Preparation method and application of DNA origami structure capable of inducing disordered-ordered rapid transformation at normal temperature

Abstract

The invention discloses a preparation method and application of a DNA origami structure capable of inducing disordered-ordered rapid transformation at normal temperature, and the preparation method comprises the following steps: a) pre-folding a support chain of the DNA origami structure and a part of staple chain subset to form a metastable disordered structure; and b) adding a complete staple chain into the metastable-state disordered structure constructed in the step a), deducting the rest staple chain subsets of the partial staple chain subsets added in the step a), and inducing the disordered structure to be converted into an ordered paper folding structure at normal temperature. According to the invention, staple chains are added in stages to induce a DNA origami structure to realize transformation from disorder to order at room temperature, so that synthesis conditions are simplified, and synthesis time is shortened. The DNA origami structure prepared by the method can simulate the induced folding behavior of natural disordered protein, and has important application value in the fields of nanoelectronics, biosensing and medical diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of DNA nanotechnology, and particularly relates to a method for preparing a DNA origami structure that induces a rapid disorder-order transition at room temperature and its application. Background Art

[0002] DNA origami structures have programmability and addressability at the nanoscale and can achieve dynamic structure-function conversion, which provides great potential for the development of molecular machines and nanorobots. For example, robots with switchable nano-containers can be developed to expose or release payloads under external molecular stimuli, ultimately realizing the integration of therapy and diagnosis. Although traditional DNA origami technology has shown potential in constructing molecular machines, there are still technical bottlenecks in achieving controllable conformational transitions in physiological environments. Traditional DNA origami preparation requires an activation step, heating to the denaturation temperature to avoid kinetic traps. Although some methods for preparing origami structures at room temperature have been reported recently, they require the use of denaturants or extremely long assembly times (at least 24 hours).

[0003] Therefore, it is necessary to develop a new method for preparing DNA origami to synthesize DNA origami structures in physiological environments and achieve controllable disorder-order transitions. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a DNA origami structure that induces a rapid disorder-order transition at room temperature and its application, so as to solve the problems of long time consumption, the need for heat denaturation, and the addition of denaturants when constructing molecular machines with DNA origami structures.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] According to the first aspect of the present invention, there is provided a method for preparing a DNA origami structure that induces a rapid disorder-order transition at room temperature, including the following steps: a) pre-folding the scaffold strand of the DNA origami structure with a partial staple strand subset to form a metastable disordered structure; b) adding the remaining staple strand subset of the complete staple strand minus the partial staple strand subset added in step a) to the metastable disordered structure constructed in step a) to induce the disordered structure to transform into an ordered origami structure at room temperature.

[0007] According to the method provided by the present invention, its working principle is as follows: the scaffold strand is pre-folded with a partial staple strand subset to form a metastable disordered structure with significant free energy fluctuation characteristics. Then, the remaining staple strands are added, and the high-energy metastable disordered structure will bind to the staple strands and approach the global energy minimum state to form an ordered and regular DNA origami structure at room temperature.

[0008] Among them, the DNA origami structure is a DNA nanostructure constructed using DNA origami technology.

[0009] Preferably, the DNA origami structure is a triangular DNA origami. It should be understood that this origami structure is only exemplified as a preferred embodiment and is not used for limitation. In fact, DNA nanostructures constructed using DNA origami technology are all applicable to the present invention, such as: DNA rectangular origami, DNA wireframe origami, and DNA polyhedron origami.

[0010] The synthesis of the metastable disordered structure is a conventional method. Preferably, it is: mixing the scaffold strand and a partial staple strand subset and annealing, and the metastable disordered structure can be synthesized.

[0011] In step a), the annealing method is a conventional method in the art. Preferably, the scaffold strand and a partial staple strand subset are mixed at a molar concentration ratio of 1:(2 - 100), heated at 65 - 95 °C for 3 - 20 min, and then cooled to 25 °C at a cooling rate of 0.1 - 1 °C / min.

[0012] According to a preferred embodiment of the present invention, in step a), the scaffold strand and a partial staple strand subset are mixed at a molar concentration ratio of 1:5, heated at 80 °C for 3 minutes, and cooled to 25 °C at a cooling rate of 0.5 °C / min.

[0013] Among them, in step a), the staple strand subset includes a combination of any part of all the staple strands of the DNA origami structure, preferably any one of a short-range subset, a long-range subset, or a random subset.

[0014] In step a), the staple strand includes DNA strands modified with functional molecules such as unmodified DNA strands, thiol-modified DNA strands, fluorescent dye-modified DNA strands, biotin / streptavidin-modified DNA strands, etc. Preferably, it is an unmodified DNA strand.

