Preparation method of DNA (Deoxyribose Nucleic Acid) fiber

Abstract

The invention belongs to the technical field of DNA (deoxyribonucleic acid) fibers, and relates to a preparation method of DNA fibers, in particular to a preparation method of DNA fibers based on a water-n-butyl alcohol reverse technology, when n-butyl alcohol is added into a water phase containing DNA, the water phase can be quickly diffused into the n-butyl alcohol through magnetic stirring treatment, and amphiphilic DNA is assembled within several minutes to obtain the DNA fibers. The preparation method is simple, easy to control, short in preparation time and convenient for industrial production. According to the DNA fiber preparation method based on the water-n-butyl alcohol reverse technology, single DNA fibers and multi-strand DNA fibers with different diameters can be prepared, and the method is of great importance to application in the fields of nanotechnology and biomedicine.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA fibers and relates to a method for preparing DNA fibers, in particular to a method for preparing DNA fibers based on a water-n-butanol reverse technology. Background Art

[0002] DNA materials have attracted much attention due to their exquisite structure, powerful programmability and biocompatibility. DNA molecules have excellent micromechanical properties at the molecular level, mainly due to their regular and periodic helical structure. However, it is still a big challenge to construct DNA bulk materials with strong macroscopic properties. For example, DNA hydrogels usually exhibit weak mechanical properties due to their loose structure. In nature, the multi-scale ordered structures in many biomaterials (such as the epidermis of crustaceans, wood fibers, and spider silk) give them excellent macroscopic mechanical properties. Inspired by this, using DNA molecules as building blocks and constructing multi-scale structures through bionic design to develop high-performance DNA materials has important research significance and use value.

[0003] DNA fiber refers to a fibrous structure formed by DNA molecules under specific conditions. This structure usually has excellent mechanical properties, and its unique programmability and biocompatibility make DNA fibers have important applications in nanotechnology and biomedicine. Although DNA fibers can be manufactured by electrospinning technology, this method can only spin single fibers with a nanometer diameter, which limits the application scenarios of DNA fibers. In addition, high voltage and limited spinning distance also make it difficult to prepare continuous long fibers. Jonathan R. Burns used DNA nanotechnology to develop a new macromolecular nanostructure assembly method to produce DNA fibers with customizable properties. This method requires complex structural design synthesis and harsh experimental conditions. Therefore, it is of great application value to develop a DNA fiber preparation method with a simple process flow and suitable for large-scale production. Summary of the invention

[0004] In view of this, the present invention discloses a method for preparing DNA fibers based on water-n-butanol reverse technology.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for preparing DNA fibers is a method for preparing DNA fibers based on a water-n-butanol reverse technology. The DNA fibers include single DNA fibers or multi-strand DNA fibers.

[0007] Specifically, the preparation of the single DNA fiber is as follows:

[0008] Dissolve 1-2OD of DNA dry powder in 100μL of deionized water and fully dissolve to obtain a uniform DNA aqueous solution; then, under magnetic stirring conditions (speed 300-500rpm / min), add 0.8-1.0mL of n-butanol to the centrifuge tube at one time, and finally let it stand for 10-15min to collect milky white single DNA fibers at the bottom of the glass bottle.

[0009] Specifically, the preparation of the DNA multi-strand fiber is as follows:

[0010] Dissolve 1-2OD of DNA dry powder in 100μL of deionized water and fully dissolve to obtain a uniform DNA aqueous solution; then, under magnetic stirring conditions (speed 300-500rpm / min), add n-butanol to the centrifuge tube in batches, each adding a volume of 10μL, with a time interval of 1-2min, until the total volume is 0.8-1.0mL of n-butanol, and finally let it stand for 10-15min to collect multiple strands of DNA fibers at the bottom of the glass bottle.

[0011] Furthermore, the DNA was purified by ULTRAPAGE, and the base sequences were TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, referred to as PolyT 40 .

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1) The present invention discloses a method for preparing DNA fibers based on a water-n-butanol reverse technology. When n-butanol is added to an aqueous phase containing DNA, magnetic stirring treatment can allow the aqueous phase to diffuse rapidly into the n-butanol, and amphiphilic DNA is assembled to obtain DNA fibers within a few minutes. The preparation method is simple and easy to control, has a short preparation time, and is convenient for industrial production.

[0014] 2) The method for preparing DNA fibers using the water-n-butanol reverse technology proposed in the present invention can prepare single DNA fibers and multiple strands of DNA fibers with different diameters, which is crucial for applications in the fields of nanotechnology and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1It is a flow chart of the experiment for preparing the DNA fiber disclosed in the present invention.

