A Csm6-based deoxyribonuclease sensor and its application in lead detection

Abstract

This invention provides a Csm6-based deoxyribonuclease sensor, characterized by comprising the following components: (1) a deoxyribonuclease enzyme chain and a deoxyribonuclease substrate chain, wherein the nucleotide sequence of the deoxyribonuclease substrate chain is shown in SEQ ID No. 1 or SEQ ID No. 2 of the sequence listing, and the nucleotide sequence of the deoxyribonuclease enzyme chain is shown in SEQ ID No. 10 of the sequence listing; (2) a Csm6 protease; and (3) a single-stranded RNA reporter probe modified with fluorescent and quenching groups, wherein the sequence of the single-stranded RNA reporter probe modified with fluorescent and quenching groups is shown in SEQ ID No. 16 of the sequence listing. This invention also provides an application of this deoxyribonuclease sensor for lead detection. The deoxyribonuclease sensor provided by this invention can improve the sensitivity and selectivity of existing biosensors in lead ion detection.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, specifically relating to a Csm6-based deoxyribonuclease sensor and its application in lead detection. Background Technology

[0002] As one of the most widely distributed toxic metallic pollutants in the ecological environment, lead ions are extensively derived from mining and vehicle exhaust emissions. The U.S. Environmental Protection Agency (EPA) has identified them as a possible human carcinogen. As a non-biodegradable element, lead ions not only have a long biological half-life but also tend to accumulate in the human body, causing irreversible damage to the immune and nervous systems, seriously threatening children's health. Even at trace levels, lead ions can have profound negative impacts on public health. Lead pollution in environments such as soil and water can easily transfer to foods such as livestock meat, eggs, and dairy products, posing a serious threat to human health through bioaccumulation and biomagnification.

[0003] Given the prevalence, high toxicity, and low threshold of lead ions, there is an urgent need to develop methods for the reliable, highly sensitive, and accurate quantitative detection of lead ions in complex matrices to ensure food and environmental safety. However, while traditional lead ion analysis techniques such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption / fluorescence spectroscopy, and reversed-phase high-performance liquid chromatography (RP-HPLC) offer satisfactory performance, they require expensive and complex equipment, demanding sample pretreatment procedures, and operators with research-skilled expertise, making them difficult to widely adopt and further develop into portable detection devices.

[0004] Deoxyribozymes, as ideal recognition elements, have been widely used in the development of various nanomaterials and molecular tools, including the detection of lead ions. A deoxyribozyme consists of two oligodeoxynucleotide chains: the deoxyribozyme chain and the deoxyribozyme substrate chain carrying RNA bases. Lead ions can intercalate into the deoxyribozyme complex, then catalyze the cleavage of the phosphodiester bond at the RNA base site of the deoxyribozyme substrate chain, producing two short substrate fragments. This conformational change can be monitored using various methods, such as fluorescently labeled probes and nucleic acid dyes.

[0005] Emerging CRISPR tools have demonstrated powerful functionality in developing analyses targeting nucleic acid biomarkers, and by combining them with appropriate biorecognition and signal transduction elements, they can also play a similar role in targeting non-nucleic acid biomarkers. Csm6, a subunit effector from the type III-A CRISPR / Cas system, can act as an RNA endonuclease to improve the accuracy of RNA detection. As a ribonuclease, Csm6 can be activated by either A4>p or A6>p, and activated Csm6 preferentially cleaves adenosine- or cytidine-rich RNA molecules. Compared to using Cas13a alone, cascading Csm6 in the Cas13a reaction can achieve significant signal amplification, shorter sample response time, and higher detection sensitivity. Furthermore, the deoxyribonuclease can generate linear oligoadenylate chains containing 2',3'-cyclic nucleotides (A4>p or A6>p), which opens a new avenue for constructing biosensors for metal ion detection using Csm6. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Csm6-based deoxyribonuclease sensor and its application in lead detection, as well as its application in actual sample detection, so as to improve the sensitivity and stability of existing biosensor methods for detecting lead ions.

