A method for preparing a DNA fiber

Abstract

The application belongs to the technical field of DNA fibers, and relates to a DNA fiber preparation method, in particular to a DNA fiber preparation method based on water-n-butanol reverse technology, when n-butanol is added to an aqueous phase containing DNA, magnetic stirring treatment can make the aqueous phase quickly diffuse into n-butanol, and amphiphilic DNA is assembled to obtain a DNA fiber in a few minutes, the preparation method is simple and easy to control, has short preparation time, and is convenient for industrialized production. The DNA fiber preparation method based on the water-n-butanol reverse technology can prepare single DNA fibers and multi-strand DNA fibers with different diameters, which is crucial for application in the fields of nanotechnology and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of DNA fiber technology and relates to a method for preparing DNA fibers, specifically a method for preparing DNA fibers based on water-n-butanol reverse technology. Background Technology

[0002] DNA materials have attracted much attention due to their intricate structure, strong programmability, and biocompatibility. DNA molecules possess excellent microscopic mechanical properties at the molecular level, primarily attributed to their regular and periodic helical structure. However, constructing bulk DNA materials with robust macroscopic properties remains a significant challenge. For example, DNA hydrogels typically exhibit weak mechanical properties due to their loose structure. In nature, the multi-scale ordered structures in many biomaterials (such as crustacean skin, lignocellulose, and spider silk) endow them with outstanding macroscopic mechanical properties. Inspired by this, utilizing DNA molecules as building blocks and constructing multi-scale structures through biomimetic design to develop high-performance DNA materials has significant research and practical value.

[0003] DNA fibers are fibrous structures formed by DNA molecules under specific conditions. These structures typically possess excellent mechanical properties, and their unique programmability and biocompatibility make DNA fibers crucial for applications in nanotechnology and biomedicine. While DNA fibers can be manufactured using electrospinning, this method can only produce single fibers with nanometer-scale diameters, limiting their applications. Furthermore, high voltage and limited spinning distance make it difficult to produce continuous long fibers. Jonathan R. Burns developed a novel macromolecular nanostructure assembly method using DNA nanotechnology to produce DNA fibers with customizable properties. This method requires complex structural design, synthesis, and stringent experimental conditions. Therefore, developing a DNA fiber preparation method with a simple process suitable for large-scale production has significant application value. 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] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing DNA fibers, the method being a method for preparing DNA fibers based on water-n-butanol reverse technology, wherein the DNA fibers comprise single DNA fibers or multi-stranded DNA fibers.

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

[0008] Dissolve 1-2 OD of DNA powder in 100 μL of deionized water until fully dissolved to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring (300-500 rpm / min), add 0.8-1.0 mL of n-butanol to the centrifuge tube at once. Finally, let it stand for 10-15 min to collect the milky white DNA single fiber at the bottom of the glass bottle.

[0009] Specifically, the DNA multistranded fibers are prepared as follows:

[0010] Dissolve 1-2 OD of DNA powder in 100 μL of deionized water until fully dissolved to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring (300-500 rpm / min), add n-butanol in portions of 10 μL at 1-2 min intervals to the centrifuge tube until the total volume of n-butanol is 0.8-1.0 mL. Finally, let stand for 10-15 min to collect DNA multistrands at the bottom of the glass bottle.

[0011] Furthermore, the DNA was purified using ULTRAPAGE, and the base sequence was TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, abbreviated as PolyT. 40 .

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] 1) This invention discloses a method for preparing DNA fibers based on water-n-butanol reverse technology. When n-butanol is added to an aqueous phase containing DNA, magnetic stirring can cause the aqueous phase to diffuse rapidly into the n-butanol. The amphiphilic DNA is assembled into DNA fibers within a few minutes. This preparation method is simple and easy to control, with 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 this invention can prepare single DNA fibers and multi-stranded DNA fibers of different diameters, which is crucial for applications in nanotechnology and biomedicine. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1This is a flowchart of the DNA fiber preparation experiment disclosed in this invention.

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

[0018] Figure 3 It is (ac)PolyT 20 PolyT 40 and PolyT 60 Scanning electron microscope image of the assembled DNA fibers; (df) PolyT 20 PolyT 40 and PolyT 60 A statistical graph showing the diameter of the assembled DNA fibers.

[0019] Figure 4 These are scanning electron microscope images of DNA single fibers obtained by adding (ac) n-butanol in amounts of 0.8 mL, 0.9 mL, and 1.0 mL, respectively.

[0020] Figure 5 (a) is a low-power scanning electron microscope image of DNA multistrands; (b) is a high-power scanning electron microscope image of DNA multistrands.

[0021] Figure 6 This is a scanning electron microscope image of the assembly obtained by adding 0.6 mL of n-butanol to a centrifuge tube in a single step.

