Deoxyribozyme probe for recognizing strontium ion and application thereof

Abstract

This invention discloses a deoxyribozyme probe for recognizing strontium ions and its applications, belonging to the field of deoxyribozyme probe technology. The nucleotide sequence of the deoxyribozyme probe provided by this invention is any one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:9. Specifically, it is a cis-structured deoxyribozyme probe composed of a deoxyribozyme and a substrate chain with a fluorescent group and a quenching group. The substrate chain sequence is shown in SEQ ID NO:10, and the deoxyribozyme sequence is any one of the sequences shown in SEQ ID NO:17 to SEQ ID NO:25. The deoxyribozyme probe provided by this invention can efficiently and specifically recognize strontium ions, thereby achieving accurate detection of the target ion, with a strontium ion response concentration of 2 mM or higher. This probe has good application prospects in the fields of biosensing and ion detection.

Description

Technical Field

[0001] This invention belongs to the field of deoxyribozyme probe technology, specifically relating to a deoxyribozyme probe for recognizing strontium ions and its application. Background Technology

[0002] The application of nuclear energy inevitably brings the potential risk of nuclear accidents, which pose a huge threat to humanity and the environment. Strontium-90 (Sr-235) is one of the fission products of uranium-235 (U-235). 90 Strontium (Sr) is a radioactive nuclide with a radioactive half-life of up to 29 years, which can cause long-term harm. Because Sr is very similar to calcium (Ca) in chemical properties, studies have shown that Sr is an essential element for the human body and can replace calcium in playing an important role in various physiological activities, including promoting bone growth and preventing fractures.

[0003] However, 90 The beta-ray radiation produced by Sr poses a serious threat to human health, potentially leading to bone cancer and leukemia. Furthermore, studies have shown that inhaling excessive amounts of strontium can also trigger cardiovascular and neurological disorders, including severe respiratory distress, allergic reactions, and extreme tachycardia. Currently, radioactive strontium-90 has been detected in soil, water, food, and even organisms. Given the harmful effects of radioactive strontium on human health and its widespread distribution in the natural environment, developing methods for detecting strontium and even future removal techniques is of great significance.

[0004] To achieve strontium detection, various techniques have been developed, including inductively coupled plasma atomic emission spectrometry (ICP-AES), atomic absorption spectrometry (AAS), X-ray fluorescence spectrometry (XRF), chemo-paper sensors, and potentiometric methods. However, these methods typically require complex sample pretreatment processes and large-scale instrumentation, making them unsuitable for on-site, real-time detection and large-scale monitoring. More importantly, due to the presence of numerous interfering metal ions in the environment and the generally low concentration of strontium, the detection specificity and sensitivity of these traditional methods are insufficient for practical applications.

[0005] Deoxyribonucleases (DNAzymes) possess catalytic functions similar to proteases. They are single-stranded oligonucleotides with catalytic functions screened using the Systematic Evolutionary Ligand Enrichment (SELEX) technique, with RNA-cleaving DNAzymes that cleave RNA-containing substrates being the most extensively studied. DNAzyme biosensors, leveraging their specific recognition sites for particular substances, provide a rapid, convenient, and economical method for detecting strontium ions (Sr). 2+ It provides a highly promising alternative. Summary of the Invention

[0006] To achieve rapid and sensitive detection of strontium ions, this invention provides a deoxyribozyme probe for identifying strontium ions and its application.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] This invention provides a deoxyribozyme probe for recognizing strontium ions, wherein the nucleotide sequence of the deoxyribozyme probe is any one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:9; specifically, the deoxyribozyme probe is a cis-structured deoxyribozyme probe composed of a deoxyribozyme and a substrate chain, wherein the substrate chain sequence is shown in SEQ ID NO:10, and the nucleotide sequence of the deoxyribozyme is any one of the following sequences (1) to (9):

[0009] (1) 5'-AGACCACAACGGTTTCCCCCACGGGGAACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0010] (2) 5'-AGACCACAACGGTTTCCCCCACGGGGAGCTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0011] (3) 5'-AGACCACAACGGTTTCCCCCAGCGGGGAACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0012] (4) 5'-AGACCACAACGGTTTCCCCCACGGGGGACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0013] (5) 5'-AGACCACAACGGTTTCCCCCACGGGGTACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0014] (6) 5'-AGACCACAACGGTTTTCCCCCCAGCAGGGAACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0015] (7) 5'-AGACCACAACGGTTTCCCTCCAGCGGGGAACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0016] (8) 5'-AGACCACAACGGTTTCCCCCACGGGGAATTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0017] (9) 5'-AGACCACAACGGTTTCCCCCATGGGGAACTAGTTGGAGAGCTAGAATGCGCTTTCATAGCATAACCCCTTG-3';

[0018] Where R represents RNA base A, F represents T base containing a fluorescent group, and Q represents T base containing a quenching group.