[0007] This invention first provides a Csm6-based deoxyribozyme sensor and its application in lead detection, comprising the following parts:

[0008] (1) Deoxyribonuclease enzyme chain and deoxyribonuclease substrate chain;

[0009] (2) Csm6 protein; when the substrate chain described in (1) is cleaved to produce A4>p, the Csm6 protease is TtCsm6; when the substrate chain described in (1) is cleaved to produce A6>p, the Csm6 protease is EiCsm6, SeCsm6 or MtCsm6.

[0010] (3) Single-stranded RNA reporter probes modified with fluorescent and quenching groups.

[0011] The deoxyribonuclease enzyme chain can be activated by intercalated lead ions, catalyzing the cleavage of phosphodiester bonds at RNA base sites in the deoxyribonuclease substrate chain, producing a substrate fragment molecule containing a linear oligoadenylate (A4>p or A6>p) containing 2',3'-cyclic nucleotides. The nucleotide sequence of the deoxyribonuclease substrate chain is shown in SEQ ID No. 1 or SEQ ID No. 2 in the sequence listing, and the nucleotide sequence of the deoxyribonuclease enzyme chain is shown in SEQ ID No. 10 in the sequence listing.

[0012] Csm6 is a ribonuclease that can be activated by A4>p or A6>p. Activated Csm6 is biased towards cleaving RNA molecules rich in adenosine or cytidine.

[0013] The single-stranded RNA reporter probe modified with fluorescent and quenching groups is composed of adenosine- or cytidine-rich RNA, and the sequence of the single-stranded RNA reporter probe modified with fluorescent and quenching groups is shown in SEQ ID No. 16 in the sequence listing.

[0014] Furthermore, the deoxyribonuclease sensor is applied to lead detection.

[0015] The application of the deoxyribozyme sensor, as described above, is characterized by the following steps:

[0016] Step 1: Using lead ions in the sample as a cofactor, react at 4~40℃ to activate the deoxyribonuclease enzyme chain to cleave the deoxyribonuclease substrate chain, generating 5' end fragments and 3' end fragments respectively.

[0017] Step 2: Add the CRISPR system to the reaction mixture from Step 1 and perform a CRISPR reporter reaction at 20-50°C. The CRISPR system includes the Csm6 protein and a single-stranded RNA reporter probe modified with fluorescent and quenching groups.

[0018] Furthermore, the application of the deoxyribonuclease sensor is characterized in that the concentration of the deoxyribonuclease enzyme chain in the coexisting system is 0.01-10 μM, the concentration of the deoxyribonuclease substrate chain is 0.01-10 μM, the concentration of Csm6 is 0.01-2 μM, and the concentration of the reporter probe is 0.01-5 μM.

[0019] Furthermore, the application of the deoxyribozyme sensor is characterized by the following reaction procedures: the deoxyribozyme cleavage process is carried out at 4~40℃ for more than 1 minute, and the CRISPR reporting process is carried out at 20~50℃ for more than 5 minutes.

[0020] Furthermore, the deoxyribonuclease sensor is characterized in that the fluorescent group modified on the reporter probe can be Alexa flour series, 6-FAM, ATTO series, cy3, cy5, cy5.5, cy7, TAMRA, etc., and the modified quenching group can be BHQ1 series, Dabcyl, etc.

[0021] Furthermore, the deoxyribonuclease sensor is characterized in that the Csm6 protein can be... Thermus thermophilus Csm6 (TtCsm6) Staphylococcus epidermidis (SeCsm6) Methanothermobacter thermautotrophicus (MtCsm6) and Enterococcus italicus Csm6 (EiCsm6), etc.

[0022] This invention demonstrates through experiments that the deoxyribonuclease sensor with endogenous double amplification provided by this invention has extremely high detection sensitivity; at the same time, the detection of lead ions using this sensor is not affected by interference from other heavy metal ions and has high specificity and selectivity for lead ions.

