Method for preparing functional DNA gel and application thereof

Abstract

This invention relates to a method for preparing a functional DNA gel and its applications. Addressing the problems of existing methods for immobilizing functional nucleic acids requiring DNA modification, which increases immobilization costs, and the reduction of functionality due to chemical fixation, this invention mixes functional circular DNA with a gel monomer solution, and adds an initiator and a coagulant to initiate a cross-linking polymerization reaction. During the reaction, the gel monomers pass through the functional circular DNA, polymerizing into long chains and then cross-linking into a network structure. This allows the functional circular DNA to be enriched on the network structure in a suspended, fixed form, resulting in a functional DNA gel. The functional circular DNA is circular DNA containing functional nucleic acid sequences. This invention immobilizes functional circular DNA on the gel network structure without chemical modification, and the preparation method is simple. It can be used for the efficient and specific removal of pollutants such as heavy metal ions, toxins, veterinary drugs, and pesticides from solutions, as well as the enrichment of small molecule nucleic acids in solutions.

Description

Technical Field

[0001] This invention relates to a method for preparing a functional DNA gel and its application. Background Technology

[0002] Nucleic acids, as carriers of genetic information storage and transfer, not only serve as the material basis for biological heredity but also demonstrate enormous application potential in the construction of biomaterials due to their programmability, functional diversity, and biodegradability. Functional nucleic acids refer to nucleic acid sequences with special functions, such as the ability to specifically bind to ligands and catalytic activity. Aptamers, deoxyribozymes, modified nucleic acids, luminescent nucleic acids, and riboswitches can all be called functional nucleic acids (Xu Wentao, Yang Min, Zhu Longjiao, et al. The connotation and extension of the concept of functional nucleic acids [J]. Progress in Biotechnology, 2021, 11(4):446). The immobilization of functional nucleic acids is more conducive to the performance of their functions, including the removal of harmful substances such as heavy metals and toxins and the enrichment of other small molecules.

[0003] Currently, the immobilization of functional nucleic acids mostly relies on linking modified nucleic acids to a vector. For example, Hu et al. used CNBr (cyanogen bromide) activated agarose to immobilize 5'-amino-modified DNA aptamers, thereby developing a novel aptamer chromatography column that can be used to remove trace amounts of drugs from drinking water (Hu X, Mu L, Zhou Q, et al. Ssdna aptamer-based column for simultaneous removal of nanogram per liter level of illicit and analgesic pharmaceuticals in drinking water[J].Environmental ScienceTechnology,2011,45(11):4890). Romero-Reyes et al. developed an aptamer-functionalized membrane in which the carboxylic acid functional group of polymethacrylic acid (PMAA) can be linked to an amine-functionalized aptamer. This membrane can remove the small-molecule pollutant bisphenol A from water (Romero-Reyes MA, Heemstra J M. Small-molecule sequestration using aptamer-functionalized membranes[J]. ACS Materials Letters,2019,1(5):568-572). Immobilized functional nucleic acids are easy to separate from samples and have broad application prospects in adsorption, enrichment, and detection. However, the modification of functional nucleic acids increases the immobilization cost and makes the operation more cumbersome.

[0004] Directly preparing nucleic acids into gels can achieve the immobilization of unmodified nucleic acids. Luo et al. prepared DNA gels by using sticky ends to form polymers under the action of ligases, realizing the transformation of DNA from a dispersion solution to a polymer solid (Um SH, Lee JB, Park N, et al. Enzyme-catalyzed assembly of DNA hydrogel[J]. Nat Mater. 2006; 5(10):797-801). However, the amount of DNA used reached millimoles, resulting in high cost and poor mechanical properties of the prepared gel. Li et al. used the addition reaction between bases and acrylamide under freeze-concentration conditions to immobilize DNA in polyacrylamide gels, and used the prepared gels to verify the functionality of lead ion adsorption (Li Y, Gao H, Qi Z, et al. Freezing-Assisted Conjugation of Unmodified Diblock DNA to Hydrogel Nanoparticles and Monoliths for DNA and Hg). 2+ Sensing[J].Angewandte Chemie,2021,133(23):13095-13101). This method does not involve chemical modification, but the reaction conditions are extremely harsh, and the chemical reaction causes some damage to the bases, reducing the utilization efficiency of DNA. Summary of the Invention

[0005] To address the problems of high cost and cumbersome fixation steps required by existing functional nucleic acid immobilization methods, such as the need for DNA modification, and the reduced functionality due to chemical fixation reactions, this invention provides a method for preparing functional circular DNA gels and their applications. This invention uses functional circular DNA and gel monomers as raw materials, and through a method of mixing followed by polymerization and cross-linking, fixes the functional circular DNA onto the gel's network structure. This method requires no chemical modification, is simple to prepare, and can be used for the efficient and specific removal of pollutants such as heavy metal ions, toxins, veterinary drugs, and pesticides from solutions, as well as the enrichment of small-molecule nucleic acids in solutions.

