CN118006617A

CN118006617A - Taq enzyme aptamer

Info

Publication number: CN118006617A
Application number: CN202211406545.3A
Authority: CN
Inventors: 蔡统聪; 范凌云; 孟媛; 卢庆庆; 林钰琼
Original assignee: Guangdong Fapon Biotech Co Ltd
Current assignee: Guangdong Fapon Biotech Co Ltd
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2024-05-10

Abstract

The invention provides an aptamer for sealing or partially sealing Taq enzyme, which relates to the field of biotechnology, has the function of inhibiting the activity of Taq enzyme at room temperature, and can effectively control the generation of primer dimer or non-specific products in the initial stage of PCR reaction.

Description

Taq enzyme aptamer

Technical Field

The invention relates to the field of biotechnology, in particular to an aptamer of Taq enzyme.

Background

Taq enzyme is a key to a number of techniques in recombinant DNA research and medical diagnosis of disease. In particular for diagnostic applications, the target nucleic acid sequence may be only a small portion of the DNA or RNA under investigation, and thus the presence of the target nucleic acid sequence may be difficult to detect without amplification. During the PCR process, since Taq enzyme is not inactivated in the denaturation step (about 94 ℃) and can directly enter the second cycle, new enzyme is not needed to be added in each cycle, which makes Taq enzyme a unique enzyme in the PCR reaction.

Because the conventional Taq enzyme active site is directly exposed and active at normal temperature, the non-specific product is easy to generate, and the PCR product contains more non-specific products or no target products. However, if the active site of Taq enzyme is blocked, the nonspecific phenomenon can be significantly improved. At present, the modification method conventionally used comprises monoclonal antibody modification, micromolecular chemical group modification and the like, but a plurality of antigen epitopes exist on the surface of Taq enzyme protein, so that a large number of antibody epitopes aiming at the sites of polymerase activity are required to be screened in the early stage, and the PCR performance of a plurality of monoclonal antibodies is required to be quantitatively verified, so that the whole operation period is longer, and the operation process is complex. Secondly, in the amplified production of the later antibody, the animal needs to be immunized, and the monoclonal antibody is isolated by collecting blood or ascites. However, as the animal itself contacts other antigens in the environment during the raising process, a certain amount of non-target antibodies are produced, resulting in low purity of the purified antibodies; and the health of the animal will also affect the activity of the final antibody. In addition, the cell lines themselves are degraded in the process of passage and storage, and the activity of the antibodies is reduced, so that the activity of the antibodies is different among batches, and the activity difference among abzyme batches is caused. Chemically modified Taq enzymes also suffer from certain disadvantages: the chemical groups achieve the purpose of modification by reacting with amino acid residues in Taq enzyme, but the uncertainty of modification is caused by different types and positions of the amino acid residues of the same protein. At the same time, the difficulty of controlling the chemical reaction is high, and by-products are often generated, and the uncertainty of the reaction is increased.

In view of this, the present invention has been made.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention particularly provides the following technical scheme:

An aptamer comprising the nucleotide sequence:

5'-GATCATCTC (X1) ₆TTCTTAGCGTTT(X2)₆ TGTGTATGATC-3', wherein X1 and X2 independently comprise 6 arbitrary nucleotides.

In one possible implementation, X1 and X2 are complementary matched in reverse.

In one possible implementation, there is a perfect match between X1 and X2.

In one possible implementation, up to 1 mismatch is allowed between X1 and X2.

In one possible implementation, the mismatch between X1 and X2 is not located at the interface with the main frame.

Preferably, X1 comprises: NNDDHH.

In one possible implementation, X1 comprises ATGACA, ACGATA, AGAACA, ATAGCA, ATTACC, GAAACA, ACGTTA, TTAGCA, CGAATA, AGAGAA, AGTATC or CAGAAT.

Preferably, X2 comprises: DDHHNN.

In one possible implementation, X2 comprises TGTCAT, TATCGT, TGTTCT, TGCTAT, GGTAAT, TGTTTC, TAACGT, TGCTAA, TATTCG, TTCTCT, GATACT or ATTCTG.