[0023] The basic principle of this invention is as follows: Figure 1 As shown, the entire workflow of the Csm6 deoxyribozyme sensor and its application in lead detection consists of two parts: (1) a deoxyribozyme digestion reaction for lead ion recognition and target cycle amplification; and (2) a CRISPR fluorescence reporter process for further endogenous signal amplification. This dual amplification enables high sensitivity for lead ion detection, particularly by achieving amplification without nucleotide polymerization. The deoxyribozyme cleavage region consists of two chains: a deoxyribozyme substrate chain modified with 4 or 6 riboadecanosides at the 5' end, and a deoxyribozyme enzyme chain without any modification. The nucleotide sequence of the deoxyribozyme substrate chain is shown in SEQ ID No. 1 or SEQ ID No. 2 in the sequence listing, and the nucleotide sequence of the deoxyribozyme enzyme chain is shown in SEQ ID No. 10 in the sequence listing. In this stage, the deoxyribozyme can generate A4>p, which can act as a Csm6 activation ligand, to trigger the subsequent CRISPR fluorescence reporter. Activated Csm6 exhibits extremely high enzymatic activity, capable of cleaving single-stranded RNA reporter probes modified with fluorescent and quenching groups. The sequence of the single-stranded RNA reporter probe modified with fluorescent and quenching groups is shown in SEQ ID No. 16 of the sequence listing. Finally, quantitative analysis of lead ions is achieved by fluorescence detection.

[0024] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0025] (1) This method also provides an application of the above-mentioned Csm6-based deoxyribozyme sensor in lead ion detection. The sensor provided by this invention can be used to detect lead ions. A portable handheld fluorescence spectrometer can be used to perform rapid quantitative analysis of lead ions. It has the advantages of simple operation, high specificity, high sensitivity and low cost.

[0026] (2) The deoxyribozyme sensor used in this invention can achieve endogenous double amplification without nucleotide polymerization, which significantly improves the detection sensitivity of lead ions.

[0027] (3) The deoxyribozyme sensor used in this invention is simple to operate, has a fast response speed, and high reliability.

[0028] (4) The deoxyribonuclease sensor used in this invention has extremely high selectivity for lead ions and can effectively resist interference from other metals in the detection environment.

[0029] This invention proposes a Csm6-based deoxyribonuclease sensor and its application in lead detection. It establishes, improves, and develops a biosensor analysis method for lead ion pollutants, which can not only provide a reference for environmental health risk assessment, such as analyzing lead pollution in water and freshwater, but also further provide a reference for food health risk assessment, such as lead content in egg samples, and further analyze biotoxicological risk assessment, such as the toxicological effects of lead ions on mice. Simultaneously, it develops a new, rapid, highly sensitive, and low-cost detection method, which is of great significance and application value for improving existing detection technologies. This invention establishes a Csm6-based deoxyribonuclease sensor and its application in lead detection. This fluorescence method has the advantages of simple preparation, rapid detection, low cost, small sample volume, and high sensitivity, providing a new detection method for food health risk assessment and biotoxicological risk assessment. This is of great significance in improving existing lead ion pollutant detection technologies. Attached Figure Description

[0030] Figure 1 The working principle of the deoxyribozyme sensor provided by this invention.

[0031] Figure 2 The signal-to-noise ratio test results show the effect of hybridization stability of deoxyribozyme enzyme chain and deoxyribozyme substrate chain on fluorescence intensity in Examples 1 and 2.

[0032] Figure 3 This is the preparation of the standard curve of the relationship between lead ion concentration and fluorescence intensity in Example 3.

[0033] Figure 4 This is the test result of the specificity selection for lead ion detection compared to other heavy metal ions in Example 4.

[0034] Figure 5 This is the test result of the toxicological effects of lead ions on mice in Example 6.

[0035] Figure 6 The results show the signal-to-noise ratio of the response capabilities of Csm6 to lead ions in different host species in Comparative Example 1. Detailed Implementation

[0036] The following embodiments are provided to further explain the present invention, but should not be used to limit the scope of the invention. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0037] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0038] The nucleotide sequences of the deoxyribonuclease substrate chain described in the following examples are shown in SEQ ID No. 1-2 of the sequence listing; the nucleotide sequences of the deoxyribonuclease enzyme chain are shown in SEQ ID No. 3-13 of the sequence listing; the nucleotide sequences of TtCsm6 are shown in SEQ ID No. 14 of the sequence listing; the nucleotide sequences of EiCsm6 are shown in SEQ ID No. 15 of the sequence listing; and the nucleotide sequences of the C5 reporter probe are shown in SEQ ID No. 16 of the sequence listing.

