CN116425816A - A kind of isoTAT unnatural base triphosphate and its preparation method and application - Google Patents

Abstract

The invention discloses an isocratic acid non-natural base triphosphate, a preparation method and application thereof, wherein the isocratic acid non-natural base triphosphate has the structural formula:

the invention also specifically discloses that in the presence of isoTATTP, TPT3-NaM can be specifically converted into C-G base through simple PCR amplification, and has high readthrough rate and low sequence dependence, and double positioning of a plurality of TPT3-NaM sites in DNA can be realized. The invention provides a general and convenient method, which realizes the TPT3-NaM unnatural base with unlimited site and quantity for the first timeAccurate localization, tracking and sequencing of DNA samples.

Description

A kind of isoTAT unnatural base triphosphate and its preparation method and application

technical field

The invention belongs to the technical field of synthesis of unnatural base triphosphate and reading and sequencing of artificial gene codes, and in particular relates to an isoTAT type unnatural base triphosphate and its preparation method and application.

Background technique

Genetic information in nature is stored in the A/T/G/C genetic alphabet consisting of four natural bases. Recent studies have found that some artificially designed and synthesized unnatural base pairs have excellent functions of amplifying natural base gene letters, among which P-Z, Ds-Px and TPT3-NaM unnatural base pairs (UBPs) exhibit excellent gene Letter amplification performance. These unnatural base pairs are unique in their ability to replicate and store genetic information as bioorthogonal genetic letters. For example, systematic evolution of ligands by exponential enrichment (SELEX) of DNA libraries carrying multiple UBPs can obtain unnatural base aptamers with pmol level affinity. The codon and anticodon system carrying UBP (A-NaM-C as codon and G-TPT3-T as anticodon) can encode three non-codon amino acids and was successfully used to express sfGFP protein. Inserting more unnatural base pairs can expand the capacity, diversity and function of classical DNA, however, it is still highly challenging to accurately sequence DNA with multiple UBPs through simple and convenient methods.

TPT3-NaM is one of the unnatural base pairs with the best replication ability. So far, biotin and sequencing assays have been developed to monitor TPT3-NaM-containing DNA methods. However, biotin shift assays cannot detect closely spaced multiple encoding UBPs or UBPs with non-default loci, nor can they detect mutations at sites near UBPs. The current TPT3-NaM sequencing analysis can be divided into Sanger sequencing and nanopore sequencing. Sanger sequencing can detect individual UBPs, where the signal end can give the correct position of the unnatural base. However, the main obstacle to sequencing multiple UBPs in DNA is the disappearance of the Sanger sequencing signal after the first UBP site, which makes it difficult to obtain subsequent base sequence information. The nanopore sequencing method can distinguish each unnatural base based on its different shape, but the method involves tedious modifications to the UBP structure, specialized protein preparation procedures, and special instrumentation. So far, it is still impossible to accurately map and efficiently sequence complex DNA products with two or more TPT3-NaM-UBPs in adjacent coding regions or non-default positions.

Contents of the invention

The object of the present invention is to provide an isoTAT type unnatural base triphosphate and its preparation method, the isoTAT type unnatural base triphosphate can specifically recognize the unnatural base NaM and natural base G to form a complementary base Yes, for the first time, accurate positioning, tracking and sequencing of DNA samples containing sites and an unlimited number of TPT3-NaM unnatural bases have been realized.

The present invention adopts following technical scheme for realizing the above object, a kind of isoTAT unnatural base triphosphate, is characterized in that its structural formula is:

The preparation method of isoTAT unnatural base triphosphate of the present invention is characterized in that concrete steps are:

Step S1: Add 5-thiazole formaldehyde, pyridine, malonic acid and piperidine in sequence to the reaction vessel and mix them uniformly, and react under reflux at 100°C to generate compound a. The reaction equation of the synthesis process is as follows:

Step S2: Add compound a, tetrahydrofuran and triethylamine in sequence to the reaction vessel and mix evenly. Add DPPA dropwise under ice bath conditions. After the dropwise addition, remove the ice bath and continue to stir to form compound b. The reaction equation of the synthesis process is as follows:

Step S3: Add compound b to diphenyl ether, heat to 250°C to react to generate compound c, the reaction equation of the synthesis process is as follows:

Step S4: Add compound c, dichloromethane and N,O-bis(trimethylsilyl)acetamide in sequence to the reaction vessel and mix uniformly, stir the reaction at room temperature, and then dissolve 3, Add 5-di-o(p-tolyl)-2-deoxy-ribofuranoyl chloride to the reaction system, add anhydrous tin tetrachloride under ice bath conditions, and continue to react to form compound d after removing the ice bath, compound d is in the β configuration , the reaction equation of the synthesis process is as follows:

Step S5: Add compound d, Lawson's reagent and tetrahydrofuran into the reaction vessel in sequence and mix well, and react at 80°C to form compound e. The reaction equation of the synthesis process is as follows:

Step S6: adding compound e, methanol and sodium methoxide into the reaction vessel in sequence and mixing evenly, stirring and reacting at room temperature to form compound f, the reaction equation of the synthesis process is as follows:

Step S7: Add compound f and 1,8-bisdimethylaminonaphthalene to the eggplant-shaped bottle in sequence, then add trimethyl phosphate to dissolve, put the eggplant-shaped bottle at -15°C and add phosphorus oxychloride to react, and then put The DMF solution of tri(tetrabutylammonium) hydropyrophosphoric acid and the DMF solution of tri-n-butylamine are added to the reaction system simultaneously, and the temperature is raised to room temperature under a nitrogen atmosphere to react to generate compound g. The reaction equation of the synthesis process is as follows:

The unnatural base pair formed by the specific pairing of isoTAT unnatural base triphosphate and NaM and derivatives thereof according to the present invention are characterized in that: the non-natural base pair formed by the specific pairing of isoTAT unnatural base triphosphate and NaM The natural base pairs and their derivatives exist in the form of nucleosides, nucleotides or oligonucleotides containing the unnatural base pairs.

