Self-hybridization chain reaction hairpin probe combination for lead ion detection and kit
By designing a single-channel probe combination for lead ion detection, the structure of the traditional HCR system is simplified, the cost and complexity are reduced, high sensitivity detection is achieved, and broad application prospects are demonstrated.
Patent Information
- Application Number
- CN202411982882.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-18
AI Technical Summary
The traditional hybrid chain reaction (HCR) architecture is complex in design and contains a large number of sequences, resulting in increased reaction costs and limited reduction in design complexity.
A combination of self-hybrid chain reactions for lead ion detection is designed, using a single DNA chain probe, and triggering HCR reactions with trigger strands, simplifying structure and reducing costs. The probe combination includes GR-5 DNAzyme and a hairpin probe consisting of a sticky end, a hairpin stem of a palindromic sequence and a hairpin ring.
By simplifying the HCR architecture, the reaction cost and complexity of sequence design and operation process are reduced, high-sensitivity lead ion detection is achieved, and broad application prospects are shown in the detection of other metal ions.
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Figure CN119842879A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of DNA amplification and detection, and in particular, to a hairpin probe combination, a kit and an application thereof for self-hybridization chain reaction for lead ion detection. Background Art
[0002] Lead ion (Pb 2+ ) is a typical heavy metal pollutant. Due to its difficult biodegradation, it poses a potential risk to the ecological environment and human health and safety that cannot be ignored. In the natural environment, it accumulates in soil and water, thus affecting the entire food chain. As far as human health is concerned, even trace amounts of Pb 2+ It may also cause a series of adverse health effects. In order to effectively solve the problem of lead pollution, it is necessary to develop highly sensitive and selective Pb 2+ Detection technology is particularly important.
[0003] The hybridization chain reaction (HCR) is a DNA circuit first reported by Pierce in 2004. It usually consists of a trigger strand DNA and two metastable hairpin DNAs (H1 and H2). Before the trigger strand is added, the two hairpins cannot hybridize with each other and remain in a metastable state. Once the trigger strand is added, H1 is opened by the trigger strand through toehold-mediated strand displacement (TMSD), and then the exposed sequence can hybridize with the H2 sticky end, opening a single-stranded region with the same sequence as the trigger strand, thereby continuing to open another H1 and repeating the above strand displacement reaction. Finally, the resulting chain reaction leads to the formation of a gapped DNA nanowire.
[0004] The DNA hairpin probe used in traditional HCR usually has 48 bases, including 6 sticky terminal bases, 6 hairpin loop bases and 36 hairpin stem bases. Such a design not only increases the complexity of sequence design, but also increases the reaction cost accordingly. In order to simplify the HCR system, people have designed DNA hairpin probes with base numbers between 36 and 42. However, this simplified strategy has caused a significant increase in background signal leakage due to excessive reduction in the number of bases in the hairpin stem. To overcome this problem, Asanuma et al. developed an HCR system based on left-handed acyclic D-threitol nucleic acid (D-aTNA), which successfully shortened the length of the hairpin probe to 26 bases. Winssinger et al. also reported an HCR system based on peptide nucleic acid (PNA), which only requires a 20-base hairpin probe to achieve signal amplification.
[0005] Although the above method shortens the number of bases in the hairpin sequence to a certain extent, it is inevitable that two hairpin probes are still used, which results in a limited reduction in cost and design complexity brought about by this simplification. Therefore, the HCR system still needs to be further simplified.
[0006] Application Contents
[0007] The current HCR system architecture is complex and contains many sequences. The system is mainly composed of two DNA hairpin probes (H1 and H2) and a trigger DNA chain. The two DNA hairpin probes each have three key domains: sticky ends, hairpin stems and loops. In the absence of a trigger DNA chain, the complementary bases inside the hairpin structures of H1 and H2 interact with each other, allowing them to coexist in a metastable state in the solution.
[0008] Since the traditional HCR system requires two DNA hairpin probes and has a complex structural design, the cost of the entire reaction increases; in response to the current problem, the present application discloses a hairpin probe combination for self-hybridization chain reaction for lead ion detection, which simplifies the structure and reduces costs by designing a single DNA hairpin probe and initiating the HCR reaction with a trigger chain.
