CN116008546A - Application of DNA strand displacement tetrahedral nano probe in preparation of reagent for detecting ATP based on entropy driving, method and product - Google Patents
Application of DNA strand displacement tetrahedral nano probe in preparation of reagent for detecting ATP based on entropy driving, method and product Download PDFInfo
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 7
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 38
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 38
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention discloses application of a tetrahedral nano probe for DNA strand displacement in preparing a reagent for detecting ATP based on entropy driving, a method and a product thereof, wherein double-strand complex and fuel strand are designed through precise programming, so that a series of cascade strand displacement reactions of the double-strand complex and the fuel strand can be started by the existence of a trigger sequence, the trigger sequence is promoted to be recycled in the hybridization-strand displacement-dissociation-rehybridization process, an entropy driving DNA strand displacement dynamic loop is organically combined with the tetrahedron, and the high-sensitivity and high-specificity detection of ATP is realized under the condition of enzyme-free constant temperature, thereby having great advantages in terms of simplicity of operation and cost performance.
Description
Technical Field
The invention relates to the field of biological detection, in particular to application of a tetrahedral nano probe for DNA strand displacement in preparation of a reagent for detecting ATP based on entropy driving, and also relates to a method and a kit for detecting ATP.
Background
Adenosine Triphosphate (ATP) is an indispensable biological small molecule in organisms and plays a vital role in regulating cellular metabolism as an energy provider. ATP can be used for a variety of enzymatic reactions and to perform vital activities of cells and tissues. The ATP level often indirectly reflects the health status of the human body. For example, hypoglycemia, parkinson's and alzheimer's disease, tumors, cardiovascular disease, etc. are all associated with abnormal ATP levels. Thus, ATP detection is of great importance for various biological processes.
Traditional ATP detection methods include fluorescence analysis, mass spectrometry, high Performance Liquid Chromatography (HPLC) and the like, but the methods have the disadvantages of complex operation, time consumption, expensive instrument and reagent, high cost, low sensitivity, incapability of in-situ detection in cells and limited application range.
Therefore, there is a need to establish an ATP detection method that is enzyme-free, highly sensitive, and highly specific.
Disclosure of Invention
In view of this, the present invention establishes a programmed nucleic acid self-assembly process with entropy driven DNA strand displacement dynamic loop for energy kinetic control, namely: the driving force of the whole reaction is derived from free energy released by enthalpy change of a system during DNA hybridization, and a double-chain complex and a fuel chain are designed through precise programming, so that a series of cascade strand displacement reactions of the double-chain complex and the fuel chain can be started by the existence of a trigger sequence, and the cyclic utilization of the trigger sequence in the hybridization-strand displacement-dissociation-rehybridization process is promoted. The entropy driving DNA strand displacement dynamic loop is organically combined with tetrahedron, one side of the DNA tetrahedron is used as a double-chain complex, an aptamer/trigger sequence complex is self-assembled at one vertex, ATP molecules can be specifically combined with the ATP aptamer, and the trigger sequence is released, so that the entropy driving DNA strand displacement dynamic loop is started, the DNA tetrahedron is used as a membrane-penetrating carrier, the entropy driving DNA strand displacement dynamic loop is used for realizing enzyme-free constant temperature cascade amplification of intracellular ATP, and further, in-situ detection of intracellular ATP in micro-nano scale can be realized, and the specific principle is shown in figure 1.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. the application of a tetrahedron nano probe of DNA strand displacement in preparing a reagent for detecting ATP based on entropy driving is that the tetrahedron nano probe is integrally a tetrahedron, a double-chain compound formed by modifying a fluorescent group and a quenching group on one double-chain of the tetrahedron is also provided with a self-assembled ATP aptamer/trigger sequence compound and a fuel chain on the vertex of the tetrahedron;
the self-assembled ATP aptamer/trigger sequence complex is capable of specifically binding ATP under ATP-containing molecules and releasing the trigger sequence; the trigger sequence is capable of complementarily binding to a double-stranded complex of a modifying fluorescent group and a quenching group and releasing fluorescence by entropy driving under the action of a fuel chain.
Preferably, the double-stranded complex is formed by complementarily binding a DNA single strand modified with a fluorescent group with the 5' end of the fluorescent trigger strand and the 5' end of the Waste strand, and the 5' end of the fluorescent trigger strand on the double-stranded complex is modified with a quenching group.
Preferably, one vertex of the tetrahedron double-stranded complex strand is a free sequence, the other vertex is a trigger sequence recognition linker, the trigger sequence recognition linker is complementary to the 5 'end of the trigger sequence, and the 3' end of the trigger sequence is complementary to the DNA single-stranded part of the double-stranded complex.
In the present invention, the proximal end of the fuel strand is preferably complementary to a sequence complementary to the Waste strand and the fluorescence trigger strand on the single strand of DNA.