The isoTAT unnatural base triphosphate and G-specific pairing of unnatural base and natural base hybridization pairing and derivatives thereof according to the present invention are characterized in that: the isoTAT unnatural base triphosphate and G specific The hybrid base pairs formed by sexual pairing and their derivatives exist in the form of nucleosides, nucleotides or oligonucleotides containing the unnatural base pairs.

The isoTAT unnatural base triphosphate of the present invention and the unnatural base pair formed by specific pairing with NaM or G, the hybrid base pair and its derivatives have at least one function in the following 1)-9) Applications in products:

1) Recognition of unnatural bases NaM and TPT3 in DNA;

2) Detection of unnatural bases NaM and TPT3 in DNA;

3) Sequencing of unnatural bases NaM and TPT3 in DNA;

4) PCR amplification of DNA comprising NaM and TPT3 unnatural bases using isoTAT triphosphate;

5) Using isoTAT for dual-positioning sequencing of unknown sites containing unnatural bases NaM and TPT3;

6) Using isoTAT to detect the replication of semi-synthetic organisms containing unnatural bases NaM and TPT3;

7) Quantitative evaluation of the plasmid replication ability of semi-synthetic organisms containing unnatural bases NaM and TPT3 using isoTAT;

8) Accurate sequencing of AP damage at multiple sites of genes using isoTAT;

9) Precise sequencing of DNA aptamers using isoTAT.

It is further defined that the product is a kit or a detection product that uses any one of the above-mentioned unnatural base pairs and its derivatives as a component.

Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention uses unnatural base nucleotides capable of recognizing NaM-TPT3 and specific natural bases in DNA as the creation target, through pre-steady-state dynamics and The double-pairing function of isoTAT unnatural bases was determined by the steady-state kinetic method, revealing that isoTAT can pair with NaM bases with high ^specificity . Highly specific pairing, the pairing ability of isoTAT and G is Km/Vmax=2.14×10 ⁸ . DNA containing NaM-TPT3 unnatural bases is amplified by PCR in the presence of isoTAT unnatural base nucleoside triphosphate disoTATTP and four natural base nucleoside triphosphates dNTPs, and the NaM-TPT3 base in the DNA chain can be effectively are converted to GC bases, while other natural bases remain completely unchanged.

Using the function of isoTAT to convert NaM-TPT3 bases into G-C bases and the sequence preference of NaM bases, one or more unknown sites of NaM-TPT3 bases in the DNA chain can be amplified by two PCRs. Incremental and Sanger sequencing methods for accurate positioning. isoTAT directional conversion of NaM-TPT3 bases to G-C bases has high read-through rate and low sequence dependence.

Utilizing the function of isoTAT to convert NaM-TPT3 bases into G-C bases, it can also realize accurate sequencing of NaM-TPT3 unnatural base plasmids and accurate assessment of unnatural base gene mutations in semi-synthetic organisms. By enzymatically inserting TPT3-NaM base into the AP site, and then using isoTAT-mediated PCR to locate its precise position, the sequencing of DNA samples containing multiple damage sites is realized.

Utilizing the function of isoTAT to convert NaM-TPT3 bases into G-C bases and the combination with NaM sequence preference, it is also possible to accurately sequence nucleic acid aptamers containing multiple TPT3 non-natural bases. The above-mentioned isoTAT is capable of directional conversion of NaM-TPT3 bases to G-C bases, providing a general and convenient method, and for the first time, the DNA samples containing TPT3-NaM unnatural bases with unlimited sites and quantities are realized. Accurately locate, track and sequence.

Description of drawings

Figure 1 shows the structural design of the unnatural base isoTAT and the function of converting NaM-TPT3 bases into G-C bases.

Figure 2 shows the kinetic parameters of the unnatural base isoTAT paired with NaM and G in the pre-steady state and steady state.

Fig. 3 is the PCR reaction and Sanger sequencing of converting NaM-TPT3 bases into G-C bases by isoTAT.

Fig. 4 is the preference analysis of isoTAT converting NaM-TPT3 base into G-C base sequence.

Figure 5 is the precise sequencing of the NaM-TPT3 non-natural base plasmid and the accurate assessment of the non-natural base gene mutation using isoTAT in semi-synthetic organisms.

Figure 6 shows that the enzymatic TPT3-NaM base is inserted into the AP site, and then its precise position is positioned using isoTAT-mediated PCR to realize the sequencing of DNA samples containing multiple damaged AP sites.

