6,1. 6Hz,1H),5.03(q,J=3.7,2.5Hz,1H),4.82(t,J=5.3Hz,1H),4.29(tt,J=5.8,3.0Hz,1H),3.88(dq,J=23.0,3.6Hz,1 H),3.54(tq,J=11.9,7.5,6.0Hz,2H),2.84-2.69(m,2H),2.17(ddt,J=13.3,5.9,2.9Hz,1H),1.92(dddd,J=50.3,1 3.5,7.8,6.1Hz,1H),1.62-1.44(m,2H),1.17(d,J=4.9Hz,3H),1.10(d,J=4.2Hz,3H),0.81(td,J=7.4,2.8Hz,3H).

[0061] 4-T:1H NMR (400MHz, DMSO-d6) δ11.29(s,1H),8.45-8.35(m,1H),7.68(dd,J=2.4,1.3Hz,1H),7.31-7.15(m,5H),6.16-6.02(m,1 H),5.09(td,J=5.1,3.1Hz,1H),4.83(t,J=5.2Hz,1H),4.32(ddd,J=7.6,5.3,3.0Hz,1H),3.86(dq,J=21.1,3.5Hz,1H),3. 65-3.47(m,2H),3.18(d,J=5.2Hz,1H),2.81-2.71(m,2H),2.32(dt,J=17.0,9.2Hz,1H),2.18-1.90(m,5H),1.78(dd,J=3 .8,1.2Hz,3H),1.61-1.45(m,2H),1.37(s,3H),1.17(d,J=3.8Hz,3H),1.10(d,J=3.7Hz,3H),0.82(td,J=7.4,2.1Hz,3H).

[0062] 3. Preparation of the 5'-position of the 3'-substituted deoxyribonucleoside by triphosphorylation

[0063] Add 2-chloro-1,3,2-benzodioxaphosphorinane-4-one (1.07g) in N.N'-dimethylformamide (6.4mL) to a single-mouth bottle containing 0.5M tripentylamine pyrophosphate (13.5mL), tripentylamine (6.4mL), and 1,8-bisdimethylaminonaphthalene (1.07g), and stir the reaction for 1h under ice bath. Then add 4-A / G / C / T (1.45mmol / 1.40mmol / 1.53mmol / 1.48mmol), transfer the reaction system to room temperature, and continue to stir the reaction for 2h. After the reaction is completed, add iodine solution (0.58g of elemental iodine, 17.3mL of pyridine, and 2mL of water) until the system no longer fades for 30min, then add ammonia water with pH=11.0 to the system, and continue the reaction for 1h. Add saturated sodium chloride solution (10.9 mL) to the system, stir the reaction for 15 minutes, pour the system into acetone (320 mL) and place it in a -20°C environment for precipitation for 2 hours. Skim off the supernatant and centrifuge the lower white powder solid. Dissolve the lower solid with 50 mL, 1.0 M, pH = 8.0 triethylamine carbonate aqueous solution for ion purification. Desalt the purified effluent and then desolventize it. Dissolve the solid with 10-4 M sodium glycine aqueous solution to 50 mM to obtain the final sample. The final yields of IA / G / C / T sodium salts are 53.4%, 43.5%, 66.4%, and 68.8%, respectively, and the purity is more than 99%. IA: MS (m / z) = 680 [MH] - IG: MS (m / z) = 696 [MH] -, IC: MS (m / z) = 656 [MH] -, IT: MS (m / z) = 676 [MH] -. The HPLC chromatogram of IA / G / C / T sodium salt is attached. Figure 5-9 Chromatographic conditions: C18 as filler; pure water-acetonitrile (30:70) as mobile phase; detection wavelength of 260nm; column temperature of 30℃; injection volume of 20μL; instrument model: Agilent 1260. IA retention time: 39.186, 39.451min, IG retention time: 27.161min, IC retention time: 29.310, 29.717min, IT retention time: 34.285, 34.865min.

[0064] The sodium salts of compounds II-A / G / C / T, III-A / G / C / T, IV-A / G / C / T, VA / G / C / T, VI-A / G / C / T, and VII-A / G / C / T were prepared in substantially the same manner as the sodium salt of IA / G / C / T. The structures of II-A / G / C / T, III-A / G / C / T, IV-A / G / C / T, VA / G / C / T, VI-A / G / C / T, and VII-A / G / C / T are shown below:

[0065]

[0066] Example 2: In the PCR amplification reaction system: 5 μL 10x Taq HS Buffer (Cat. No.: Vazyme P132), 1 μL dCTP (10mM), 44 μL Rnase-free ddH2O, total system 50 μL. Modified dCTP compounds IC, II-C, III-C, IV-C, VC, VI-C, VII-C were used at the hot start temperature (95°C) for 0 / 0.5 / 1 / 3 / 5 / 10 min, respectively. The peak area change was quantified by HPLC, and the dCTP release rate was calculated (release rate = release peak area / total peak area * 100%). By comparing the release rate, it was found that the thermal release effect of modified dCTP compounds IC and VII-C was better.

Claims

1. A modified dNTP, the structure of which is as follows: in, B is any "universal base" or "degenerate base" selected from substituted or unsubstituted purine or pyrimidine, any aza or deaza derivative thereof, or any NTP analogue; R is selected from the following groups 2. The modified dNTP according to claim 1, wherein the R group is 3. The modified dNTP according to claim 1, selected from 3'-substituted dATP, 3'-substituted dTTP, 3'-substituted dGTP, 3'-substituted dCTP or 3'-substituted dUTP.

4. A method for replicating a nucleic acid, the method comprising: Incubating a replication reaction mixture at 37° C. to 100° C., the mixture comprising the nucleic acid, at least one modified dNTP, and a replication reaction buffer without a chemical reagent for removing a 3′-substituent of the modified dNTP; and replicating the nucleic acid in the replication reaction mixture, Wherein the at least one modified dNTP is the modified dNTP of claim 1.

5. The method of claim 4, wherein the modified dNTPs impair or prevent nucleic acid extension by a polymerase. The method according to claim 4 , wherein the modified dNTP is a non-substrate NTP.

7. The method of claim 4, wherein the modified dNTP is a stop NTP.

8. The method of claim 4, wherein the modified dNTP is converted into an open 3'-hydroxyl group during nucleic acid replication.

9. The modified dNTP according to claim 1 or the method according to claim 4, wherein the modified dNTP is selected from IV-A, IV-G, IV-C, IV-T.

10. Use of the modified dNTP according to claim 1 in nucleic acid amplification.

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