CN112522373B

CN112522373B - Preparation method of spider-web-shaped self-assembly functional nucleic acid hydrogel

Info

Publication number: CN112522373B
Application number: CN202110144181.5A
Authority: CN
Inventors: 许文涛; 黄昆仑; 宋欢; 张洋子
Original assignee: China Agricultural University
Current assignee: Beijing Lihuan Biotechnology Co ltd
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2021-11-26
Anticipated expiration: 2041-02-03
Also published as: CN112522373A

Abstract

The invention provides a preparation method of a spider-web-shaped self-assembly functional nucleic acid hydrogel. According to the method, a target response unit is introduced on the basis of self-assembly of the functional nucleic acid hydrogel, and the visual detection of the target is realized through the controllable release of the embedding substance. According to the invention, the self-assembly between the soft brush hydrogel and the DNA super sandwich structure increases the DNA double-chain area, improves the rigidity of the hydrogel network, and provides a chimeric site for an embedded object; the microscopic morphology is a spider-web structure formed by the mutual cross-linking of the nanoflowers, and the cross-linking density and the particle size of the nanoflowers can be adjusted by the concentration of the probe and the DNA super sandwich structure and the applied centrifugal force. The preparation method of the spider web-shaped functional nucleic acid hydrogel constructed by the invention has the advantages of high speed, controllable appearance, target response, controllable release of embedded substances, strong universality and the like, and has good application prospects in the aspects of molecular detection, drug loading and delivery and the like.

Description

Preparation method of spider-web-shaped self-assembly functional nucleic acid hydrogel

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a preparation method of a spider-web-shaped self-assembly functional nucleic acid hydrogel.

Background

Traditional methods for preparing nucleic acid hydrogels mostly rely on the formation of basic unit structures through the artificial synthesis of high-concentration oligonucleotide strands through complementary pairing and further self-assembly to build a cross-linked hydrogel network. The method has the advantages of complex operation, high nucleic acid consumption, dependence on a precise instrument and high synthesis cost. On one hand, the obtained nucleic acid hydrogel has single micro-morphology, and the size and the morphology structure of the nucleic acid hydrogel are difficult to regulate and control in the preparation process; on the other hand, since the basic unit has a simple structure, it is difficult to innovate in functional design to prepare a nucleic acid hydrogel by a conventional method, resulting in limited applications thereof. The Rolling Circle Amplification (RCA) technology is an economic and effective nucleic acid hydrogel preparation strategy because long nucleic acid chains can be generated and hydrogel-like products with microscopic nanoflower structures are formed in the reaction process, and the problems that the consumption of nucleic acid is large and the length of artificially synthesized DNA chains is limited in the traditional method are solved. However, research on the aspects of mechanical strength improvement, functionality enhancement, controllable microstructure and the like of nucleic acid hydrogel prepared based on the RCA technology is still very limited.

Disclosure of Invention

The novel method for preparing the nucleic acid hydrogel overcomes the defects of the existing hydrogel preparation method, and realizes the preparation of the nucleic acid hydrogel which is rapid, simple, efficient, controllable in appearance, target-responsive and controllable in release of the embedded substances.

The invention aims at providing a preparation method, which is based on an in vitro isothermal nucleic acid amplification technology, wherein a reaction system of the in vitro isothermal nucleic acid amplification technology comprises a padlock probe and a connecting primer, and is characterized in that the 5' end of the padlock probe is subjected to phosphorylation modification and contains a region complementary with the connecting primer; the connecting primer can be hybridized with the 5 'end and the 3' end of the padlock probe to form 2 adjacent base complementary pairing regions;

the complementation includes complementation or reverse complementation defined by the prior art or the common general knowledge and/or complementation or reverse complementation according to the complementation principle defined by the prior art or the common general knowledge.

The polymerases include polymerases useful in vitro nucleic acid amplification techniques.

The ligase includes a ligase that can be used in an in vitro nucleic acid amplification technique.

The sequence in the amplification reaction system comprises a sequence defined by the prior art or common general knowledge, can be directly obtained by artificial synthesis by the public, and the preparation method belongs to the prior art; the design includes the design methods described in the prior art or the common general knowledge.