Figure 7 is the standard curve of sample A mixed with B sequencing, and the signal intensity is calculated by reverse C.

Figure 8 is a sample C mixed B sequencing standard curve, calculated using the reversed three C signals.

Figure 9 shows the mutation rate of G on the sense strand of DNA in deep sequencing of DNA fragments after amplification by isoTAT-assisted PCR.

Detailed ways

The above-mentioned contents of the present invention are described in further detail below through the embodiments, but this should not be interpreted as the scope of the above-mentioned themes of the present invention being limited to the following embodiments, and all technologies realized based on the above-mentioned contents of the present invention all belong to the scope of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

Example 1

Synthesis, purification and structure identification of unnatural base isoTAT nucleoside triphosphate

Add 5-thiazoleformaldehyde (10.0g, 88.4mmol), pyridine 50mL, malonic acid (13.8g, 132.7mmol), piperidine 1.3mL (1.1g, 13.3mmol) into a 250mL eggplant-shaped bottle in sequence, place in oil Reflux at 100° C. for 8 hours under bath conditions, monitor the reaction with TCL until the raw materials are completely reacted, then stop the reaction. After cooling to room temperature, the reaction solution was poured into 150 mL of ice water, and 3M hydrochloric acid was added dropwise with constant stirring. During the dropwise addition, a white solid was continuously precipitated. After the precipitation was complete, the solid was filtered with suction and washed with cold water, and dried in vacuo to obtain 12.0 g White solid compound 9, yield 87.5%.

Compound 9 (12.0g, 77.3mmol), 50mL of dry tetrahydrofuran, and 12.9mL of dry triethylamine (9.4g, 92.8mmol) were successively added to a 250mL round-bottomed flask, and 18.3mL of DPPA was slowly added dropwise under ice bath conditions ( 23.4g, 85.1mmol), after the addition, the ice bath was removed to continue the stirring reaction, and the reaction was monitored by TCL until the reaction of compound 9 was complete, and then the reaction was stopped. The solvent was removed by a rotary evaporator, and 10.9 g of white solid compound 10 was obtained by column chromatography, with a yield of 78.4%.

Add 200mL of diphenyl ether into a 500mL round bottom flask and heat to 250°C. Dissolve compound 10 (5.0g, 27.7mmol) in an appropriate amount of dry dichloromethane and slowly add it to the reaction system. Monitor the reaction with TLC until the compound 10 Stop the reaction after the reaction is complete. After separation by column chromatography, 1.9 g of tan solid 11 was obtained, with a yield of 45.2%.

Under the protection of nitrogen, compound 11 (340mg, 2.24mmol), dry dichloromethane 10mL, N,O-bis(trimethylsilyl)acetamide (500mg, 2.5mmol) were sequentially added into a 100mL eggplant-shaped flask, The reaction was stirred at room temperature for 40 min, and 3,5-di-o-(p-tolyl)-2-deoxy-ribofuranoyl chloride (956.8 mg, 2.46 mmol) was dissolved with an appropriate amount of dry dichloromethane and added to the reaction system. Anhydrous tin tetrachloride (290.8 mg, 1.12 mmol) was slowly added dropwise to the system in an ice bath, and the ice bath was removed to continue stirring. The reaction was monitored by TLC. After compound 11 was completely reacted, an appropriate amount of saturated sodium bicarbonate solution was added to quench the reaction, extracted with dichloromethane and saturated brine, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was removed by a rotary evaporator. After separation by column chromatography, 484 mg of white solid compound 12 (β-configuration) was obtained, with a yield of 42.9%.

The chemical synthesis route of isoTAT nucleoside triphosphate

Under nitrogen protection, compound 12 (56mg, 0.11mmol), Lawson’s reagent (67.4mg, 0.17mmol), and 8mL tetrahydrofuran were successively added to a 50mL round-bottomed flask, and reacted overnight in an oil bath at 80°C, and the reaction was detected by TLC The solvent was spin-dried by a rotary evaporator, and 29 mg of light yellow solid compound 13 was obtained by column chromatography, with a yield of 45.6%. Add compound 13 (120mg, 0.1mmol), 5mL of methanol, and sodium methoxide (49.8mg, 0.9mmol) to a 50mL eggplant-shaped flask in sequence, and stir the reaction at room temperature until the reaction solution is clear. The reaction is detected by TLC, and the reaction of compound 13 is complete. Finally, the solvent was evaporated to dryness by a rotary evaporator and separated by column chromatography to obtain 46 mg of light yellow solid compound 14 with a yield of 70.7%. Add compound 14 (20mg, 0.07mmol) and 1,8-bisdimethylaminonaphthalene (20mg, 0.07mmol) into a 10mL eggplant-shaped flask in sequence, pump and exchange air three times, and add 320μL trimethyl phosphate under nitrogen atmosphere The reactants were dissolved, and the reaction bottle was placed in an ice-salt bath at -15°C, then phosphorus oxychloride (14 mg, 0.07 mmol) was added and reacted at -15°C for 3 hours. Tris(tetrabutylammonium)hydropyrophosphoric acid (364mg, 0.36mmol dissolved in 760μL dry DMF) and tri-n-butylamine (77.8mg, 0.4mmol) were simultaneously added to the reaction system, and heated at -10°C and -5°C React for 10 minutes respectively, react at 0° C. for 5 minutes, and finally add 0.3 mL of 1.2 M TEAB buffer solution to quench the reaction. The crude product was first separated by DEAEsephdexA25, then separated by high performance liquid chromatography and freeze-dried to obtain 3.6 mg of pale yellow solid compound disoTATTP with a yield of 10%.

