阅读说明：本技术 硒原子修饰在降低dNTP亲和力和DNA解链温度中的应用 (Application of selenium atom modification in reducing dNTP affinity and DNA melting temperature ) 是由 黄震 罗光成 张军 于 2021-06-24 设计创作，主要内容包括：本发明公开了一种硒原子修饰在降低dNTP亲和力和DNA解链温度中的应用,采用硒原子修饰的dNTP(即硒代磷酸dNTP,dNTPαSe)作为反应底物,用以直接降低dNTP与DNA聚合酶之间的亲和力,以及降低产物DNA的Tm值,从而提升了DNA聚合反应的准确性。本发明不额外添加外源性化合物、不改变反应步骤、不增加反应体系复杂程度的前提下,仅通过对dNTP进行硒修饰即可实现dNTP亲和力的降低和产物DNA的Tm值降低。(The invention discloses an application of selenium atom modification in reducing dNTP affinity and DNA melting temperature, wherein dNTP (namely selenophosphoric acid dNTP, dNTP alpha Se) modified by selenium atoms is used as a reaction substrate to directly reduce the affinity between the dNTP and DNA polymerase and reduce the Tm value of product DNA, thereby improving the accuracy of DNA polymerization reaction. According to the invention, the reduction of dNTP affinity and the Tm value of product DNA can be realized only by selenium modification of dNTP on the premise of not additionally adding an exogenous compound, not changing reaction steps and not increasing the complexity of a reaction system.)

1. The application of selenophosphate modification in reducing the affinity of dNTP and DNA polymerase is characterized in that the affinity of the DNA polymerase is reduced by adopting the dNTP modified by selenium atoms.

2. The use of claim 1, wherein a selenium atom modified dNTP is used as a reaction substrate.

3. The use according to claim 1, wherein the DNA polymerase is a reverse transcription functional polymerase.

4. Use of a phosphoroselenoate modification to lower the melting temperature of a DNA, wherein said DNA is a DNA-DNA duplex or a DNA-RNA duplex.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to application of selenium atom modification in reducing dNTP affinity and DNA melting temperature.

Background

Nucleic acid amplification technology is one of the most widely used biotechnology in the life science field, and plays an important role in scientific research, disease diagnosis and infectious disease prevention and control. The basic reaction step for both in vivo genetic material replication and in vitro DNA amplification is DNA polymerization.The DNA polymerization reaction has high accuracy in vivo, and the error rates of the bacterial system and the mammalian system are respectively 5X 10^-10And 5X 10^-11. However, the accuracy of in vitro DNA polymerization decreases by 4-5 orders of magnitude, and polymerases such as pol α, pol δ, pol ε, and pol III have in vitro error rates of 10^-4To 10^-5In the meantime. The reason for this is that the reaction environment in vitro is changed greatly and a repair system is lacking. A decrease in polymerase accuracy will lead to an increased risk of non-specific amplification of the in vitro DNA amplification reaction, which presents a huge challenge for the development of high performance nucleic acid detection techniques.

In order to improve the specificity of in vitro DNA polymerization, PCR additives and chemically modified nucleotides are widely used in nucleic acid detection technology. PCR additives are generally low molecular weight compounds such as betaine, dimethyl sulfoxide (DMSO), formamide, and the like. These PCR additives can lower the melting temperature (Tm) of nucleic acids and act as a physical barrier preventing hybridization between nucleic acids, thereby enhancing the specificity of the PCR reaction. Another strategy to improve detection performance is to use chemically modified nucleotides such as Peptide Nucleic Acids (PNA), Locked Nucleic Acids (LNA), AEGIS nucleotides, and 2-selenomethyl thymine. These modified nucleotides have a function of reducing electrostatic repulsion between DNA strands, or increasing the accuracy of base pairing, thereby changing the analytical performance of the nucleic acid amplification technique.