[0022] Figure 7 (a) is based on PolyA 40 (a) is a scanning electron microscope image of the assembly obtained by constructing the primitives; (b) is a scanning electron microscope image of the assembly obtained by PolyC. 40 Scanning electron microscope image of the assembly obtained by constructing the primitives. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0025] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0026] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0027] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

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

[0029] In this invention, all DNA single strands used were synthesized by Shanghai Sangon Biotech Co., Ltd., purified by ULTRAPAGE, and their base sequences are TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, abbreviated as PolyT. 40 .

[0030] The experimental procedure for preparing DNA single fibers is as follows:

[0031] Dissolve 1-2 OD of DNA powder in 100 μL of deionized water until fully dissolved to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring (300-500 rpm / min), add 0.8-1.0 mL of n-butanol to the centrifuge tube at once. Finally, let it stand for 10-15 min and collect the milky white DNA single fiber at the bottom of the glass bottle.

[0032] The experimental procedure for preparing DNA multistranded fibers is as follows:

[0033] Dissolve 1-2 OD of DNA powder in 100 μL of deionized water until fully dissolved to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring (300-500 rpm / min), add n-butanol in portions of 10 μL at 1-min intervals until the total volume of n-butanol added is 0.8-1.0 mL. Finally, let stand for 10-15 min to collect DNA multistrands at the bottom of the glass bottle.

[0034] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0035] Example 1:

[0036] 1OD of PloyT 40 The dry powder was dissolved in 100 μL of deionized water to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring at 400 rpm / min, 0.9 mL of n-butanol was added to the centrifuge tube at once. After standing for 15 min, milky white DNA single fibers were collected from the bottom of the centrifuge tube. To characterize the microstructure of the DNA single fibers, 2 μL of the n-butanol solution containing the DNA fibers was dropped onto a clean silicon wafer substrate. After drying at room temperature, the microstructure was characterized using a scanning electron microscope (SEM).

[0037] Results analysis:

[0038] First, the microstructure of single DNA filaments was characterized using scanning electron microscopy. Figure 2 The results clearly show that the water-n-butanol reverse technique can prepare a large number of single DNA fibers ranging in length from tens to hundreds of micrometers in just a few minutes. (SEM magnification) Figure 2 b shows that the DNA single fibers have smooth surfaces and uniform diameters. Next, statistical analysis of their diameters was performed using nanometer software. Figure 2 c) It can be seen that the DNA fibers prepared by this technique have a diameter of approximately 140 nm. When n-butanol is added to the DNA solution, a clear phase interface exists between the two, and the DNA building blocks are bound in the aqueous phase and cannot assemble. Magnetic stirring can cause the aqueous phase containing DNA building blocks to form microdroplets in the n-butanol phase. Then, water molecules in the aqueous phase spontaneously and rapidly diffuse into the n-butanol phase, and the amphiphilic properties of DNA molecules drive them to migrate towards the water-oil interface. Due to the strong hydrophobicity of thymine (T), it cannot be stabilized at the two-phase interface, thus obtaining a single DNA fiber. The above research results demonstrate that a large number of smooth-surfaced single DNA fibers can be rapidly prepared using the water-n-butanol reverse technique.

[0039] Example 2:

[0040] The influence of DNA single-strand length on its assembly behavior was investigated using the following procedures:

[0041] DNA single strands with 20, 40, and 60 thymine (T) bases were designed and synthesized, and named PolyT. 20PolyT 40 and PolyT 60 Dissolve the 1OD DNA powder in 100μL of deionized water to obtain a homogeneous DNA aqueous solution. Then, add 0.9mL of n-butanol to the centrifuge tube at once, sonicate for 10s, and finally let 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 a clean silicon wafer substrate. After drying at room temperature, the assembly morphology of DNA single strands of different lengths was characterized using scanning electron microscopy (SEM).

[0043] Results analysis:

[0044] Research has found that PolyT 20 PolyT 40 and PolyT 60 All three types of DNA single strands can be assembled into a large number of DNA single fibers using the water-n-butanol reverse osmosis 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 DNA single fiber decreases significantly. Further statistical analysis was conducted on the diameters of the three types of DNA single fibers obtained from the above assembly. Figure 3 d-3f). With T 20 When constructing the building blocks, the assembled DNA single-fiber has a diameter of approximately 170 nm. However, when the number of T bases is increased to 60, the diameter of the DNA single-fiber decreases to about 100 nm. This shows that the diameter of the DNA single-fiber decreases as the length of the DNA single strand increases. Therefore, by flexibly changing the number of T bases, the diameter of the DNA single-fiber can be effectively controlled, allowing for the customization of DNA single-fibers of different thicknesses according to the requirements of the application.