Structural identification data of disoTATTP and its key intermediates:

Compound 9: 1H NMR (400MHz, MeOD) δ9.04(s,1H),8.11(s,1H),7.89-7.85(d,J=16,1H),6.33-6.29(d,J=16,1H ). 13C NMR (101 MHz, MeOD) δ 168.04, 155.98, 145.47, 135.06, 133.66, 121.07. Compound 10: 1HNMR (400MHz, CDCl3) δ8.82(s, 1H), 8.03(s, 1H), 7.88-7.84(d, J=16,1H), 6.21-6.17(d, J=16,1H) .13C NMR (101 MHz, CDCl3) δ 171.14, 155.59, 147.42, 135.35, 134.44, 121.53. Compound 11: 1H NMR (400MHz, DMSO) δ11.70(s, 1H), 9.14(s, 1H), 7.37-7.35(d, J=8, 1H), 6.96-6.95(d, J=4, 1H ). 13C NMR (101 MHz, DMSO) δ 158.00, 153.54, 144.78, 144.51, 131.46, 100.06. Compound 12: 1H NMR (400MHz, CDCl3) δ = 8.83 (s, 1H), 7.98-7.96 (d, J = 8, 2H), 7.89-7.87 (d, J = 8, 2H), 7.70-7.68 (d ,J=8,1H),7.29-7.27(d,J=8,2H),7.21-7.19(d,J=8,2H),6.85-6.82(q,J=4,1H),6.65-6.63 (d,J=4,1H),5.66-5.63(m,1H),4.79-4.68(m,2H),4.64-4.62(q,J=4,1H),3.07-3.02(m,1H), 2.43(s,3H),2.39(s,3H),2.36-2.29(m,1H).13C NMR(101MHz,CDCl3)δ166.20,166.16,157.15,151.82,144.51,144.31,144.15,143.09,129.91,129. 57, 129.33, 129.30, 127.99, 126.64, 126.39, 100.06, 86.08, 83.28, 75.10, 67.79, 64.34, 46.85, 39.43, 33.55, 21.76, 21.71, 20.62. Compound 13: 1H NMR (400MHz, CDCl3) δ8.99(s, 1H), 8.17-8.16(d, J=4, 1H), 7.99-7.97(m, 2H), 7.89-7.87(m, 2H), 7.45-7.42(q,J=4,1H),7.29-7.26(d,J=12,2H),7.22-7.20(d,J=8,2H),7.04-7.02(d,J=8,H ),5.65-5.64(m,1H),4.87-4.74(m,2H),4.71-4.68(m,1H),3.48-3.42(m,1H),2.44(s,3H),2.40(s,3H ),2.30-2.22(m,1H).13C NMR(101MHz,CDCl3)δ166.16,158.08,154.71,154.44,154.07,150.09,144.58,144.47,140.83,138.36,131.38,130.06,129 .93, 129.59, 129.39, 129.33, 127.50, 126.53, 126.31, 109.76, 105.61, 97.58, 91.40, 84.42, 83.81, 74.55, 64.08, 38.79, 29.71, 21.78, 21.72, 1.03. Compound 14: 1HNMR (400MHz, MeOD) δ9.19(s, 1H), 8.58-8.56(d, J=8, 1H), 7.44-7.42(d, J=8, 1H), 7.31-7.28(t, J=4,1H),4.45-4.41(m,1H),4.09-4.06(q,J=4,1H),3.96-3.82(m,1H),2.83-2.77(m,1H),2.17-2.11 (m,1H).13C NMR (101 MHz, MeOD) δ 174.35, 155.92, 153.70, 138.96, 131.42, 105.74, 91.13, 88.25, 69.84, 60.79, 41.13. disoTATTP: 31PNMR (162MHz, D2O) δ-9.10, -11.27, -11.39, -22.72, -22.85, -22.97.

Example 2

Recognition and pairing of unnatural base isoTAT with NaM base and G base (as shown in Figure 2)

1. Pre-steady state kinetics study Single nucleotide insertion experiment

1. The 45-mer DNA template containing NaM, TPT3 or a natural base at the 24th base and the 23-mer primer labeled with a fluorescent label at the 5' end are annealed and combined to form a template/primer complex (2.25 pmol). 2. Mix the template/primer complex with 4.5U KF(exo-) DNA polymerase, and pre-stabilize at 37°C for 1 min. 3. Add the triphosphate of isoTAT to start the reaction, and the reaction time is 15s. 4. Add 0.05M EDTA with pH 8.0 to terminate the reaction, and spin dry to remove water. 5. Add 4 μL of 1×single-strand sample loading buffer to the reaction residue, perform quantitative analysis on 15 wt% deformed polyacrylamide gel electrophoresis, Amersham Imager 680 imaging, and AI600 imaging analysis software. 6. Calculate the ratio of n+1 (primer 24-mer product) product to the total amount of primers, that is, the yield of n+1 product.