[0006] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0007] This invention provides a method for preparing a functional DNA gel, comprising mixing functional circular DNA with a gel monomer solution, and adding an initiator and a coagulant to carry out a cross-linking polymerization reaction. During the reaction, the gel monomer passes through the functional circular DNA and polymerizes into long chains, which then cross-link into a network structure, thereby enriching the functional circular DNA on the network structure in a suspended and fixed form, thus obtaining a functional DNA gel; wherein the functional circular DNA is a circular DNA containing a functional nucleic acid sequence.

[0008] In this invention, the gel monomer has the ability to polymerize into a network structure. Smaller gel monomer molecules more easily pass through circular DNA, and linear gel monomers pass through circular DNA more easily than non-linear ones. Acrylamide monomers are preferred, and acrylamide and N,N-dimethylacrylamide monomers are more preferred. The mass fraction ratio of acrylamide to N,N-dimethylacrylamide solution is (25-33):1, more preferably 29:1.

[0009] In this invention, when acrylamide monomers are used, the initiator is one or more of ammonium persulfate, azobisisobutyronitrile, benzoyl peroxide, diisopropylbenzene peroxide, and azobisisoheptanenitrile; the coagulant is tetramethylethylenediamine or 3-dimethylaminepropionitrile.

[0010] In this invention, the concentration of the gel monomer affects the fixation efficiency of circular DNA. In some embodiments of this invention, the concentration of the acrylamide monomer solution is preferably ≥12%, more preferably 12%-30%.

[0011] After adding the initiator and coagulant, stirring is performed. Stirring can improve the efficiency of the cross-linking reaction and also facilitates the uniform distribution of circular DNA in the acrylamide monomer.

[0012] In this invention, the circular DNA cannot be too small; otherwise, the probability of gel monomers passing through will decrease. Taking acrylamide monomer as an example, acrylamide monomer molecules can pass through circular DNA up to 30 nt. In some embodiments of this invention, circular DNA larger than 59 nt can be fixed to a certain extent by polyacrylamide, indicating that acrylamide monomers can pass through during gel formation. The larger the circular DNA, the easier it is for gel monomers to pass through, and the better the fixation effect. From a cost perspective, if the circular DNA is too large, it will increase the preparation cost. Preferably, the length of the circular DNA is 59-177 nt. Some embodiments of this invention demonstrate that 177 nt of circular DNA is the ideal size for circular DNA fixation efficiency.

[0013] In this invention, circular DNA preferably maintains a simple secondary structure at room temperature. Complex secondary structures can affect the fixation efficiency of circular DNA. For example, if there are many complementary DNA sequences, the space of the circular DNA will shrink. Other examples include hairpin structures with long stem loops and G-quadruplexes. For circular DNA with complex secondary structures, methods such as adding complementary strands and increasing the amount of gel monomers can be used to improve its fixation rate.

[0014] In this invention, the functional nucleic acid sequence on the circular DNA is one or more of the following: aptamers, deoxyribonucleases, and antisense nucleic acids of miRNA.

[0015] The length of the circular DNA is 30-300 nt, preferably 59-177 nt.

[0016] The functional circular DNA can be prepared using existing technologies without particular limitations. Preferably, it is prepared using the following method: First, three DNA strands containing functional nucleic acid sequences are designed, and different helper DNA sequences are designed to link the three DNA strands into a circular shape; exonucleases are used to remove linear and double-stranded DNA contaminating the circular DNA; alcohol precipitation is performed to remove magnesium ions and proteins generated during the circular DNA preparation process, yielding pure circular DNA.

[0017] Some embodiments of the present invention demonstrate that metal ions during the preparation of circular DNA affect the subsequent fixation of circular DNA, necessitating extraction and ethanol precipitation of the prepared circular DNA. Specific procedures include: adding an organic solvent to the circularized sample to remove protein impurities; precipitating with ice-cold ethanol, concentrating and recovering the circular DNA; washing the product with 75% ethanol; and allowing the product to evaporate moisture in the air to obtain pure circular DNA.

[0018] Another aspect of the present invention provides the application of the aforementioned functional DNA gel in the removal of pollutants such as heavy metal ions, toxins, veterinary drugs or pesticides, and the enrichment of small molecule nucleic acids.

[0019] The functional DNA gel is immersed in an aqueous solution containing the target, and the functional nucleic acid sequence on the circular DNA strand specifically binds to the target substance; the gel adsorbed with the target substance is then separated from the aqueous solution.

[0020] The gel in this invention is insoluble in water but easily swells in water. Its main chain contains polar bonds with strong hydrophilicity, which can realize the mutual exchange of internal and external water, thereby achieving the purpose of treating aqueous solutions.