[0015] Among them, the synthesis of the ordered origami structure in step b) is a conventional method. Preferably, it is: adding the remaining staple strands to the metastable disordered structure prepared in step a), and the ordered origami structure can be synthesized within 2 hours through room temperature incubation. In step b), the complement of the added subset is added, that is, among a set of staple strands, the remaining staple strands after adding in step a).

[0016] In step b), the room temperature incubation method is a conventional method in the art. Preferably, the metastable disordered structure and the remaining staple strands are mixed at a molar concentration ratio of 1:(2 - 100) and incubated at 15 - 40 °C for 10 min - 2 h.

[0017] According to a preferred embodiment of the present invention, the metastable disordered structure and the remaining staple strands are mixed at a molar concentration ratio of 1:5 and incubated at 25 °C for 2 hours.

[0018] Preferably, in step a), the scaffold strand and a partial subset of staple strands are assembled isothermally at 55 °C.

[0019] Preferably, in step b), the metastable disordered structure and the remaining staple strands are incubated and assembled at 25 °C or lower (5 - 24 °C).

[0020] Among them, the staple strands described in step b) include DNA strands modified with functional molecules such as unmodified DNA strands, thiol - modified DNA strands, fluorescent dye - modified DNA strands, biotin / streptavidin - modified DNA strands, etc. Preferably, they are unmodified DNA strands.

[0021] According to the second aspect of the present invention, there is also provided an application of a method for preparing a DNA origami structure that can be rapidly transformed from disorder to order at room temperature in synthesizing a DNA origami structure in a physiological environment and realizing its controllable disorder - to - order transformation.

[0022] The above - mentioned application includes the rapid disorder - to - order transformation of the DNA origami structure at room temperature in biosensing, drug delivery, or molecular computing.

[0023] According to the present invention, the DNA origami structure that can be rapidly transformed from disorder to order at room temperature is preferably a DNA triangle origami structure. It should be understood that the above - mentioned DNA triangle origami structure is only used as a preferred embodiment to illustrate the present invention and is not used for limitation. In fact, according to the differences in the scaffold strand and staple strands, other DNA origami structures that can be rapidly transformed from disorder to order at room temperature can also be provided.

[0024] As is well known, it is very difficult to form an ordered structure for a folding structure with a large number of components (such as the co - folding of hundreds of staple strands) in a high - salt environment at room temperature. Most mixed systems will fall into kinetic traps under this condition. Conventional techniques avoid kinetic traps by programmed temperature changes to form ordered structures. Most research and conventional understanding lead to the belief that these structures are all in kinetic traps under this condition, so that neither pre - folded nor non - pre - folded structures can undergo ordered assembly, which is a technical prejudice. However, the present invention proves through experiments that these partially folded structures can also undergo an ordered folding process of subsequently adding a subset containing a large number of components under such conditions. Even an arbitrary subset can undergo an ordered folding process, which has generality.

[0025] The inventive point of the present invention lies in that, for the first time, a method for realizing the rapid disorder-order transformation of a DNA origami structure at room temperature by regulating the DNA origami folding path is proposed, and the DNA origami structure is induced to transform from disorder to order at room temperature by adding staple strands in stages. Specifically, pre-folding is carried out through a scaffold strand and a partial staple strand subset to form a metastable disordered structure, and then the remaining staple strands are added. The high-energy metastable disordered structure will bind to the staple strands and approach the global energy minimum state, and an ordered and regular DNA origami structure can be formed at room temperature. The present invention also confirms through experiments that, regardless of using any staple strand subset to construct the metastable disordered structure and supplementing the remaining staple strands at room temperature, the metastable structure can be induced to transform into the ordered DNA origami structure with the lowest global energy within 2 hours. The AFM images of the ordered steady-state structure transformed from the disordered metastable structure according to the method of the present invention and the directly constructed ordered steady-state structure are both regular and ordered DNA triangular origami structures, with no obvious difference.

[0026] The positive and progressive effects of the present invention compared with the prior art are as follows:

[0027] The preparation method of the DNA origami structure proposed by the present invention can realize the rapid disorder-order transformation of the DNA origami structure at room temperature, simplifies the synthesis conditions, shortens the synthesis time, achieves the effect of rapid structure transformation at room temperature, and is beneficial to the development of molecular machines and biomedical applications. In practical applications, optimally, the synthesis time of the DNA triangular origami structure at room temperature is greatly shortened to 2 hours, which is much lower than the current literature level (24 hours).