[0017] Figure 2 This is a scanning electron microscope image of a single DNA fiber prepared in Example 1 of the present invention.

[0018] Figure 3 Yes (ac)PolyT 20 , PolyT 40 and PolyT 60 Scanning electron microscopy images of assembled DNA fibers; (df) PolyT 20 , PolyT 40 and PolyT 60 Diameter statistics of assembled DNA fibers.

[0019] Figure 4 (ac) are scanning electron microscope images of single DNA fibers obtained when 0.8 mL, 0.9 mL, and 1.0 mL of n-butanol were added, respectively.

[0020] Figure 5 (a) is a low-magnification scanning electron micrograph of a DNA multi-strand fiber; (b) is a high-magnification scanning electron micrograph of a DNA multi-strand fiber.

[0021] Figure 6 This is a scanning electron micrograph of the assembly obtained by adding 0.6 mL of n-butanol to a centrifuge tube at one time.

[0022] Figure 7 (a) PolyA 40 (a) is a scanning electron micrograph of the assembly obtained by building the basic unit; (b) is a scanning electron micrograph of the assembly obtained by building the basic unit; (c) is a scanning electron micrograph of the assembly obtained by building the basic unit; (d) is a scanning electron micrograph of the assembly obtained by building the basic unit; (e) is a scanning electron micrograph of the assembly obtained by building the basic unit; (f) is a scanning electron micrograph of the assembly obtained by building the basic unit; (g) is a scanning electron micrograph of the assembly obtained 40 This is a scanning electron microscope image of the assembly obtained by building the basic unit. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0025] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0026] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0027] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0028] The invention discloses a method for preparing DNA fiber based on water-n-butanol reverse technology.

[0029] In the present invention, the DNA single strands used were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and purified by ULTRAPAGE. The base sequences were TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, referred to as PolyT 40 .

[0030] The experimental process of preparing single DNA fiber is as follows:

[0031] Dissolve 1-2OD of DNA dry powder in 100μL of deionized water and fully dissolve to obtain a uniform DNA aqueous solution; then, under magnetic stirring conditions (speed 300-500rpm / min), add 0.8-1.0mL of n-butanol to the centrifuge tube at one time, and finally let it stand for 10-15min to collect milky white single DNA fibers at the bottom of the glass bottle.

[0032] The experimental procedure for preparing DNA multi-strand fibers is as follows:

[0033] Dissolve 1-2OD of DNA dry powder in 100μL of deionized water and fully dissolve to obtain a uniform DNA aqueous solution; then, under magnetic stirring conditions (speed 300-500rpm / min), add n-butanol to the centrifuge tube in batches, each adding a volume of 10μL, with a time interval of 1min, until the total volume of n-butanol added is 0.8-1.0mL. Finally, let it stand for 10-15min to collect multiple strands of DNA fibers at the bottom of the glass bottle.

[0034] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0035] Embodiment 1:

[0036] Place 1OD of PolyT 40 The dry powder was dissolved in 100 μL of deionized water and fully dissolved to obtain a uniform DNA aqueous solution. Then, 0.9 mL of n-butanol was added to the centrifuge tube at a time under magnetic stirring at a speed of 400 rpm / min. Finally, milky white DNA single fibers were collected at the bottom of the centrifuge tube after standing for 15 minutes. In order to characterize the microscopic morphology of single DNA fibers, 2 μL of n-butanol solution containing DNA fibers was added dropwise to the surface of a clean silicon wafer substrate. After drying at room temperature, the microstructure was characterized using a scanning electron microscope (SEM).

[0037] Result analysis:

[0038] First, a scanning electron microscope was used to characterize the microstructure of a single DNA fiber. Figure 2 a The results clearly show that a large number of single DNA long fibers ranging from tens to hundreds of microns in length can be prepared in just a few minutes using the water-n-butanol reverse technology. SEM magnification Figure 2 b shows that the surface of a single DNA fiber is smooth and the diameter is uniform. Then, the diameter is statistically analyzed using nano measure software ( Figure 2 c), it can be seen that the diameter of the DNA fiber prepared by this technology is about 140nm. When n-butanol is added to the DNA solution, there is an obvious phase interface between the two, and the DNA building blocks are bound in the water phase and cannot be assembled. Magnetic stirring can cause the water phase containing the DNA building blocks to form microdroplets in the n-butanol phase. Then, the water molecules in the water phase spontaneously and rapidly diffuse into the n-butanol phase, and the amphiphilic properties of the DNA molecules drive themselves to migrate to the water-oil interface. Since thymine (T) has strong hydrophobicity, it cannot be stabilized at the interface between the two phases, thereby obtaining a single DNA fiber. The above research results show that a large number of single DNA fibers with smooth surfaces can be quickly prepared by the water-n-butanol reverse technology.

