An oligonucleotide hydrogel, its preparation method and application

Abstract

The application discloses an oligonucleotide hydrogel and a preparation method and application thereof, and comprises the following steps: sequentially adding oligonucleotide and PBS buffer into a metal salt solution to obtain a mixed solution; placing the mixed solution in a shaking table and oscillating and incubating; after incubation, centrifuging, discarding the supernatant and reserving the precipitate; resuspending the precipitate by adding deionized water, centrifuging, discarding the supernatant and washing to obtain the hydrogel; the application only needs single-stranded oligonucleotide, has no special requirements on the sequence composition and structure of the oligonucleotide, does not need special design, can be assembled with divalent and trivalent metal ions, has a simple process, is easy to operate, and can wrap biological enzymes in the hydrogel, realizes enzyme immobilization and recycling.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to an oligonucleotide hydrogel, its preparation method, and its applications. Background Technology

[0002] Oligonucleotide hydrogels are a class of hydrogel networks formed by the self-assembly or chemical cross-linking of short-chain DNA or RNA. They combine the molecular recognition capabilities of nucleic acids with the biomimetic properties of hydrogels, giving them unique advantages in drug delivery, tissue engineering, and biosensing. Currently, the design or synthesis methods based on oligonucleotide hydrogels mainly achieve reversible synthesis through base complementary pairing (such as AT, GC) or specific sequence design (such as i-motif, G-tetramer). They have the advantage of biocompatibility, meaning they are natural nucleic acid materials with low immunogenicity, making them suitable for in vivo applications. They also have environmental responsiveness, being sensitive to pH, temperature, ionic strength, or specific nucleic acid chains. For example, the pH-responsive i-motif structure forms a quadruple chain under acidic conditions.

[0003] The preparation methods of oligonucleotide hydrogels mainly include physical cross-linking methods, which mainly include: (1) two partially complementary oligonucleotide chains forming a three-dimensional network through hybridization (such as the self-assembly of Y-shaped DNA units); (2) cross-linking hydrogels based on nucleic acid structural motifs: using special structures such as G-tetramers and i-motifs as cross-linking points to form hydrogels; at the same time, chemical cross-linking methods based on oligonucleotides are also commonly used in the preparation of hydrogels, such as forming hydrogels by linking oligonucleotides with polymers (such as polyethylene glycol and hyaluronic acid) through click chemistry or enzymes through covalent modification. Hydrogels can also be synthesized through hybridization chain reaction (HCR): triggering long-chain DNA cross-linking to form a gel through the initiator and two hairpin probes (H1, H2);

[0004] Current methods for designing and preparing oligonucleotide hydrogels are complex, have high requirements for the sequence and structure of oligonucleotides, and are generally costly, which greatly limits their application. Therefore, considering the safety of oligonucleotides, designing a simple and low-cost method for preparing oligonucleotide hydrogels has important application value. Summary of the Invention

[0005] The purpose of this invention is to provide an oligonucleotide hydrogel, its preparation method, and its application, in order to solve the above-mentioned technical problems.

[0006] This invention provides a method for preparing oligonucleotide hydrogels, comprising the following steps:

[0007] S1: Add oligonucleotides and PBS buffer sequentially to the metal salt solution, mix, and obtain a mixture;

[0008] S2: Place the mixture in a shaker and incubate by shaking;

[0009] S3: After incubation, centrifuge, discard the supernatant, and retain the precipitate to obtain the precipitate;

[0010] S4: Add deionized water to the precipitate to resuspend the precipitate, centrifuge, discard the supernatant, wash, and obtain hydrogel.

[0011] Preferably, the mass ratio of the metal salt solution: oligonucleotide:PBS buffer is 1×10. 6 ~2x10 6 :1:5x10 5 .

[0012] Preferably, the PBS buffer is composed of disodium hydrogen phosphate, sodium dihydrogen phosphate, and deionized water, wherein the concentrations of disodium hydrogen phosphate and sodium dihydrogen phosphate are both 50 mmol / L.

[0013] Preferably, in S1, the pH value of the PBS buffer is 7.0 to 7.5.