[0019] Furthermore, the deoxyribozyme probe is chain-like.

[0020] Furthermore, the fluorescent group is FAM.

[0021] Furthermore, the quenching group is Quench.

[0022] The present invention also provides a kit for identifying strontium ions, comprising the aforementioned deoxyribozyme probe.

[0023] Furthermore, the kit includes a cofactor, and the cofactor is Na. + .

[0024] The present invention also provides the application of the deoxyribonuclease probe or the kit in biosensing for the specific identification of strontium ions.

[0025] Furthermore, the conditions for specific recognition of strontium ions are: pH 7-8, temperature 4-25℃, and Na+. + As a cofactor.

[0026] The beneficial effects of this invention are:

[0027] (1) The present invention provides multiple chain-like deoxyribonuclease probes, which can efficiently and specifically identify strontium ions, thereby achieving accurate detection of target ions.

[0028] (2) The deoxyribozyme probe of the present invention has good structural stability, high sensitivity and excellent selectivity, which can effectively improve the accuracy and reliability of detection results. This probe can be widely used in the detection of strontium ions in environmental samples and has important application prospects in the fields of biosensing, environmental monitoring and radioactive pollution assessment. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0030] Figure 1 This is a schematic diagram of the chain deoxyribonuclease screening process.

[0031] Figure 2 This is a statistical chart of the cut rate for each round of screening.

[0032] Figure 3 This is a verification of the cleavage activity of sequences RCD-Sr1, RCD-Sr9, RCD-Sr10, RCD-Sr12, RCD-Sr13, RCD-Sr15, RCD-Sr16, RCD-Sr18, and RCD-Sr19.

[0033] Figure 4 This is a characterization experiment of the RCD-Sr1 sequence at different pH values.

[0034] Figure 5 These are characterization experiments of the RCD-Sr1 sequence at different temperatures.

[0035] Figure 6 This is a kinetic characterization of the RCD-Sr1 sequence at 4°C, 15°C, and 25°C.

[0036] Figure 7 This is a characterization of the dependence of the RCD-Sr1 sequence on different strontium ion concentrations. Detailed Implementation

[0037] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] The examples include the construction of DNA libraries, the ligation of libraries and substrates, reverse screening, forward screening, PCR amplification, etc. The names and sequences of nucleic acids involved in the examples are shown in Table 1.

[0039] Table 1. Nucleic Acid Names, Sequences, and Uses

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Example 1: Construction of DNA Library

[0046] The synthesized DNA L2 library (SEQ ID NO:11), PNK enzyme (T4 polynucleotide kinase), and ATP were mixed and incubated at 37°C for 40-60 min. Then, 1.2 molar amounts of substrate strand FQ30 (SEQ ID NO:10) and 1.5 molar amounts of linker strand (SEQ ID NO:12) were added, and the mixture was heated at 90°C for 2-5 min. After cooling to room temperature for at least 10 min, T4 DNA ligase was added, and the mixture was thoroughly mixed. The mixture was then reacted at 37°C for 2-3 h. After the reaction, 2.5 volumes of 100% cold ethanol, 0.1 volumes of 3 mol / L sodium acetate, and 2 µL of 1 µg / µL glycogen were added, and the mixture was thoroughly mixed. The mixture was then frozen at -20°C for at least 30 min to precipitate DNA. The precipitated mixture was centrifuged at low temperature and high speed (4°C, 14000 rpm, 20 min), the supernatant was removed, and the solid product was obtained by vacuum drying for 5-10 min. The DNA was reconstituted with ultrapure water and purified by 10% denaturing polyacrylamide gel electrophoresis (dPAGE). The location of the target band was determined using a gel imaging system, and the gel was excised and recovered. Elution solution was added to the recovered target band for DNA elution. The elution buffer was precipitated with cold ethanol, vacuum dried, and reconstituted with ultrapure water to obtain the desired library for screening. The sequence of the screened library L2-FQ30 is shown in SEQ ID NO:16.

[0047] The elution solution is an Elution Buffer: 5 M NaCl, 1 M Tris (pH 7.5), 0.5 M EDTA (pH 8.0).