In one possible implementation, the above-mentioned aptamer includes any one of the following nucleotide sequences:

5’-GATCATCTCATGACATTCTTAGCGTTTTGTCATTGTGTATGATC-3’。

5’-GATCATCTCACGATATTCTTAGCGTTTTATCGTTGTGTATGATC-3’。

5’-GATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATC-3’

5’-GATCATCTCATAGCATTCTTAGCGTTTTGCTATTGTGTATGATC-3’。

5’-GATCATCTCATTACCTTCTTAGCGTTTGGTAATTGTGTATGATC-3’。

5’-GATCATCTCGAAACATTCTTAGCGTTTTGTTTCTGTGTATGATC-3’。

5’-GATCATCTCACGTTATTCTTAGCGTTTTAACGTTGTGTATGATC-3’。

5’-GATCATCTCTTAGCATTCTTAGCGTTTTGCTAATGTGTATGATC-3’。

5’-GATCATCTCCGAATATTCTTAGCGTTTTATTCGTGTGTATGATC-3’。

5’-GATCATCTCAGAGAATTCTTAGCGTTTTTCTCTTGTGTATGATC-3’。

5’-GATCATCTCAGTATCTTCTTAGCGTTTGATACTTGTGTATGATC-3’。

5’-GATCATCTCCAGAATTTCTTAGCGTTTATTCTGTGTGTATGATC-3’。

In one possible implementation, the aptamer is a stem-loop structure.

In one possible implementation, the nucleotides of the aptamer are modified.

In one possible implementation, the modification occurs at the 5 'and/or 3' end of the nucleotide.

In one possible implementation, the modification occurring at the 5' end includes a thio modification, amino modification, sulfhydryl modification, phosphorylation modification, dideoxy modification, or biotin modification.

In one possible implementation, the modification occurring at the 3' end includes a thio modification, a phosphorylation modification, a dehydroxy modification, a sulfhydryl modification, a dideoxy modification, an amino modification, or a biotin modification.

In one possible implementation, the aptamer binds to Taq enzyme.

The invention also provides application of the aptamer in sealing or partially sealing Taq enzyme activity.

In one possible implementation, the aptamer blocks or partially blocks Taq enzyme activity at a temperature below the enzyme reaction temperature.

In one possible implementation, the aptamer may block or partially block Taq enzyme activity at 35-60 ℃.

Also provided by the present invention are aptamer combinations comprising at least 2 nucleotide sequences of the above-described aptamers.

The invention also provides an enzyme mixture comprising the above-mentioned aptamer or aptamer combination, and Taq enzyme.

The invention also provides a kit comprising the above-mentioned aptamer, or an aptamer combination, or an enzyme mixture.

The invention also provides application of the aptamer, or the aptamer combination, or the enzyme mixture in sealing Taq enzyme activity, nucleic acid amplification, preparing a nucleic acid extension reaction mixture and preparing a nucleic acid amplification kit.

The invention also provides a nucleic acid extension reaction mixture comprising the above enzyme mixture, at least one primer, dNTPs.

Also provided by the present invention is a method of nucleic acid amplification comprising: the nucleic acid extension reaction mixture reacts with a template to be detected, and under the condition that nucleic acid amplification is allowed to occur, the aptamer is separated from Taq enzyme, so that a primer extension product is formed.

Compared with the prior art, the invention has the following beneficial effects:

The Taq enzyme aptamer provided by the invention has the function of inhibiting the activity of Taq enzyme at room temperature, so that the normal temperature stability of the Taq enzyme is effectively improved, compared with the Taq enzyme singly used, the Taq enzyme aptamer has greatly enhanced anti-interference capability on PCR, and the generation of primer dimer or non-specific products in the initial stage of PCR reaction can be effectively controlled. The aptamer cannot be inactivated or degraded in the reaction process, renaturates after the temperature is reduced, can be refolded and combined on Taq enzyme to seal the activity of the aptamer, and protects the activity of the Taq enzyme. In addition, the preparation of the aptamer based on the specific sequence can ensure the stability among different batches.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of binding and inhibiting the function of Taq enzyme at a temperature of 35℃at 40℃at 45℃at 50℃at 55℃with the aptamer provided in example 2 of the present invention;

FIG. 2 is a graph showing the results of binding and inhibiting the function of Taq enzyme at a temperature of 35℃at 40℃at 45℃at 50℃at 55℃with a commercially available Taq enzyme antibody provided in example 2 of the present invention;

FIG. 3 is a graph showing the results of binding and inhibiting the function of Taq enzyme at 50℃at 55℃at 60℃with the aptamer provided in example 2 of the present invention;

FIG. 4 is a graph showing the results of the binding and inhibition of Taq enzyme function at 55℃of the aptamer mixture provided in example 2 of the present invention;

FIG. 5 is a graph showing the result of fluorescent quantitative PCR provided in example 3 of the present invention.