[0028] In summary, the present invention provides a preparation method of a DNA origami structure that can induce rapid disorder-order transformation at room temperature. The DNA origami structure prepared according to this method can simulate the induced folding behavior of natural intrinsically disordered proteins (IDPs) and has important application values in the fields of nanoelectronics, biosensing, and medical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a coarse-grained simulation diagram of metastable structures constructed by different staple strand subsets in coarse-grained molecular dynamics simulation (oxDNA software);

[0030] Figure 2 is an AFM image of metastable structures constructed by different staple strand subsets;

[0031] Figure 3 is the area distribution diagram of metastable structures constructed by different staple strand subsets;

[0032] Figure 4 is an AFM image of the steady-state structures constructed by adding subsequent staple strand subsets to different metastable structures and the directly constructed steady-state structures;

[0033] Figure 5 It is the area distribution diagram of the steady-state structure formed by adding different metastable structures to the subsequent staple strand subsets and the direct steady-state structure;

[0034] Figure 6 The yields of the steady-state structure formed by adding different metastable structures to the subsequent staple strand subsets and the direct steady-state structure. Specific Embodiments

[0035] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are carried out according to conventional methods and conditions, or are selected according to the product specifications.

[0036] In this embodiment, DNA origami is mainly selected as a representative to construct a DNA origami structure that can rapidly transform from disorder to order at room temperature. The following embodiments specifically illustrate the implementation effects of the present invention.

[0037] The DNA sequences (5'-3') used in the embodiments of the present invention:

[0038] Table 1. Digital serial numbers of the DNA sequences of the short-range subsets of the DNA origami structure in step a Domain A Serial Number Domain B Serial Number Domain C Serial Number 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8 17 17 17 21 21 21 27 27 27 31 31 31 33 33 33 34 34 34 35 35 35 36 36 36 38 38 38 39 39 39 40 40 40 41 41 41 50 50 50 54 54 54 60 60 60 64 64 64 Link-A1C Link-B1A Link-C1B Link-A2C Link-B2A Link-C2B Link-A3C Link-B3A Link-C3B Link-A4C Link-B4A Link-C4B

[0039] Table 1 shows the digital serial numbers of the DNA sequences of the short-range subsets in the DNA origami structure. These short-range subsets are part of the staple strands, which span relatively short distances in the DNA origami structure and connect regions on the backbone strands that are relatively close to each other.

[0040] Table 2. Digital serial numbers of the DNA sequences of the long-range subsets of the DNA origami structure in step a Domain A Serial Number Domain B Serial Number Domain C Serial Number 9 9 9 10 10 10 11 11 11 12 12 12 13 13 13 14 14 14 15 15 15 16 16 16 18 18 18 19 19 19 20 20 20 22 22 22 23 23 23 24 24 24 25 25 25 26 26 26 28 28 28 29 29 29 30 30 30 32 32 32 37 37 37 42 42 42 43 43 43 44 44 44 45 45 45 46 46 46 47 47 47 48 48 48 49 49 49 51 51 51 52 52 52 53 53 53 55 55 55 56 56 56 57 57 57 58 58 58 59 59 59 61 61 61 62 62 62 63 63 63 65 65 65

[0041] Table 2 shows the digital serial numbers of the DNA sequences of the long-range subsets in the DNA origami structure. These long-range subsets are part of the staple strands, which span relatively long distances in the DNA origami structure and connect regions on the backbone strands that are relatively far from each other.

[0042] Table 3. Digital serial numbers of the DNA sequences of the random subsets of the DNA origami structure in step 1

[0043] Table 3 shows the digital serial numbers of the DNA sequences of the random subsets in the DNA origami structure. Random subsets refer to those staple strands with certain randomness in sequence and position. They may be used to increase the complexity of the assembly path, explore new self-assembly paths, or study the dynamic behavior of DNA origami.

[0044] It should be understood that the long-range subset, short-range subset, random subset and long single-stranded backbone together constitute a complete DNA origami structure. They interact through base complementary pairing to fold the long single-stranded backbone into a designed two-dimensional or three-dimensional nanostructure. This combination enables DNA origami technology to achieve highly complex nanostructure designs.

[0045] According to this embodiment, the staple chain subset includes a short-range subset (123 staples, spanning a short distance region of the scaffold), a long-range subset (84 staples, spanning a long distance region of the scaffold), and a random subset (104 staples, randomly distributed on the scaffold).

[0046] Table 4. Numerical sequence numbers of staple chain DNA sequences (SEQ ID No. 1-208) in the DNA origami structure

[0047] Example 1: Prefolding of the disordered metastable structure of the DNA origami structure

[0048] In this example, M13mp18 is used as the scaffold chain, and the short-range, long-range and random subsets are used as the staple chain subsets to illustrate the pre-folding of the disordered metastable structure.

[0049] Prefolding of the disordered metastable structure of the DNA triangular origami structure. M13mp18 as the scaffold chain was purchased from CNBioRuler (cnbioruler.cn) company, with 7249 bases. 5 μL of 100 nM single-stranded M13mp18 DNA was mixed with 10 μL of 250 nM staple chain subset (long-range / short-range / random subset) and 5 μL of preparation buffer (5.0 mM Tris, 1.0 mM EDTA and 125 mM MgAc2, pH = 8.5), and 27 μL of deionized water was added, and two-step annealing-isothermal assembly was carried out in a PCR thermal cycler. First, it was isothermally heated at 80 °C for 3 minutes, and then annealed to 25 °C at a rate of 0.5 °C / min.