The method further comprises at least one of the following 1) -3):

1) the in vitro nucleic acid amplification technology comprises a rolling circle amplification reaction, wherein the reaction process of the rolling circle amplification reaction comprises the following steps: connecting reaction and amplifying reaction;

2) the ligation reaction comprises a process of hybridizing the padlock probe with the primer, and the reaction process comprises the following steps: slowly cooling at 80-100 ℃ for 5-10 min; and (3) allowing the hybridization product to generate a cyclized template by the padlock probe under the action of ligase, wherein the reaction process comprises the following steps: at the temperature of 16-30 ℃, 20 min-3 h;

3) the amplification reaction comprises a process of amplifying the circularized template and the primer, and the reaction process comprises the following steps: 30-37 ℃ for 10-30 h.

4) The padlock probe comprises a compound which has a long-chain structure and the 5' end of which is modified by phosphorylation.

More specifically, the chemical structure of the 5' end phosphorylation modification is as follows:

specifically, the method further comprises at least one of the following 1) to 6):

1) the padlock probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 1, and carrying out phosphorylation modification on the 5' end of the nucleotide sequence shown in the figure to obtain a primer;

2) the connecting primer comprises a sequence shown in SEQ ID NO: 2;

3) the padlock probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 1 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 1, the 5' end of the nucleotide sequence with the same function is modified by phosphorylation to obtain a primer;

4) the connection primer comprises a primer formed by combining SEQ ID NO: 2 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 2 has the same function;

5) carrying out agarose electrophoresis analysis on the product of the rolling circle amplification reaction to obtain a DNA long single chain of which the length is more than 5000 bp;

6) the products of the rolling circle amplification reaction are in a hydrogel state with certain viscoelasticity.

Another object of the present invention is to provide a preparation method comprising preparation of a spider web-like self-assembly functional nucleic acid hydrogel by a preparation system comprising an RCA product, a pair of L-type probes A, B and a pair of

probes

1 and 2. The A, B and 1, 2 are used only to distinguish between different complementary sequences and are not used for sequencing.

Another object of the present invention is to provide a method for preparing a spider web-like self-assembly functional nucleic acid hydrogel, comprising a long single-stranded DNA product, a pair of L-shaped probes A, B, and a pair of

probes

1, 2;

the long single-stranded DNA product and L-probe A, B are capable of self-assembling into a soft brush DNA hydrogel with a nanoflower microstructure;

the

probes

1 and 2 can form a DNA super sandwich structure through complementary pairing, and are further assembled with the soft brush DNA hydrogel through physical interaction to form a spider-web-shaped microstructure embedded with nanoflowers, and finally form the spider-web-shaped self-assembly functional nucleic acid hydrogel.

Optionally, in the above preparation method, the spider web-like self-assembly functional nucleic acid hydrogel may be disassembled when a target is added, where the target is a target to which the aptamer sequence of probe 1 is correspondingly bound.

Optionally, in the above preparation method, the pair of L-shaped probes A, B has the following structure:

1) the L-type probe A, B sequence includes a complementary region and a non-complementary region to the RCA long single-stranded DNA product;

2) the complementary region in the L-type probe A sequence is a nucleic acid sequence which is arbitrarily complementary with each unit sequence in the RCA long single-strand DNA product and has the length of 20 nt;

3) the complementary region in the L-type probe B sequence is a nucleic acid sequence which is arbitrarily complementary with each unit sequence in the RCA long single-strand DNA product, has the length of 20nt and has no repeat with A;

4) the non-complementary region in the L-type probe A, B sequence is a non-complementary nucleic acid sequence that does not bind to the RCA long single-stranded DNA product and is identical to probe 2;

5) the L-type probe A sequence is a nucleic acid sequence which is arranged from a non-complementary region to a complementary region from 5 'end to 3' end;

6) the L-type probe B sequence is a nucleic acid sequence arranged from a complementary region to a non-complementary region in the direction from 5 'end to 3' end.

Optionally, in the above preparation method, the probe 1 has the following structure:

1) the probe 1 sequence is a specific aptamer sequence capable of binding a specific target, and comprises A, B two partial sequences, wherein the lengths of the two partial sequences are the same or differ by 1nt, and the sum of the lengths is the length of the probe 1 sequence;

2) the probe 1 sequence is a nucleic acid sequence arranged from 5 'end to 3' end according to the sequence from the sequence A to the sequence B;

3) the specific aptamer sequence of the probe 1 enables the spider-web hydrogel to have target responsiveness and the effect of controllably releasing the inclusion through disassembly.