[0021] Some embodiments of the present invention provide effective removal of heavy metal ions (lead, cadmium, and chromium) and ochratoxin A, as well as enrichment of small nucleic acids. When the functional DNA sequence is an aptamer sequence or deoxyribozyme sequence with a specific binding site, the functional DNA / polyacrylamide gel is immersed in the contaminated liquid. The functional nucleic acid sequence on the circular DNA strand specifically binds to the contaminant, and the gel adsorbed with the contaminant is separated from the aqueous solution. When the functional DNA sequence is a complementary sequence to the small nucleic acid to be enriched, the functional DNA / polyacrylamide gel is immersed in the enrichment solution. The functional nucleic acid sequence on the circular DNA strand specifically binds to the small nucleic acid, achieving enrichment of the functional small nucleic acid.

[0022] Preferably, the prepared functional DNA gel is soaked in water before use to leach out small molecule chemicals.

[0023] It is preferable to cut the functional DNA gel into small pieces for the adsorption of heavy metal ions in the solution, so as to increase the contact area.

[0024] Preferably, the functional DNA gel is immersed in an aqueous solution contaminated with pollutants such as heavy metals and mixed at low speed using a mixer at room temperature.

[0025] Preferably, the functional DNA / polyacrylamide gel has NH- bonds in the main chain that have strong hydrophilicity, enabling the exchange of internal and external water.

[0026] The functional DNA gel in this invention can be reused repeatedly after desorption treatment. The desorption method is as follows: the adsorbent material saturated with adsorbents is immersed in hot water, filtered, taken out, washed and collected, and can then be used for cyclic adsorption of pollutants.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The present invention can achieve the fixation of DNA in polyacrylamide gel without modification, which greatly reduces the fixation cost of functional nucleic acids caused by chemical modification.

[0029] (2) The DNA sequence containing functional nucleic acid used in this invention is relatively short and is suspended and fixed in polyacrylamide gel in the form of circular monomers. This avoids the adverse effects on the functionality of functional nucleic acid sequence caused by secondary structures generated by excessively long sequences during the traditional DNA gel preparation process. The prepared DNA gel has more ideal functionality.

[0030] (3) This invention innovatively utilizes the process of polymerizing circular nucleic acid monomers to immobilize functional nucleic acid sequences in a network gel, which is universally applicable to the immobilization of circular nucleic acids. It can achieve the immobilization of natural circular DNA (such as eccDNA) in polyacrylamide gel, which is difficult to achieve with traditional immobilization methods.

[0031] (4) The nucleic acid fixation method in this invention relies on the gel monomers to pass through the circular DNA when they polymerize into a network structure to achieve suspension fixation. The selection of gel monomers is relatively wide and the application range is broad.

[0032] (5) This invention can achieve the immobilization of functional nucleic acid monomers in gels without chemical modification. It can be used as a low-cost desorbent for the specific removal of heavy metal ions and other pollutants, and has great application potential in the fields of desorption and adsorption.

[0033] (6) The specific adsorption element selected in this invention is a circular DNA containing the adsorption properties of heavy metals or other pollutants. This element is chemically stable, has sequence designability and substitutability. Different functional nucleic acids can be selected to achieve the adsorption of different kinds of heavy metal ions and other pollutants, and has broad application potential in the fields of desorption and adsorption.

[0034] (7) This invention fixes circular DNA in a suspended form to a gel network, preventing it from falling off and allowing for recycling. When used for the adsorption of heavy metal ions and other pollutants, it maintains a high adsorption efficiency even after multiple cycles of adsorption. When used as an adsorbent material, the recycling cost is low.

[0035] (8) The preparation method of the functional DNA gel of the present invention is simple, the reaction conditions are mild, the process is safe, and no specific reaction device is required.

[0036] (9) In this invention, functional nucleic acid sequences with specific binding ability are immobilized in polyacrylamide gel, which can achieve specific adsorption and removal of target substances. When used as an adsorbent material, it reduces the probability of non-specific adsorption compared with traditional adsorbents. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the preparation of functional DNA / polyacrylamide gel.

[0038] Figure 2 This image shows the effect of fixing functional circular DNA on polyacrylamide gel.

[0039] Figure 3 This is a diagram showing the adsorption effect of functional DNA / polyacrylamide gel on lead ions in Example 1.

[0040] Figure 4This is a diagram showing the specific adsorption effect of functional DNA / polyacrylamide gel on lead ions in Example 1.

[0041] Figure 5 This is a diagram showing the adsorption effect of the functional DNA / polyacrylamide gel during cyclic regeneration in Example 1.

[0042] Figure 6 The images show the adsorption and enrichment effects of functional DNA / polyacrylamide gels on cadmium ions, chromium ions, ochratoxin A, and miR-206 in Examples 2-5. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments.