Previous studies on the structural function of polymerases have shown that: the accuracy of DNA polymerization reaction is closely related to dNTP recognition, polymerization rate, mismatch correction and the like. dNTP recognition is an important link for ensuring the accuracy of polymerization reaction, and the link is completed by a finger structural domain of DNA polymerase. Correctly paired dntps have a higher affinity for the polymerase finger domain and are polymerized onto the primer, and incorrectly paired dntps are knocked out by the polymerase finger domain due to weaker binding. Therefore, the affinity between dNTP and polymerase, i.e., dNTP affinity, is a key factor affecting the accuracy of DNA polymerization. Furthermore, the Melting temperature (Tm) of DNA is also a key factor affecting the accuracy of DNA polymerization, and lowering the Tm value of nucleic acids is expected to improve the accuracy of DNA polymerization. It is known that increasing the annealing temperature of PCR can promote melting of double-stranded DNA, thereby reducing the generation of non-specific hybridization and enhancing the specificity of PCR. This suggests that, when the reaction temperature is kept constant, lowering the Tm value of the double-stranded DNA may reduce the occurrence of non-specific hybridization and enhance the specificity of PCR. Based on the above theoretical basis, we propose the hypothesis: the accuracy of the in vitro DNA polymerization reaction can be enhanced by properly reducing the affinity of dNTP and the Tm value of DNA.

In the prior art, the Tm value of DNA is reduced and the amplification reaction specificity is increased by adding betaine, and in general, an AT-rich DNA sequence has a lower dissolution temperature than GC-rich DNA. Therefore, this difference in thermal stability between different base sequences allows different DNA sequences to have completely different Tm values. However, a high concentration of betaine eliminates the influence of the difference in base sequence of DNA on the Tm value, and lowers the Tm value of DNA, particularly GC-rich DNA. However, in order to increase the accuracy of the DNA amplification reaction, betaine is often added to the reaction buffer as an exogenous additive, which significantly increases the complexity of the amplification system and also significantly reduces the amplification efficiency of the DNA amplification reaction. Moreover, betaine has a single function, and betaine only can reduce the Tm value of DNA but cannot reduce the affinity between a substrate (dNTP) and DNA polymerase.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a technical scheme for reducing dNTP affinity and a DNA Tm value through selenium atom modification, and the reduction of the dNTP affinity and the Tm value of a product DNA can be realized only through selenium modification on dNTP on the premise of not additionally adding an exogenous compound, not changing reaction steps and not increasing the complexity of a reaction system.

In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:

the invention provides application of selenophosphate modification in reducing the affinity of dNTP and DNA polymerase.

The invention adopts dNTP (namely selenophosphoric acid dNTP, dNTP alpha Se) modified by selenium atoms as a reaction substrate to directly reduce the affinity between the dNTP and DNA polymerase.

The polymerase is DNA polymerase, and also includes polymerase with reverse transcription function.

Meanwhile, when dNTP α Se is polymerized into Se-DNA, the Tm value of Se-dsDNA is lower than that of natural dsDNA of the same sequence.

Thus, selenophosphate modification can be used to lower the melting temperature of DNA to improve the accuracy of DNA polymerization reactions.

The mechanism by which dNTP α Se increases the accuracy of DNA polymerization is shown in FIG. 1. When a natural dNTP is used as a substrate, since the affinity of the natural dNTP to a polymerase is high, even in the presence of a mismatch in a primer-template, the dNTP can be rapidly polymerized onto the mismatched primer, resulting in non-specific amplification. When dNTP α Se is used as a substrate, since its affinity for polymerase is relatively low, when a mismatch exists in a primer-template, dNTP α Se is difficult to be polymerized onto the mismatched primer, thereby avoiding the occurrence of non-specific amplification. However, in the case of perfect primer-template matching, the polymerization reaction proceeds smoothly despite the reduced affinity of dNTP α Se for the polymerase.

The invention has the beneficial effects that:

the invention provides a technical scheme for reducing dNTP and DNA affinity and reducing DNA melting temperature through selenophosphoric acid modification, compared with natural dNTP, the affinity of dNTP alpha Se and DNA polymerase is obviously reduced (about 660 times, dNTP K)_d＝1.51×10^-3M and dNTPαSe K_d＝9.90×10^-5M). The affinity of Se-DNA for DNA polymerase is reduced by a factor of 2.8 (DNA K) compared to the corresponding native DNA_d＝3.46×10^-3M and Se-DNA K_d＝9.76×10^-3M). In addition, the stability (Tm value) of the Se-DNA double strand is much lower than that of the corresponding native dsDNA. Under the combined action of the above factors, the accuracy of the DNA polymerization reaction is obviously increased, so that the specificity of the DNA amplification reaction is obviously increased.