[0039] Embodiment 2:

[0040] To explore the effect of DNA single-strand length on its assembly behavior, the specific operations are as follows:

[0041] We designed and synthesized DNA single strands with 20, 40, and 60 thymine (T) bases, respectively, and named them PolyT. 20, PolyT 40 and PolyT 60 Dissolve the above 1OD DNA dry powder in 100μL deionized water to obtain a uniform DNA aqueous solution. Then, add 0.9mL of n-butanol to the centrifuge tube at one time, ultrasonicate for 10s, and let it stand for 30min to collect the milky white DNA assembly at the bottom of the centrifuge tube.

[0042] Finally, 2 μL of n-butanol solution containing DNA assemblies was dropped onto the surface of a clean silicon wafer substrate. After drying at room temperature, the assembly morphology of DNA single chains of different lengths was characterized using a scanning electron microscope (SEM).

[0043] Result analysis:

[0044] The study found that PolyT 20 , PolyT 40 and PolyT 60 All three DNA single strands can be assembled into a large number of DNA single fibers through the water-n-butanol reverse technique, such as Figure 3 As shown in a-3c, it can be clearly seen that as the number of T bases increases, the diameter of the single DNA fiber decreases significantly. The diameters of the three single DNA fibers assembled above are further statistically analyzed ( Figure 3 d-3f). 20 When the motif is constructed, the diameter of the assembled single DNA fiber is about 170nm. However, when the number of T bases is increased to 60, the diameter of the single DNA fiber is reduced to about 100nm. It can be seen that as the length of the DNA single chain increases, the diameter of the single DNA fiber decreases. Therefore, by flexibly changing the number of T bases, the diameter of the single DNA fiber can be effectively regulated, so that the single DNA fiber of different thicknesses can be customized according to the needs of the place of use.

[0045] Embodiment 3:

[0046] To explore the effect of the amount of n-butanol added on the assembly behavior, the specific operations are as follows:

[0047] 1OD of PolyT 40 The dry powder was dissolved in 100 μL of deionized water and fully dissolved to obtain a uniform DNA aqueous solution. Then, under the condition of magnetic stirring at a speed of 400 rpm / min, 0.80 mL, 0.9 mL, and 1.0 mL of n-butanol were added to the centrifuge tube at one time. Finally, after standing for 15 minutes, the milky white DNA assemblies were collected from the bottom of the centrifuge tube. 2 μL of n-butanol solution containing DNA assemblies was added dropwise to the surface of a clean silicon wafer substrate. After drying at room temperature, its microstructure was characterized using a scanning electron microscope (SEM).

[0048] The study found that the amount of n-butanol added is a key factor affecting the water-n-butanol reverse technology DNA assembly. On the one hand, it is necessary to ensure that the water phase is completely diffused into the n-butanol phase, and on the other hand, a suitable diffusion rate is required. For example, at room temperature, the solubility of water in n-butanol is approximately 20.4wt%. Therefore, for 100μL of water phase, at least 605μL of n-butanol volume is required to ensure that the water can be completely diffused into the oil phase. In addition, the amount of n-butanol added will affect the diffusion rate of the water phase, thereby significantly affecting the morphology and structure of the DNA fiber. Figure 4 As shown in Figures 4a and 4c, when the volume of n-butanol added was 0.8 mL and 1.0 mL respectively, the faster and slower diffusion rates of the aqueous phase resulted in poor uniformity of the assembled DNA fibers and the presence of obvious nodules. Only when the volume of n-butanol added was 0.9 mL, DNA fibers with uniform thickness were obtained at a moderate diffusion rate of the aqueous phase. Therefore, strictly controlling the amount of n-butanol added is a key factor in preparing uniform single DNA fibers.

[0049] Embodiment 4:

[0050] To investigate the effect of the addition method of n-butanol on its assembly behavior, different from the one-time addition of n-butanol in the preparation of single DNA fibers, the 1OD PolyT 40 The dry powder was dissolved in 100 μL of deionized water to obtain a uniform DNA aqueous solution. Then, under magnetic stirring conditions (speed 300 rpm / min), n-butanol was added to the centrifuge tube in batches, with each addition volume of 10 μL and a time interval of 1 min until the total volume of n-butanol added was 0.9 mL. Finally, the milky white DNA assembly was observed at the bottom of the glass bottle after standing for 10-15 min.