[0050] Result: As Figure 1 shown, coarse-grained molecular dynamics simulations show that the metastable structures formed by the three staple subsets are in a disordered state, and atomic force microscopy results show that the images of the three metastable structures are all incomplete triangles ( Figure 2), the area distribution statistics of the three metastable structures is 794±1095nm 2 、3750±1137nm 2 and 1250±350nm 2 ( Figure 3 ), in summary, the structure composed of three staples is a disordered metastable structure.

[0051] Example 2: Transformation of disordered metastable structure into ordered stable structure under the induction of staple chain subset

[0052] 3 μL of the remaining stapler subset corresponding to 834 nM was added to the synthesized disordered metastable structure to ensure that the final system stapler chain concentration was 50 nM and the scaffold chain was 10 nM. Incubate isothermally at 25°C for 120 minutes. At the same time, the steady-state structure was directly constructed according to the traditional DNA triangle origami synthesis method (mix 5 μL of 100 nM single-stranded M13mp18 DNA, 10 μL of 250 nM stapler chain, 5 μL of preparation buffer and 30 μL of deionized water, first isothermally heated at 80°C for 3 minutes in a PCR thermal cycler, and then annealed to 25°C at a rate of 0.5°C / min). Then take 5 μL of each sample and dissolve it in 20 μL of 0.5×TAEMg 2+ AFM imaging was performed in a buffer (5 mM Tris, pH=8.5, 1 mM EDTA, 12.5 mM MgAc2), and coarse-grained molecular dynamics simulations were performed on the scaffold chain, the ordered stable structure transformed from the disordered metastable structure, and the ordered stable structure directly constructed.

[0053] Results: Figure 4 As shown in Figure 2, the AFM images of the ordered stable structure transformed from the disordered metastable structure and the directly constructed ordered stable structure are all regular and orderly triangles. The area distribution statistics of the four ordered stable structures are 7250±1050nm 2 、6250±1071nm 2 、5750±1488nm 2 and 6250±1299nm 2 ( Figure 5 ), with no significant difference. The yields of the ordered stable structures synthesized by the four methods were 62.7%, 66.0%, 48.2% and 61.6% ( Figure 6 ), indicating that the short-range and long-range subset metastable structures are transformed into stable structures with a yield comparable to that of the traditional direct synthesis method.

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made in accordance with the claims and the content of the specification of the present invention application fall within the scope of protection of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a DNA origami structure that induces a rapid disorder-order transition at room temperature, characterized in that, Comprising the following steps: a) Pre-folding the scaffold strand of the DNA origami structure with a partial staple strand subset to form a metastable disordered structure; b) Adding the remaining staple strand subset of the complete staple strand minus the partial staple strand subset added in step a) to the metastable disordered structure constructed in step a), and inducing the transformation of the disordered structure into an ordered origami structure at room temperature.

2. The preparation method according to claim 1, characterized in that, The DNA origami structure includes: DNA triangular origami, DNA rectangular origami, DNA wireframe origami, and DNA polyhedron origami.

3. The preparation method according to claim 1, wherein In step a), the partial staple strand subset is any one of a short-range subset, a long-range subset, and a random subset.

4. The preparation method according to claim 1, characterized in that, In steps a) and b), the staple strand includes: an unmodified DNA strand, a thiol-modified DNA strand, a fluorescent dye-modified DNA strand, and a biotin / streptavidin-modified DNA strand.

5. The preparation method according to claim 1, characterized in that, In step a), the scaffold strand and the partial staple strand subset are mixed at a molar concentration ratio of 1:(2 - 100), heated at 65 - 95 °C for 3 - 20 min, and then cooled to 25 °C at a cooling rate of 0.1 - 1 °C / min.

6. The preparation method according to claim 1, characterized in that, In step b), the metastable disordered structure and the remaining staple strand are mixed at a molar concentration ratio of 1:(2 - 100) and incubated at 15 - 40 °C for 10 min - 2 h.

7. The preparation method according to claim 1, characterized in that, In step a), the scaffold strand and the partial staple strand subset are assembled isothermally at 55 °C.

8. The preparation method according to claim 1, wherein In step b), the metastable disordered structure and the remaining staple strand are incubated and assembled at 25 °C or a lower temperature.

9. An application of the preparation method of the DNA origami structure that induces rapid disorder-order transformation at room temperature according to any one of claims 1 - 8 in synthesizing the DNA origami structure in a physiological environment and achieving its controllable disorder-order transformation.

10. The application according to claim 9, characterized in that, The application includes the rapid disorder-order transformation of the DNA origami structure at room temperature in biosensing, drug delivery, or molecular computing.

Images