[0009] The present application discloses a hairpin probe combination for self-hybridization chain reaction for lead ion detection, the hairpin probe combination comprising GR-5 DNAzyme and a hairpin probe;
[0010] GR-5 DNAzyme contains Pb 2+ A specific DNA enzyme chain and a substrate chain, wherein the substrate chain contains a trigger chain sequence;
[0011] The hairpin probe consists of sticky ends at both ends, a hairpin stem with a palindromic sequence, and a hairpin loop.
[0012] In certain optimized embodiments, the sum of the cytosine bases and the guanine bases in the hairpin stem portion of the palindromic sequence is not less than two-thirds of the total number of bases;
[0013] After optimization, the number of cytosine bases and guanine bases contained in the hairpin stem part of the palindrome sequence is exactly two-thirds of the total number of bases.
[0014] In certain optimized embodiments, the hairpin stem portion of the palindromic sequence is 18 bases in length.
[0015] In certain optimized embodiments, the length of the hairpin loop is 12 bases, and the total number of bases of the sticky ends at both ends is consistent with the total number of bases of the hairpin loop.
[0016] In certain optimized embodiments, the sticky end sequences at both ends are identical and are complementary to the hairpin loop sequence bases.
[0017] In certain optimized embodiments, the GR-5 DNAzyme comprises Pb 2+ The invention relates to a specific DNA enzyme chain and a substrate chain, wherein a trigger chain sequence is designed in the substrate chain; the trigger chain sequence includes a hairpin sticky end, a hairpin stem and a free segment; the hairpin sticky end and the free segment sequence are the same, the hairpin sticky end can be complementary to the base of the hairpin probe sticky end, and the hairpin stem can be complementary to the base of the hairpin probe stem.
[0018] In certain optimized embodiments, the sequence of the hairpin probe is shown as SEQ ID NO.1, the sequence of the DNA enzyme chain is shown as SEQ ID NO.2, and the sequence of the substrate chain is shown as SEQ ID NO.3.
[0019] The principle of the traditional HCR system is as follows Figure 1 As shown in the figure, in the presence of a trigger DNA chain, the sticky end (a) of the hairpin probe H1 will hybridize with the base-paired sequence a* in the trigger chain (I), causing the hairpin structure of H1 to unfold and generate the intermediate product I·H1. Then, the newly exposed sequence c of H1 will undergo base-pairing with the sticky end (c*) of H2, thereby opening the hairpin structure of H2 and generating the complex I·H1·H2. At this time, a sequence (a*b*) identical to that of I is exposed on H2, which can further trigger and unfold the new H1 hairpin structure, thereby initiating a series of cascade reactions and ultimately generating a long-chain DNA nanowire with a cut.
[0020] In a specific embodiment of the present application, the number of cytosine bases and guanine bases contained in the hairpin stem portion of the palindromic sequence is exactly two-thirds of the total number of bases.
[0021] At the same time, the hairpin probe is designed so that its two sticky ends form a complementary pair with the hairpin loop region. Specifically, the two sticky end regions each contain 6 thymine bases (T), while the hairpin loop region is composed of 12 adenine bases (A). This design is intended to ensure the formation of a stable H hairpin structure. At the same time, under the action of trigger chain I, once the hairpin structure of H is opened, the exposed sticky end can effectively open another H hairpin structure, so that the process can be repeated, eventually forming a long-chain DNA nanowire with a cut. Therefore, the probe combination designed in this application is named self-hybridization chain reaction based on a single hairpin, abbreviated as SHCR.
[0022] Based on the above single-hair probe SHCR system, it is used for Pb 2+ The reaction principle of the test is as follows Figure 2The hairpin probe H proposed in the present application has a symmetrical structural design so that the 5' end or 3' end of the trigger strand T can hybridize with the 3' end or 5' end of the hairpin H, respectively, to improve the efficiency of the hybridization reaction.
[0023] In another aspect, the present application provides a detection kit, which includes any of the aforementioned hairpin probe combinations for self-hybridization chain reaction for lead ion detection.
[0024] In certain optimized embodiments, the SHCR reaction uses a buffer solution of 40 mM Tris-HCl (including 20 mM magnesium chloride, pH 7.4).