Optionally, in the above preparation method, the probe 2 has the following structure:

1) the probe 2 sequence is a specific aptamer sequence of a target and comprises C, D sequences which are respectively complementary with A, B sequences in the probe 1, and the sum of the lengths of C, D sequences is the length of the probe 2 sequence;

2) the probe 2 sequence is a nucleic acid sequence arranged from 5 'end to 3' end according to the sequence C to the sequence D, wherein the C sequence is the same as the B sequence of the probe 1 in length, namely the B, C sequence is completely complementary; the D sequence is increased by at least 1nt length in the 3' end direction compared with the A sequence, namely, a spacer region of at least 1nt exists between the A, D sequence complementary region and the B, C complementary region.

Optionally, in the above preparation method,

probes

1 and 2 of the L-type probe A, B are the following sequences:

1) the L-type probe A comprises SEQ ID NO: 3 and/or the nucleic acid sequence of SEQ ID NO: 3 by substitution and/or deletion and/or addition of one or more nucleotides.

2) The L-type probe B comprises SEQ ID NO: 4 and/or the nucleic acid sequence of SEQ ID NO: 4 through substitution and/or deletion and/or addition of one or more nucleotides.

3) The probe 1 comprises a nucleotide sequence shown as SEQ ID NO: 5 and/or the nucleic acid sequence of SEQ ID NO: 5 by substitution and/or deletion and/or addition of one or more nucleotides.

4) The probe 2 comprises SEQ ID NO: 6 and/or the nucleic acid sequence of SEQ ID NO: 6 by substitution and/or deletion and/or addition of one or more nucleotides.

Optionally, in the above preparation method, the method for preparing a spider web-like self-assembly functional nucleic acid hydrogel further comprises the steps of:

1) before the L-type probe and the RCA product are subjected to self-assembly, the hydrogel state of the RCA product needs to be disturbed in a stirring mode until the viscoelasticity of the RCA product disappears;

2) the L-type probe was added to the RCA product after the stirring, and the L-type probe was partially complementarily hybridized with the RCA product by a short-time stirring for 1 minute.

On the other hand, the probe 1 of the present invention may be an ATP-specific aptamer sequence, or may be any aptamer sequence. Any aptamer sequence includes those obtained by screening in a variety of ways and capable of specifically binding to a target. The probe 1 selects any aptamer sequence to be combined with the probe 2 with the corresponding structure, and when no specific target exists, the probe 1 and the probe 2 can complete the spider-web self-assembly function nucleic acid hydrogel; when a specific target exists, the probe 1 can be specifically combined with the target, so that the spider-web-shaped hydrogel is disassembled and the embedded matter is released.

The application of the spider-web-shaped self-assembly functional nucleic acid hydrogel can lead to the disassembly of the hydrogel network by combining the target with the specific aptamer sequence of the target, thereby realizing the release of embedding substances such as fluorescent dye, medicine and the like in the hydrogel. Further, the hydrogel of the present invention can be visually detected by target-responsive release of the inclusion. Further, the hydrogel of the invention is applied to molecular detection, drug loading and delivery.

Therefore, the limitation of the aptamer is not limited to the ATP aptamer in the embodiment of the application, and other aptamers can achieve self-assembly and controlled release of the spider-web hydrogel through self-assembly action and target binding.

The invention also aims to provide a preparation method, which comprises the loading and purification of the inclusion in the spider-web self-assembly functional nucleic acid hydrogel and is characterized in that the visual detection is carried out by target response release inclusion, and the complex components introduced by a nucleic acid amplification reaction system in the preparation process and the excessive inclusion after loading are removed.

Specifically, the preparation method further comprises the following steps:

1) incubating the aqueous solution of the embedding medium and the transparent spider web-shaped DNA hydrogel in a blending instrument at normal temperature and in dark, wherein the rotating speed is 150 rpm, the incubation time is 90min, and then separating the supernatant from the colloid;

2) immersing the separated spider web-shaped self-assembly functional nucleic acid hydrogel into ddH₂O for a certain time;

3) and removing the supernatant to obtain the purified spider web-shaped self-assembly functional nucleic acid hydrogel.