[0014] Preferably, in step S1, the metal salt solution includes one of a divalent metal and a trivalent metal.

[0015] Preferably, in S2, the conditions for the oscillation incubation are: oscillation temperature of 10℃~37℃, oscillation frequency of 150~220rpm, and oscillation time of 30~60min.

[0016] Preferably, in step S3, the centrifugation conditions are: a centrifugation speed of 9000–15000 rpm and a centrifugation time of 5–10 min.

[0017] Preferably, in step S4, the deionized water is 200–1000 μL, and the centrifugation conditions are: centrifugation speed 9000–15000 rpm, centrifugation time 5–10 min, the supernatant is discarded, and the washing step is repeated 3 times.

[0018] The oligonucleotide hydrogel prepared by the method described above.

[0019] Application of the oligonucleotide hydrogel described herein on immobilized biological enzymes.

[0020] This invention discloses the following technical effects: This invention provides an oligonucleotide hydrogel, its preparation method, and its application. By forming coordinate bonds between divalent or trivalent metal cations and oxygen atoms on the phosphate groups of adjacent oligonucleotide molecules, multiple oligonucleotides can be cross-linked to form a hydrogel network. The oligonucleotide hydrogel prepared by this invention only requires single-stranded oligonucleotides, and there are no special requirements for the oligonucleotide sequence composition (the number and composition of A / T / C / G) and structure. No special design is required. At the same time, both divalent and trivalent metal ions can be assembled with it. The process is simple and easy to operate. Furthermore, biological enzymes can be encapsulated in the hydrogel to achieve enzyme immobilization and recycling. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the assembly principle of the oligonucleotide-metal ion hydrogel network of the present invention.

[0023] Figure 2 This is a schematic diagram of the hydrogel centrifugation and dehydration precipitation of the present invention;

[0024] Figure 3 This is a schematic diagram of the hydrogel of the present invention being resuspended in water;

[0025] Figure 4 This is a schematic diagram of the hydrogels formed by different oligonucleotides and calcium ions according to the present invention;

[0026] Figure 5 This is a schematic diagram of the nucleotide-immobilized asparaginase hydrogel of the present invention;

[0027] Figure 6 This is a schematic diagram of the morphology and structure of the oligonucleotide and calcium ion hydrogel immobilized asparaginase of the present invention.

[0028] Figure 7 This is a schematic diagram of the enzyme activity of the free enzyme (control) and the immobilized asparaginase of the present invention;

[0029] Figure 8 This is a schematic diagram of the enzyme activity after the oligonucleotide hydrogel immobilized enzyme of the present invention has been recycled. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] This invention provides oligonucleotide hydrogels, their preparation methods, and applications. The hydrogels have application value in the field of bio-enzyme immobilization.

[0033] Example 1:

[0034] A method for preparing an oligonucleotide hydrogel, the method comprising the following steps:

[0035] First, take 80 μL of 10 mg / mL CaCl2 solution, and add 5 μL of 20 nmol / L oligonucleotide and 500 μL of pH 7 PBS buffer to this solution and mix thoroughly. Place the mixture in a shaker and incubate at 10℃ and 150 rpm for 30 min. After incubation, centrifuge at 9000 rpm for 5 min, discard the supernatant, and retain the precipitate. Then, add 200 μL of deionized water to resuspend the precipitate, centrifuge at 9000 rpm for 5 min, discard the supernatant, and repeat the washing step 3 times to form a hydrogel.

[0036] In this embodiment, the specific preparation method of PBS buffer is as follows: 17.91g of disodium hydrogen phosphate and 7.80g of sodium dihydrogen phosphate are weighed, added to 1000mL of deionized water and diluted to obtain a standard solution. Then, 61mL of disodium hydrogen phosphate standard solution and 39mL of sodium dihydrogen phosphate solution are mixed to obtain a PBS buffer with pH 7.0. The final concentrations of disodium hydrogen phosphate and sodium dihydrogen phosphate in the solution are both 50mmol / L.