The pairing efficiency of isoTAT and NaM is 90.63%, and the pairing efficiency of isoTAT and G base is 94.94%. The pairing efficiencies of isoTAT with TPT3 and other natural bases A, T, and C are all lower than 5%. The results are shown in Figures 2B and 2C. The results of single nucleotide insertion experiments showed that isoTAT can specifically recognize and pair with NaM and G bases without interfering with the recognition and pairing of other natural and unnatural bases.

2. Determination of steady-state kinetic parameters

In order to further test the ability of isoTAT to pair with NaM, G and its natural and unnatural bases, the kinetic parameters of single nucleotide insertion were determined. The determination of kinetic parameters was carried out under steady state conditions.

1. Dissolve 9 pmol template and primer in 1× reaction buffer, anneal and combine to form template/primer complex. 2. Mix the template/primer complex with 0.225U KF(exo-)DNA polymerase, and pre-stabilize at 37°C for 1min. 3. Add different concentrations of nucleotides to start the reaction, and the reaction time is 10s. 4. Immediately add 0.05M EDTA with pH 8.0 to terminate the reaction, and spin dry to remove water. 5. Add 4 μL of 1×single-strand sample loading buffer to the reaction residue, perform quantitative analysis on 15 wt% deformed polyacrylamide gel electrophoresis, Amersham Imager 680 imaging, and AI600 imaging analysis software. 6. Calculate the ratio of the n+1 (primer 24-mer product) product to the total amount of primers, that is, the yield of the n+1 product, and calculate the initial speed of the single-base insertion reaction of different concentrations of nucleotides. 7. Use GraphPad Prism 8 to fit the Michaelis-Menten curve, and calculate the kinetic parameters such as Vmax, Km and Vmax/Km of the reaction.

The results are shown in Figure 2. The Km/Vmax value of isoTAT and NaM recognition pairing is 0.77×10 ⁸ , and the Km/Vmax value of isoTAT and G recognition pairing is 2.14×10 ⁸ . In contrast, under the same conditions, the reaction rate of isoTAT and T, A, C recognition pairing was lower than the detection limit. Therefore isoTAT can specifically recognize and pair with NaM and G bases.

Example 3

Utilize unnatural base IsoTAT unnatural base pair to comprise the PCR amplification and sequencing of TPT3-NaM base DNA (as shown in Figure 3)

① Preparation of DNA double-stranded template containing dNaM-PTP3

To verify the base specificity of disoTAT and dNaM substitution PCR, we first prepared a double strand containing dNaM-PTP3 using a custom-made single-stranded DNA template, and used this as a template for substitution PCR.

DNA template sequence (single strand containing dNaM):

CACACAGGAAACAGCTATGACCCGGGTTATTACATGCGCTAGCACTTGGAATTCACAACCGGNaMATCCCGAGGAAACCATAGTAAATCTCTTCTTAAAGTTAAGCTTAACCCTATAGTGAGTCGTATTAATTTC

Primers:

Fend T-F

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACACAGGAAACAGCTATGAC

Fend T-R

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAATTAATACGACTCACTATAGG

2 x OneTaq DNA polymerase, dNaMTP and disoTATTP

Reaction system: 12.5 μL DNA polymerase, 100 μM unnatural base triphosphate dNaMTP and disoTATTP, 0.4 μM primer, 0.4ng template, ultrapure water was added to the reaction system to 25 μL. Subsequent PCR amplification reaction systems all refer to this reaction system, and only the template and unnatural base triphosphate species are different.

Mix the reaction system and put it into the PCR machine. The operation program is as follows (the subsequent PCR operation program is consistent with this program): a: denaturation at 96°C for 10 seconds; b: annealing at 60°C for 15 seconds; c: extension at 68°C for 1 minute; d: Repeat steps a, b, and c for 35 cycles; e: extend at 68°C for 5 minutes. Take 3 μL of the reaction system and add 0.5 μL of loading buffer to run agarose gel electrophoresis. The target band is displayed at 250 bp, and the remaining samples are subjected to Sanger sequencing (sequencing in both forward and reverse directions).

② disoTATTP base substitution PCR

Using the DNA double-stranded template containing dNaM-dTPT3 obtained in ①, name the template 1N template, then add disoTATTP-dNaMTP to the PCR reaction system, and base substitution results through sequencing results.

1N template:

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCA

CACAGGAAACAGCTATGACCCGGGTTATTACATGCGCTAGCACTTGGAATTCACAA

CCGGNaMATCCCGAGGAAACCATAGTAAATCTCCTTCTAAAGTTAAGCTTAACCC

TATAGTGAGTCGTATTAATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAA

Reaction system: 12.5 μL OneTaq DNA polymerase, 0.4 μM primer, 0.4 ng template 1N, 100 μM dNaMTP, 100 μM disoTATTP, ultrapure water added to the reaction system to 25 μL.

Sample 1: 1N Template, One Taq DNA Polymerase, dNaMTP

Sample 2: 1N Template, One Taq DNA Polymerase, disoTATTP and dNaMTP

Analysis of experimental results: In the PCR reaction system with only dNaMTP added, the position of the unnatural base dNaM will be read as T. This is because dNaM is more likely to pair with A when there is no dTPT3 in the system, resulting in dNaM being read during sequencing In the PCR system adding disoTATTP, the position of NaM unnatural base will be replaced by G. The DNA sequence analysis with disoTATTP-dNaMTP shows that isoTAT will replace most or all of the complementary paired unnatural base sites in DNA with natural bases.