The beneficial effects of the invention include:

1. on the basis of forming soft brush DNA hydrogel by fast self-assembling a DNA long single chain obtained by RCA and an L-shaped probe, a DNA super sandwich structure formed by complementary pairing of the

probes

1 and 2 is introduced, and a novel spider-web-shaped microstructure embedded with DNA nanoflowers is formed by further high-efficiency self-assembling;

2. the invention adds a rigid DNA double-chain structure due to the introduced DNA super sandwich structure, obviously improves the mechanical strength of the DNA hydrogel and provides a chimeric site for embedding substances such as dye, medicine and the like;

3. the probe 1 introduced in the invention can be changed into other target specific nucleic acid aptamer sequences according to the analysis and detection requirements, when the target is added, the release of the embedded substances such as dye, medicine and the like in the hydrogel is caused through the strong affinity action between the target and the specific nucleic acid aptamer thereof, and the controllable release of the embedded substances is realized through the concentration of the added target;

4. according to the invention, the crosslinking density of the spider web microstructure can be realized by adjusting the concentration of the probe and the DNA super sandwich structure and the applied centrifugal force.

Drawings

FIG. 1 shows the principle of self-assembly of spider web-like DNA hydrogels and the effect of high speed centrifugation on hydrogels. (A) Soft brush DNA hydrogel formed by self-assembly of RCA product with L-type probe A, B; (B) based on a soft brush DNA hydrogel and a DNA super sandwich structure (formed by a pair of

probes

1 and 2 through complementary pairing and spontaneous formation), self-assembling to form a spider-web-shaped DNA hydrogel; (C) under high speed centrifugation, the spider web-like DNA hydrogel is disrupted.

FIG. 2 shows spider web-like DNA hydrogels before (A) and after (B) high-speed centrifugation.

FIG. 3 is a graph demonstrating the cross-linking state between RCA products and DNA super sandwich structures and the effect of high speed centrifugation (scale bar =2 μ M) by (A) agarose gel electrophoresis, (B-C) laser scanning confocal microscopy (CLSM) and (D-I) Scanning Electron Microscopy (SEM).

Fig. 4 is the microstructure of the spider web-like DNA hydrogel before (a), after (B) high speed centrifugation (scale bar =2 μ M).

FIG. 5 is a graph of the effect of the concentration of L-probe A, B and DNA super sandwich on the microstructure (A-H) and rheological properties (I-L) of spider web DNA hydrogels.

FIG. 6 is a schematic representation of ATP-responsive spider web-like DNA hydrogels. (A) Preparation of ATP response type spider web-shaped DNA hydrogel. (B) Methylene Blue (MB) loaded spider web DNA hydrogels. (C) ATP triggers the disassembly of the spider web-like DNA hydrogel and the controlled release of the inclusion.

Fig. 7 is the MB loading and washing steps of the spider web-like DNA hydrogel. (A) The original state of the spider web-like DNA hydrogel; (B) incubating the spider web-like DNA hydrogel with MB for 0min and 90min respectively; (C) immersing the MB-loaded spider web-like DNA hydrogel in ddH₂Washing in O; (D) washed state of the MB-loaded spider web-like DNA hydrogel.

FIG. 8 is ATP triggered release of MB from a spider web-like DNA hydrogel. (A) Loading of MB with spider-web DNA hydrogel and application of ddH₂And O washing. (B) Release process of loaded MB in spider web DNA hydrogel at 0 mM (left) or 80 mM ATP (right) and different reaction times including 6s, 1min and 3min, respectively. (C) The spider web-like DNA hydrogel was incubated with ATP for 20 minutes. (D) Separating the supernatant and the DNA hydrogel from the sample in (C).

FIG. 9 is the effect of time (A-C) and ATP concentration (D-F) on release of MB from a spider web-like DNA hydrogel.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples further illustrate the contents and embodiments of this invention, which are described in more detail and detail, but are not to be construed as limiting the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Example 1 preparation and characterization of spider-web DNA hydrogels

(I) test materials

The information of the experimental reagents used in this example is shown in Table 1, and the nucleotide sequences of the designed primers are shown in Table 2 and the sequence Listing.

The experimental water was obtained from a Milli-Q pure water system, except for the experimental reagents in Table 1. Other reagents were purchased from the national pharmaceutical group.

In Table 2, the 5' end of the padlock probe is phosphorylated and modified and has the chemical structure:

in Table 2, underlined sequences indicate free sequences remaining after the RCA product was hybridized with the L-type probe. The number of nucleotides in the free sequence is indicated in parentheses.

The sequences listed in Table 2 were all artificially synthesized.