[0044] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0045] The functional DNA / polyacrylamide gel synthesized in this embodiment of the invention was analyzed by polyacrylamide electrophoresis to determine the immobilization effect. The method is as follows: 2 μL of 2 μM circular DNA was mixed with 8 μL of 30% acrylamide solution (acrylamide mass: N,N-dimethylacrylamide mass = 29:1), and 10 μL of water was added. Then, 0.3 μL of ammonium persulfate and 0.3 μL of tetramethylethylenediamine were added, and the mixture was quickly added to the sample wells of the polyacrylamide gel. Electrophoresis was performed at 350 V and 15 mA for 1.5 h, and the bands in the lanes were observed to determine the level of immobilization of the circular DNA in the acrylamide gel.

[0046] Figure 2 Electrophoresis images showing the immobilization effect of circular DNA of different sizes at different gel concentrations are provided. C represents circular DNA, and L represents linear DNA; 59, 118, and 177 represent the length of circular DNA in nt; 8%, 10%, 12%, etc., represent gel concentrations. Experimental results show that the larger the size of the circular DNA, the greater the probability of it being penetrated by the long chains of polyacrylamide, and the better the immobilization effect. After preliminary quantification using Image Lab software, the immobilization rate was obtained by calculating the amount of DNA in the gel wells / (amount of DNA in the gel wells + amount of DNA in the lanes), thus determining the immobilization efficiency of three sizes of circular DNA at different gel concentrations. For a 177nt DNA loop, the immobilization rate reached 90% with a 14% PAGE gel, while the immobilization rates for 59nt and 118nt DNA loops were around 50%.

[0047] The adsorption efficiency of functional DNA / polyacrylamide gel for heavy metal ions in liquid was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The adsorption efficiency of functional DNA / polyacrylamide gel for small molecule pollutants such as ochratoxin in liquid was analyzed by high-performance liquid chromatography (HPLC). The adsorption efficiency of functional DNA / polyacrylamide gel for small molecule nucleic acids in liquid was analyzed by ultraviolet-visible spectrophotometry (UV-VIS). The test method was as follows: Gel fragments containing 4000 pmol of circular DNA were immersed in 8 mL of a solution containing 0.5 μM of target substance. The mixture was stirred and adsorbed in a water bath for 24 h at room temperature. The concentration of the target substance in the solution before and after adsorption was determined using equipment such as inductively coupled plasma mass spectrometry. The removal rate (removal percent) was calculated using the following formula:

[0048]

[0049] Where Co is the initial concentration and Ce is the concentration after removal.

[0050] The specific recycling process of the functional DNA / polyacrylamide gel is as follows: the adsorbent material saturated with adsorbents is immersed in water at 90°C for 10 minutes, filtered out, washed, and collected for recycling adsorbents.

[0051] Example 1: Methods for removing lead ions (heavy metal)

[0052] 1. Design of functional circular DNA

[0053] GR5 DNAzyme, which has a strong affinity for lead ions, was selected as a functional nucleic acid for the specific adsorption of lead ions. Three DNA sequences containing GR5 DNAzyme were designed and linked end-to-end to prepare a 177nt DNA loop. The main design method is as follows:

[0054] (1) The GR5 DNAzyme is 26nt in length. To avoid the impact of circularization on the secondary structure of the functional nucleic acid sequence and to provide binding arms for circularization, it is extended by 19nt on the left side and 14nt on the right side of the functional nucleic acid sequence, resulting in a long chain of 59nt containing one functional nucleic acid region, named Pb-1.

[0055] (2) In order to prepare it into a large-sized circular DNA, two 59nt long chains containing the same GR5 DNAzyme sequence but with different binding arms were designed and named Pb-2 and Pb-3.

[0056] (3) Based on the three 59nt long chains, design three ring-forming auxiliary chains Pbsplint-1, Pbsplint-2, and Pbsplint-3 for ring-forming connections (lengths of 13nt, 20nt, and 20nt, respectively).

[0057] (4) The three 59nt long chains and their end-to-end connected 177nt sequences were subjected to ring formation simulation in M-fold (temperature 37℃, Na + Concentration 1mM, Mg 2+ The concentration was 10 mM to ensure that it had no complex secondary structure and that the functional nucleic acid sequence region was not interfered with by the binding arm.

[0058] 2. Preparation of functional circular DNA

[0059] The designed DNA strands Pb-1, Pb-2, and Pb-3 containing functional nucleic acids were phosphorylated, and then the three DNA strands were ligated end-to-end to form a circular structure using DNA ligase. The circularization conditions were as follows: 5 μM of 59 nt DNA strand Pb-1, 5 μM of 59 nt DNA strand Pb-2, 5 μM of 59 nt DNA strand Pb-3, 1×T4 Buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8 @ 25℃), 10 μM of Pbsplint-1, 10 μM of Pbsplint-2, and 10 μM of Pbsplint-3 were added, and the mixture was reacted at 90℃ for 3 min, then cooled to 25℃ at 0.1℃ / s and maintained at 25℃ for 20 min. Then, 1 U / μL of T4 DNA ligase was added, and the ligation was carried out for 5 h. The total volume was 25 μL. The enzymes were inactivated at 75°C for 10 minutes. Uncircularized linear nucleic acid strands and added circular auxiliary strands were removed from the circular DNA using exonulease I and exonulease III. Exonulease I and exonulease III were then inactivated at 85°C. The circularization and enzyme digestion status could be determined by polyacrylamide gel electrophoresis.