Example 4

Accurate sequencing of NaM-TPT3 unnatural base plasmids and accurate assessment of unnatural base gene mutations in semi-synthetic organisms using isoTAT (as shown in Figure 5)

Experiments have proved that disoTAT can replace the unnatural base NaM in the template with G, and mix the replaced DNA solution with other natural templates in proportion for sequencing. The ratio and the ratio of the G-C template were used as a standard curve. The establishment of a standard curve can be used to calculate the retention rate of unnatural bases in cells containing unnatural base pair amplification.

① DNA template and primers used in the experiment

1N template with one NaM:

CACACAGGAAACAGCTATGACCCGGGTTATTACATGCGCTAGCACTTGGAATTCAC

AACCGGNaMATCCCGAGGAAACCATAGTAAATCTCCTTCTTAAAGTTAAGCTTAAC

CCTATAGTGAGTCGTATTAATTTC

Natural template:

CACACAGGAAACAGCTATGACCCGGGTTATTACATGCGCTAGCACTTGGAATTCAC

AATACTTTCTTTAAGGAAACCATAGTAAATCTCTTCTTAAAGTTAAGCTTAACCCT

ATAGTGAGTCGTATTAATTTC

3N template:

CACACAGGAAACAGCTATGACCCGGGTTATTACATGCGCTAGCACTTGGNaMATTC

ACAATACTNaMTCTTTAAGGAAACCNaMTAGTAAATCTCTCTTCTTAAAGTTAAGCT

TAACCCTATAGTGAGTCGTATTAATTTC

Note: The difference between the 1N template and the natural template is underlined. The unnatural base NaM corresponds to the natural base T in the natural template; the first and third unnatural bases of the 3N template correspond to A in the natural template. Marked in bold italics.

Primers: long primers containing poly-T, consistent with the PCR amplification primers in Example 2. 2 x OneTaq DNA polymerase, disoTATTP and dNaMTP.

②Experimental methods and results

reaction system:

Sample A: 12.5 μL 2×OneTaq DNA polymerase, 0.4 ng non-native template 1N, 0.4 μM primer, 100 μM disoTATTP and dNaMTP, add water to 25 μL.

Sample B: 12.5 μL 2×OneTaq DNA polymerase, 0.4 ng native template, 0.4 μM primer, add water to 25 μL.

Sample C: 12.5 μL 2×OneTaq DNA polymerase, 0.4 ng non-native template 3N, 0.4 μM primer, 100 μM disoTATTP and dNaMTP, add water to 25 μL.

The PCR reaction procedure was the same as above.

After the reaction, the PCR products were recovered by agarose gel electrophoresis and gel recovery kit to obtain samples A, B and C containing the target bands. (Because the sequencing sample requires a certain concentration, the above reactions are more than one group). Samples A, B, and C were quantified to the same concentration and then mixed uniformly in different proportions and sent for sequencing.

Sequencing sample ①: 100% A Sequencing sample ②: 90% A and 10% B

Sequencing sample ③: 80% A and 20% B Sequencing sample ④: 70% A and 30% B

Sequencing sample ⑤: 60% A and 40% B Sequencing sample ⑥: 50% A and 50% B

Sequencing sample ⑦: 40% A and 60% B Sequencing sample ⑧: 30% A and 70% B

Sequencing sample ⑨: 20% A and 80% B Sequencing sample ⑩: 100% B

Samples B and C (sequencing samples 11-20) were mixed in the same mixing method, and the sequencing results are as follows:

The unnatural base pair in sample A was replaced by isoTAT with G-C, and the corresponding position of the natural base pair in sample B was T-A; the corresponding positions of the three unnatural base pairs in sample C were all replaced with G-C, and The corresponding ones are A-T, T-A, A-T respectively. As the B samples gradually increased, the signal value of T corresponding to the unnatural base gradually increased. With the gradual reduction of A samples, the signal of unnatural base replaced by G gradually decreased. The ratio of sample A/C is different, and the ratio of unnatural base signal intensity is also different.

Calculation of the signal intensity ratio of unnatural bases (all based on the original sequencing signal): Take the 1N reverse unnatural base site C as an example: the signal intensity ratio of C = the sequencing signal of C / all after the unnatural base Mean signal value of C. The signal intensity ratio and the actual sample ratio were used as a standard curve as shown in Figure 7.

From the standard curve of the 3N template (Figure 8), it can be seen that even if it is the same sequence, the standard curve obtained by not the same unnatural base is different, which may be due to the different sequencing signal intensities at different sites. The establishment of a standard curve helps to calculate the retention rate of unnatural base pairs in cells. Using semi-synthetic organisms containing unnatural bases as templates for PCR amplification and sequencing, by calculating the signal intensity ratio of unnatural bases in the sequence, the retention rate is calculated according to the standard curve.