(II) RCA reaction

1) Ligation reaction

The first step of the RCA reaction is to ligate the padlock probes with the help of the ligation primer by T4 ligase to form a circular amplification template. The composition of the rolling circle amplification ligation system is shown below (Table 3). Firstly, the components in the table 3 are mixed and then placed in a PCR instrument for heating at 90 ℃ for 5min, and the temperature is slowly cooled to room temperature at the speed of 1 ℃ per min. Subsequently, 2. mu. L T4 DNA ligase (40U/. mu.L) was added to the system, mixed by gentle pipetting with a pipette tip, and incubated at room temperature for 1 h.

2) Amplification reaction

The second step of the RCA reaction is to perform rolling circle amplification reaction on the ligation product under the action of phi29 DNA polymerase and dNTPs to obtain a large amount of amplification products of long single-stranded DNA (ssDNAs). The composition of the amplification system for the rolling circle amplification reaction is shown below (Table 4). First, the components in Table 4 were mixed and incubated at 30 ℃ for 24 h. Subsequently, the amplification reaction was terminated by inactivating the phi29 DNA polymerase by incubation at 65 ℃ for 10 min.

(III) crosslinking of spider web-like DNA hydrogels

The RCA product was first agitated with a tip to break its pre-gel state until the viscoelasticity disappeared. As shown in FIG. 1A, B, a soft-brush DNA hydrogel was formed by stirring with 6. mu.L of 100. mu.M L-type probe A, B for 1 minute and ddH was used₂And O washing. Then, 6. mu.L of 100. mu.M probes 1, 2 were added and stirring was continued for 1min to obtain a spider web-like DNA hydrogel.

(IV) characterization of spider web-like DNA hydrogels

The prepared spider web-like DNA hydrogel is characterized by four modes of optical photograph, agarose gel electrophoresis, CLSM and SEM.

1) Optical photographs recording the macroscopic morphology of spider web-like DNA hydrogels

As shown in FIG. 2A, the spider web-like DNA hydrogel exhibited a uniform micellar state. When the release agent is released from the pipette head, the release agent is in a micelle shape with uniform thickness and larger viscoelasticity.

2) Agarose gel electrophoresis to verify the formation of spider web-like DNA hydrogel

Replacing the probe 2 for preparing the spider web-shaped DNA hydrogel with the probe 2 which is fluorescently labeled by FAM, and preparing the spider web-shaped DNA hydrogel with the FAM fluorescence label according to the same steps so as to indicate the formation of a DNA super sandwich structure and the action in the spider web-shaped DNA hydrogel.

The results of agarose gel electrophoresis demonstrate cross-linking between the DNA super sandwich structure and RCA products (fig. 3A). The diffusion band at the bottom of lane 1 indicates that the DNA super sandwich is formed by probe 1 and FAM-probe 2 and cannot be ligated to RCA product without the addition of L-type probe A, B. After addition of L-probe A, B, binding of the DNA super-sandwich to the RCA product was achieved, and the sample was trapped directly in the well of lane 3, showing a bright band, due to the large enough size of the nucleic acid structure formed.

3) CLSM verifies formation of spider web-like DNA hydrogels

As shown in fig. 2B, the different size of the separate green regions demonstrated that the nanoflower was covered by the DNA super sandwich structure via the L-probe (fig. 3B), which is quite distinct compared to the large number of free green dot images presented by the control sample without the addition of L-probe A, B (fig. 3C). The construction of the spider web-like DNA hydrogel is not possible for the RCA product, the L-type probe A, B, and the

probes

1 and 2.

4) SEM characterization of the microstructure of spider Web-like DNA hydrogels

The samples were first snap frozen with liquid nitrogen and then placed into a freeze dryer for complete drying. Platinum was sprayed for 6 min at 20 mA and electron microscopy was performed at 5 kV.

As shown in FIG. 3D, after RCA reaction for 24h, a number of nanoflower structures with a diameter of 1-2 μm were observed. On the other hand, many bent fibrous structures were obtained in the assembly of

probes

1, 2 to form a DNA super sandwich structure (FIG. 3G). By mixing the 24h RCA product with the formed DNA super sandwich structure, the nanoflowers were randomly scattered on the bent fibers (fig. 3E). L-type probes A, B were further added to the mixture of RCA product and ultrasandwich, achieving mutual cross-linking and acting as a bridge between the mixtures. Thus, the fibers bent in the microscopic morphology are straightened and cross-linked with the nanoflower to form a regular network structure, such as a spider web (fig. 3F and fig. 4A).

