CN107653297B - Detection kit based on aptamer - Google Patents

Abstract

The invention discloses a detection kit based on a nucleic acid aptamer, which comprises stem-loop structure nucleic acid H1, DNA1, DNA2 and DNA 3. The stem-loop structure nucleic acid contains aptamer which is divided into two sections, and the aptamer is used as a molecular recognition element to realize specific binding to molecules to be detected, so that the catalytic activity of DNA enzyme is activated, the substrate chain is cut circularly, a strong fluorescence detection signal is generated, and high-sensitivity detection is realized. The limit of ATP detection is 0.2nM, the linear range of detection is wider, 1nM to 10 μ M; meanwhile, the detection system has good specificity, and common analogues including CTP, GTP and UTP do not interfere with the detection result. The detection process does not need a separation and purification process, can be detected by simple mixing, has the advantages of simple operation, low cost, quick response and the like, and can be used for quickly detecting and analyzing the ATP content in an environment or food sample.

Description

Detection kit based on aptamer

Technical Field

The invention belongs to the field of biosensors, and relates to a detection kit based on a nucleic acid aptamer.

Background

Aptamer (aptamer) is an oligonucleotide fragment which is screened by in vitro screening technology SELEX (systematic evolution of exponential enrichment ligands) and can specifically bind to proteins or other small molecular substances. The aptamer serving as a novel molecular recognition tool has the advantages of high affinity, good specificity, easiness in synthesis, modification, good stability and the like compared with the traditional antibody, and has good application potential in the field of protein and molecular detection. In recent years, by combining the molecular characteristics of the aptamer, the detection of protein is converted into the detection of the aptamer with specific recognition, so that the defects of complex operation, frequent need of labeling of protein, difficulty in keeping activity and the like in the traditional protein detection method are avoided, and the efficiency and the sensitivity of protein detection are effectively improved.

The existing detection method based on the aptamer is generally complex, and is difficult to realize the detection of different substances by changing the sequence of the aptamer, so that the design is difficult, and the application range of the detection method is limited. The development of a detection kit with good universality is of great significance.

DNAzymes (deoxyribozymes) are single-stranded DNA fragments with catalytic function synthesized by in vitro molecular evolution technology, and have high catalytic activity and structure recognition capability. Since the first discovery of deoxyribozymes in 1994, several tens of deoxyribozymes have been discovered so far. They can be classified into 7 categories according to their functions: has RNA cutting activity, DNA ligase activity, porphyrin metalating enzyme and peroxidase activity, DNA hydrolyzing activity, DNA kinase activity, N-glycosylase activity and DNA capping activity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a detection kit based on the aptamer, which has the advantages of good specificity, high sensitivity, simple operation and low cost.

The technical scheme adopted by the invention is as follows:

a nucleic acid aptamer-based detection kit comprising stem-loop structure nucleic acid H1, DNA1, DNA2 and DNA3, wherein:

h1 consisting of 7 parts a, b, c, d, c, e and f in sequence, wherein b and e are aptamers divided into two parts, and b and e bind to each other and approach a and f in the presence of a substrate for the aptamers; c and c are complementary paired to form a stem portion; d is a ring moiety;

DNA1 and DNA2 are DNases that are divided into two parts, which have catalytic activity when they are brought close together and cleave substrates; DNA1 contains three parts a, I, g, a is complementary paired with H1 and part a; DNA2 contains three parts, f, II, H, f is complementary paired with parts H1 and f; i and II are the core of the catalytic part of the DNase;

DNA3 is a substrate for catalytic cleavage by dnase, DNA3 has fluorophores and corresponding quenchers attached to each end, and DNA3 has g and h portions that are complementary pairs of g portion of DNA1 and h portion of DNA 2.

As a further improvement of the detection kit, the number of the bases of the parts a and f is 9-18 independently.

As a further improvement of the detection kit, the number of bases in the part c is 7-12.

As a further improvement of the detection kit, the number of bases in the part d is 12-18.

As a further improvement of the detection kit, the number of the bases of the g part and the h part is 5-8 independently.