[0051] Finally, 2 μL of n-butanol solution containing DNA assemblies was dropped onto the surface of a clean silicon wafer substrate. After drying at room temperature, the microscopic morphology of the DNA assemblies was characterized using a scanning electron microscope (SEM).

[0052] Result analysis:

[0053] The study found that when n-butanol was added in batches to induce assembly, PolyT 40 The obtained coarse fibers are several hundred micrometers in length and have an average diameter of 9.24 μm ( Figure 5 a). It can be clearly seen from the high-magnification SEM image that the coarse fiber is composed of about 6-10 strands of single fibers, and the diameter of each single fiber is about 2.13μm ( Figure 5b). This is because, when a small amount of n-butanol is added to the DNA solution, although the water molecules in the aqueous phase will quickly diffuse into the n-butanol phase, since the solution is mainly based on the aqueous phase, DNA cannot be significantly assembled. When n-butanol is continued to be added, along with the rapid diffusion of water molecules, magnetic stirring can make the aqueous phase containing the DNA building blocks form unstable micro-droplets in the n-butanol phase, at which time the droplets will fuse, thereby obtaining a chain structure of multiple droplet solutions. When the amount of n-butanol added is more, most of the water molecules in the aqueous phase diffuse into the n-butanol phase, and these chain structures further directionally fuse. Finally, when the water molecules are completely diffused into the n-butanol phase, the amphiphilic DNA molecules are assembled to obtain DNA multi-strand fibers.

[0054] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following comparative examples are used to further illustrate the technical features disclosed in the present invention, but they should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above invention content without inventive work are also considered to fall within the scope of protection of the present invention.

[0055] Comparative Example 1

[0056] The effect of the amount of n-butanol added on the DNA assembly structure was investigated. The specific details were basically the same as Example 1, except that 0.6 mL of n-butanol was added to the centrifuge tube at one time.

[0057] The study found that when the amount of n-butanol added was 0.6 mL, single-stranded DNA could not assemble into fibers, but instead obtained irregular block materials, such as Figure 6 This is because, when the amount of n-butanol added is small, the water molecules cannot diffuse into the n-butanol phase completely, causing the DNA to be dissolved in the water phase and unable to assemble in a directional manner.

[0058] Comparative Example 2

[0059] The effect of DNA base types on its assembly behavior structure was explored. The specific details were basically the same as in Example 1, except that the DNA single strand was composed of 40 adenine (A) and cytosine (C) polyA. 40 , PolyC 40 .

[0060] The study found that PolyA 40 When building primitives, micron-sized spherical vesicles can be assembled ( Figure 7 a); and PolyC 40 When constructing primitives, irregular rod-like structures can be assembled ( Figure 7 b).

[0061] It can be seen from this that the assembly behavior of DNA in the water-n-butanol reverse system mainly depends on the type of bases. Only DNA homopolymers composed of thymine (T) can construct DNA fibers, which is caused by the different hydrophilic and hydrophobic properties of different types of bases.

[0062] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a DNA fiber, characterized in that: The method is a method for preparing DNA fibers based on the water-n-butanol reverse technology. The DNA fibers include single DNA fibers or multi-strand DNA fibers.

2. The method for preparing a DNA fiber according to claim 1, characterized in that: The preparation of the DNA single fiber is as follows: Dissolve 1-2 OD of DNA dry powder in 100 μL of deionized water and fully dissolve to obtain a uniform DNA aqueous solution; then, under magnetic stirring, add 0.8-1.0 mL of n-butanol at one time, and finally let stand to obtain a milky white single DNA fiber.

3. The method for preparing DNA fiber according to claim 1, characterized in that: The preparation of the DNA multi-stranded fiber is as follows: Dissolve 1-2OD of DNA dry powder in 100μL of deionized water and fully dissolve to obtain a uniform DNA aqueous solution; then, under magnetic stirring, add n-butanol in portions, each adding a volume of 10μL, with a time interval of 1-2min, until the total volume added is 0.8-1.0mL of n-butanol, and finally let it stand to obtain multi-strand DNA fibers.

4. The method for preparing a DNA fiber according to claim 2 or 3, characterized in that: The DNA was purified by ULTRAPAGE, and the base sequences were TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, referred to as PolyT 40 .

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