Example 2 micro-topography control of spider-web DNA hydrogels

The micro-morphology of the spider web-like DNA hydrogel is mainly controlled by two ways: firstly, high-speed centrifugation; and secondly, adjusting the concentration of the L-shaped probe and the DNA super sandwich structure.

(I) influence of high speed centrifugation on spider web-like DNA hydrogel

1) High-speed centrifugal treatment of spider web-like DNA hydrogel

Immersing the prepared spider web DNA hydrogel in 500. mu.L of ddH₂O for 5min, then centrifuged at 16000 g for 10min and the supernatant fraction removed.

2) Recording the influence of high-speed centrifugation on macroscopic morphology of spider web-like DNA hydrogel by optical photos

After high-speed centrifugation, the spider web DNA hydrogel is directly observed to have uneven gel texture, and when the gel is released from a pipette tip, the gel drops of the DNA hydrogel can be more clearly observed to be uneven micelles (FIG. 2B), which shows that the high-speed centrifugation can destroy the network structure of the spider web DNA hydrogel (FIG. 1C).

3) Agarose gel electrophoresis characterizes the effect of high speed centrifugation on the structure of spider web DNA hydrogel

After high speed centrifugation of the samples in

lanes

1 and 3, the diffusion bands corresponding to the formation of the DNA super sandwich structure almost disappeared (FIG. 3A, lanes 2 and 4), indicating that high speed centrifugation destroys the cross-linked network of the spider web-like DNA hydrogel.

4) SEM characterization of the Effect of high speed centrifugation on the microstructure of spider web-like DNA hydrogels

After high speed centrifugation of the mixture of 24H RCA product and DNA super sandwich, the bent fibers were reduced on the microscopic scale (FIG. 3H). Similarly, after subjecting the obtained spider web-like DNA hydrogel to high-speed centrifugation treatment, it was found that the hydrogel network structure was decomposed into short fibers (FIG. 3I). At the same time, the nanoflower fell off the network structure, and the broken fibers between different nanoflowers are shown in fig. 4B. This indicates that the cross-linking between the RCA product and the DNA super sandwich is achieved by L-probe A, B and is easily broken by external forces such as high speed centrifugation.

(II) influence of L-type probe and DNA super sandwich structure concentration on spider web-like DNA hydrogel

1) SEM characterization of the influence of DNA super sandwich structure concentration on the microstructure of spider web-like DNA hydrogel

At a concentration of 50 μ M of the DNA super sandwich structure, the fibers of the rough and irregular surface in the microstructure of the formed spider web-like DNA hydrogel tended to shorten and stick together (FIG. 5A). When the concentration reached 100. mu.M, the fibers became smooth, separated and elongated (FIG. 5B). Further increasing the concentration of the DNA super-sandwich to 150. mu.M (FIG. 5C) and 300. mu.M (FIG. 5D) resulted in a significant increase in the degree of cross-linking and average diameter of the fibers. The fibers become thicker and more swollen, resulting in an increase in the narrow space between the different fibers (fig. 5D).

2) SEM characterization of the Effect of L-type Probe concentration on the microstructure of spider Web-like DNA hydrogels

As the concentration of L-type probes and DNA super sandwich was increased from 50 μ M to 300 μ M, the density of the cross-linked network and fiber diameter increased significantly (FIGS. 5E-H), as the gap between the different fibers decreased from 16.3 μ M to 3.2 μ M. In addition, when the concentration of L-type probe and DNA super sandwich reached 300. mu.M, the fiber was very bent as shown in FIG. 3H. Therefore, the concentrations of L-type probes and DNA super sandwich structure significantly affect the internal structure of spider web-like DNA hydrogels.

3) Rheological test characterizes the effect of the concentration of L-type probes and DNA super sandwich structures on the mechanical strength of spider web-like DNA hydrogels

The parameters of the rheological test were set according to table 5.