Detailed Description

The application as claimed may be practiced with reference to this disclosure by those skilled in the art, and it is expressly noted that all such similar alterations and modifications are apparent to those skilled in the art and are deemed to be included within the application. While the methods and applications of this application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the application can be practiced and practiced with modification and alteration of the methods and applications described herein, or with appropriate modification and combination, without departing from the spirit and scope of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The following basic terms or definitions are provided solely to aid in the understanding of the application. These definitions should not be construed to have a scope less than understood by those skilled in the art. Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to include at least a certain number of embodiments, this should also be understood to disclose a group that optionally consists of only those embodiments.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

The terms "about" and "substantially" in this application mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operation in other sequences than described or illustrated herein.

Reference now will be made in detail to embodiments of the application, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the application. It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the scope or spirit of the application. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment. Accordingly, it is intended that the present application cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present application.

In a specific embodiment of the present invention, the comparative tests were provided in which the same primordial conditions were used for each treatment group, and the test conditions were consistent for each group except for the differences. The reagents and the like according to the present invention can be obtained by commercially available methods unless otherwise specified.

The term "aptamer" as used herein refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) which itself has a stable tertiary structure and has the property of being able to bind with high affinity and specificity to a target molecule, e.g. the aptamer herein specifically binds to Taq enzyme.

The term "nucleotide" generally refers to a compound formed by linking a nucleoside to an acidic molecule or group via an ester linkage, e.g., a phosphate ester of a nucleoside, typically having one, two or three phosphate groups covalently attached at position 5 of the sugar group of the nucleoside. In some cases, the definition of a nucleotide also includes homologs or analogs of some typical nucleotides. DNA polymerase typically synthesizes DNA using 2' deoxynucleotides triphosphate.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, "nucleic acids" may be synthetic, naturally occurring or non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.

The term "complementary" refers to pairing between nucleoside or nucleotide sequences according to Watson-Crick base pairing rules, i.e., guanine (G) -cytosine (C) and adenine (A) -thymine (T)/uracil (U). Complementarity generally includes complete complementarity and partial complementarity, and it is understood that partial complementarity may exist with some mismatched or unpaired bases. As used herein, "matching" or "pairing" of bases refers to the formation of a reverse complementary double-stranded structure of the corresponding bases in the two nucleotide sequences, following the principle of pairing a with T, G with C. "match" refers to a complete complementarity or, although not complete complementarity, only the presence of an appropriate base mismatch (e.g., a 1 base mismatch). "perfect match" means that the sequences are perfectly complementary, without any base mismatches.

The term "amplification" refers to a process in which the number of nucleic acid fragments of interest is increased by the action of a nucleic acid polymerase, including, but not limited to, polymerase Chain Reaction (PCR), ligase Chain Reaction (LCR), nucleic acid sequence-based amplification (NASBA)), and the like. In embodiments of the invention, amplification refers to the Polymerase Chain Reaction (PCR). The template is denatured and melted, the oligonucleotide primer hybridizes with the template by annealing, and the oligonucleotide primer is extended with the addition of nucleotides, and the number of cycles is repeated to increase the target nucleotide fragments.

The term "Taq enzyme" refers to a hot start DNA polymerase. In some embodiments, the Taq enzyme is from thermus, including wild-type or mutant or modified versions thereof. Wherein wild-type Taq enzyme refers to Taq enzyme directly purified from Thermus species such as Thermus aquaticus; mutant Taq enzyme refers to Taq enzyme obtained by mutating amino acids at one or more sites in the amino acid sequence of wild-type Taq enzyme; the modified Taq enzyme is Taq enzyme in which a mutant protein or a functional group is introduced into an amino acid residue of the wild-type Taq enzyme to realize a specific function.

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

According to one aspect of the present invention there is provided an aptamer for inhibiting Taq enzyme comprising the nucleotide sequence 5'-GATCATCTC (X1) ₆TTCTTAGCGTTT(X2)₆ TGTGTATGATC-3' (SEQ ID NO: 1), wherein X1 and X2 independently comprise 6 arbitrary nucleotides.