[0039] The specific methods for expressing and purifying the Csm6 protein described in the following examples are as follows: First, plasmids containing DNA sequences encoding TtCsm6 and EiCsm6 were constructed and sequenced. The nucleotide sequence of TtCsm6 is shown in SEQ ID No. 14 of the sequence listing, and the nucleotide sequence of EiCsm6 is shown in SEQ ID No. 15 of the sequence listing. Then, the plasmids corresponding to TtCsm6 and EiCsm6 were transformed into E. coli competent cells (BL21 Rosetta2(DE3)) and grown in LB medium supplemented with appropriate concentrations of chloramphenicol and kanamycin until the absorbance at 600 nm reached 0.6. Protein expression was triggered by adding 0.2 mM IPTG (isopropyl-β-d-thiogalactopyranoside) and cultured at 18°C ​​for 16 h. Cells expressing the protein were collected and resuspended in lysis buffer containing 20 mM Tris-HCl (pH=8.0), 300 mM KCl, and 10% glycerol. The cell suspension was lysed by sonication and centrifuged at 15,000 rpm for 30 minutes to remove bacterial cells. Next, unwanted bacterial lysates were filtered through a 5 mL Ni NTA column (GE Healthcare), washed with 10 volumes of lysis buffer, and then eluted with 5 volumes of imidazole lysis buffer at a series of concentrations (10, 20, 50, 100, 200 mM; pH=8.0). The eluted proteins were dialyzed overnight with lysis buffer containing 20 mM HEPES (pH=7.5), 300 mM KCl, 5 mM MgCl2, and 10% glycerol under TEV protease conditions. The dialyzed proteins were filtered through a 5 mL Ni NTA column, and the filtrate was further concentrated and purified using an S200 (16 / 600) column. Finally, the purified protein was concentrated to 5-75 mg / mL using a cutoff centrifuge filter with a molecular weight of 30,000, and then rapidly frozen in liquid nitrogen and stored in a -80°C freezer.

[0040] Example 1

[0041] In this embodiment, the effect of hybridization stability between the deoxyribozyme substrate chain and the deoxyribozyme enzyme chain on fluorescence intensity was investigated in the deoxyribozyme sensor. The steps are as follows:

[0042] First, the deoxyribonuclease digestion reaction was carried out in a 36 μL solution containing 1× buffer (20 mM HEPES (pH=7.5), 50 mM KCl, 5 mM MgCl2), 400 nM deoxyribonuclease substrate chain (Sub-C), 400 nM deoxyribonuclease enzyme chain (Dz-C), and 150 nM lead ions, and reacted at 25 °C for 1 h. Next, 4 μL of CRISPR reporter solution containing 100 nM TtCsm6 and 500 nM C5 reporter probe was added to the deoxyribonuclease digestion reaction solution, and then incubated at 37 °C for 30 min. Finally, fluorescence intensity was measured with an excitation wavelength of 480 nm, an emission range of 510–600 nm, and a step size of 1 nm.

[0043] The invention is characterized by the design of 11 deoxyribonuclease chains, each with different arm lengths at the t-terminus and non-t-terminus, including Dz-C9-4t, Dz-C9-6t, Dz-C9-8t, Dz-C9-10t, Dz-C9-12t, Dz-C9-14t, and Dz-C8-10t, Dz-C10-10t, Dz-C11-10t, Dz-C12-10t, and Dz-C13-10t, which are mixed in a coexisting system, and fluorescence intensity data are measured. The nucleotide sequence of the deoxyribonuclease substrate chain (Sub-C) is shown in SEQ ID No. 1 of the sequence listing, and the nucleotide sequences of the deoxyribonuclease chains Dz-C9-4t, Dz-C9-6t, Dz-C9-8t, Dz-C9-10t, Dz-C9-12t, Dz-C9-14t and Dz-C8-10t, Dz-C10-10t, Dz-C11-10t, Dz-C12-10t, and Dz-C13-10t are shown in SEQ ID No. 3-13 of the sequence listing, respectively.

[0044] The fluorescence intensity measured for each mixture system is as follows: Figure 2 As shown in (A and B). The results indicate that shortening the t-terminal arm length from 14 to 10 nucleotides and the non-t-terminal arm length from 13 to 10 nucleotides produces the optimal signal-to-noise ratio, while further shortening reduces the hybridization stability of the deoxyribozyme substrate chain and the deoxyribozyme chain. This suggests that both excessively low and excessively high stability of the deoxyribozyme chain binding to the deoxyribozyme substrate chain limit the multiple inversion cleavage reaction. Examples 2-6 below all use deoxyribozyme chains with both t-terminal and non-t-terminal chain lengths of 10t (Dz-C), i.e., the nucleotide sequence of the deoxyribozyme chain is shown in SEQ ID No. 10 of the sequence listing.