In-cell experiment verification standard curve:

Plasmid-expressed PtNTT2 was synthesized using GenScript. At the same time, the 134-mer template containing dNaM was amplified by PCR with OneTaq DNA polymerase, including a 1N template containing a pair of unnatural bases and a 3N template containing three pairs of unnatural bases. A linear fragment was amplified from pBLUE-T. The product was purified using a DNA Gel Extraction Kit. The linear fragment was combined with the 134-mer template using circular overlap extension PCR.

The PCR operation procedure is the same as above. The PCR products were analyzed by restriction endonuclease digestion and directly used for Escherichia coli transformation. The dTPT3 was incubated with E. coli with different templates alone for 17 hours, and the cell growth was detected. Finally, polymerase chain reaction was performed using Escherichia coli cells as a template, and the reaction system was sequenced.

The sequencing results are shown in the figure:

In vivo sequencing retention rate calculation

Analysis of experimental results: It can be seen from Figure 5 that the sequencing results of adding unnatural base pair dTPT3-dNaM have attenuation, which proves that the unnatural base pair has been successfully incorporated into the cell. Base sequence retention and fidelity. The fidelity is calculated from the sequencing raw data, and the fidelity is calculated using the standard curve. The retention rate ① was calculated from the standard curve, and the retention rate ② was calculated from the sequencing data. It is currently impossible to calculate the retention rate of multiple pairs of unnatural bases, but it can be calculated through the standard curve. It is normal that there is a difference between the actual calculation and the formula calculation. Unnatural bases will be amplified in the body and some bases will be lost, which is the reason for the high calculation results. The establishment of the standard curve is helpful to calculate the retention rate of sequences containing multiple pairs of unnatural bases in E. coli cells.

Example 5

By enzymatically inserting TPT3-NaM base into the AP site, and then using isoTAT-mediated PCR to locate its precise position, the sequencing of DNA samples containing multiple damaged AP sites is realized (Figure 6)

Using TPT3 to mark the generated AP site after dU cleavage in DNA

KRAS-1U-F or 2U-F and KRAS-R or 134-2U-F and 134-2U-R (0.2 μM, 50 μL) were annealed in AB cloning buffer B to form double-stranded DNA. UDG (1 U) was added to the reaction mixture and incubated at 37°C for 30 minutes. APE1 (10 U) was then added to the reaction mixture, incubated at 37°C for 1 hour, and heated at 95°C for 10 minutes. Next, dTPT3TPbiotin (30 μM) and Kf(exo-) DNA polymerase (7 U) were added to the reaction mixture, reacted at 37° C. for 1 h, and heated to 95° C. to terminate the reaction. Finally, T4-DNA ligase (200 U), dimethyl sulfoxide (10% (v/v)) and ATP (0.2 mM) were added to the reaction system, and incubated at 25° C. for 1 h. The above reaction steps were monitored by 20% denaturing PAGE gel.

The processes of gap formation reaction and ligation reaction were analyzed by denaturing PAGE gel. Two shorter DNA strands were found after treatment of template DNA with APE1, and the full-length product could be detected after adding T4-DNA ligase. Then, the reaction product was used for PCR amplification with dTPT3TPbiotin and NaM, and the PCR product was conveniently enriched by biotin-streptavidin-based strand translocation.

Isolation of Marked DNA and PCR Amplification of TPT3 Marked AP Site DNA

The above final reaction solution (KRAS-1U or 134-2U) was used as a template for further PCR assays. First, the labeled DNA was further labeled and amplified by PCR, template (final reaction solution, 1 μL), dNTP (400 μM), dTPT3TPbiotin (20 μM), dNaMTP (50 μM) and MgSO4 (2.2 mM), primers (400 nM each) OneTaq DNA Polymerase (0.018U/μL) and DeepVent DNA polymerase (0.007U/μL), in 1× reaction buffer (total 25 μL), under thermocycling conditions: 20× (96°C, 30s; 50°C, 10s ; 68°C, 4min), and a final extension at 68°C, 5min. The product (5 μL) was incubated with streptavidin (1 μg, Solarbio) at 37° C. for 30 min. Samples were then mixed with loading dye and separated by 6% (134-2U) or 10% (KRAS-1U) native polyacrylamide gel electrophoresis. The shifted strips were eluted by shaking and soaking at 37°C for 2 hours, ^TM OneC was quantified by NanoDrop, and used as template for PCR (0.5-2 ng per sample) for PCR amplification.

For DNA samples containing one dU site, the dU site substituted with TPT3 biotin was transferred to C, while the original dU site was transferred to T. For DNA samples containing two dU sites, the dU sites substituted with TPT3 biotin were both transferred to C, while the original dU sites were both transferred to T. The results demonstrate that TPT3 biotin can insert into dU lesion sites and transfer to C by bridging PCR, and dU can be identified in this model system.

Preparation of plasmids containing multiple AP damage sites

Transform the pUC-19 plasmid into E. coli DH5α cells, grow a single colony in 2 mL of LB medium overnight at 37 °C, then dilute 10 μL of the culture with fresh LB medium and incubate at 37 °C at 230 rpm until OD ₆₀₀ =1 for H ₂ O ₂ treatment. _H2O2 was added to the culture at a final concentration _of 1 mM to generate AP sites. After standing at ambient temperature for 30 min, cells were harvested by centrifugation at ambient temperature and plasmid DNA was extracted by a plasmid isolation kit following the manufacturer's protocol (OMEGA). Plasmids from cells not treated with _H2O2 were _used as controls.