In some embodiments, "5'-GATCATCTC … … TTCTTAGCGTTT … … TGTGTATGATC-3'" in the aptamer provided by the invention is defined as the main framework, wherein X1 can be selected from 6 arbitrary nucleotide combinations, for example X1 ₁X1₂X1₃X1₄X1₅X1₆, and X1 ₁、X1₂、X1₃、X1₄、X1₅ and X1 ₆ can be identical, partially identical, or different; similarly, X2 may be selected from any combination of 6 nucleotides, for example X2 ₁X2₂X2₃X2₄X2₅X2₆, where X2 ₁、X2₂、X2₃、X2₄、X2₅ and X2 ₆ may be identical, partially identical, or different. The specific selection of X1 and X2 is not limited, the Taq enzyme can be selected in a targeted manner according to the Taq enzyme to be modified, and under the condition that the main framework is kept unchanged, the aptamer for inhibiting the Taq enzyme can inhibit the activity of the Taq enzyme below 60 ℃, and has good hot start performance, so that the generation of primer dimer or non-specific products in the initial stage of the PCR reaction can be effectively controlled.

In some embodiments, X1 and X2 are reverse complementary matches, which may be perfect matches or allow up to 1 mismatch. When there is a mismatch between X1 and X2, the mismatch site is not located at the interface with the main frame, i.e., X1 ₁、X1₆、X2₁ and X2 ₆ are not mismatch sites.

In some embodiments, X1 comprises: NNDDHH. The X1 contains degenerate bases, N represents any deoxyribonucleotide degenerate base, D represents A, G or T, and H represents A, C or T. Specifically, X1 may be selected from ATGACA, ACGATA, AGAACA, ATAGCA, ATTACC, GAAACA, ACGTTA, TTAGCA, CGAATA, AGAGAA, AGTATC or CAGAAT. According to the matching principle of X1 and X2, X2 comprises: DDHHNN. The X2 contains degenerate bases, N represents any deoxyribonucleotide degenerate base, D represents A, G or T, and H represents A, C or T. Specifically, X2 may be selected from TGTCAT, TATCGT, TGTTCT, TGCTAT, GGTAAT, TGTTTC, TAACGT, TGCTAA, TATTCG, TTCTCT, GATACT or ATTCTG.

In some embodiments, preparation based on defined nucleic acid sequences can further ensure stability between different batches, typical aptamers include any of the following nucleotide sequences:

5’-GATCATCTCATGACATTCTTAGCGTTTTGTCATTGTGTATGATC-3’(SEQ ID NO:2)。

5’-GATCATCTCACGATATTCTTAGCGTTTTATCGTTGTGTATGATC-3’(SEQ ID NO:3)。

5’-GATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATC-3’(SEQ ID NO:4)。

5’-GATCATCTCATAGCATTCTTAGCGTTTTGCTATTGTGTATGATC-3’(SEQ ID NO:5)。

5’-GATCATCTCATTACCTTCTTAGCGTTTGGTAATTGTGTATGATC-3’(SEQ ID NO:6)。

5’-GATCATCTCGAAACATTCTTAGCGTTTTGTTTCTGTGTATGATC-3’(SEQ ID NO:7)。

5’-GATCATCTCACGTTATTCTTAGCGTTTTAACGTTGTGTATGATC-3’(SEQ ID NO:8)。

5’-GATCATCTCTTAGCATTCTTAGCGTTTTGCTAATGTGTATGATC-3’(SEQ ID NO:9)。

5’-GATCATCTCCGAATATTCTTAGCGTTTTATTCGTGTGTATGATC-3’(SEQ ID NO:10)。

5’-GATCATCTCAGAGAATTCTTAGCGTTTTTCTCTTGTGTATGATC-3’(SEQ ID NO:11)。

5’-GATCATCTCAGTATCTTCTTAGCGTTTGATACTTGTGTATGATC-3’(SEQ ID NO:12)。

5’-GATCATCTCCAGAATTTCTTAGCGTTTATTCTGTGTGTATGATC-3’(SEQ ID NO:13)。

In some embodiments, the aptamer is in a stem-loop structure, the aptamer can not be inactivated or degraded in the reaction process by limiting the sequence, meanwhile, the stem-loop structure can ensure that the aptamer can be refolded in the renaturation process and is combined with Taq enzyme to seal the activity of the aptamer, so that the activity of the Taq enzyme in a reaction system is ensured to be sufficient.