[0045] Example 2

[0046] In this embodiment, the effects of the concentration of the deoxyribozyme substrate chain and the ratio of the deoxyribozyme substrate chain to the deoxyribozyme chain on the fluorescence intensity were investigated. The steps are as follows:

[0047] The detection steps described in Example 1 are characterized by the following: the concentrations of the deoxyribonuclease substrate chain (Sub-C) are 50, 100, 200, 400, 600, and 800 nM, and the concentration of the deoxyribonuclease enzyme chain (Dz-C) is 400 nM. These are mixed in a coexisting system, and the fluorescence intensity data are measured. Then, based on the optimal concentration of the deoxyribonuclease substrate chain according to the fluorescence data, the substrate chain to deoxyribonuclease enzyme chain is mixed in a coexisting system at ratios of 1:2, 1:1, 2:1, 4:1, 6:1, and 8:1, and the fluorescence intensity data are measured.

[0048] The fluorescence intensity measured for each mixture system is as follows: Figure 2 As shown in (C and D). The results indicate that the optimal signal-to-noise ratio is achieved when the substrate concentration is 400 nM and the ratio of the deoxyribozyme substrate chain to the deoxyribozyme chain is 1:1.

[0049] Example 3

[0050] In this embodiment, the preparation of a standard curve for studying the relationship between lead ion concentration and fluorescence intensity in the deoxyribozyme sensor is carried out through the following steps:

[0051] The detection steps described in Example 1 are characterized by using a large number of standard concentration lead ion solutions (0, 0.1, 1, 5, 10, 25, 50, 100, 150, 200 nM) to be mixed in the coexistence system, and measuring the fluorescence intensity data.

[0052] The fluorescence intensity measured for each mixture system is as follows: Figure 3 As shown in the figure. The results indicate that the fluorescence intensity gradually increases with the increase of lead ion content (0-200 nM). When the lead ion concentration is in the range of 0.1-100 nM, a linear regression equation for the relationship between lead ion concentration and fluorescence intensity can be obtained: y=553.06x+3192.90, R 2 =0.9971, where y, x and R 2 Representing fluorescence intensity, lead ion concentration, and correlation coefficient, the limit of detection (LOD) is 212 pM, which significantly meets the requirement of the National Health Commission of China for the maximum lead ion pollution level of 0.01 mg / L (48 nM) in drinking water as stipulated in the "Standards for Drinking Water Quality (GB 5749-2022)".

[0053] Example 4

[0054] In this embodiment, the specificity of the deoxyribozyme sensor for detecting lead ions relative to other heavy metal ions is studied, and the steps are as follows:

[0055] The detection steps described in Example 1 are characterized by the inclusion of seven metal ions (Mn, lead, and phosphate groups) in the design. 2+ Ni 2+ Cd 2+ Al 3+ Cu 2+ Co 2+ and Pb 2+ The fluorescence intensity data were measured by mixing the sample in a coexisting system at three different concentration gradients (10, 100, and 1000 nM).

[0056] The fluorescence intensity measured for each mixture system is as follows: Figure 4 As shown in the figure. The results indicate that, compared with the blank control group, no metal ions other than lead ions showed significant changes in fluorescence intensity, which suggests that the deoxyribozyme sensor still has good ability to distinguish lead ions even in an interference environment.

[0057] Example 5

[0058] In this embodiment, the detection of lead ions in food samples using the deoxyribonuclease sensor follows these steps:

[0059] The detection steps described in Example 1 are characterized by preparing sample reagents with lead ion contents of 10, 30, and 50 nM, respectively, using tap water and fresh eggs as solvents. These reagents are then mixed in a coexisting system, replacing the lead ion solution, and the fluorescence intensity data is measured. The sample reagents using fresh eggs as solvents require digestion before using the deoxyribozyme sensor.