Comparing the effect of different concentrations of DNA super sandwich structures on the rheological properties of spider web-like DNA hydrogels, it was found that the storage modulus (G') increases with increasing concentration at 25 ℃, demonstrating that elasticity changes due to the concentration of DNA super sandwich structures (fig. 5I-L). In all samples, G' gradually increased with increasing frequency (fig. 5J) and gradually decreased with decreasing strain (fig. 5K). As shown in fig. 5L, when the temperature exceeded 45 ℃, G' rapidly decreased, indicating that the temperature was the gel-to-sol transition point. At the same time, as the temperature increased from 25 ℃ to 75 ℃, G' decreased by 33.2% (300. mu.M), 30.9% (100. mu.M) and 19.4% (0. mu.M), respectively. This indicates that high temperature can disrupt the network structure, resulting in a decrease in the mechanical properties of the hydrogel. Therefore, the DNA super sandwich structure plays an important role in the network structure of the spider-web DNA hydrogel, and the elasticity of the hydrogel is enhanced by crosslinking the L-type probe and the RCA product.

Example 3 controlled Release of Encapsulated ATP-responsive spider Web-like DNA hydrogels

The pre-designed super sandwich, consisting of ATP aptamer (probe 1) and its complementary single stranded DNA (probe 2), provides a reliable loading site for inclusion loading and ATP-responsive release (fig. 1 and 6). As shown in FIG. 6A, spider web-like DNA hydrogels have fluid-like properties that can flow freely in tubes. The cationic dye MB was then selected for loading into ATP-responsive DNA super sandwich duplexes in spider web-like DNA hydrogels. Subsequently, the volume of the spider web-like DNA hydrogel was significantly contracted and became a small disk-like solid (FIG. 6B). In the presence of ATP, the affinity between probe 1 and ATP is stronger than the stability of the probe 1/probe 2 structure, which results in specific binding of probe 1/ATP and release of probe 2, eventually leading to hydrogel disassembly (FIG. 6C).

(I) test materials

The information on the reagents used in the present example is shown in Table 6.

TABLE 6

Experimental reagent	Manufacturer of the product	Rank of
			ATP	Solaibao	Biological reagent
MB	Sigma	Biological reagent

The experimental water was obtained from a Milli-Q pure water system, except for the experimental reagents in Table 6. Other reagents were purchased from the national pharmaceutical group.

(II) embedding and releasing of MB in ATP response type spider web-shaped DNA hydrogel

1) Embedding MB with spider web-shaped DNA hydrogel

After the spider web-shaped DNA hydrogel and the 8 mg/mLMB aqueous solution are incubated for 90min, the color of the hydrogel is changed from transparent to dark blue or even close to black, while the original dark blue MB aqueous solution is changed to light blue, which shows that MB is successfully migrated into the spider web-shaped DNA hydrogel from the original aqueous solution, and simultaneously shows that the hydrogel has strong adsorption capacity to MB molecules (fig. 7A-C). Subsequently, the hydrogel was washed to remove excess MB, i.e., the hydrogel was incubated in 1 mL of ddH2O for 5min, and the washing solution was removed, at which time a spider web-like DNA hydrogel loaded with high concentration of MB was obtained (fig. 7D).

2) Release of MB from spider-web-like DNA hydrogel

The release of MB from the hydrogel was carried out by adding 80. mu.L of 0 mM and 80. mu.L of 80 mM ATP to the MB-embedded spider web-like DNA hydrogel (FIG. 8A), as shown in FIGS. 8B-D. After 0 mMATP is added, MB is slowly released, and the solution is light blue; after addition of 80 mM ATP, MB is released rapidly and after 3min the solution turns dark blue in color. After 20min, the color of the sample solutions to which 0 mM ATP and 80 mM ATP were added was darker than that at 3min (FIG. 8C). After separation of the supernatant and the hydrogel, it was found that the volume of the hydrogel sample to which 80 mM ATP was added was greatly reduced, while the volume of the hydrogel control sample to which 0 mM ATP was added did not change much (FIG. 8D). Shows that the spider web-shaped DNA hydrogel can make strong response to ATP and simultaneously realize the controlled release of MB.