[0025] The reaction system using the buffer solution is as follows: the DNA enzyme chain and the substrate chain (including the trigger chain sequence) are pre-mixed together using the buffer solution, and then the mixture and the hairpin probe are annealed at 95°C for 5 minutes and naturally cooled to room temperature for 2 hours. 2+ and hairpin probes were mixed in buffer solution and incubated at room temperature.
[0026] Compared with the prior art, the beneficial effects of the present application include: the self-hybridization chain reaction SHCR (self-hybridization chain reaction) provided by the present application successfully simplifies the hybridization chain reaction (HCR) that traditionally requires two DNA hairpin probes into a SHCR system that only requires a single DNA hairpin probe by designing the stem of the DNA hairpin probe to embed a palindromic sequence, and combines it with GR-5 DNAzyme for Pb 2+ This design effectively reduces the reaction cost and the complexity of sequence design and operation process. In addition, the system can be further applied to the detection of other metal ions by designing specific DNA enzymes for different metal ions, showing broad application prospects.
[0027] Description of the accompanying tables and graphs
[0028] Figure 1 It is a reaction schematic diagram of the traditional hybridization chain reaction HCR provided in this application;
[0029] Figure 2 The SHCR-based Pb detection method provided in this application 2+ Schematic diagram of the principle;
[0030] Figure 3 is a schematic diagram of the simulated structure of the hairpin probe H in the SHCR provided in Example 1;
[0031] Figure 4is a schematic diagram of the analysis results of SHCR before and after the reaction using fluorescence detection, gel electrophoresis and atomic force microscopy imaging technology provided in Example 3;
[0032] Figure 5 is a schematic diagram of the results of the analysis of the optimized experimental conditions for the SHCR reaction provided in Example 5;
[0033] Figure 6 The SHCR for Pb provided in Example 6 2+ Schematic diagram of the results of sensitivity testing;
[0034] Figure 7 The SHCR for Pb provided in Example 7 2+ Schematic diagram of the results of specific detection; DETAILED DESCRIPTION
[0035] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0036] The features and performance of the present application are further described in detail below in conjunction with the embodiments.
[0037] Example 1
[0038] This embodiment designs a sequence of a self-hybridization chain reaction SHCR (self-hybridization chain reaction) based on a single hairpin.
[0039] In this embodiment, lead ions (Pb 2+ ) as a model molecule and introduced the specific recognition of Pb 2+ The GR-5 DNAzyme is combined with the SHCR system. The designed hairpin probe combination includes GR-5 DNAzyme (including the trigger chain sequence) and the hairpin probe, wherein the sequence of the hairpin probe is shown in SEQ ID NO.1, the sequence of the DNA enzyme chain is shown in SEQ ID NO.2, and the sequence of the substrate chain is shown in SEQ ID NO.3.
[0040] By simulating the DNA sequence of the hairpin probe, the results are as follows Figure 3 shown. Figure 3A shows the expected structure of the hairpin probe, where the hairpin stem region consists of 18 pairs of complementary bases and the hairpin loop region contains 12 bases. This single hairpin structure is relatively stable and is the main product after annealing. In addition, due to the introduction of the palindromic sequence, Figure 3 The double hairpin structure shown in B is a byproduct after annealing.
[0041] Example 2
[0042] Based on the hairpin probe combination designed in Example 1, the two sides of the hairpin probe stem are modified with a fluorescent group FAM and a quenching group BHQ1, and the hairpin probe sequence with a fluorescent group and a quenching group is shown in SEQ ID NO.4; the present design uses fluorescence intensity as a signal output mode, thereby designing a Pb based on a self-hybridization chain reaction of a single hairpin 2+ Detection of fluorescent biosensors.
[0043] Example 3
[0044] In order to compare the effects of the hairpin probe of the hairpin probe combination designed in the present application and GR-5 DNAzyme, lead ions (Pb 2+ ) as a model molecule to specifically recognize Pb 2+ The DNA enzyme is combined with the SHCR system, and the fluorescence intensity is used as the signal output mode. The fluorescent group FAM and the quenching group BHQ1 are modified on both sides of the stem of the hairpin probe, respectively, so as to design a Pb-based self-hybridization chain reaction based on a single hairpin. 2+ Detection of fluorescent biosensors.