[0048] DNA library preparation system:

[0049] 1) Linking substrate phosphorylation

[0050] Table 2. List of components in the phosphorylation reaction of the library.

[0051]

[0052] 2) Library connecting substrate

[0053] Table 3. Components of the Connecting Reaction

[0054]

[0055] Example 2 DNA preservation and strontium ion preparation

[0056] Store the DNA ligation product at -20°C. Dissolve strontium chloride in ultrapure water to prepare a 1 M solution, and use immediately to ensure the stability and accuracy of the solution.

[0057] Example 3 In vitro screening

[0058] In vitro screening involves steps including reverse screening, forward screening, PCR amplification, and library ligation. Cloning and sequencing are performed after 12 rounds of screening. Figure 1 As shown, the specific screening steps include:

[0059] 1. Positive screening

[0060] The library L2-FQ30 screened in Example 1 was dissolved in 10 µL of ultrapure water, and after adding 2× reaction buffer, it was heated at 90°C for 2 min and then cooled to room temperature. The reaction was carried out in a reaction buffer containing 50 mM strontium ions for 12 h (the reaction time was reduced to 2 h starting from the 9th round of screening).

[0061] Table 4. Components of Positive Sieve Reaction

[0062]

[0063] The 2× reaction buffer solution consisted of 100 mM HEPES (pH 7.5) and 50 mM NaCl. The product was purified by 10% denaturing polyacrylamide gel electrophoresis, followed by cold ethanol precipitation to obtain the cleavable DNA sequence.

[0064] 2. PCR amplification

[0065] Using DNA sequences capable of cleavage recovered through forward screening as templates, the target sequences were amplified in large quantities via two-step PCR using upstream primer FP and downstream primers RP1 and RP2. The PCR products were purified by 10% dPAGE. Because downstream primer RP2 is modified with a steric ion, PCR amplification yielded sense and antisense strands of different lengths. The sense strand was recovered by gel excision and its concentration was determined. The sequence of upstream primer FP is shown in SEQ ID NO:13, and the sequences of downstream primers RP1 and RP2 are shown in SEQ ID NO:14 and SEQ ID NO:15, respectively.

[0066] PCR amplification conditions:

[0067] Table 5. PCR amplification conditions

[0068]

[0069] PCR reaction system:

[0070] 1) PCR1

[0071] Table 6. PCR1 reaction conditions

[0072]

[0073] 2) PCR2

[0074] Table 7. PCR2 reaction conditions

[0075]

[0076] 3. Connect into a database

[0077] The PCR2 product was mixed with PNK enzyme (T4 polynucleotide kinase) and incubated at 37°C for 40-60 minutes. Then, an equimolar amount of substrate strand FQ30 (SEQ ID NO:10), 1.2 times the molar amount of linker strand (SEQ ID NO:12), and T4 ligase were added, mixed thoroughly, and reacted at room temperature for 2-3 hours. After separation and purification, the DNA library for the next round of screening was obtained.

[0078] The cut percentage (Clv%) is calculated using the following formula to characterize the enrichment level of the DNA library after each round of screening:

[0079]

[0080] Where Clv% represents the cleavage percentage, Clv is the amount of cleaved bands in the dPAGE gel image, and Unclv is the amount of uncleaved bands in the dPAGE gel image. After RNA site breakage, the fluorescence intensity of the fluorescent group in the cleavage band is amplified by 6 times; therefore, when calculating the cleavage rate, the fluorescence intensity of the cleavage band needs to be divided by 6.

[0081] After multiple rounds of screening, the cut percentage of positive screening showed a significant increasing trend, and after 10 rounds of screening, the positive screening cut rate was ≥1%.

[0082] 4. Reverse Filtering

[0083] When the DNA library shows signs of enrichment (i.e., a forward selection cut rate ≥1%), the screening library obtained from the previous round is taken and dissolved in ultrapure water. The reaction is then carried out in 2× reaction buffer. Before the reaction, the dissolved screening library is heated at 90°C for 2 min, cooled to room temperature, and then a reaction buffer containing 100 mM sodium ions is added. The reaction time is 12 h. The products after the reaction are purified using a 10% polyacrylamide gel electrophoresis to separate and purify DNA of different lengths, and uncleaved DNA sequences are recovered.

[0084] Table 8. Components of the Reverse Sieving Reaction

[0085]

[0086] The DNA sequences recovered after reverse screening are then subjected to forward screening, PCR amplification, and ligation into a library. This process is repeated twice, resulting in a total of 12 rounds of screening. The cleavage rate of the chain library in the 12th round of forward screening is approximately 5.8%. Figure 2 As shown, DNA library enrichment is complete.

