阅读说明：本技术 一种构建结构开关型核酸适配体的通用方法及应用其的通用型传感器和其构建方法 (general method for constructing structure switch type aptamer, general sensor using same and construction method thereof ) 是由 娄新徽 黄旸 于 2018-07-21 设计创作，主要内容包括：本发明涉及一种构建结构开关型核酸适配体的通用方法,以及应用其检测多种类型靶标的通用型传感器。通过将核酸适配体的两端分别连接富含鸟嘌呤核苷酸(G)的序列来构建具有结构开关性能的探针(Apt-G4)。当没有能够与核酸适配体特异性结合的靶标存在时,Apt-G4形成G-四聚体结构(G4)；当有靶标存在时,靶标与核酸适配体结合,G4被破坏。Apt-G4设计对不同类型靶标的核酸适配体具有普适性。利用G4可以显著增强氯化血红素催化H<Sub>2</Sub>O<Sub>2</Sub>与无色的2’-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)反应生成绿色ABTS<Sup>+·</Sup>的性质,分别实现对汞离子、凝血酶和磺胺地索辛的信号减型的比色检测。(The invention relates to general methods for constructing structure switch type aptamers and a general sensor for detecting multiple types of targets by applying the methods, wherein the two ends of the aptamers are respectively connected with sequences rich in guanine nucleotide (G) to construct a probe (Apt-G4) with structure switch performance, when no target capable of being specifically combined with the aptamers exists, Apt-G4 forms a G-tetramer structure (G4), when the target exists, the target is combined with the aptamers, G4 is damaged, Apt-G4 is designed to have universality for the aptamers of different types of targets, and G4 can remarkably enhance chlorhematin catalysis H catalysis 2 O 2 Reacting with colorless 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS) to generate green ABTS +· The colorimetric detection of signal reduction of mercury ions, thrombin and sulfadimethoxine is realized respectively.)

1, general methods for constructing a structure switch type aptamer, comprising the steps of designing a structure switch type aptamer by ligating a sequence rich in guanine nucleotide G to each of both ends of the aptamer to construct a structure switch type probe Apt-G4.

2. The universal method for constructing a structure-switched aptamer according to claim 1, wherein the probe Apt-G4 with structure-switching property is A-SULF-A; A-TBA 29-A; and A-T11-A.

3, construction method of universal sensor, comprising the steps of (1) designing nucleic acid aptamer with structure switch performance, connecting the two ends of the nucleic acid aptamer with sequences rich in guanine nucleotide G to construct probe Apt-G4 with structure switch performance, and (2) heating Apt-G4 at 95 ℃ and slowly cooling to room temperature.

4. The method for constructing the universal sensor according to claim 3, further comprising the steps of (3) adding substances to be tested with different concentrations to incubate with Apt-G4 to form a mixed solution; adding hemin (hemin) and incubating the mixed solution; and (5) simultaneously adding hydrogen peroxide and 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic acid, measuring the ultraviolet absorption at 418nm of the selected time, and drawing a working curve by taking the ultraviolet absorption value as a vertical coordinate and the concentration of the substance to be measured as a horizontal coordinate.

5. The method for constructing a universal sensor according to claim 3, wherein in the step (2), the temperature is kept at 95 ℃ for 6 minutes, and then the temperature is slowly cooled to 25 ℃.

6. The method for constructing a universal sensor according to claim 4, further comprising the step (6) of selectively testing: and (5) replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiments in the steps (1) to (5).

7. The method of claim 6, wherein the probe Apt-G4 with structure switching performance is configured as a solution with a concentration of 0.375 μ M.

8, general-purpose sensor for constructing structure-switched aptamers, wherein the sensor comprises aptamers with structure-switched performance, and the two ends of each aptamer are respectively connected with a sequence rich in guanine nucleotide G to construct a probe Apt-G4 with structure-switched performance.

9. The sensor of claim 8, wherein the probe Apt-G4 with structural switching capability is A-SULF-A; A-TBA 29-A; and A-T11-A.

10. The sensor of claim 8 or 9, wherein the probe Apt-G4 with structural switching properties is configured as a 0.375 μ M solution.

Technical Field

The invention relates to general methods for constructing structure switch type aptamer, a general sensor for detecting various types of targets (metal ions, proteins and organic small molecules) by applying the general method and a construction method thereof, belonging to the technical field of biology.

Background

Aptamers are obtained from in vitro synthesized oligonucleotide libraries through multiple rounds of affinity Enrichment, polymerase chain reaction and other steps by means of in vitro screening technology, namely, systematic evolution of Ligands by amplification and evolution, SELEX (Nature,1990,346(6287), 818-.

In recent years, SSA-based sensors have been developed with great variety of advantages, including simple structure, easy operation, high sensitivity and good specificity, but currently screening SSA has very few methods and low efficiency (Proc. Natl. Acad. Sci. U.S.A.2010,107, 14053-), requires 15-20 screening cycles, and obtains nucleic acid aptamers with poor selectivity and low affinity.