[0045] SHCR system detection of Pb 2+ The principle is as follows Figure 2 As shown in Figure 1, the designed GR-5 DNAzyme consists of a DNA enzyme chain sequence E and a substrate chain sequence S (including a trigger chain sequence T), which hybridize with each other. 2+ In the case of , the E strand in GR-5 DNAzyme partially blocks T and thus cannot trigger the SHCR reaction. 2+ Added into the system, GR-5 DNAzyme specifically recognizes Pb 2+ , so that the E chain is catalyzed, which in turn cuts the S chain at the adenine ribonucleotide (rA), causing the T chain to be released and open H through a chain displacement reaction, thereby triggering the SHCR reaction.
[0046] The buffer solution used in this system is: 40mM tris(hydroxymethyl)aminomethane hydrochloride (including 20mM magnesium chloride, pH 7.4). The DNA enzyme chain E and the substrate chain S are pre-mixed together using the buffer, and then the mixture and the hairpin probe are annealed at 95°C for 5 minutes and naturally cooled to room temperature for 2 hours to form a DNA enzyme complex SE and a stable hairpin H. One-pot method for detecting Pb 2+ , that is, different concentrations of SE, Pb 2+ Mix with H and incubate at room temperature. When using a fluorescence spectrophotometer to measure, according to the fluorescence characteristics of FAM, the excitation wavelength is set to 487nm, the emission spectrum range is set to 505-600nm, and the slit width is set to 5nm. The relative fluorescence intensity F / F0 is used to measure the Pb 2+ Quantitative determination was performed, where F and F0 represent Pb 2+ Fluorescence intensity in the presence and absence of the SHCR system.
[0047] Example 4
[0048] To verify the feasibility of the SHCR system, fluorescence detection, gel electrophoresis, and atomic force microscopy were used to characterize it. In fluorescence detection, we used hairpins labeled with a fluorophore (FAM) and a quencher (BHQ1). 2+ In the system, the hairpin probe H remains in a metastable state, and the fluorescence is quenched due to the proximity of FAM to BHQ1. 2+ After being added into the system, GR-5 DNAzyme specifically recognizes Pb 2+ , so that the E chain is catalyzed, and then the S chain is cut off, causing the T chain to be released and open H, thereby triggering the SHCR reaction. At this time, due to the separation of FAM and BHQ1, the fluorescence signal is restored. Figure 4 As shown in A, in the absence of Pb 2+ When Pb 2+ The fluorescence signal increased significantly after the reaction, indicating that SHCR was triggered and signal amplification was achieved. Next, we performed gel electrophoresis experiments to verify the results. Figure 4 As shown in B. By adding Pb 2+ DNA nanowires of different lengths can be observed, which is consistent with the fluorescence results. In addition, AFM was used to characterize the morphology of the SHCR system. 2+ In the SHCR system, only a small amount of DNA nanowires were formed ( Figure 4 C), while in the presence of Pb 2+ In the SHCR system, a large number of long DNA nanowires ( Figure 4 D).
[0049] Example 5
[0050] In order to obtain the best sensing performance, we performed optimization experiments.
[0051] Pb 2+ As the detection object, we optimized the reaction time, the concentration ratio of E chain to S chain (E / S), S, H and Mg 2+ The concentration of Figure 5 As shown, the results show that when the reaction time is 50 min, the S concentration is 20 nM, E / S = 2, H and Mg 2+ When the concentrations of Pb are 300 nM and 20 mM, respectively, F / F0 reaches the maximum value (F and F0 are the 2+ and the absence of Pb 2+ The above optimized experimental conditions were used in subsequent experiments.
[0052] Example 6
[0053] Under the above optimized experimental conditions, we further studied the detection performance of the SHCR system.
[0054] Pb 2+ As the detection object, Figure 6 As shown in A, the fluorescence intensity increases with the Pb 2+ The concentration of Figure 6 As shown in B, in the range of 100-500nM, F / F0 and Pb 2+ There is a good linear relationship between the concentrations (y = 0.00282x + 0.71978, R 2 =0.992), and the detection limit was calculated to be 24.8 nM according to σ=3S / N (S is the standard deviation of background fluorescence intensity, N is the linear slope).