[0087] High-throughput sequencing was used to select the top 20 sequences for experimental verification, identifying nine deoxyribozyme sequences BG1–BG9 that met the target requirements. Their nucleotide sequences are shown in SEQ ID NO:17–SEQ ID NO:25, respectively. After ligation of the nine deoxyribozyme sequences BG1–BG9 with the substrate chain FQ30, the corresponding sequences are RCD-Sr1 (SEQ ID NO:1), RCD-Sr9 (SEQ ID NO:2), RCD-Sr10 (SEQ ID NO:3), RCD-Sr12 (SEQ ID NO:4), RCD-Sr13 (SEQ ID NO:5), RCD-Sr15 (SEQ ID NO:6), RCD-Sr16 (SEQ ID NO:7), RCD-Sr18 (SEQ ID NO:8), and RCD-Sr19 (SEQ ID NO:9).

[0088] Example 4 Characterization of deoxyribozyme performance

[0089] 1. Characterization of cutting ability

[0090] Nine sequences BG1-BG9 obtained from sequencing were ligated into a chain with substrate strand FQ30 and incubated in a reaction buffer containing strontium ions. Samples of different enzyme chains were separated by 10% polyacrylamide gel electrophoresis, and the cleavage rate was calculated. Figure 3 As shown in the image, gel electrophoresis revealed that the deoxyribozyme sequence BG1 (SEQ ID NO:17) exhibited the best cleavage ability.

[0091] 2. pH characterization

[0092] Taking the sequence RCD-Sr1, the result of a chain deoxyribonuclease linked to a substrate, as an example, in Na... + Seven reaction buffers with different pH gradients (pH 4.0-8.0) were prepared under conditions where the ion concentration was 25 mM. Strontium ions at a concentration of 50 mM were added to each reaction buffer, and the mixture was incubated for 2 h. Separation was then performed using a 10% polyacrylamide gel electrophoresis. Figure 4 As shown in the image, gel electrophoresis reveals that RCD-Sr1 exhibits a strong response to strontium ions at pH 7.0-8.0, indicating that this chain-like deoxyribonuclease has high activity in a neutral environment.

[0093] 3. Characterization of reaction temperature

[0094] Taking RCD-Sr1 as an example, after incubation for 2 hours at different temperatures in a buffer solution containing 50 mM strontium ions, it was separated by 10% polyacrylamide gel electrophoresis. Figure 5 As shown in the image, gel electrophoresis revealed that RCD-Sr1 exhibited strong responses to strontium ions at 4°C, 15°C, and 25°C. Furthermore, the kinetics at these three temperatures were analyzed (e.g., ...). Figure 6 The results showed that RCD-Sr1 exhibited high reactivity at 4°C, 15°C, and 25°C.

[0095] 4. Concentration dependence

[0096] Taking RCD-Sr1 as an example, buffer solutions containing different concentrations of strontium ions were prepared. Under the same conditions, RCD-Sr1 was incubated in these buffer solutions for 2 hours and then separated using a 10% dPAGE gel. Figure 7 As shown, RCD-Sr1 can detect strontium ions at concentrations of 2 mM and above.

[0097] The experimental sequences RCD-Sr10, RCD-Sr12, RCD-Sr13, RCD-Sr15, RCD-Sr16, RCD-Sr18, and RCD-Sr19 all exhibit similar properties.

[0098] The above description represents a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A deoxyribozyme probe for recognizing strontium ions, characterized in that, The nucleotide sequence of the deoxyribonuclease probe is shown in SEQ ID NO:1; Where R represents RNA base A, the 15th base is a T base containing a fluorescent group, and the 13th base is a T base containing a quenching group.

2. The deoxyribozyme probe according to claim 1, characterized in that, The deoxyribozyme probe is chain-like.

3. The deoxyribozyme probe according to claim 1, characterized in that, The fluorescent group is FAM.

4. A kit for identifying strontium ions, characterized in that, The deoxyribonuclease probe includes any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The kit includes a cofactor, which is Na. + .

6. The application of the deoxyribonuclease probe according to any one of claims 1-3 or the kit according to claim 4 or 5 in biosensing, characterized in that, Used for the specific recognition of strontium ions.

7. The application according to claim 6, characterized in that, The conditions under which the deoxyribozyme probe specifically recognizes strontium ions are: pH 7-8, temperature 4-25℃, and Na+. + As a cofactor.

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