Preferably, the tetrahedral double-stranded complex is assembled by SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO. 6.
Preferably, the tetrahedron consists of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.7 and double-stranded complex.
2. The method for detecting ATP based on entropy driving is to mix the ATP to be detected with tetrahedron nano probe uniformly, then add entropy driving working solution, incubate for 2 hours at 37 ℃, and detect fluorescence signal value.
Preferably, the entropy driving working solution comprises the following components: 10mM Tris,5mM MgCl 2 ，pH 8.0。
3. The kit for detecting ATP based on entropy driving comprises a tetrahedron nano probe, wherein the tetrahedron nano probe is integrally tetrahedron, a double-chain of the tetrahedron is modified with a fluorescent group and a quenching group to form a double-chain complex, and a self-assembled ATP aptamer/trigger sequence complex and a fuel chain are also arranged on the vertexes of the tetrahedron;
the self-assembled ATP aptamer/trigger sequence complex is capable of specifically binding ATP under ATP-containing molecules and releasing the trigger sequence; the trigger sequence is capable of complementarily binding to a double-stranded complex of a modifying fluorescent group and a quenching group and releasing fluorescence by entropy driving under the action of a fuel chain.
Preferably, the kit further comprises entropy driving working solution, and the components are as follows: 10mM Tris,5mM MgCl 2 ，pH 8.0。
The invention has the beneficial effects that: the double-stranded complex and the fuel chain are designed through accurate programming, so that a series of cascade strand displacement reactions of the double-stranded complex and the fuel chain can be started by the existence of one trigger sequence, and the trigger sequence is promoted to be recycled in the hybridization-strand displacement-dissociation-rehybridization process. The method can complete the high-sensitivity and high-specificity detection of the intracellular and extracellular of ATP under the condition of no enzyme constant temperature, and has great advantages in terms of simplicity and cost performance of operation.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a schematic diagram of the detection of the present invention;
FIG. 2 is a graph of ATP fluorescence at various concentrations (a.fwdarw.f: blank,1pM,10pM,100pM,1nM,10 nM);
FIG. 3 is a graph showing peak values of ATP fluorescence curves at different concentrations;
FIG. 4 is a linear fit curve;
FIG. 5 shows the results of the specificity detection.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
The nucleotide chains required for the materials of the examples of the present invention were synthesized and modified by Sangon (Shanghai) Biotechnology Co., ltd. And the specific sequences used are shown in Table 1.
TABLE 1 nucleic acid sequences and modifications required for experiments
Example 1
The method for assembling the DNA tetrahedral nano probe comprises the following specific steps:
(1) Entropy driven working duplex of assembled DNA tetrahedra (p3+123+456): mu. L P3 (10. Mu.M), 1. Mu.L 123 (10. Mu.M), 1. Mu.L 456 (10. Mu.M) were added to 7. Mu.L TM buffer (20mM Tris,50mM MgCl) 2 pH 8.0), the reaction conditions are: 95 ℃/5min, 72 ℃/5min, 65 ℃/5min, 55 ℃/5min, 37 ℃/10min, 25 ℃/10min, and 4 ℃.
(2) The other three sides of the assembled DNA tetrahedron and ATP aptamer sequence (p1+p2+p4+67): 1. Mu. L P1 (10. Mu.M), 1. Mu. L P2 (10. Mu.M), 1. Mu. L P4 (10. Mu.M), 1. Mu.L 67 (10. Mu.M) were added to 6. Mu.L of TM buffer under the following reaction conditions: 95 ℃/5min, 80 ℃/5min, 72 ℃/5min, 60 ℃/5min, 50 ℃/5min.
(3) Assembled entropy driven DNA tetrahedral nanoprobe (p1+p2+p3+p4+123+456): mixing 20. Mu.L of the solutions of (1) and (2) above together, and adding 5. Mu.L of TM buffer under the following reaction conditions: 50 ℃/5min, 45 ℃/10min, 40 ℃/5min, 37 ℃/30min, 30 ℃/5min, 25 ℃/30min, 20 ℃/5min, 15 ℃/5min, 10 ℃/5min, 4 ℃.
Example 2 detection of ATP by assembled DNA tetrahedral nanoprobes
(1) Detection sensitivity of DNA tetrahedral nano probe based on entropy driving DNA strand displacement
Target ATP (2 mu L) with different concentrations is uniformly mixed with synthesized tetrahedral nano probe (25 mu L), 23 mu L of entropy driving working solution (10mM Tris,5mM MgCl2,pH 8.0) is added, the final concentration of ATP is 1pM,10pM,100pM,1nM and 10nM respectively, after incubation for 2 hours at 37 ℃, absorbance values in the wavelength range of 500 to 600nM are measured at excitation wavelength of 475 nM. And meanwhile, detecting the absorbance value of a blank sample (equivalent buffer solution), and calculating to obtain a detection limit and a standard curve.