Drawings

FIG. 1 is a scheme of the present invention dNTP α Se to increase the accuracy of DNA polymerization reaction;

FIG. 2 is the effect of selenophosphoric acid modification on dNTP, DNA affinity;

FIG. 3 is a Tm analysis of Se-dsDNA and dsDNA of the present invention;

FIG. 4 shows the amplification of HPV16-DNA by SEA of the present invention in the presence of high background DNA concentrations; A. real-time fluorescence amplification curve diagram; B. gel electrophoresis analysis of the amplification product in A;

FIG. 5 shows the non-specific amplification of a sample containing a large amount of background DNA according to the present invention by LAMP; A. carrying out real-time fluorescence monitoring on LAMP amplification reaction; B. and (3) performing gel electrophoresis analysis on the LAMP amplification product.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isothermal titration analysis of the Effect of selenophosphoric acid modification on dNTP, DNA affinity

The affinity of natural dNTPs, natural DNA, dNTP alpha Se, Se-DNA and Bst DNA polymerase is analyzed by an ITC method. For ITC analysis, the above material was dissolved in 1 × Isoth ermal Amplification Buffer, and Bst DNA polymerase was titrated with dNTP, DNA, dNTP α Se, Se-DNA. Bst DNA polymerase (15. mu.M) was added to the sample cell of the ITC200 isothermal titrator, native dNTP, native DNA, dNTP. alpha. Se, Se-DNA (150. mu.M) was added to the syringe of the ITC200 isothermal titrator, and the titration reaction was performed at 25 ℃.

As a result, it was found (fig. 2A and 2B): the natural dNTP has a high affinity with DNA polymerase (Kd: 1.51X 10)^-7M), whereas the affinity between dNTP. alpha. Se and DNA polymerase is relatively weak (Kd: 9.90X 10)^-5M). After dNTP α Se was polymerized into Se-DNA by polymerization, it was further found (fig. 2C and 2D): Se-dsDNA (Kd 9.76X 10)^-3M) also has significantly lower affinity for DNA polymerase than native dsDNA (Kd 3.46X 10)^-3M). These results show that: selenophosphoric acid modification obviously reduces dNTP, DNA and DNA polymeraseAffinity between the two.

Example 2 analysis of the Effect of selenophosphate modification on the Tm value of DNA by the dissolution Curve method

Based on the basic thermodynamic property of dsDNA melting by heat, the present study used the fluorescent dye method (SYBR Green I) to analyze Tm values of native dsDNA and Se-dsDNA. The experiment is that96 systems, i.e., raising the temperature from 55 ℃ to 95 ℃ (0.07 ℃/s). During the temperature change, the melting curve and Tm value are obtained by continuously monitoring the change of fluorescence.

The natural dsDNA is synthesized directly by Shanghai Biotechnology Limited, and the sense strand sequence is as follows: 5'-tctgaagtagatatggcagcacatagttactgttgttgatactacacg-3' are provided. Se-dsDNA is obtained by enzymatic synthesis (with dNTP. alpha. Se as substrate). Wherein the antisense strand Se-DNA is obtained by: synthesizing natural sense strand DNA terminated by 3 'end phosphate as a template by a manufacturer, and then using an oligonucleotide with poly T at the 5' end as a primer to enzymatically synthesize an antisense strand Se-DNA (primer sequence: 5 '-tttttttttt-cgtgtagtat-3') under the catalysis of DNA polymerase; then, purified Se-DNA (which is slower in migration of the 5' end due to poly-T) was separated by 12% denaturing PAGE electrophoresis. Se-DNA of the sense strand is obtained in the same manner. Finally, Se-DNA of the positive strand and the antisense strand are mixed together to prepare Se-dsDNA.