[0060] The prepared sample was treated with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1) to remove proteins. The product was then washed with 75% ethanol and anhydrous ethanol, respectively, and the water was evaporated at 37°C to obtain pure circular DNA. The DNA was reconstituted with water, and the concentration of the final circular DNA was determined quantitatively by polyacrylamide gel electrophoresis.

[0061] 3. Preparation of functional DNA / polyacrylamide gel

[0062] 100 μL of 40 μM circular DNA was mixed with 75 μL of 30% acrylamide solution, and 12.5 μL of water was added to obtain a 12% polyacrylamide gel solution rich in 4000 pmol of circular DNA. After stirring and mixing, 3 μL of ammonium persulfate and 0.5 μL of coagulating agent were added, and the mixture was allowed to solidify to obtain a functional DNA / polyacrylamide gel containing 4000 pmol of DNA.

[0063] This gel preparation method can be used in 8 mL of 0.5 μM Pb-containing solutions. 2+ Removal of heavy metal ions from aqueous solutions.

[0064] The functional DNA / polyacrylamide gel prepared above exhibits an adsorption rate of up to 97% for lead ions. After 20 cycles of adsorption, the sample shows a high adsorption rate for heavy metal ions (Pb). 2+ The adsorption rate can still reach 87.2%.

[0065] Table 1. Oligonucleotide single-stranded sequences used in Example 1

[0066]

[0067]

[0068] Note: The underlined portion represents the active site of the functional nucleic acid.

[0069] Example 2: Methods for removing heavy metal cadmium ions

[0070] 1. Design of functional circular DNA

[0071] BN-Cd16 DNAzyme, which has a strong affinity for cadmium ions, was selected as a functional nucleic acid for the specific adsorption of cadmium ions. Three DNA sequences containing BN-Cd16 DNAzyme were designed and linked end-to-end for the preparation of a 177nt DNA loop. The main design method is as follows:

[0072] (1) The active site sequence of BN-Cd16 DNAzyme is 13nt in length. In order to avoid the influence of circularization on the secondary structure of the functional nucleic acid sequence and to provide binding arms for circularization, 23nt was added to the left and 23nt to the right of the functional nucleic acid sequence, resulting in a long chain of 59nt containing one functional nucleic acid region, named Cd-1.

[0073] (2) To prepare it into a ring, two 59nt long chains containing the same sequence BN-Cd16 DNAzyme but with different binding arms were designed and named Cd-2 and Cd-3.

[0074] (3) Based on the three 59nt long chains, three ring-forming auxiliary chains Cdsplint-1, Cdsplint-2, and Cdsplint-3 are designed for ring-forming connections.

[0075] (4) The three 59nt long chains and their end-to-end connected 177nt sequences were subjected to ring formation simulation in M-fold (temperature 37℃, Na + Concentration 1mM, Mg 2+ The concentration was 10 mM to ensure that it had no complex secondary structure and that the functional nucleic acid sequence region was not interfered with by the binding arm.

[0076] 2. Preparation of functional circular DNA

[0077] The designed DNA strands Cd-1, Cd-2, and Cd-3 containing functional nucleic acids were phosphorylated, and then the three DNA strands were ligated end-to-end to form a circular structure using DNA ligase. The circularization conditions were as follows: 5 μM of 59 nt DNA strand Cd-1, 5 μM of 59 nt DNA strand Cd-2, 5 μM of 59 nt DNA strand Cd-3, 1×T4 Buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8 @ 25℃), 10 μM of Cdsplint-1, 10 μM of Cdsplint-2, and 10 μM of Cdsplint-3 were added, and the mixture was reacted at 90℃ for 3 min, then cooled to 25℃ at 0.1℃ / s and maintained at 25℃ for 20 min. Then, 1 U / μL of T4 DNA ligase was added, and the ligation was carried out for 5 h. The total volume was 25 μL. The enzymes were inactivated at 75°C for 10 min. The circular DNA was then treated with exonuleases I and III to remove the uncirculated linear nucleic acid strands and any added circularization accessory strands. Exonuleases I and III were then inactivated at 85°C. The circularization and enzyme digestion status were determined by polyacrylamide gel electrophoresis.

[0078] The prepared sample was treated with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1) to remove proteins. The product was then washed with 75% ethanol and anhydrous ethanol, respectively, and the water was evaporated at 37°C to obtain pure circular DNA. The DNA was reconstituted with water, and the concentration of the final circular DNA was determined quantitatively by polyacrylamide gel electrophoresis.