The fluorescence curves corresponding to the ATP concentrations are shown in FIG. 2, and the peak fluorescence curves are shown in FIG. 3. In the detection strategy, when the target concentration is in the range of 1pM-1nM, the fluorescence signal response is in good linear relationship, as shown in FIG. 4The sum equation is F= 0.2762Lg (C) +1.344, R 2 = 0.9225, the lower detection Limit (LOD) reached 0.56pM.
(2) Detection specificity based on entropy driving DNA strand displacement DNA tetrahedral nano probe
Different NTP molecules (2. Mu.L) including ATP, GTP, CTP and TTP were mixed with the synthesized tetrahedral nanoprobe (25. Mu.L) and 23. Mu.L of entropy driving working solution (10mM Tris,5mM MgCl2,pH 8.0) was added to make the final concentration of ATP 1nM, and after incubation at 37℃for 2 hours, absorbance values at an excitation wavelength of 475nM were measured at emission wavelengths in the range of 500 to 600 nM. At the same time, absorbance values were measured for the blank samples (equivalent amount of buffer), and the results are shown in fig. 5. The results show that the DNA tetrahedral nano probe has a specific fluorescence response signal for detecting the target ATP.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
- The application of a tetrahedral nano probe for DNA strand displacement in preparing a reagent for detecting ATP based on entropy driving is characterized in that: the tetrahedron nano probe is integrally a tetrahedron, a double-chain of the tetrahedron is modified with a fluorescent group and a quenching group to form a double-chain complex, and a self-assembled ATP aptamer/trigger sequence complex and a fuel chain are also arranged on the vertex of the tetrahedron;the self-assembled ATP aptamer/trigger sequence complex is capable of specifically binding ATP under ATP-containing molecules and releasing the trigger sequence; the trigger sequence is capable of complementarily binding to a double-stranded complex of a modifying fluorescent group and a quenching group and releasing fluorescence by entropy driving under the action of a fuel chain.
- 2. The use according to claim 1, characterized in that: the double-chain complex is formed by complementarily binding a DNA single chain modified with a fluorescent group with the 5' end of a fluorescent trigger chain and the 5' end of a Waste chain, and the 5' end of the fluorescent trigger chain on the double-chain complex is modified with a quenching group.
- 3. The use according to claim 1, characterized in that: one vertex of the tetrahedron double-stranded complex strand is a free sequence, the other vertex is a trigger sequence recognition joint, the trigger sequence recognition joint is complementary with the 5 'end of the trigger sequence, and the 3' end of the trigger sequence is complementary with the DNA single-stranded part of the double-stranded complex.
- 4. The use according to claim 1, characterized in that: the near-vertex end of the fuel strand is complementary to a sequence on the single strand of DNA that is complementary to the Waste strand and the fluorescence trigger strand.
- 5. The use according to claim 1, characterized in that: the tetrahedral double-chain complex is assembled by SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO. 6.
- 6. The use according to claim 5, characterized in that: the tetrahedron consists of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.7 and double-stranded complex.
- 7. The method for detecting ATP based on entropy driving is characterized in that: uniformly mixing the sample to be detected with the tetrahedral nano probe, adding entropy driving working solution, incubating for 2 hours at 37 ℃, and detecting a fluorescence signal value;the tetrahedron nano probe is integrally a tetrahedron, a double-chain of the tetrahedron is modified with a fluorescent group and a quenching group to form a double-chain complex, and a self-assembled ATP aptamer/trigger sequence complex and a fuel chain are also arranged on the vertex of the tetrahedron;the self-assembled ATP aptamer/trigger sequence complex is capable of specifically binding ATP under ATP-containing molecules and releasing the trigger sequence; the trigger sequence is capable of complementarily binding to a double-stranded complex of a modifying fluorescent group and a quenching group and releasing fluorescence by entropy driving under the action of a fuel chain.
- 8. The method according to claim 7, wherein: the entropy driving working solution comprises the following components: 10mM Tris,5mM MgCl 2 ，pH 8.0。
- 9. Kit for detecting ATP based on entropy driving, characterized in that: the kit comprises a tetrahedron nano probe, wherein the tetrahedron nano probe is integrally a tetrahedron, a double-chain of the tetrahedron is modified with a fluorescent group and a quenching group to form a double-chain complex, and a self-assembled ATP aptamer/trigger sequence complex and a fuel chain are also arranged on the vertexes of the tetrahedron;the self-assembled ATP aptamer/trigger sequence complex is capable of specifically binding ATP under ATP-containing molecules and releasing the trigger sequence; the trigger sequence is capable of complementarily binding to a double-stranded complex of a modifying fluorescent group and a quenching group and releasing fluorescence by entropy driving under the action of a fuel chain.
- 10. The kit of claim 9, wherein: the kit also contains entropy driving working solution, and the components are as follows: 10mM Tris,5mM MgCl 2 ，pH 8.0。
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