In some embodiments, the aptamer can be provided with different characteristics by performing different modifications to the aptamer, e.g., a thio modification can prevent degradation by nucleases, a phosphorylation modification can be resistant to exonuclease digestion, etc. Preferably, at least one of the 5 'end or the 3' end of the aptamer inhibiting Taq enzyme has a modification. For example, the modification at the 5' end may be one of a thio modification, an amino modification, a thiol modification, a phosphorylation modification, a dideoxy modification, and a biotin modification; the modification at the 3' end may be one of thio modification, phosphorylation modification, dehydroxy modification, mercapto modification, dideoxy modification, amino modification and biotin modification.

In some embodiments, the aptamer provided by the invention is capable of binding to Taq enzyme, thereby blocking the activity of Taq enzyme, and in some embodiments, when the temperature is raised to, for example, 60 ℃ or above, 62 ℃ or above 65 ℃, the aptamer is capable of dissociating from the bound Taq enzyme and initiating Taq enzyme activity, thereby effectively reducing the production of primer dimers or non-specific products at the initial stage of the PCR reaction.

In some embodiments, the invention provides the use of an aptamer to block or partially block Taq enzymatic activity.

In some embodiments, the aptamers provided based on the invention are capable of inhibiting the activity of Taq enzyme at temperatures below the enzyme reaction temperature. For example, at 35-60℃ (e.g., 35℃, 40℃, 45℃, 50℃, 55℃, 60℃), the aptamer blocks Taq enzymatic activity, and when the temperature is raised to hot start conditions, the aptamer is able to cleave from bound Taq enzyme and start Taq enzymatic activity, reaching or exceeding the same level as the chemically modified control from both quantitative and qualitative result analysis.

It will be appreciated that the aptamer provided by the invention may be a single aptamer or a mixture of aptamers: when a single sequence is selected as the aptamer provided by the invention, the aptamer is a single sequence aptamer; when any two or more single sequences are selected as the aptamer provided by the invention, the aptamer is a mixture of aptamers of different sequences. Whether the sequence aptamer is single or a mixture of different sequence aptamers, the Taq enzyme activity can be effectively blocked, and the thermal start function is realized.

Based on this, the invention also provides an aptamer combination comprising at least two different selected sequences of SEQ ID NO. 1 or comprising at least two sequences selected from SEQ ID NO. 2-SEQ ID NO. 13.

Notably, when a combination of aptamers of different sequences is selected as an aptamer for inhibiting Taq enzyme, the sequence that can be complementarily paired should not be included in the combination, and preferably the number of complementary bases does not exceed 50% of the total number of bases, in order to ensure the functional efficiency of the aptamer.

The invention also provides an enzyme mixture comprising the aptamer or the aptamer combination provided by the invention, and Taq enzyme.

In addition, the invention also provides a preparation method of the enzyme mixture, which comprises the step of incubating the aptamer provided by the invention with Taq enzyme to obtain the enzyme mixture with low-temperature activity blocking, wherein in some embodiments, the low temperature is below 60 ℃.

In some embodiments, the unit concentration ratio of the aptamer to Taq enzyme is 1-10:1, typical concentration ratios may be 10:1, 8:1, 5:1, 2:1, or 1:1. The unit concentration ratio of the aptamer to the Taq enzyme means the concentration of the aptamer: taq enzyme unit, wherein the aptamer concentration is pmol/. Mu.l, and Taq enzyme unit is U. In some embodiments, the temperature of co-incubation is 4-50 ℃, typical co-incubation temperatures may be 4 ℃,10 ℃,15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃,40 ℃,45 ℃, or 50 ℃. In some embodiments, the incubation time is 0.5-24h, and typical incubation times may be 0.5h, 1h, 5h, 10h, 15h, 20h, or 24h. In some embodiments, the method further comprises the step of purifying the active blocked enzyme mixture. In some embodiments, the enzyme mixture is treated using column purification to remove unbound aptamer and hot Taq enzyme, improving purity. In some embodiments, the method further comprises a step of concentrating after purification such that the concentration of the purified enzyme mixture is not less than the concentration of Taq enzyme prior to active blocking. Through concentration of the enzyme mixture after concentration and purification, the addition amount of the enzyme mixture in the actual use process can be ensured not to be too large, unnecessary components in a reaction system are reduced, and the accuracy of a detection result is ensured. It will be appreciated that the concentration of Taq enzyme prior to active blocking is that prior to co-incubation with the aptamer.