[0079] 3. Preparation of functional DNA / polyacrylamide gel

[0080] 100 μL of 40 μM circular DNA was mixed with 75 μL of 30% acrylamide solution, and 12.5 μL of water was added to obtain a 12% polyacrylamide gel rich in 4000 pmol of circular DNA. After stirring and mixing, 3 μL of ammonium persulfate and 0.5 μL of a coagulating agent were added, and the mixture was allowed to solidify to obtain a functional DNA / polyacrylamide gel containing 4000 pmol. This gel can be used in 8 mL of 0.5 μM Cd-containing solution. 2+ Removal of cadmium ions from aqueous solutions.

[0081] The functional DNA / polyacrylamide gel prepared above has good resistance to heavy metal ions (Cd). 2+ The adsorption rate is as high as 98.3%.

[0082] Table 2 shows the oligonucleotide single-stranded sequences used in Example 2.

[0083]

[0084] Note: The underlined portion represents the active site of the functional nucleic acid.

[0085] Example 3: Methods for removing chromium ions

[0086] 1. Design of functional circular DNA

[0087] Literature review revealed that there is currently no Cr-specific DNAzyme. Most studies currently use Ce13d DNAzymes for chromium ion detection. In this process, lanthanide ions such as Ce can be masked using phosphate buffer. Therefore, this study selected Ce13d DNAzymes for Cr detection. 3+ Adsorption. Three DNA sequences containing the Ce13d DNAzyme were designed and linked end-to-end for the preparation of a 177nt DNA loop. The main design method is as follows:

[0088] (1) The active site sequence of Ce13d DNAzyme is 32nt in length. In order to avoid the influence of circularization on the secondary structure of the functional nucleic acid sequence and to provide binding arms for circularization, 14nt was added to the left side of the functional nucleic acid sequence and 13nt to the right side, resulting in a long chain of 59nt containing one functional nucleic acid region, named Cr-1.

[0089] (2) To prepare it into a ring, two 59nt long chains containing the same Ce13d DNAzyme sequence but with different binding arms were designed and named Cr-2 and Cr-3.

[0090] (3) Based on the three 59nt long chains, three ring-forming auxiliary chains Crsplint-1, Crsplint-2, and Crsplint-3 are designed for ring-forming connections.

[0091] (4) The three 59nt long chains and their end-to-end connected 177nt sequences were subjected to ring formation simulation in M-fold (temperature 37℃, Na + Concentration 1mM, Mg 2+ The concentration was 10 mM to ensure that it had no complex secondary structure and that the functional nucleic acid sequence region was not interfered with by the binding arm.

[0092] 2. Preparation of functional circular DNA

[0093] The designed DNA strands containing functional nucleic acids, Cr-1, Cr-2, and Cr-3, were phosphorylated, and then the three DNA strands were ligated end-to-end into a circular state using DNA ligase. The circularization conditions were as follows: 5 μM of 59 nt DNA strand Cr-1, 5 μM of 59 nt DNA strand Cr-2, 5 μM of 59 nt DNA strand Cr-3, 1×T4 Buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8 @ 25℃), 10 μM of Crsplint-1, 10 μM of Crsplint-2, and 10 μM of Crsplint-3 were added. The mixture was reacted at 90℃ for 3 min, then cooled to 25℃ at 0.1℃ / s and maintained at 25℃ for 20 min. Then, 1 U / μL of T4 DNA ligase was added, and ligation was carried out for 5 h, with a total volume of 25 μL. The enzyme was inactivated at 75℃ for 10 min. The circular DNA was treated with exonuleases I and III to remove the uncircularized linear nucleic acid strands and any added circularization accessory strands. Exonuleases I and III were inactivated at 85°C. The circularization and digestion status were determined by polyacrylamide gel electrophoresis.

[0094] The prepared sample was treated with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1) to remove proteins. The product was then washed with 75% ethanol and anhydrous ethanol, respectively, and the water was evaporated at 37°C to obtain pure circular DNA. The DNA was reconstituted with water, and the concentration of the final circular DNA was determined quantitatively by polyacrylamide gel electrophoresis.

[0095] 3. Preparation of functional DNA / polyacrylamide gel

[0096] 100 μL of 40 μM circular DNA was mixed with 75 μL of 30% acrylamide solution, and 12.5 μL of water was added to obtain a 12% polyacrylamide gel rich in 4000 pmol of circular DNA. After stirring and mixing, 3 μL of ammonium persulfate and 0.5 μL of a coagulating agent were added, and the mixture was allowed to solidify to obtain a functional DNA / polyacrylamide gel containing 4000 pmol. This gel can be used in 8 mL of 0.5 μM Cr-containing... 3+ Removal of chromium ions from aqueous solutions.

[0097] The functional DNA / polyacrylamide gel prepared above is effective against heavy metal ions (Cr). 3+ The adsorption rate is as high as 97.6%.

[0098] Table 3 shows the oligonucleotide single-stranded sequences used in Example 3.

[0099]

[0100] Note: The underlined portion represents the active site of the functional nucleic acid.