[0060] The specific digestion steps are as follows: Take 0.1-0.5g of sample containing different amounts of lead ions, add it to an acidic solution containing 10mL concentrated HNO3 and 5mL HClO4, place it in a fume hood, react at 180℃ for 40min, then at 200℃ for 4h, and finally at 220℃ for 1h. Dissolve the digested solid in 8mL of ultrapure water, and adjust the pH to 7 using 1mol / L NaOH and 0.1mol / L HCl, finally maintaining a constant volume of 13mL to obtain the digest solution for subsequent analysis.

[0061] The fluorescence intensities of each mixture are shown in Table 1. The results show that the recoveries of lead ions in water and eggs ranged from 90.09% to 112.18%, with relative standard deviations (RSD) remaining within 3.14%. This indicates that the described deoxyribozyme sensor has potential application value in the detection of lead contamination in complex matrices such as food and the environment.

[0062] Table 1. Detection of lead ions added to fresh eggs and tap water samples.

[0063]

[0064] Example 6

[0065] In this embodiment, the toxicological effects of lead ions on mice are estimated using the deoxyribozyme sensor, and the steps are as follows:

[0066] (1) All experimental animals (10 mice) were divided into two groups: 5 control mice (treated with sterile PBS) and 5 experimental mice (treated with sterile 1000 mg / kg lead ion solution). First, healthy mice (BALB / c, 6-8 weeks old) without pathogenic infection were acclimatized for 3 days. Then, they were orally administered with sterile 1000 mg / kg lead ion solution or sterile PBS for 3 days. Finally, infected mice were euthanized, and their kidneys, livers, colons, and feces were collected, weighed, and then digested for analysis. The digestion process followed the steps in Example 5.

[0067] (2) The detection steps described in Example 1 are employed, characterized in that digestive fluids from different parts of the experimental mice are used instead of lead ion solution in the coexisting system, and fluorescence intensity data are measured. We use the aforementioned deoxyribonuclease sensor and ICP to evaluate the toxicological effects of lead ions on mice in parallel.

[0068] The fluorescence intensity measured for each mixture system is as follows: Figure 5 As shown. Figure 5 (A) is a schematic diagram of the mouse experimental research protocol. Figure 5 (B) and (C) show the determination of lead ion content in mice in different infection groups using the described deoxyribonuclease sensor and ICP method, respectively. The results indicate that acute lead poisoning leads to lead ion accumulation in the tissues of all experimental groups of mice. The accumulation levels in in vivo tissues (25-280 μg / g wet tissue) followed the order: colon > kidney > liver, significantly lower than fecal excretion (20000-30000 μg / g wet tissue). The fluorescence sensing detection results were consistent with the ICP detection results, indicating that the described deoxyribonuclease sensor can be used to study the toxicological effects of lead ions in vivo.

[0069] Comparative Example 1

[0070] In this comparative example, the responsiveness of Csm6 to lead ions in different host species of the deoxyribozyme sensor was studied, and the steps are as follows:

[0071] The detection steps described in Example 1 are characterized in that the Csm6 are TtCsm6 and EiCsm6, respectively, which are mixed in a coexisting system with the deoxyribonuclease substrate chain (Sub-C) and the deoxyribonuclease substrate chain (Sub-CE), respectively, and the fluorescence intensity data are measured. The nucleotide sequence of the deoxyribonuclease substrate chain (Sub-C) is shown in SEQ ID No. 1 of the sequence listing, the nucleotide sequence of the deoxyribonuclease substrate chain (Sub-CE) is shown in SEQ ID No. 2 of the sequence listing, the nucleotide sequence of TtCsm6 is shown in SEQ ID No. 14 of the sequence listing, and the nucleotide sequence of EiCsm6 is shown in SEQ ID No. 15 of the sequence listing.

[0072] The fluorescence intensity measured for each mixture system is as follows: Figure 6 As shown in the figure. The results show that the signal-to-noise ratio obtained using TtCsm6 is 26.7, which is 3.5 times that of EiCsm6.