[0055] Example 7
[0056] In order to study the SHCR system for Pb 2+ The selectivity of the assay allowed the detection of other potentially interfering metal ions under the same experimental conditions.
[0057] like Figure 7 As shown, select Ni 2+ , Mn 2+ 、Zn 2+ 、Co 2+ , Sr 2+ , Cu 2+ 、Cd 2+ , Ba 2+ and Ag + As a control. Only when Pb 2+When ions are present, the F / F0 of the SHCR system is significantly enhanced, while other metal ions cannot increase the fluorescence signal, indicating that the SHCR system has good selectivity.
[0058] Example 8
[0059] The SHCR system can also be used to detect Pb in real samples. 2+ Concentration detection.
[0060] In order to verify the detection capability of the SHCR system for real samples, the recovery experiment of different samples (Jinjiang River water, Yangtze River water and liquor) was carried out by standard addition method. 2+ The recovery rate was 94.88-105.05%, and the relative standard deviation (RSD) was 0.50-5.77%, indicating that the SHCR system provided in this application can be used for Pb in actual samples. 2+ Detection, for Pb in environmental monitoring and food safety 2+ Testing offers a potential approach.
[0061] The specific results are shown in Table 1
[0062]
[0063] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.
Claims
1. A hairpin probe assembly for self-hybridization chain reaction for lead ion detection, characterized in that: The hairpin probe combination includes GR-5 DNAzyme and a hairpin probe; The GR-5 DNAzyme contains Pb 2+ A specific DNA enzyme chain and a substrate chain, wherein the substrate chain contains a trigger chain sequence; The hairpin probe consists of sticky ends at both ends, a hairpin stem with a palindromic sequence, and a hairpin loop.
2. The hairpin probe assembly for self-hybridization chain reaction for lead ion detection according to claim 1, characterized in that: The sum of the cytosine bases and guanine bases in the hairpin stem portion of the palindromic sequence is not less than two-thirds of the total number of bases; After optimization, the number of cytosine bases and guanine bases contained in the hairpin stem portion of the palindromic sequence is exactly two-thirds of the total number of bases.
3. The hairpin probe assembly for self-hybridization chain reaction for lead ion detection according to claim 1, characterized in that: The hairpin stem portion of the palindromic sequence is 18 bases in length.
4. The hairpin probe assembly for self-hybridization chain reaction for lead ion detection according to claim 1, characterized in that: The length of the hairpin loop is 12 bases, and the total number of bases of the sticky ends at both ends is consistent with the total number of bases of the hairpin loop.
5. The hairpin probe assembly for self-hybridization chain reaction for lead ion detection according to claim 1, characterized in that: The sticky end sequences at both ends are identical and are complementary to the bases of the hairpin loop sequence.
6. The hairpin probe assembly for self-hybridization chain reaction for lead ion detection according to claim 1, characterized in that: The GR-5 DNAzyme contains Pb 2+ A specific DNA enzyme chain and a substrate chain, wherein a trigger chain sequence is designed in the substrate chain; The trigger chain sequence includes a hairpin sticky end, a hairpin stem and a free segment; the hairpin sticky end and the free segment have the same sequence, the hairpin sticky end can be complementary to the base of the hairpin probe sticky end, and the hairpin stem can be complementary to the base of the hairpin probe stem.
7. The hairpin probe assembly for self-hybridization chain reaction for lead ion detection according to claim 1, characterized in that: The sequence of the hairpin probe is shown in SEQ ID NO.1, the sequence of the DNA enzyme chain is shown in SEQ ID NO.2, and the sequence of the substrate chain is shown in SEQ ID NO.
3.
8. A detection kit, characterized in that The detection kit comprises a hairpin probe combination for self-hybridization chain reaction for lead ion detection as described in any one of claims 1-7.
9. The detection kit according to claim 8, characterized in that: The buffer solution used in the self-hybridization chain reaction was 40 mM Tris hydrochloride (including 20 mM magnesium chloride, pH 7.4).