As a further improvement of the above-mentioned detection kit, the base of part I which is 5'-AGCGATTAAC-3', II is 5'-GTTACACCCATGT-3', DNA3 and has the corresponding recognition site-TrAG-base.

As a further improvement of the detection kit, the number of bases of DNA3 is 13-21.

As a further improvement of the detection kit, the sequences of the nucleic acids H1, DNA1, DNA2 and DNA3 are as follows:

H1：5'-CGAATCAAGAGT-ACCTGGGGAGTAT-CACCGTATC-CTATGCCTGCAACGT-GATACGGTG-TGCGGAGGAAGGT-CTACAAATAAGC-3'(SEQ ID NO：1)

DNA1：5'-GATATC-AGCGATTAAC-ACTCTTGATTCG-3'(SEQ ID NO：2)

DNA2：5'-GCTTATTTGTAG-GTTACACCCATGT-TACTCT-3'(SEQ ID NO：3)

DNA 3: 5 '-fluorophore-AGAGTA-TrAG-GATATC-quencher-3' (SEQ ID NO: 4).

As a further improvement of the above-mentioned detection kit, the reaction buffer of the detection kit comprises 10mM Tris-HCl, pH 7.5, and contains 50mM NaCl, 35mM MgCl₂。

The invention has the beneficial effects that:

the detection kit has expansibility and universality, and can detect a series of substances including but not limited to ATP, cocaine, mercury ions, silver ions, thrombin and the like by modifying the two segments of divided aptamer sequences.

The detection kit disclosed by the invention has the advantages that the enzyme is not required to be added, the operation is simple, the cost is low, the washing and separation processes are not required, the temperature control is not required, and the detection can be finished at room temperature;

the detection kit provided by the invention can greatly improve the detection signal intensity and greatly improve the detection sensitivity by utilizing the circular cutting action of the DNA enzyme.

Drawings

FIG. 1 is a schematic diagram of the detection kit of the present invention;

FIG. 2 is a graph showing the results of ATP detection at various concentrations;

FIG. 3 shows the results of the specificity experiment.

Detailed Description

A nucleic acid aptamer-based detection kit comprising stem-loop structure nucleic acid H1, DNA1, DNA2 and DNA3, wherein:

h1 consisting of 7 parts a, b, c, d, c, e and f in sequence, wherein b and e are aptamers divided into two parts, and b and e bind to each other and approach a and f in the presence of a substrate for the aptamers; c and c are complementary paired to form a stem portion; d is a ring moiety;

DNA1 and DNA2 are DNases that are divided into two parts, which have catalytic activity when they are brought close together and cleave substrates; DNA1 contains three parts a, I, g, a is complementary paired with H1 and part a; DNA2 contains three parts, f, II, H, f is complementary paired with parts H1 and f; i and II are the core of the catalytic part of the DNase;

DNA3 is a substrate for catalytic cleavage by dnase, DNA3 has fluorophores and corresponding quenchers attached to each end, and DNA3 has g and h portions that are complementary pairs of g portion of DNA1 and h portion of DNA 2.

As a further improvement of the above-mentioned detection kit, the number of bases in the parts a and f is independently 9 to 18, preferably 12. Thus, the method can not only ensure that the nucleic acid sequences have better affinity, but also avoid the influence of overlong sequences on the reaction.

As a further improvement of the above-mentioned detection kit, the number of bases in the part c is 7 to 12, preferably 9. This allows the formation of a stable stem structure.

As a further improvement of the above-mentioned detection kit, the number of bases in the d-portion is 12 to 18, preferably 15. This may form a stable ring structure.

As a further improvement of the detection kit, the number of bases in the g and h parts is 5 to 8 independently, preferably 6.

As a further improvement of the above-mentioned detection kit, the base of part I which is 5'-AGCGATTAAC-3', II is 5'-GTTACACCCATGT-3', DNA3 and has the corresponding recognition site-TrAG-base.

As a further improvement of the above detection kit, the number of bases of DNA3 is 13 to 21, preferably 15. Therefore, the fluorescent group and the quenching group can be well interacted, the overhigh background fluorescence value is avoided, and the detection sensitivity is improved.