[0073] sequence list

[0074] <110> Sichuan University

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[0182] gtttacggcc tggcgcgtac gctgctgcac ctggcacagg aagctaaaga agaagcggcg 960

[0183] gtgctggccg cactgtatgc ataccgcgct ctggaactgc tgctgcagga acgtctggcg1020

[0184] ctgctgggcc gtcgtgctga agctccgggt ctgagcccgg aagaagccga agccctgcgt1080 ctgctgggcc gtcgtgctga agctccgggt ctgagcccgg aagaagccga agccctgcgt1080

[0185]

[0185] aaagctctgg cggaactgct gggcgtcctg ccggaagaag tgcgcctgcc ggcgaaactg1140 aaagctctgg cggaactgct gggcgtcctg ccggaagaag tgcgcctgcc ggcgaaactg1140

[0186]

[0186] ggtctgctgg acctgctggc attcctgcgc ctgaaaggcg acgaagctct gggccgcctg1200 ggtctgctgg acctgctggc attcctgcgc ctgaaaggcg acgaagctct gggccgcctg1200

[0187]

[0187] agcctggctg aactgcgcgg tcttgcaggc gcgctgaaag gtcgtaactc cgcgctgctg1260 agcctggctg aactgcgcgg tcttgcaggc gcgctgaaag gtcgtaactc cgcgctgctg1260

[0188]

[0188] gtgcacggct ttgacgtgcc gagcccgaaa gcagtagaag gcatcgcacg cctggcgcag1320 gtgcacggct ttgacgtgcc gagcccgaaa gcagtagaag gcatcgcacg cctggcgcag1320

[0189]

[0189] ggcctgctgc aggacctgga agcgcgcacc gcgctgggtc cgctgtctcc ggaaccggtg1380 ggcctgctgc aggacctgga agcgcgcacc gcgctgggtc cgctgtctcc ggaaccggtg1380

[0190]

[0190] ccgctgggtt tctaa 1395 ccgctgggtt tctaa 1395

[0191]

[0191] <210>15<210>15

[0192]

[0192] <211>1317 <211>1317 <00,00420>

[0193] <212>DNA <212>DNA

[0194]

[0194] <213>人工序列(Artificial Sequence) <213>Artificial Sequence

[0195]

[0195] <400>15 <400>15

[0196]

[0196] catatgaaaa ttctattttc acccatagga aatacggacc cgtggcgtaa cgaccgcgac 60 catatgaaaa ttctattttc acccatagga aatacggacc cgtggcgtaa cgaccgcgac 60

[0197]

[0197] ggcgcaatgc tgcacattgt tcgtcactat cagccagaca gagtcgtgct gtttttcacc 120 ggcgcaatgc tgcacattgt tcgtcactat cagccagaca gagtcgtgct gtttttcacc 120

[0198]