[0037] In this embodiment, the specific preparation method of the metal salt solution is as follows: accurately weigh 0.1g of CaCl2, ZnCl2, FeCl3, FeSO4 or CuSO4. All of the above metal salts are analytical grade with a purity between 95% and 99%. Dissolve them in deionized water and make up to 10mL for later use. This yields CaCl2 solution, ZnCl2 solution, FeCl3 solution, FeSO4 solution or CuSO4 solution, respectively.

[0038] Among them, oligonucleotides are 16 to 40 ribonucleotides or deoxyribonucleotides randomly synthesized by chemical methods.

[0039] In this invention, divalent or trivalent metal cations form coordination bonds with oxygen atoms on phosphate groups in adjacent oligonucleotide molecules, thereby crosslinking multiple oligonucleotides to form a hydrogel network. The specific principle is as follows: Figure 1 As shown.

[0040] This invention explores the combinations of oligonucleotides with different metal ions to verify the feasibility of the invention, such as... Figure 2 and 3 As shown:

[0041] Combination 1: 80 μL CaCl2 solution (10 mg / mL) and oligonucleotide sequence 1: TAATACGACTCACTATAGGG.

[0042] Combination 2: 80 μL ZnCl2 solution (10 mg / mL) and oligonucleotide sequence 1: TAATACGACTCACTATAGGG.

[0043] Combination 3: 80 μL FeCl3 solution (10 mg / mL) and oligonucleotide sequence 1: TAATACGACTCACTATAGGG.

[0044] Combination 4: 80 μL CuSO4 solution (10 mg / mL) and oligonucleotide sequence 1: TAATACGACTCACTATAGGG.

[0045] Combination 5: 80 μL FeSO4 solution (10 mg / mL) and oligonucleotide sequence 1: TAATACGACTCACTATAGGG.

[0046] This invention also explored the combinations of different random oligonucleotides with calcium ions to verify the feasibility of the invention, such as... Figure 4 As shown:

[0047] Combination 1: 80 μL CaCl2 solution (10 mg / mL) and oligonucleotide sequence 1; TAATACGACTCACTATAGGG.

[0048] Combination 2: 80 μL CaCl2 solution (10 mg / mL) and oligonucleotide sequence 2: CATTAGGATCCCACCACCACCACCA.

[0049] Combination 3: 80 μL CaCl2 solution (10 mg / mL) and oligonucleotide sequence 3: CTGCATATGAGTACTAAATTAGTT.

[0050] Combination 4: 80 μL CaCl2 solution (10 mg / mL) and oligonucleotide sequence 4: TATAATGGATCCGGCTC.

[0051] Combination 5: 80 μL CaCl2 solution (10 mg / mL) and oligonucleotide sequence 5: GATCAGTCAAGTCTCGGATCG.

[0052] This invention also explores the application of oligonucleotide hydrogels in immobilized biological enzymes, and the specific technical solution is as follows:

[0053] The selected bioenzyme, E. coli-derived asparaginase, was purchased from Sigma-Aldrich (EC 3.5.1.1). The enzyme activity was detected using the following method:

[0054] 1. Add 20 μL of enzyme solution to 200 μL of substrate (L-asparagine 50 mM) solution and react at 35 °C for 20 min.

[0055] 2. After the reaction is complete, centrifuge at 12,000 rpm for 5 minutes and retain the supernatant.

[0056] 3. Take 100 μL of supernatant and add 900 μL of deionized water.

[0057] 4. Add 200 μL Nessler's reagent, mix well, and let stand at room temperature for 20 min.

[0058] 5. Use an ultraviolet spectrophotometer to measure the absorbance value at a wavelength of 420 nm.

[0059] This invention relates to a method for immobilizing L-asparaginase with oligonucleotide calcium ion hydrogel and a method for detecting enzyme activity.

[0060] 1. Take 80 μL of CaCl2 solution (10 mg / mL), add 5 μL of oligonucleotide sequence 1 (20 nM), 500 μL of PBS buffer (pH 7), and 20 μL of asparaginase solution in sequence, and mix thoroughly.

[0061] 2. Place the mixture in a shaker and incubate at 10°C and 150 rpm for 30 minutes.