As a further improvement of the detection kit, the sequences of the nucleic acids H1, DNA1, DNA2 and DNA3 are as follows:

H1：5'-CGAATCAAGAGT-ACCTGGGGAGTAT-CACCGTATC-CTATGCCTGCAACGT-GATACGGTG-TGCGGAGGAAGGT-CTACAAATAAGC-3'

DNA1：5'-GATATC-AGCGATTAAC-ACTCTTGATTCG-3'

DNA2：5'-GCTTATTTGTAG-GTTACACCCATGT-TACTCT-3'

DNA 3: 5 '-fluorophore-AGAGTA-TrAG-GATATC-quencher-3'.

The detection kit has no particular requirement for the reaction buffer, and generally speaking, it is sufficient if it can maintain the reaction pH stably, has ions required for the DNase reaction, and does not react with the substance to be detected. As a further improvement of the above-mentioned detection kit, the reaction buffer of the detection kit contains 10mM Tris-HCl, pH 7.5, and contains 50mM NaCl, 35mM MgCl₂。

The detection principle of the detection kit of the present invention is shown in FIG. 1, taking ATP detection as an example.

1) The stem-loop structure nucleic acid H1 comprises 7 portions, a, b, c, d, c, e, f, wherein the portions b and e are aptamers to ATP split into two parts, the portions c and c are complementary to form a stem portion, and d is a loop portion. When ATP is present in the detection system, the ATP is combined with parts b and c of H1, so that the distance between parts a and f is shortened and the parts a and f are close to each other;

2) DNA1 and DNA2 are DNases that are divided into two parts, and when they are not close to each other, they have no catalytic activity and cannot cleave the substrate. DNA1 comprises three parts, a, I, g; DNA2 contains three parts, f, II, h respectively. Where I and II are the core of the catalytic part of the DNase. A of DNA1 and a portion of H1; f of DNA2 is complementary to f of H1, thereby bringing DNA1 and DNAII into proximity with each other to form a dnase having catalytic activity;

3) DNA3 is a substrate for DNase catalytic cleavage, the recognition site is-TrAG-base, and DNA3 can be cleaved at rA base in a reaction buffer containing magnesium ions; one end of the DNA3 is modified with a fluorescent group FAM, and the other end is modified with a quenching group BHQ1, and before cutting, the fluorescence of the fluorescent group is quenched by the quenching group, so that the system has no fluorescence. After cutting, separating the fluorescent group from the quenching group, and enabling the system to have stronger fluorescence;

4) the formed DNase has the capability of circularly cutting a substrate and can continuously cut the substrate strand DNA3, thereby achieving the aim of signal amplification and improving the detection sensitivity.

Example 1: an ATP detection kit, comprising the following components:

(1) nucleic acid H1, DNA1, DNA2 and DNA3, the sequences of which are as follows:

H1：5'-CGAATCAAGAGT(a)-ACCTGGGGAGTAT(b)-CACCGTATC(c)-CTATGCCTGCAACGT(d)-GATACGGTG(c*)-TGCGGAGGAAGGT(e)-CTACAAATAAGC(f)-3'

DNA1：5'-GATATC(g)-AGCGATTAAC(Ⅰ)-ACTCTTGATTCG(a*)-3'

DNA2：5'-GCTTATTTGTAG(f*)-GTTACACCCATGT(Ⅱ)-TACTCT(h)-3'

DNA3：5'-FAM-AGAGTA(h*)-TrAG-GATATC(g*)-BHQ1-3'

(2) ATP standard solution

(3) Reaction buffer: comprises 10mM Tris-HCl, pH 7.5, 50mM NaCl, 35mM MgCl₂。

The using method comprises the following steps:

1) reaction buffer (10mM Tris-HCl, pH 7.5, containing 50mM NaCl, 35mM MgCl) was used first₂) Dissolving H1, DNA1, DNA2 and DNA3 respectively;

2) adding ATP with different concentrations into 200nM H1 solution, mixing well, reacting for 45 minutes at room temperature;

3) adding 200nM DNA1 and DNA2 solution, mixing well, reacting for 30 min at room temperature;

4) adding 200nM DNA3 solution, mixing, and reacting at room temperature for 90 min;

5) the intensity of the emitted light at 525nm was recorded under excitation light at an excitation wavelength of 490 nm.