[0198] gagagcatct ggcagggtaa tcagcatttt agcggccagc aagcgtttga ttgggttaag 180

[0199] attatccaaa gtatcaacga gaactgccaa atcgaaatca agtgcgatac tattgaagtt 240

[0200] gagaacgact ttgacgcata taaggacctg ttccaccagt acttggtgga ggaaaaacgt 300

[0201] aaatacccga atgcggaaat tttcctgaat gttacctctg gtacaccgca gatggaaacc 360

[0202] accctgtgtc tggaatacgt aacctatccc gacaagatgc gttgcattca ggttagcacc 420

[0203] ccgctgaaaa cgagcaatgc taaaaccaaa tatgctcagg ctgattgtca agaggttgat 480

[0204] ttggagattg ttaatgagga agagagccag cagccgagcc gttgccataa aatcgccatc 540

[0205] ctgagcttcc gcgaagccat cgtccgtaac cagattaagt ccctgctgga caattatgat 600

[0206] tatgaagccg cgttacaact ggttgcgtcg caaaagagct tccgtaacgg caaagagatc 660

[0207] cgcaagaagc tgaaagagtt gattgatgac atcaaaatgc atcgtgtttt cagctatctt 720

[0208] atcaagcagt acccgcgtaa cgagaagctg cagaaagcgc tgttgcacac cattttgtta 780

[0209] gaaatgcgtc accaacgtgg tgatatcgcg gaaactctga tccgtgtgaa atccatcgcg 840

[0210] gaatatatcg tggagcaga cattcaaaaa aactacccgt acctgatcat ttaaaagaa 900

[0211] gataagccgt acttcaacgt gtcctacagc caagagctga ccgaatctta cttggcgctg 960

[0212] atggattcac gcaacaagaa gacgaacaag aagatgaccg tcgatagcct ggaccgtatt1020

[0213] ctcggttttc cggcataccg cgacttcctg caactgctcg aggcgtcgaa cgaaatgacg1080

[0214] aacgagatga ataaggtcaa tgagattaac aacctgcgta ataaagttgc acataattta1140

[0215] gactctctga atctcgaccg cgataaaaac ggccgtaaga tcaccaacgc tgtgaccgca1200

[0216] gtgcgcacca tgctgttggc ggtgttcccg gaagtgcagg agaacgactt ccactacctt1260

[0217] aaacaattta accaatccat aaaggagttg ctctaaaagc ttgcggccgc actcgag 1317

[0218] <210> 16

[0219] <211> 5

[0220] <212> RNA

[0221] <213> Artificial Sequence

[0222] <220>

[0223] <221> modified_base

[0224] <222> (1,5)

[0225] <222> The 5' and 3' ends were modified with FAM and BHQ1, respectively.

[0226] <400> 16

[0227] ccccc 5

[0228] <210> 17

[0229] <211> 4

[0230] <212> RNA

[0231] <213> Artificial Sequence

[0232] <400> 17

[0233] aaaa 4

[0234] <210> 18

[0235] <211> 6

[0236] <212> RNA

[0237] <213> Artificial Sequence

[0238] <400> 18

[0239] aaaaaa 6

Claims

1. A Csm6-based deoxyribozyme sensor, characterized in that... Includes the following parts: (1) Deoxyribonuclease enzyme chain and deoxyribonuclease substrate chain; the deoxyribonuclease enzyme chain can be activated by intercalated lead ions, catalyzing the cleavage of phosphodiester bonds at RNA base sites of the deoxyribonuclease substrate chain to produce a substrate fragment molecule containing a linear oligoadenylate chain A4>p containing 2',3'-cyclic nucleotides, the nucleotide sequence of the deoxyribonuclease substrate chain is shown in SEQ ID No.1 in the sequence listing, the nucleotide sequence of the deoxyribonuclease enzyme chain is shown in SEQ ID No.10 in the sequence listing, and the nucleotide sequence of the linear oligoadenylate A4>p is shown in SEQ ID No.17 in the sequence listing; (2) Csm6 protein; the Csm6 is TtCsm6; the nucleotide sequence of TtCsm6 is shown in SEQ ID No. 14 in the sequence listing; (3) A single-stranded RNA reporter probe modified with fluorescent and quenching groups; the sequence of the single-stranded RNA reporter probe modified with fluorescent and quenching groups is shown in SEQ ID No. 16 in the sequence listing; The concentrations of the deoxyribonuclease enzyme chain, the deoxyribonuclease substrate chain, and Csm6 were 400 nM and 100 nM, respectively, and the reporter probe was 500 nM.

2. The deoxyribonuclease sensor according to claim 1, characterized in that, The fluorescent group modified on the report probe is selected from either the Alexa flour series or the ATTO series.

3. The deoxyribonuclease sensor according to claim 1, characterized in that, The fluorescent groups modified on the reporting probe are 6-FAM, cy3, cy5, cy5.5, cy7, or TAMRA.

4. The deoxyribonuclease sensor according to claim 1, characterized in that, The quenching group modified on the report probe is selected from one of the BHQ1 series.

5. The deoxyribonuclease sensor according to claim 1, characterized in that, The quenching group modified on the report probe is Dabcyl.

6. The application of the deoxyribonuclease sensor according to claim 1 in lead ion detection.

7. The application according to claim 6, characterized in that... Includes the following steps: Step 1: Using lead ions in the sample as a cofactor, react at 4–40°C to activate the deoxyribonuclease enzyme chain to cleave the deoxyribonuclease substrate chain, generating 5' and 3' end fragments respectively. Step 2: Add the CRISPR system to the reaction mixture from Step 1 and perform a CRISPR reporter reaction at 20–50°C. The CRISPR system includes the Csm6 protein and a single-stranded RNA reporter probe modified with fluorescent and quenching groups.

8. The application according to claim 6, characterized in that, The reaction procedure is as follows: the deoxyribozyme cleavage process is carried out at 4–40°C for more than 1 minute, and the CRISPR reporter process is carried out at 20–50°C for more than 5 minutes.

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