The AP site in plasmid DNA was tagged with the unnatural nucleotide dTPT3TPbiotin, the tagged DNA was isolated, and isoTAT-assisted PCR and deep sequencing were used as described above. We found that the mutation rates of A to C and C to A were selectively increased at certain loci. In addition, we also found that some G and T sites had significantly increased mutation rates, most of the sites with increased A or C signal overlapped well, and especially these sites increased the most, while other sites had mutation ratios close to baseline (Fig. 9 ). These suggest that AP sites may accumulate in some preferential regions, and our bridging method can be used to identify AP damage sites in DNA.

The above embodiments have described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What are described in the above embodiments and description are only to illustrate the principles of the present invention. Without departing from the scope of the principle of the present invention, there will be various changes and improvements in the present invention, and these changes and improvements all fall within the protection scope of the present invention.

Claims (6)

1. An isoTAT non-natural base triphosphate is characterized by the structural formula:

2. a method for preparing the isoctat non-natural base triphosphate according to claim 1, which is characterized by comprising the following specific steps:

step S1: sequentially adding 5-thiazole formaldehyde, pyridine, malonic acid and piperidine into a reaction vessel, uniformly mixing, and carrying out reflux reaction at 100 ℃ to generate a compound a, wherein the reaction equation in the synthesis process is as follows:

step S2: sequentially adding the compound a, tetrahydrofuran and triethylamine into a reaction vessel, uniformly mixing, dropwise adding DPPA under the ice bath condition, removing the ice bath after the dropwise adding is finished, and continuously stirring to react to generate the compound b, wherein the reaction equation in the synthesis process is as follows:

step S3: the compound b is added into diphenyl ether, and is heated to 250 ℃ to react to form the compound c, and the reaction equation of the synthesis process is as follows:

step S4: sequentially adding a compound c, methylene dichloride and N, O-bis (trimethylsilyl) acetamide into a reaction vessel, uniformly mixing, stirring at room temperature for reaction, adding 3, 5-di-O- (p-tolyl) -2-deoxy-ribofuranosyl chloride dissolved in methylene dichloride into a reaction system, adding anhydrous tin tetrachloride under ice bath condition, removing the ice bath, and continuing to react to generate a compound d which is beta-configuration, wherein the reaction equation of the synthesis process is as follows:

step S5: sequentially adding the compound d, the Lawson reagent and tetrahydrofuran into a reaction vessel, uniformly mixing, and reacting at 80 ℃ to generate a compound e, wherein the reaction equation in the synthesis process is as follows:

step S6: sequentially adding the compound e, methanol and sodium methoxide into a reaction vessel, uniformly mixing, stirring and reacting at room temperature to generate a compound f, wherein the reaction equation in the synthesis process is as follows:

step S7: sequentially adding a compound f and 1, 8-bis (dimethylamino) naphthalene into a eggplant-shaped bottle, adding trimethyl phosphate for dissolution, placing the eggplant-shaped bottle at-15 ℃ and adding phosphorus oxychloride for reaction, then simultaneously adding a DMF solution of tri (tetrabutylammonium) hydrogen pyrophosphoric acid and a DMF solution of tri (n-butylamine) into a reaction system, and heating to room temperature under nitrogen atmosphere for reaction to generate a compound g, wherein the reaction equation of the synthesis process is as follows:

3. the non-natural base pair formed by specific pairing of an isocrata non-natural base triphosphate and NaM as claimed in claim 1 and its derivatives, characterized in that: the non-natural base pair formed by specific pairing of the isocrata non-natural base triphosphate and NaM and the derivative thereof exist in the form of a nucleoside, a nucleotide or an oligonucleotide containing the non-natural base pair.

4. The non-natural base hybridization pairing and derivatives thereof formed by the non-natural base triphosphate and G-specific pairing of isoTAT according to claim 1, characterized in that: the hybridized base pair formed by the specific pairing of the isocratic acid and the G and the derivative thereof exist in the form of nucleoside, nucleotide or oligonucleotide containing the unnatural base pair.

5. Use of the non-natural base pairs formed by specific pairing of the iso tat non-natural base triphosphate and NaM or G, hybrid base pairs and derivatives thereof according to claim 1 for the preparation of a product having at least one of the following functions 1) -9):

1) Recognition of the unnatural bases NaM and TPT3 in DNA;

2) Detecting unnatural bases NaM and TPT3 in DNA;

3) Sequencing of unnatural bases NaM and TPT3 in DNA;

4) PCR amplification of DNA containing NaM and TPT3 unnatural bases using isoTAT triphosphate;

5) Double-position sequencing of unknown sites containing non-natural bases NaM and TPT3 by using isoTAT;

6) Detection of semisynthetic living plasmid replication comprising the unnatural bases NaM and TPT3 using isoTAT;

7) Quantitative assessment of the ability of semisynthetic life bodies to replicate comprising the unnatural bases NaM and TPT3 using isoTAT;

8) Precise sequencing of gene multi-site AP injury by using isoTAT;

9) Precise sequencing of DNA aptamers using isoTAT.

6. The use according to claim 5, characterized in that: the product is a kit or a detection product which takes one of any one of the unnatural base pairs and derivatives thereof as a component.

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