The influence of selenophosphoric acid modification on the DNA dissolution temperature was analyzed by a fluorescent dye dissolution curve method. The results are shown in FIG. 3: when dNTP. alpha. Se is polymerized into Se-DNA, the Tm value of Se-dsDNA is lower than that of natural dsDNA of the same sequence (Se-dsDNA,61.9 ℃ C.; natural dsDNA 70.5 ℃ C.).

Example 3 Effect on the accuracy and specificity of DNA polymerization

Because dNTP alpha Se can obviously reduce dNTP affinity and reduce the Tm value of DNA, the dNTP alpha Se has the potential of increasing the accuracy and specificity of DNA polymerization reaction. To verify the potential of dNTP alpha Se, the LAMP isothermal amplification technology (when a large amount of background DNA exists in a sample, nonspecific amplification is easy to occur) is adopted as an example for experimental verification. The LAMP technique based on dNTP α Se was named: selenucleotide-enhanced specific amplification (SEA) technology.

According to the LAMP amplification principle, primers are designed and synthesized aiming at target molecules to be detected (in the embodiment, HPV16-DNA is detected as an example), and online software is adopted to carry out primer design (http:// primer explorer. jp/lampv5e/index. html). The primers were synthesized by Shanghai Biotechnology Ltd and then formulated into a primer mixture having the following concentrations of the components: 16 μ M Forward Inner Primer (FIP), 16 μ M reverse inner primer (BIP), 2 μ M forward outer primer (F3) and 2 μ M reverse outer primer (B3). Primer compositions for SEA and LAMP are identical.

Preparing a substrate mixture: the substrate mixture is composed of dNTPs (including dATP, dTTP, dCTP, dGTP) and dNTP alpha Se (including dATP alpha Se, dTTP alpha Se, dCTP alpha Se, dGTP alpha Se), and the proportion of dNTP alpha Se in the substrate mixture is different from 5-50%. In this mixture, the concentration of (dATP + dATP. alpha. Se) was 2.5mM, the concentration of (dTTP + dTTP. alpha. Se) was 2.5mM, the concentration of (dCTP + dCTP. alpha. Se) was 2.5mM, and the concentration of (dGTP + dGTP. alpha. Se) was 2.5 mM.

SEA amplification buffer (2X) was prepared: 40mM Tris-HCl (pH 8.8), 20mM (NH4)2SO4, 100mM KCl, 16mM MgSO4, 1% Tween 20, 2.5mM dNTP/dNTP α Se mix, 1.6M betaine, and 10,000 × SYBR Green I. The LAMP amplification buffer completely conforms to the SEA amplification buffer except that the LAMP amplification buffer takes natural dNTP as a substrate.

The SEA reaction system was prepared as follows, using 20. mu.L as an example: 10 μ L SEA amplification buffer (2X), 2 μ L primer mix, 1 μ L Bst 2.0DNA polymerase (8U/. mu.L), template to be tested, then add ddH2O to 20 μ L. When detecting a sample, a positive control and a negative control are set at the same time.

The prepared reaction system is placed in a fluorescence thermostat and incubated at the constant temperature of 65 ℃ for 120 minutes, and the fluorescence change of the reaction system is monitored in real time (a fluorescence signal is collected every minute).

SEA amplification of HPV 16-DNA: clinical samples often contain large amounts of background DNA, increasing the risk of non-specific amplification. To simulate real clinical sample testing, 200ng of E.coli genomic DNA was added as background to a 20. mu.l reaction in this embodiment.

The results show that: SEA maintained good amplification even with high background DNA concentrations, as shown in FIG. 4 below, with the positive control still showing a typical "sigmoidal" amplification curve and the negative control still showing no significant amplification within 120 minutes (FIG. 4A). Electrophoretic analysis of the amplified products still showed that the positive control had ladder-like specific amplified products, and the negative control had no obvious amplified products (FIG. 4B).

LAMP amplification of HPV 16-DNA: in accordance with the amplification conditions of SEA, 200ng of E.coli genomic DNA was added as a background to a 20. mu.l reaction system in the case of LAMP amplification. As shown in FIG. 5 below, in the case where a large amount of background DNA is present in the reaction system, non-specific amplification occurs by the LAMP technique based on natural dNTPs.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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Selenium Rayne Biotechnology (Chengdu) Ltd

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