Disclosure of Invention

In view of the problems of the prior art, the invention aims to provide general methods for constructing structure switch type aptamers and general sensors for detecting various types of targets (metal ions, proteins and organic small molecules) by using the same.

The invention provides general methods for constructing structure switch type aptamers, which comprise the following steps of designing the aptamers with structure switch performance, and constructing a probe Apt-G4 with structure switch performance by respectively connecting sequences rich in guanine nucleotide G to two ends of the aptamers.

, the probe Apt-G4 with structure switch performance is A-SULF-A, A-TBA29-A and A-T11-A in the general method for constructing the structure switch type aptamer.

In addition, the invention also provides a construction method of universal sensors, which comprises the steps of (1) designing a nucleic acid aptamer with structural switch performance, connecting sequences rich in guanine nucleotide G to two ends of the nucleic acid aptamer respectively to construct a probe Apt-G4 with the structural switch performance, step (2) heating Apt-G4 at 95 ℃, slowly cooling to room temperature, step (3) adding substances to be tested with different concentrations and Apt-G4 to form mixed liquid, step (4) adding hemin (hemin) to incubate with the mixed liquid, step (5) simultaneously adding hydrogen peroxide and 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic acid, measuring ultraviolet absorption at 418nm of a selected time, and drawing a working curve by taking an ultraviolet absorption value as a vertical coordinate and the concentration of the substances to be tested as a horizontal coordinate.

In the method for constructing the general-purpose sensor, in the step (2), the temperature is maintained at 95 ℃ for 6 minutes, and then the temperature is gradually cooled to 25 ℃.

In addition, the method for constructing the general-purpose sensor further comprises the step (6) of a selectivity test: and (5) replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiments in the steps (1) to (5).

, according to the method for constructing the universal sensor, the probe Apt-G4 with structural switching performance is configured to be a solution with a concentration of 0.375. mu.M.

The invention also provides general sensors for constructing the structure switch type aptamer, which comprises the aptamer with structure switch performance, wherein the two ends of the aptamer are respectively connected with sequences rich in guanine nucleotide G to construct a probe Apt-G4. with structure switch performance, the probe Apt-G4 is A-SULF-A, A-TBA29-A, and A-T11-A, and the probe Apt-G4 with structure switch performance is configured into a solution with the concentration of 0.375 mu M.

The specific experimental steps of the invention are as follows:

(1) design of aptamers with structural switching properties: connecting sequences rich in guanine nucleotide (G) to two ends of the aptamer respectively to construct a probe (Apt-G4) with structure switch performance;

(2) heating Apt-G4 at 95 ℃, and slowly cooling to room temperature;

(3) adding substances to be detected with different concentrations and incubating with Apt-G4;

(4) adding hemin (hemin) and incubating with the above mixture;

(5) with addition of hydrogen peroxide (H)₂O₂) Measuring the ultraviolet absorption at 418nm of the selected time with 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS), and drawing a working curve by taking the ultraviolet absorption value as a vertical coordinate and the concentration of the substance to be measured as a horizontal coordinate;

(6) and (3) selective test: and replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiment in 1-5 steps.

The method and the sensor thereof have the following advantages:

1) any aptamer can be designed using the SSA design method of the present invention as an aptamer with significant structural switching properties, i.e., SSA;

2) the SSA design method has good universality, not only can convert non-SSA into SSA, but also can convert SSA with different structural switch performances into SSA with systematic conformational change, namely SSA with G-tetramer structural switch performance;

3) the SSA designed by the invention can be used for constructing a sensor with universality and can be used for detecting various different types of targets.

Drawings

FIG. 1 is a schematic diagram of the colorimetric detection of a target using SSA constructed by the method of the present invention.

FIG. 2 is a diagram (A) and a working curve (B) of the ultraviolet-visible absorption spectrum of sulfadimethoxine detected by the method of the present invention.

FIG. 3 is a graph of the results of a selectivity test for a sulfadoxine colorimetric sensor constructed according to the method of the present invention.

FIG. 4 shows a graph (A) of the UV-VIS absorption spectrum and a working curve (B) of thrombin detected according to the method of the present invention.

FIG. 5 is a graph showing the results of a selectivity test of a thrombin colorimetric sensor constructed according to the method of the present invention.

FIG. 6 is a graph (A) of the UV-VIS absorption spectrum and a working curve (B) of mercury ion detection according to the method of the present invention.

FIG. 7 is a graph of the results of a selectivity test for a mercury ion colorimetric sensor constructed according to the method of the present invention.

FIG. 8 is a circular dichroism spectrum of three SSAs constructed according to the methods of the present invention (A: A-SULF-A; B: A-TBA 29-A; and C: A-T11-A) before and after addition of heme and target (A: sulfadimethoxine; B: thrombin; C: mercuric ions).

Detailed Description

Table 1: sequence information of the DNA used in the present invention.

