CN110734957A - general method for constructing structure switch type aptamer, general sensor using same and construction method thereof - Google Patents
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Abstract
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 catalysis2O2Reacting 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.
Description
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+、Pb2 +、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.
Example 1. working curve of sulfadoxine and selectivity test of the sensor were measured according to the method of the present invention.
A-SULF-A was prepared as a 0.375 micromole per liter (. mu.M) solution (20mM Tris-HCl, 50mM NaCl, 5mM KCl, 5mM MgCl)2pH 8.0), heating at 95 deg.C for 6 min, and slowly cooling to room temperature (about 25 deg.C). Mu.l (μ L) of A-SULF-A solution was taken and 60 μ L of sulfadoxine solutions of different concentrations (20mM Tris-HCl, 50mM NaCl, 5mM KCl, 5mM MgCl)2pH 8.0) (0. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 500. mu.M, 1000. mu.M), and incubated for 30 minutes. Heme (10. mu.L, 3. mu.M) was then added and incubated for 30 minutes at room temperature in the absence of light. Finally, ABTS (20. mu.L, 3.75mM) and H were added simultaneously2O2(10. mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. And drawing a working curve.
50 μ L of the heat-treated A-SULF-A solution was added with sulfadoxine, kanamycin A, kanamycin B, ampicillin, and tetracycline (60 μ L, 2.5mM), respectively, and incubated for 30 minutes. Heme (10. mu.L, 3. mu.M) was then added and incubated at 37 ℃ for 30 minutes in the absence of light. Finally, ABTS (20. mu.L, 3.75mM) and H were added simultaneously2O2(10. mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. And calculating the relative signal change of each target by taking the difference between the absorbance of the blank experiment and the absorbance of the sample containing sulfadoxine as one hundred percent.
The obtained working curve is shown in FIG. 2, and the detection limit is 0.2. mu.M, and the detection range is 0.2. mu.M to 1000. mu.M. The sulfadoxine sensor has good selectivity (figure 3), and the selectivity for kanamycin A, kanamycin B, ampicillin and tetracycline is respectively 25.8 times, 8.1 times, 10.8 times and 26.3 times.
Example 2 working curves for the detection of thrombin and selectivity tests of the sensor according to the method of the invention.
A-TBA29-A was prepared as a 0.375. mu.M solution [25mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 20mM KNO3，200mM NaNO30.025% (w/v%) Triton X-100, 1% (v/v% (mass/volume%) dimethyl sulfoxide (DMSO), 150mM NH4Cl， pH 5.3]Heating at 95 deg.C for 6 min, and slowly cooling to room temperature. Mu.l (. mu.L) of A-TBA29-A solution was taken and 60. mu.L of thrombin solution (25mM HEPES, 20mM KNO3, 200mM NaNO3, 0.025% (w/v%) Triton X-100, 1% (v/v%) DMSO, 150mM NH4+pH 5.3) (0. mu.M, 0.1. mu.M, 0.2. mu.M, 0.5. mu.M, 1. mu.M, 10. mu.M, 50. mu.M), and incubated for 30 minutes. Heme (10. mu.L, 3. mu.M) was then added and incubated for 30 minutes at room temperature in the absence of light. Finally, ABTS (20. mu.L, 3.75mM) and H were added simultaneously2O2(10. mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. And drawing a working curve.
50 μ L of the above-mentioned heat-treated A-TBA29-A solution was added with thrombin and bovine serum albumin (60 μ L, 50 μ M), and incubated for 30 minutes. Heme (10. mu.L, 3. mu.M) was then added and incubated at 37 ℃ for 30 minutes in the absence of light. Finally, ABTS (20. mu.L, 3.75mM) and H were added simultaneously2O2(10. mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. The relative signal change of each target was calculated using the difference between the absorbance of the blank and the absorbance of the thrombin-containing sample as one hundred percent.
The obtained working curve is shown in FIG. 4, with a detection limit of 0.1. mu.M and a detection range of 0.1. mu.M to 50. mu.M. The thrombin sensor was very selective (FIG. 5), with a selectivity for bovine serum albumin of 19.2 times.
Example 3. working curve for detection of mercury ions and selectivity test of the sensor according to the method of the invention.