In some embodiments, the method further comprises the step of adding a cryoprotectant or surfactant to the concentrated enzyme mixture. The addition of cryoprotectant and surfactant can ensure that the enzyme mixture does not freeze at-20 ℃ so as to maintain the protein structure and activity of the enzyme mixture. The cryoprotectant and the surfactant are not particularly limited, and any reagent which can play a corresponding role can be used. Wherein the cryoprotectant may be, for example, but not limited to, glycerol; the surfactant may be, for example, but not limited to, tween 20, tween 80, tritonX-100, NP-40, or CA-630. In some embodiments, an equal volume of cryoprotectant is added to a final concentration of 50% (V/V) and surfactant is added to a final concentration of 0.025% (V/V).

Based on the same inventive concept as the enzyme mixture provided by the invention, the invention also provides a kit and a nucleic acid extension reaction mixture comprising the aptamer, the aptamer combination or the enzyme mixture provided by the invention, so that the kit and the nucleic acid extension reaction mixture provided by the invention have all the beneficial effects as the enzyme mixture provided by the invention, and are not repeated herein.

Based on the beneficial effects of the aptamer provided by the invention, the invention also provides application of the aptamer, the aptamer combination and the enzyme mixture in inhibiting Taq enzyme activity, nucleic acid amplification, preparing a nucleic acid extension reaction mixture and preparing a nucleic acid amplification kit.

The invention also provides a nucleic acid extension reaction mixture comprising the above enzyme mixture, at least one primer and dNTPs.

In addition, the invention also provides a method for amplifying nucleic acid, which comprises the steps of reacting the nucleic acid extension reaction mixture with a template to be detected, and separating the aptamer from Taq enzyme under the condition of allowing the nucleic acid amplification to occur to form a primer extension product.

By using the nucleic acid amplification method, the enzyme mixture in the system is not exposed to activity when the reaction temperature is not reached, and the activity is recovered when the reaction temperature is reached, so that the generation of nonspecific bands of the amplification reaction is effectively controlled.

The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or were directly commercially available unless otherwise specified.

The main reagent information used in the examples of the present invention is as follows:

Taq enzyme is Taq DNA polymerase (cat# MD 001) manufactured by Fei Peng biological Co., ltd; the aptamer, the primer for PCR detection and the TaqMan probe are synthesized by a entrusted synthesis manufacturer, so that the purity is more than or equal to 99%, and the sequence is sequenced correctly; the buffer used for the PCR reaction is 5 XPCR buffer (product number: MD 001-B) matched with Taq enzyme of the Phpeng biological Co., ltd; the PCR instrument for fluorescence quantification is ABI-real-time fluorescence quantification, and the model is ABI-7500.

Example 1 preparation of enzyme mixture

The invention obtains the aptamer with the sequence shown as SEQ ID NO. 2-SEQ ID NO. 13 through a large number of screening and design transformation, and the aptamer commonly has the nucleotide framework shown as SEQ ID NO. 1.

1. Single sequence aptamer

Annealing the aptamers shown in SEQ ID NO. 2-SEQ ID NO. 13 respectively under the following conditions: annealing from 95 ℃ to 4 ℃, wherein the temperature of each round of annealing interval is 8-10 ℃, and the time of each round of annealing is 1-2 min; the aptamer is folded into a stem-loop structure. Taking Taq enzyme and respectively mixing with the folded aptamer according to the unit concentration ratio of the aptamer to the Taq enzyme of 5:1, placing the mixture into a 5ml nut tube, and uniformly mixing by using a plasma mixer, wherein the conditions are as follows: the rotation speed was 50rpm, the temperature was 10℃and the time was 8 hours, to obtain an enzyme mixture.

2. Aptamers of different sequences

The invention can also be used for respectively annealing the aptamer with different sequences, then mixing the aptamer with Taq enzyme, and referring to the method 1, obtaining an enzyme mixture after treatment of different aptamers.

3. Purification and preservation

Purifying the enzyme mixture prepared in the step 1 or the step 2 by using a Mono Q column, separating free aptamer from Taq enzyme, and collecting Taq enzyme with the aptamer completely modified. Concentrating the obtained enzyme mixture to make its protein concentration not less than that before modification, and adding vomit and glycerol for preservation.

The enzyme mixture has good stability and the detection efficiency of the enzyme mixture is equivalent when the enzyme mixture is placed for 7 days at 37 ℃ compared with the freshly prepared enzyme mixture.