Name of probe	Sequence (5 '-3')
		A-SULF-A	AGGGACGGGACTAGAGAGGGCAACGAGTGTTTATAGAAGGGACGGGA
A-TBA29-A	AGGGACGGGAAGTCCGTGGTAGGGCAGGTTGGGGTGACTAGGGACGGGA
		A-T11-A	AGGGACGGGATTTTTTTTTTTAGGGACGGGA

FIG. 1 is a schematic diagram of the colorimetric detection of a target using SSA constructed by the method of the present invention. The two ends of any aptamer are respectively connected with a sequence rich in guanine nucleotide (G) to construct a probe (Apt-G4) with structure switch performance; when no target exists, Apt-G4 forms a G-tetramer structure which can enhance heme catalysis ABTS oxidation to form a colored product ABTS^+·Forming; when the target exists, the aptamer fragment in Apt-G4 is combined with the target, the G-tetramer structure is destroyed, and the color product ABTS formed by oxidation of ABTS catalyzed by heme cannot be enhanced^+·Is performed.

FIG. 2 is a diagram (A) and a working curve (B) of the ultraviolet-visible absorption spectrum of sulfadimethoxine detected by the method of the present invention. In FIG. 2, the left graph A is a graph of the ultraviolet-visible absorption spectrum, and the ordinate of the right graph B is the absorbance value at 418 nanometers (nm) of the test sample at the eighth minute. The SSA used was A-SULF-A (Table 1).

FIG. 3 is a graph of the results of a selectivity test for a sulfadoxine colorimetric sensor constructed according to the method of the present invention. The final concentrations of sulfadoxine, ampicillin, kanamycin A, kanamycin B, tetracycline were all 1 millimole per liter (mM). The values on the ordinate were calculated from the absorbance values at 418nm at the eighth minute for each sample and blank. The difference between the absorbance of the blank experiment and the absorbance of the sample containing sulfadoxine is one hundred percent.

FIG. 4 shows a graph (A) of the UV-VIS absorption spectrum and a working curve (B) of thrombin detected according to the method of the present invention. In FIG. 4, the left side A is a graph of the UV-visible absorption spectrum, and the ordinate of the right side B is the absorbance value at 418nm of the test sample at the eighth minute. The SSA used was A-TBA29-A (Table 1).

FIG. 5 is a graph showing the results of a selectivity test of a thrombin colorimetric sensor constructed according to the method of the present invention. The final concentrations of thrombin and bovine serum albumin were 20 micromoles per liter (. mu.M). The values on the ordinate were calculated from the absorbance values at 418nm at the eighth minute for each sample and blank. The difference between the absorbance of the blank and the absorbance of the thrombin-containing sample was one hundred percent.

FIG. 6 is a graph (A) of the UV-VIS absorption spectrum and a working curve (B) of mercury ion detection according to the method of the present invention. In FIG. 6, the left graph A is a graph of the UV-visible absorption spectrum, and the ordinate of the right graph B is the absorbance value of the test sample at 418nm at the eighth minute. The SSA used was A-T11-A (Table 1).

FIG. 7 is a graph of the results of a selectivity test for a mercury ion colorimetric sensor constructed according to the method of the present invention. Hg is a mercury vapor²⁺、Pb^{2 +}、Fe²⁺、Fe³⁺、Mg²⁺、Ca²⁺、Cd²⁺、Co²⁺、Zn²⁺、Ni²⁺All concentrations of (2) were 2. mu.M. The values on the ordinate were calculated from the absorbance values at 418nm at the eighth minute for each sample and blank. Absorbance and Hg content in blank experiment²⁺The difference in absorbance of the samples of (1) is one hundred percent.

FIG. 8 is a circular dichroism spectrum of three SSAs constructed according to the methods of the present invention (A: A-T11-A; B: A-SULF-A; and C: A-TBA29-A) before and after addition of heme and target (A: mercuric ion; B: sulfadimethoxine; C: thrombin). In FIG. 8, from left to right, A-T11-A + heme + mercuric ions; A-SULF-A + heme + sulfadimethoxine; A-TBA29-A + heme + thrombin.

The specific experimental steps of the invention are as follows:

(1) design of aptamers with structural switching properties: connecting sequences rich in guanine nucleotide (G) to two ends of the aptamer respectively to construct a probe (Apt-G4) with structure switch performance;

(2) heating Apt-G4 at 95 ℃, and slowly cooling to room temperature;

(3) adding substances to be detected with different concentrations and incubating with Apt-G4;

(4) adding hemin (hemin) and incubating with the above mixture;

(5) are added simultaneouslyHydrogen peroxide (H)₂O₂) Measuring the ultraviolet absorption at 418nm of the selected time with 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS), and drawing a working curve by taking the ultraviolet absorption value as a vertical coordinate and the concentration of the substance to be measured as a horizontal coordinate;

(6) and (3) selective test: and replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiment in 1-5 steps.