3) Reaction time of ATP with spider web-like DNA hydrogel and Effect of ATP concentration on controlled release of MB

The effect of ATP reaction time with the hydrogel on MB release was first explored. The reaction time was set at 2, 4, 6, 8, 10, 15, 20, 25, 30 min and the supernatant and hydrogel were separated immediately after the reaction was complete. As shown in FIG. 9A, the naked eye observation shows that in the ATP experimental group, the supernatant exhibited a deep blue color already at a reaction time of 2 min, and the longer the reaction time, the more MB was released, the darker the color of the supernatant, and the smaller the volume of the hydrogel due to the disintegration of the DNA super sandwich structure. The MB release amount in the supernatant increased with the reaction time as compared with the control group, but the color was much lighter than that in the ATP test group, and the volume of the hydrogel did not change significantly (FIG. 9B). In order to further quantify the above phenomenon, the supernatant was measured using an ultraviolet spectrophotometer, and it was found that the release of MB was very rapid in the time range of 0-2 min with the hydrogel added with 80 mM ATP; in the time frame of 2-30 min, the release rate of MB slowed down compared to before 2 min, but the release rate was relatively smooth and remained released up to 30 min (fig. 9C). In contrast, the absorbance of the control was in a slowly increasing state from 0-15 min to 15 min, and reached a plateau. The above results indicate that ATP is able to stimulate release of MB from spider web-like DNA hydrogels and is strongly correlated with reaction time.

Subsequently, the effect of ATP concentration on MB release in the hydrogel was further investigated. ATP concentrations were set at 0, 2.5, 5, 10, 20, 40, 80 and 160 mM and incubated with MB-embedded hydrogel for 20min (fig. 9D). As shown in FIG. 9E, the higher the ATP concentration, the more released MB, the darker the color in the supernatant, and the smaller the hydrogel volume. The absorbance of the supernatant was measured by an ultraviolet spectrophotometer after 20-fold dilution, and it was found that the larger the ATP concentration, the larger the peak value of MB at 664 nm of the absorption peak and 610 nm of the dimer absorption peak (FIG. 9F). Therefore, the concentration of ATP has a great influence on the release of MB in the spider web-shaped DNA hydrogel, namely the release rate and the release amount of MB in the hydrogel can be regulated and controlled through the ATP concentration.

The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.

Sequence listing

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Claims

1. A preparation method of a spider-web-shaped self-assembly functional nucleic acid hydrogel is characterized by comprising a long single-stranded DNA product, a pair of L-shaped probes A, B and a pair of probes 1 and 2;

the probes 1 and 2 can form a DNA super sandwich structure through base complementary pairing, the structure and the soft brush DNA hydrogel are further assembled through physical interaction to form a spider-web-shaped microstructure embedded with nanoflowers, and finally, the spider-web-shaped self-assembly functional nucleic acid hydrogel is formed;

the pair of L-shaped probes A, B has the following structure:

6) the L-type probe B sequence is a nucleic acid sequence which is arranged from a complementary region to a non-complementary region from 5 'end to 3' end;

the probe 1 has the following structure:

3) the specific aptamer sequence of the probe 1 enables the spider-web hydrogel to have target responsiveness and the effect of controllably releasing the inclusion through disassembly;

the probe 2 has the following structure:

2. The method for preparing the nucleic acid hydrogel according to claim 1, wherein the spider web-like self-assembly functional nucleic acid hydrogel can be disassembled upon addition of a target to which the aptamer sequence of probe 1 is bound.

3. The method according to claim 1 or 2, wherein the L-shaped probe A, B, probes 1 and 2 have the following sequences:

1) the L-type probe A is shown in a sequence table SEQ ID NO: 3;

2) the L-type probe B is shown in a sequence table SEQ ID NO: 4;

3) the probe 1 is shown as a sequence table SEQ ID NO: 5;

4) the probe 2 is shown as a sequence table SEQ ID NO: 6.

4. The method for producing according to claim 3, wherein the method for producing a spider web-like self-assembling functional nucleic acid hydrogel further comprises the steps of:

5. The method for preparing a spider web-like self-assembling functional nucleic acid hydrogel according to claim 3, further comprising the steps of loading an inclusion and purification, specifically:

3) and removing the supernatant to obtain the purified loaded spider web-shaped self-assembly functional nucleic acid hydrogel.

6. The method for preparing a spider web-like self-assembling functional nucleic acid hydrogel according to claim 4, further comprising the steps of loading an inclusion and purification, in particular:

7. Use of a spider web-like self-assembled functional nucleic acid hydrogel prepared by the method of any one of claims 1 to 6 for non-disease diagnosis, which is visually detectable by target-responsive release of the inclusion.

8. Use of the spider web-like self-assembled functional nucleic acid hydrogel prepared by the preparation method of any one of claims 1-6 for molecular detection, drug loading and delivery in non-disease diagnosis.