A-T11-A was prepared as a 0.375. mu.M solution (25mM HEPES, 20mM KNO3, 200mM NaNO3, 0.025% (w/v%) Triton X-100, 1% (v/v%) DMSO, 150mM NH4Cl, pH 5.3), heating at a constant temperature of 95 ℃ for 6 minutes, and then slowly cooling to room temperature. 50 μ L of A-T11-A solution was added with 60 μ L of mercuric nitrate solution (25mM HEPES, 20mM KNO3, 200mM NaNO3, 0.025% (w/v%) Triton X-100, 1% (v/v%) DMSO, 150mM NH4+，pH 5.3)(0nM、50nM、250nM、
500nM, 1000nM, 2000nM, 5000nM, 10000nM), incubated for 30 min. Heme (10. mu.L, 3. mu.M) was then added and incubated for 30 minutes at room temperature in the absence of light. Finally, ABTS (20. mu.L, 3.75mM) and H were added simultaneously2O2(10. mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. And drawing a working curve.
Hg was added to 50. mu.L of the heat-treated A-T11-A solution2+、Pb2+、 Fe2+、Fe3+、Mg2+、Ca2+、Cd2+、Co2+、Zn2+、Ni2+(60. mu.L, 5. mu.M) and incubated for 30 minutes. Heme (10. mu.L, 3. mu.M) was then added and incubated at 37 ℃ for 30 minutes in the absence of light. Finally, ABTS (20. mu.L, 3.75mM) and H were added simultaneously2O2(10. mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. The relative signal change of each target was calculated using the difference between the absorbance of the blank and the absorbance of the thrombin-containing sample as one hundred percent.
The obtained working curve is shown in FIG. 6, with a detection limit of 50nM and a detection range of 50nM to 5. mu.M. The mercury ion sensor has good selectivity (figure 7), and the selectivity to metal ions is 18.5-37.0 times.
Example 4. conformational change upon binding of a SSA constructed according to the methods of the invention to a target.
To further confirm that the SSA constructed according to the present invention ((A-SULF-A, AT11A, A-TBA29-A, Table 1) has structural switching properties, the conformation of the SSA before and after target addition was determined by binary Chromatography (CD). The experiment found that TritonX-100 affects CTesting of the D spectra, therefore, in the study of SSA of thrombin and mercury ions, the binding buffer used was depleted of this component (25mM HEPES, 20mM KNO)3，200mM NaNO3，1％(v/v％)DMSO， 150mM NH4Cl, pH 5.3). The experimental procedure was as follows: heat-treating 2 μ M SSA at 95 deg.C for 6 min, and slowly cooling to room temperature; hemoglobin (1 μ L0.4 mM) was added to SSA (100 μ L, 2 μ M) and incubated for 90 minutes; add 100. mu.L of the target molecule corresponding to SSA (100. mu.M sulfadimethoxine, 100. mu.M Hg)2+50 μ M thrombin) for 60 minutes; blank SSA, and hemoglobin incubated SSA were added to target molecule SSA for CD profiling.
As can be seen from FIG. 8, all three SSAs have a parallel G tetrameric structure with a negative peak at 240nm and a positive peak at 260 nm. The parallel G tetramer structure is enhanced after SSA is combined with heme, and is destroyed after target molecules are added, and CD characteristic peaks disappear.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention.
Claims (10)
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.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160194641A1 (en) * | 2013-05-24 | 2016-07-07 | Council Of Scientific & Industrial Research | G-RICH isoDNA SEQUENCES FORMING G-QUADRUPLEX STRUCTURE, THEIR FUNCTION AS APTAMERS AND A PROCESS FOR THE PREPARATION THEREOF |
| CN107794295A (en) * | 2017-11-09 | 2018-03-13 | 湖南工程学院 | A kind of blood coagulation enzyme assay method and kit that ring mediated isothermal amplification is opened based on double Aptamer interlayer structures |
-
2018
- 2018-07-21 CN CN201810807459.0A patent/CN110734957B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160194641A1 (en) * | 2013-05-24 | 2016-07-07 | Council Of Scientific & Industrial Research | G-RICH isoDNA SEQUENCES FORMING G-QUADRUPLEX STRUCTURE, THEIR FUNCTION AS APTAMERS AND A PROCESS FOR THE PREPARATION THEREOF |
| CN107794295A (en) * | 2017-11-09 | 2018-03-13 | 湖南工程学院 | A kind of blood coagulation enzyme assay method and kit that ring mediated isothermal amplification is opened based on double Aptamer interlayer structures |
Non-Patent Citations (2)
| Title |
|---|
| JIANGTAO REN 等: "Versatile G-quadruplex-mediated strategies in label-free biosensors and logic systems", 《ANALYST》 * |
| LONG MA 等: "Design of an aptamer – based fluorescence displacement biosensor for selective and sensitive detection of kanamycin in aqueous samples", 《RSC ADV.》 * |
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