[0045] Example 3:

[0046] The effect of n-butanol addition on assembly behavior was investigated, and the specific procedures were as follows:

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

[0048] Studies have found that the amount of n-butanol added is a key factor affecting DNA assembly in the water-n-butanol reverse engineering technique. On the one hand, it is necessary to ensure complete diffusion of the aqueous phase into the n-butanol phase; on the other hand, a suitable diffusion rate is also required. For example, at room temperature, the solubility of water in n-butanol is approximately 20.4 wt%. Therefore, for 100 μL of aqueous phase, at least 605 μL of n-butanol volume is needed to ensure complete diffusion of water into the oil phase. Furthermore, the amount of n-butanol added affects the diffusion rate of the aqueous phase, thus significantly influencing the morphology and structure of DNA fibers. Figure 4 As shown in a and 4c, when the volume of n-butanol added was 0.8 mL and 1.0 mL, respectively, the relatively fast and slow diffusion rates in the aqueous phase resulted in poor uniformity of the assembled DNA fibers, with obvious nodules. Only when the volume of n-butanol added was 0.9 mL, DNA fibers of uniform thickness were obtained under a moderate diffusion rate in the aqueous phase. Therefore, strictly controlling the amount of n-butanol added is a key factor in the preparation of uniform DNA single fibers.

[0049] Example 4:

[0050] The effect of the method of adding n-butanol on its assembly behavior was investigated. Unlike the preparation of DNA single fibers, where n-butanol is added all at once, the above-mentioned 1OD PolyT... 40 The dry powder was dissolved in 100 μL of deionized water to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring (300 rpm / min), n-butanol was added to the centrifuge tube in portions of 10 μL each time, with an interval of 1 min, until the total volume of n-butanol added was 0.9 mL. Finally, after standing for 10-15 min, milky white DNA assemblies could be observed at the bottom of the glass bottle.

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

[0052] Results analysis:

[0053] The study found that when n-butanol was added in batches to induce assembly, PolyT... 40 Coarse fibers with lengths of several hundred micrometers can be obtained, with an average diameter of 9.24 μm. Figure 5 a). High-magnification SEM images clearly show that the coarse fibers consist of approximately 6-10 individual fibers, each with a diameter of approximately 2.13 μm. Figure 5(b) This is because when a small amount of n-butanol is added to the DNA solution, although water molecules in the aqueous phase diffuse rapidly into the n-butanol phase, the DNA cannot undergo significant assembly due to the predominantly aqueous phase in the solution. With continued addition of n-butanol, the rapid diffusion of water molecules, combined with magnetic stirring, causes the aqueous phase containing DNA building blocks to form unstable microdroplets in the n-butanol phase. These droplets then fuse, resulting in a chain-like structure of multiple droplets. As more n-butanol is added, most of the water molecules in the aqueous phase diffuse into the n-butanol phase, and these chain-like structures further fuse directionally. Finally, when all water molecules have diffused into the n-butanol phase, the amphiphilic DNA molecules assemble into multistranded DNA fibers.

[0054] To further demonstrate the beneficial effects of the present invention and to better understand it, the technical features disclosed in the present invention are further illustrated by the following comparative examples, but these should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above description of the invention, without inventive effort, are also considered to fall within the protection scope 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 in 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 0.6 mL of n-butanol was added, single-stranded DNA could not assemble into fibers, but instead formed irregular, blocky materials, such as... Figure 6 This is because when a small amount of n-butanol is added, water molecules cannot diffuse completely into the n-butanol phase, causing the DNA to dissolve in the aqueous phase and fail to assemble in a directional manner.

[0058] Comparative Example 2

[0059] The study investigated the influence of DNA base types on its assembly behavior and structure, essentially the same as in Example 1, except that the DNA single strand was PolyA, composed of 40 adenine (A) and 40 cytosine (C) atoms respectively. 40 PolyC 40 .

[0060] Research has found that PolyA 40 When constructing primitives, micron-sized spherical vesicles can be assembled. Figure 7 a); while PolyC 40 When constructing primitives, irregular rod-shaped structures can be assembled. Figure 7 b).

[0061] Therefore, it can be seen that the assembly behavior of DNA in the water-n-butanol reverse system mainly depends on the type of base. 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 those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing DNA fibers, characterized in that, The method is a method for preparing DNA fibers based on water-n-butanol reverse technology, wherein the DNA fibers include single DNA fibers or multi-stranded DNA fibers. The DNA was purified using ULTRAPAGE, and its base sequence is TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, abbreviated as PolyT. 40 .

2. The method for preparing DNA fibers according to claim 1, characterized in that, The DNA single fiber is prepared as follows: Dissolve 1-2 OD of DNA powder in 100 μL of deionized water to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring, add 0.8-1.0 mL of n-butanol at a time, and finally let stand to obtain a milky white DNA single fiber.

3. The method for preparing DNA fibers according to claim 1, characterized in that, The DNA multistranded fibers are prepared as follows: Dissolve 1-2 OD of DNA dry powder in 100 μL of deionized water to obtain a homogeneous DNA aqueous solution. Then, under magnetic stirring, add n-butanol in portions of 10 μL at 1-2 min intervals until the total volume of n-butanol is 0.8-1.0 mL. Finally, allow the mixture to stand to obtain DNA multistrands.

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