Example 2, temperature sealing Performance test

1. Sealing performance test at different temperatures

The testing method comprises the following steps: the enzyme mixture prepared in example 1 (aptamer SEQ ID NO: 2) was subjected to pretreatment of 35-55deg.C (two replicates per 5 ℃), respectively. The enzyme mixture after pretreatment is respectively prepared into PCR reaction systems, the template is diluted by 5 times of human genome DNA, PCR amplification reaction is carried out, the quantity and the brightness of nonspecific bands are analyzed through agarose electrophoresis after the reaction, and the blocking effect of the aptamer on Taq enzyme at different temperatures is judged.

As can be seen from FIG. 1, the enzyme mixture under the treatment condition of 35-55 ℃ has no obvious nonspecific band, which proves that the aptamer of the invention has good active blocking effect on Taq enzyme.

2. Blocking performance comparison with abzyme

Taq enzyme can also be subjected to active blocking by combining an antibody with Taq enzyme, and the Taq enzyme is treated by using a commercial Taq enzyme antibody and is subjected to pretreatment of 35-55 ℃ (two repeats at 5 ℃), respectively. The blocking effect on Taq enzyme was evaluated in the same manner as in 1.

As can be seen from FIG. 2, there are distinct non-specific bands for antibody modified Taq enzyme at processing conditions of 35℃to 55 ℃.

3. Temperature rise test

The invention also selects the SEQ ID NO. 4 aptamer for temperature rise test, and the enzyme mixture is prepared by the method of example 1 and then tested at 50 ℃, 55 ℃ and 60 ℃ by the test method basically same as 1.

As shown in FIG. 3, the enzyme mixture has no obvious nonspecific band under the treatment condition of 50-60 ℃, which proves that the aptamer of the invention has good active blocking effect on Taq enzyme, and can realize blocking even under the treatment condition of 60 ℃.

4. 55 ℃ Test

Using the above method, this example also demonstrates 10 aptamers, other than SEQ ID NO 2 and 4, respectively, that all exert the ability to bind and block Taq enzymatic activity at 55 ℃.

5. Aptamer mixture testing

Enzyme mixtures of the aptamer compositions prepared according to method 2 of example 1 were tested at 55℃with SEQ ID NOs 3 and 4 (abbreviated as sequence 3 and sequence 4) and the results are shown in FIG. 4, M representing the marker, sequence 4 from left to right: sequence 32 replicates at a molar ratio of 3.25:3, and sequence 4: 2 replicates at a 3.25:2.75 molar ratio of sequence 3 all showed excellent blocking properties.

Example 3 fluorescent quantitative PCR Activity detection

1. Preparation of fluorescent quantitative PCR detection system

Using GAPDH gene in human genome as DNA fragment to be detected, primer probe was designed:

The GAPDH upstream primer has a sequence shown as SEQ ID NO. 14, the downstream primer has a sequence shown as SEQ ID NO. 15, the 5 '-end of the probe is modified with FAM, the 3' -end of the probe is modified with BHQ1, and the sequence shown as SEQ ID NO. 16.

An enzyme mixture was prepared with reference to example 1 using control aptamer (1 #,2 #) and the sequences are shown below:

1#:5’-CGATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATCG-3’；

2#:5’-GATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGA-3’。

The total volume of the fluorescent quantitative detection system is 50 μl, and the method comprises the following steps: 5 XPCR buffer 10. Mu.l, 25mM dNTP 0.4. Mu.l, GAPDH upstream primer (10. Mu.M) 1. Mu.l, GAPDH downstream primer (10. Mu.M) 1. Mu.l, GAPDH probe (10. Mu.M) 0.8. Mu.l, taq enzyme/aptamer enzyme mix/comparison enzyme mix/chemical modification enzyme hotspot Taq (5U/. Mu.l), template (10. Mu.3 copies/. Mu.l) 2. Mu.l, ddH ₂ O make up.

2. The reaction conditions were as follows:

3. fluorescent quantitative PCR result analysis

The above fluorescence quantification results are shown in CT values (FIG. 5), and the different aptamer-modified Taq enzymes are superior to the unmodified Taq enzymes in PCR, and have more excellent performance compared with the control, wherein the CT values are approximately 10% lower.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An aptamer comprising a nucleotide sequence

2. The aptamer of claim 1, wherein X1 is reverse complementarily matched to X2.

3. The aptamer of claim 2, wherein X1 and X2 are perfectly matched.

4. The aptamer of claim 2, wherein up to 1 mismatch is allowed between X1 and X2.

5. The aptamer of claim 4, wherein the mismatch between X1 and X2 is not located at the interface with the mainframe.

6. The aptamer of any one of claims 1-5, the X1 comprising: NNDDHH.

7. The aptamer of claim 6, X1 comprising ATGACA, ACGATA, AGAACA, ATAGCA, ATTACC, GAAACA, ACGTTA, TTAGCA, CGAATA, AGAGAA, AGTATC or CAGAAT.