[0101] Example 4: Method for removing ochratoxin A from solution

[0102] 1. Design of functional circular DNA

[0103] An aptamer with strong affinity for ochratoxin A (OTA) was selected as the functional nucleic acid sequence. Three DNA sequences containing the OTA-aptamer were designed and linked end-to-end for the preparation of a 177 nt DNA loop. The main design method is as follows:

[0104] (1) The OTA-aptamer sequence is 36nt in length. To avoid the impact of looping on the secondary structure of the functional nucleic acid sequence and to provide binding arms for looping, 12nt was added to the left side of the functional nucleic acid sequence and 11nt to the right side, resulting in a long chain of 59nt containing one functional nucleic acid region, named OTA-1.

[0105] (2) To prepare it into a ring, two 59nt long chains containing the same OTA-aptamer sequence but with different binding arms were designed and named OTA-2 and OTA-3.

[0106] (3) Based on the three 59nt long chains, three ring-forming auxiliary chains OTAsplint-1, OTAsplint-2, and OTAsplint-3 are designed for ring-forming connections.

[0107] (4) The three 59nt long chains and their end-to-end connected 177nt sequences were subjected to ring formation simulation in M-fold (temperature 37℃, Na + Concentration 1mM, Mg2+ The concentration was 10 mM to ensure that it had no complex secondary structure and that the functional nucleic acid sequence region was not interfered with by the binding arm.

[0108] 2. Preparation of functional circular DNA

[0109] The designed DNA strands OTA-1, OTA-2, and OTA-3 containing functional nucleic acids were phosphorylated, and then the three DNA strands were ligated end-to-end into a circular state using DNA ligase. The circularization conditions were as follows: 5 μM of 59 nt DNA strand OTA-1, 5 μM of 59 nt DNA strand OTA-2, 5 μM of 59 nt DNA strand OTA-3, 1×T4 Buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8 @ 25℃), 10 μM OTAsplint-1, 10 μM OTAsplint-2, and 10 μM OTAsplint-3 were added. The mixture was reacted at 90℃ for 3 min, then cooled to 25℃ at 0.1℃ / s and maintained at 25℃ for 20 min. Then, 1 U / μL of T4 DNA ligase was added, and ligation was carried out for 5 h, with a total volume of 25 μL. The enzyme was inactivated at 75℃ for 10 min. The circular DNA was treated with exonuleases I and III to remove the uncircularized linear nucleic acid strands and any added circularization accessory strands. Exonuleases I and III were inactivated at 85°C. The circularization and digestion status were determined by polyacrylamide gel electrophoresis.

[0110] The prepared sample was treated with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1) to remove proteins. The product was then washed with 75% ethanol and anhydrous ethanol, respectively, and the water was evaporated at 37°C to obtain pure circular DNA. The DNA was reconstituted with water, and the concentration of the final circular DNA was determined quantitatively by polyacrylamide gel electrophoresis.

[0111] 3. Preparation of functional DNA / polyacrylamide gel

[0112] 100 μL of 40 μM circular DNA was mixed with 75 μL of 30% acrylamide solution, and 12.5 μL of water was added to obtain a 12% polyacrylamide gel rich in 4000 pmol of circular DNA. After stirring and mixing, 3 μL of ammonium persulfate and 0.5 μL of coagulating agent were added, and the mixture was allowed to solidify to obtain a functional DNA / polyacrylamide gel containing 4000 pmol. This gel can be used for the removal of ochratoxin A from 8 mL of 0.5 μM aqueous solution containing OTA.

[0113] The functional DNA / polyacrylamide gel prepared above exhibits an adsorption rate of up to 97.3% for ochratoxin A (OTA).

[0114] Table 4 shows the oligonucleotide single-stranded sequences used in Example 4.

[0115]

[0116] Note: The underlined part is the functional nucleic acid sequence used.

[0117] Example 5: Enrichment methods for small-molecule nucleic acids in solution

[0118] 1. Design of functional circular DNA

[0119] The complementary sequence of the characteristic miRNA (miR-206) of turbot was selected as the functional nucleic acid sequence. Three DNA sequences containing the complementary sequence of miR-206 were designed and concatenated end-to-end for the preparation of a 177nt DNA circle. The main design method is as follows:

[0120] (1) The complementary sequence of miR-206 is 22nt in length. In order to avoid the influence of looping on the secondary structure of the functional nucleic acid sequence and to provide binding arms for looping, 19nt was added to the left side of the functional nucleic acid sequence and 18nt to the right side, resulting in a long chain of 59nt containing one functional nucleic acid region, named 206-1.

[0121] (2) To prepare it into a ring, two 59nt long chains containing the same miR-206 complementary sequence but with different binding arms were designed and named 206-2 and 206-3.

[0122] (3) Based on the three 59nt long chains, three ring-forming auxiliary chains 206splint-1, 206splint-2, and 206splint-3 are designed for ring-forming connections.