When the system has no ATP, the background value is very low; when ATP exists in the system, the fluorescence intensity increases along with the ATP concentration, and the ATP have good correlation, so that the aim of detecting ATP is fulfilled.

Detection experiments for different concentrations of ATP:

ATP standard solutions were prepared at concentrations of 1nM, 10nM, 100nM, 1. mu.M, 10. mu.M, 20. mu.M, and 60. mu.M, respectively

ATP solutions were added to the reaction systems described in example 1, respectively, and the fluorescence intensity was measured after the reaction was sufficiently performed. As shown in FIG. 2, the fluorescence intensity gradually increases with the increase of ATP concentration, and when the ATP concentration exceeds 10. mu.M, the system gradually reaches saturation, and the fluorescence intensity tends to level off. Taking logarithm of ATP concentration (lgC) as an abscissa and fluorescence intensity as an ordinate, drawing a standard curve, wherein the logarithm of ATP concentration (lgC) and the fluorescence intensity have a good linear relation, the linear relation is from 1nM to 10 μ M, and the linear equation is as follows: f-50 +73lgC (R)²0.989) (F for fluorescence intensity, C for ATP concentration) with a limit of 0.2nM according to 3-fold signal-to-noise ratio standard (3S/N).

Specific experiments:

and selecting structural analogues CTP, GTP and UTP of ATP for detection, and investigating the specific selectivity of a detection system.

The 100nM CTP, GTP and UTP standard solutions and the 100nM ATP standard solution were added to the reaction system described in example 1, respectively, and the fluorescence intensities were measured after sufficient reaction, as shown in FIG. 3, the fluorescence intensities of the 100nM CTP, GTP and UTP were not changed much compared with the blank sample, and the measurement was not affected. The fluorescence intensity is obviously increased only when ATP is added, which proves that the method has good specificity for detecting ATP, and other structural analogues have no influence on the detection.

Sequence listing

<110> institute of ecological environment and technology in Guangdong province

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cgaatcaaga gtacctgggg agtatcaccg tatcctatgc ctgcaacgtg atacggtgtg 60

cggaggaagg tctacaaata agc 83

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gatatcagcg attaacactc ttgattcg 28

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gcttatttgt aggttacacc catgttactc t 31

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agagtatrag gatatc 16

Claims (2)

1. A nucleic acid aptamer-based detection kit comprising stem-loop structure nucleic acid H1, DNA1, DNA2 and DNA3, wherein:

h1 consisting of 7 parts a, b, c, d, c, e and f in sequence, wherein b and e are aptamers divided into two parts, and b and e bind to each other and approach a and f in the presence of a substrate for the aptamers; c and c are complementary paired to form a stem portion; d is a ring moiety;

DNA1 and DNA2 are DNases that are divided into two parts, which have catalytic activity when they are brought close together and cleave substrates; DNA1 contains three parts a, I, g, a is complementary paired with H1 and part a; DNA2 contains three parts, f, II, H, f is complementary paired with parts H1 and f; i and II are the core of the catalytic part of the DNase;

DNA3 is a substrate for catalytic cleavage by dnase, DNA3 has fluorophores and corresponding quenchers attached to each end, DNA3 has g and h portions that are complementary pairs of g portion of DNA1 and h portion of DNA2, wherein: the sequences of the nucleic acids H1, DNA1, DNA2 and DNA3 are as follows:

H1：5'-CGAATCAAGAGT-ACCTGGGGAGTAT-CACCGTATC-CTATGCCTGCAACGT-GATACGGTG-TGCGGAGGAAGGT-CTACAAATAAGC-3'

DNA1：5'-GATATC-AGCGATTAAC-ACTCTTGATTCG-3'

DNA2：5'-GCTTATTTGTAG -GTTACACCCATGT-TACTCT-3'

DNA 3: 5 '-fluorophore-AGAGTA-TrAG-GATATC-quencher-3'.

2. The detection kit according to claim 1, characterized in that: the reaction buffer of the assay kit contained 10mM Tris-HCl, pH 7.5, 50mM NaCl, 35mM MgCl₂。

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