8. The aptamer of any one of claims 1-5, the X2 comprising: DDHHNN.

9. The aptamer of claim 8, X2 comprising TGTCAT, TATCGT, TGTTCT, TGCTAT, GGTAAT, TGTTTC, TAACGT, TGCTAA, TATTCG, TTCTCT, GATACT or ATTCTG.

10. The aptamer of any one of claims 1-9, comprising any one of the following nucleotide sequences:

5’-GATCATCTCATGACATTCTTAGCGTTTTGTCATTGTGTATGATC-3’；

5’-GATCATCTCACGATATTCTTAGCGTTTTATCGTTGTGTATGATC-3’；

5’-GATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATC-3’；

5’-GATCATCTCATAGCATTCTTAGCGTTTTGCTATTGTGTATGATC-3’；

5’-GATCATCTCATTACCTTCTTAGCGTTTGGTAATTGTGTATGATC-3’；

5’-GATCATCTCGAAACATTCTTAGCGTTTTGTTTCTGTGTATGATC-3’；

5’-GATCATCTCACGTTATTCTTAGCGTTTTAACGTTGTGTATGATC-3’；

5’-GATCATCTCTTAGCATTCTTAGCGTTTTGCTAATGTGTATGATC-3’；

5’-GATCATCTCCGAATATTCTTAGCGTTTTATTCGTGTGTATGATC-3’；

5’-GATCATCTCAGAGAATTCTTAGCGTTTTTCTCTTGTGTATGATC-3’；

5’-GATCATCTCAGTATCTTCTTAGCGTTTGATACTTGTGTATGATC-3’；

5’-GATCATCTCCAGAATTTCTTAGCGTTTATTCTGTGTGTATGATC-3’。

11. the aptamer of any one of claims 1-10, which is a stem-loop structure.

12. The aptamer of any one of claims 1-11, wherein the nucleotide of the aptamer is modified.

13. The aptamer of claim 12, wherein the modification occurs at the 5 'and/or 3' end of the nucleotide.

14. The aptamer of claim 13, wherein the modification at the 5' end comprises a thio modification, an amino modification, a thiol modification, a phosphorylation modification, a dideoxy modification, or a biotin modification.

15. The aptamer of claim 13, wherein the modification at the 3' end comprises a thio modification, a phosphorylation modification, a dehydroxy modification, a sulfhydryl modification, a dideoxy modification, an amino modification, or a biotin modification.

16. The aptamer of any one of claims 1-15 that binds to Taq enzyme.

17. Use of an aptamer according to any one of claims 1 to 16 for blocking or partially blocking Taq enzymatic activity.

18. The use of claim 17, wherein the aptamer occludes or partially occludes Taq enzyme activity at a temperature below the enzyme reaction temperature.

19. The use according to claim 17, wherein the aptamer is capable of blocking or partially blocking Taq enzyme activity at 35-60 ℃.

20. An aptamer combination comprising at least 2 nucleotide sequences selected from the group consisting of the aptamers of any of claims 1 to 16.

21. An enzyme mixture comprising the aptamer of any one of claims 1-16 or the aptamer combination of claim 20, and Taq enzyme.

22. A kit comprising the aptamer of any one of claims 1-16, or the aptamer combination of claim 20, or the enzyme mixture of claim 21.

23. Use of the aptamer of any one of claims 1-16, or the aptamer combination of claim 20, or the enzyme mixture of claim 21 for blocking Taq enzymatic activity, nucleic acid amplification, preparation of a nucleic acid extension reaction mixture, preparation of a nucleic acid amplification kit.

24. A nucleic acid extension reaction mixture comprising the enzyme mixture of claim 21, at least one primer, dNTPs.

25. A method of nucleic acid amplification comprising: reacting the nucleic acid extension reaction mixture of claim 24 with a template to be tested, under conditions that allow nucleic acid amplification to occur, the aptamer is free of Taq enzyme, forming a primer extension product.