[0062] 3. After incubation, centrifuge at 9000 rpm for 5 minutes, discard the supernatant, and retain the precipitate.

[0063] 4. Resuspend the precipitate in 200 μL of deionized water, centrifuge at 9000 rpm for 5 min, and discard the supernatant. Repeat the washing step 3 times, freeze-dry the immobilized enzyme, plate it with gold, and observe its morphology using a scanning electron microscope.

[0064] 5. Add 200 μL of substrate solution to the precipitate and react at 35 °C for 20 min.

[0065] 6. After the reaction is complete, centrifuge at 12,000 rpm for 5 minutes and retain the supernatant.

[0066] 7. Take 100 μL of supernatant and add 900 μL of deionized water.

[0067] 8. Add 200 μL Nessler's reagent, mix well, and let stand at room temperature for 20 min.

[0068] 9. Use an ultraviolet spectrophotometer to measure the absorbance value at a wavelength of 420 nm.

[0069] 10. Centrifuge to remove the reaction buffer, then add the enzyme reaction buffer again.

[0070] 11. Repeat the use 8 times and measure the enzyme activity after each use.

[0071] This invention Figure 1 The black lines in the middle represent oligonucleotide chains, M n+ Represents metal cations (n=2, 3);

[0072] Figure 2 , Figure 3 These correspond to the precipitates formed after dehydration of hydrogels formed by different metal ions and oligonucleotides, and the hydrogel networks formed after rehydration.

[0073] Figure 4 Hydrogel networks formed after watering CaCl2 and different oligonucleotides;

[0074] Figure 6 SEM image of hydrogel after immobilization of asparaginase with oligonucleotides and calcium ions.

[0075] from Figure 7 As can be seen, the hydrogel of the present invention significantly reduces the enzyme activity of immobilized asparaginase.

[0076] from Figure 8 As can be seen from the present invention, after immobilizing asparaginase with hydrogel, the enzyme can be recycled multiple times and its activity decreases slowly.

[0077] The hydrogel prepared by the method of the present invention has good application value in the field of bio-enzyme immobilization.

[0078] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0079] 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 an oligonucleotide hydrogel, characterized in that, Includes the following steps: S1: Oligonucleotides and PBS buffer are added sequentially to the metal salt solution and mixed to obtain a mixture; S2: Place the mixture in a shaker and incubate by shaking; S3: After incubation, centrifuge, discard the supernatant, and retain the precipitate to obtain the precipitate; S4: Add deionized water to the precipitate to resuspend the precipitate, centrifuge, discard the supernatant, wash, and obtain hydrogel; In step S1, the PBS buffer is composed of disodium hydrogen phosphate, sodium dihydrogen phosphate, and deionized water; the mass ratio of the metal salt solution: oligonucleotide:PBS buffer is 1×10. 6 ~2x10 6 :1:5x10 5 The pH value of the PBS buffer is 7.0–7.

5. In step S2, the conditions for the oscillation incubation are: oscillation temperature of 10℃~37℃, oscillation frequency of 150~220rpm, and oscillation time of 30~60min. The metal salt solution contains one or more of divalent and trivalent metals.

2. The method for preparing an oligonucleotide hydrogel according to claim 1, characterized in that: The concentrations of both disodium hydrogen phosphate and sodium dihydrogen phosphate are 50 mmol / L.

3. The method for preparing an oligonucleotide hydrogel according to claim 1, characterized in that: In S3, the centrifugation conditions are: centrifugation speed of 9000-15000 rpm and centrifugation time of 5-10 min.

4. The method for preparing an oligonucleotide hydrogel according to claim 1, characterized in that: In step S4, the deionized water is 200–1000 μL, and the centrifugation conditions are: centrifugation speed 9000–15000 rpm, centrifugation time 5–10 min, discard the supernatant, and repeat the washing step 3 times.

5. An oligonucleotide hydrogel prepared by a method according to any one of claims 1-4.

6. The application of an oligonucleotide hydrogel according to claim 5 on immobilized biological enzymes.

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