[0123] (4) The three 59nt long chains and their end-to-end connected 177nt sequences were subjected to ring formation simulation in M-fold (temperature 37℃, Na + Concentration 1mM, Mg 2+ The concentration was 10 mM to ensure that it had no complex secondary structure and that the functional nucleic acid sequence region was not interfered with by the binding arm.

[0124] 2. Preparation of functional circular DNA

[0125] The designed DNA strands 206-1, 206-2, and 206-3 containing functional nucleic acids were phosphorylated, and then the three DNA strands were ligated end-to-end to form a circular structure using DNA ligase. The circularization conditions were as follows: 5 μM of 59 nt DNA strand 206-1, 5 μM of 59 nt DNA strand 206-2, 5 μM of 59 nt DNA strand 206-3, 1×T4 Buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8 @ 25℃), 10 μM of 206splint-1, 10 μM of 206splint-2, and 10 μM of 206splint-3 were added, and the mixture was reacted at 90℃ for 3 min, then cooled to 25℃ at 0.1℃ / s and maintained at 25℃ for 20 min. Then, 1 U / μL of T4 DNA ligase was added, and the mixture was ligated for 5 h. The total volume was 25 μL. The enzymes were inactivated at 75°C for 10 minutes. The circular DNA was then treated with exonuleases I and III to remove the uncirculated linear nucleic acid strands and any added circularization accessory strands. Exonuleases I and III were then inactivated at 85°C. The circularization and enzyme digestion status were determined by polyacrylamide gel electrophoresis.

[0126] The prepared sample was treated with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1) to remove proteins. The product was then washed with 75% ethanol and anhydrous ethanol, respectively, and the water was evaporated at 37°C to obtain pure circular DNA. The DNA was reconstituted with water, and the concentration of the final circular DNA was determined quantitatively by polyacrylamide gel electrophoresis.

[0127] 3. Preparation of functional DNA / polyacrylamide gel

[0128] 100 μL of 40 μM circular DNA was mixed with 75 μL of 30% acrylamide solution, and 12.5 μL of water was added to obtain a 12% polyacrylamide gel rich in 4000 pmol of circular DNA. After stirring and mixing, 1.5 μL of ammonium persulfate and 0.15 μL of coagulating agent were added, and the mixture was allowed to solidify to obtain a functional DNA / polyacrylamide gel containing 4000 pmol of DNA. This gel can be used for the removal of miR-206 characteristic miRNA from 8 mL of 0.5 μM aqueous solution.

[0129] The functional DNA / polyacrylamide gel prepared above showed an enrichment rate of up to 96.3% for miR-206.

[0130] Table 5 shows the oligonucleotide single-stranded sequences used in Example 5.

[0131]

[0132]

[0133] Note: The underlined part is the complementary sequence of miR-206.

[0134] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a functional DNA gel, characterized by, The functional circular DNA is mixed with a gel monomer solution, and an initiator and a coagulant are added to perform a cross-linking polymerization reaction, during which the gel monomer penetrates through the functional circular DNA and polymerizes into a network structure, so that the functional circular DNA is enriched on the gel network structure in a suspended and fixed form, thereby obtaining a functional DNA gel; the functional circular DNA is a circular DNA containing a functional nucleic acid sequence; the length of the circular DNA is 59-177 nt; and the gel monomer is an acrylamide monomer.

2. The production method according to claim 1, characterized by, The functional circular DNA maintains a simple secondary structure at room temperature.

3. The preparation method according to claim 1, characterized in that, The concentration of the gel monomer solution is 8%-30%.

4. The production method according to claim 1, characterized by, The acrylamide monomer is acrylamide and N,N-dimethyl acrylamide, and the mass ratio of the two is (25-33):

1.

5. The production method according to claim 4, characterized by, The initiator is one or more of ammonium persulfate, azobisisobutyronitrile, dibenzoyl peroxide, dicumyl peroxide and azobisisoheptyl nitrile; and the coagulant is tetramethyl ethylenediamine or 3-dimethylamine propionitrile.

6. The method of claim 1, wherein, The functional circular DNA is prepared by the following method: first, three DNA chains containing a functional nucleic acid sequence are designed, and auxiliary DNA with different sequences is designed for connecting the three DNA chains into a ring; an exonuclease is used to remove linear and double-stranded DNA mixed in the circular DNA; and magnesium ions and proteins in the preparation process of the circular DNA are removed by alcohol extraction, so as to obtain pure circular DNA.

7. Use of the functional DNA gel according to claim 1 for removing or enriching target substances from a solution, characterized in that, The functional DNA has a target substance binding function, the functional DNA gel is immersed in an aqueous solution containing a target substance, and the functional nucleic acid sequence on the circular DNA chain specifically binds to the target substance; and the gel adsorbed with the target substance is separated from the aqueous solution.

8. Use according to claim 7, characterized in that, The application is used for removing heavy metal ions, toxins, veterinary drugs or pesticides in a solution, or enriching small molecule nucleic acids in a solution.

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