Logic element based on DNA nanostructure and application thereof

Abstract

The invention belongs to the field of biological computer technology and DNA nanotechnology, and particularly relates to a logic element based on a DNA nanostructure and application of the logic element in manufacturing of a true random number generator. According to the logic element based on the DNA nano-structure, a two-dimensional cross DNA nano-structure is used as a main body of the element, and the logic element bearing different information is constructed through site programming by utilizing the basic characteristics that the DNA nano-structure is uniform in size, accurate in site programming and rich in extensible side chains; the extensible side chain serves as a sticky tail end to achieve a stable and reliable coaxial physical connection relation between the logic elements, and only when the sticky tail ends between the logic elements have a complementary relation, a dimer or a multimer can be formed through self-assembly, so that when the logic element is applied to manufacturing of a random number generator, the random number generator can be manufactured through self-assembly, and the reliability of the random number generator is greatly improved. It can be guaranteed that the polymer formed by connecting a plurality of cross-shaped origami still has good rigidity, and the correctness of a random generation result is guaranteed.

Description

Technical Field

[0001] The invention belongs to the fields of biocomputer technology and DNA nanotechnology, and specifically relates to a logic element based on a DNA nanostructure and its application in making a true random number generator. Background Art

[0002] With the rapid development of information technology, the demand for random numbers is increasing day by day. Traditional computer random number generators are mostly pseudo-random number generators, whose results are determined by a certain algorithm and initial seed, and have a certain degree of predictability. Since all software algorithms used to generate random values ​​work based on mathematical formulas, their output will appear in a pseudo-random form, and even the strongest formula will repeat the output after a period of time. In some fields with extremely high requirements for randomness, such as cryptography, quantum physics research, secure communications, and Monte Carlo simulations, this pseudo-randomness may bring security risks or affect the accuracy of simulation results. Therefore, it is urgent to find a method for generating true random numbers.

[0003] The true random number generation process is truly random and does not allow the generated sequence to be predicted in any way. These generators are called true random number generators (TRNG). In order to generate true random values, the need for a natural noise source means that the noise source can be reliably used in a random number generator if and only if it comes from the physical environment. For example, the patent with publication number CN106687916B discloses a random number generator that uses a quantum mechanism to generate photons from a light source to obtain a true random number sequence. It has the uncertainty of the measurement event as an inherent property of the quantum system itself. However, its random number generation formula first obtains the photon source through an electronic sampling device, and then processes the binary sequence through the von Neumann algorithm. It also relies on the algorithm to judge randomness, and the random number generation result cannot be directly observed.

[0004] DNA origami technology uses the special structure of DNA molecules and the rules of complementary base pairing to fold specific areas of the long DNA chain and fix it with short chains to construct the expected structure; reference "Folding DNA to create nanoscale shapes and patterns. Nature, 2006, 440: 297-302", in 2006, the research team of California Institute of Technology successfully used DNA origami to use the genome DNA of bacteriophage M13 as a long chain and more than 200 short single-stranded DNAs through the principle of complementary base pairing to assemble into a variety of two-dimensional plane patterns such as rectangles, triangles, five-pointed stars and smiley faces. DNA origami technology has unique properties and provides new ideas for random number generation. The self-assembly process of DNA molecules is affected by many factors, including temperature, ion concentration, solution pH, etc. Slight changes in these factors may lead to different assembly results, which means that structures with high uncertainty can be generated based on DNA origami technology, so it is possible to obtain true random numbers from them. In addition, DNA, as a biological molecule, has high stability and operability, providing a reliable material basis for the generation and storage of random numbers. Summary of the invention

[0005] In order to solve the above technical problems, the present invention mainly provides a logic element based on DNA nanostructure, which takes a two-dimensional cross DNA nanostructure as the main body of the element, utilizes the basic characteristics of DNA nanostructure, such as uniform size, precise site programming and rich extendable side chains, and constructs logic elements carrying different information through site programming. The extendable side chains serve as sticky ends to achieve stable and reliable physical connection relationships between logic elements. The cross origami of the connection relationship is coaxial, and only when there is a complementary relationship between the sticky ends of the logic elements can they be self-assembled to form dimers or polymers through one-pot annealing. Based on the above logic element, the present invention also provides its application in generating random numbers.

[0006] In the first aspect, a logic element based on a DNA nanostructure is provided, wherein the logic element is a two-dimensional cross DNA nanostructure. Currently, most two-dimensional DNA nanostructures are prepared by DNA origami. The two-dimensional cross DNA nanostructure, as a type of two-dimensional DNA nanostructure, is also assembled from a DNA template chain and a DNA stapler chain by DNA origami. The DNA template chain serves as the main chain, and excess DNA stapler chains serve as auxiliary chains. The main chain and the auxiliary chain hybridize and complement each other at specific positions, and then assemble to form the two-dimensional cross DNA nanostructure. In the selection of the DNA template chain, the M13mp18 phage genome DNA sequence provided in GenBank: X02513.1 is preferred; the DNA stapler chain is used as an auxiliary chain, wherein part of the DNA stapler chain is a programmable auxiliary chain, i.e., a functional DNA stapler chain, and the remaining DNA stapler chain is a non-functional DNA stapler chain; a part of the functional DNA stapler chain is used to generate a sticky end chain after subsequent assembly, and this part of the functional DNA stapler chain is recorded as a sticky DNA chain. After assembly, a plurality of free sticky end chains extend from one side or both sides of the main body edge of the two-dimensional cross DNA nanostructure, and these sticky end chains constitute a sticky end chain group, and the base sequences of the sticky end chains in the sticky end chain group are all different from each other, so as to ensure that the two-dimensional cross DNA nanostructure maintains a high specific recognition ability and self-assembly yield; and preferably, the sticky end chains in the sticky end group There are 6 terminal chains that are evenly distributed on one side edge of the main body of the two-dimensional cross DNA nanostructure; the other part of the functional DNA staple chain is hereinafter referred to as the DNA capture chain, and the DNA capture chain includes two blocks, namely, one part of the blocks is used as a staple chain for the assembly of the two-dimensional cross DNA nanostructure main body, and the other part is used to extend at least one capture sequence for capturing the marking structure on the surface of the two-dimensional cross DNA nanostructure main body after assembly, and the captured position is the site of the logic element for carrying information; preferably, the capture sequence is a nucleotide sequence added to the 5' end of the DNA staple chain, and further preferably, the capture sequence is a 10-nucleotide length sequence, so as to accurately control the number and relative position of the marking structure on the two-dimensional cross DNA nanostructure; the marking structure is selected from any one of nanogold-thiolated oligonucleotide, streptavidin SA and a fluorescent group. If the labeling structure is a nanogold-thiolated oligonucleotide, the capture structure is a sequence complementary to the oligonucleotide in the nanogold-thiolated oligonucleotide; preferably, in order to take advantage of the strong affinity and fast binding speed between SA and biotin, the labeling structure is preferably SA, the capture sequence is a biotin-modified capture sequence, and the DNA capture chain is a biotin-modified DNA capture chain;

[0007] The two-dimensional cross DNA nanostructure has the property that all sites are programmable and can be used as a carrier of information. Its cross-shaped structural feature is conducive to clearly distinguishing different sites. For example, as a preferred embodiment of the logic element described in the present application, Figure 2a As shown, the specific sites on the two-dimensional cross DNA nanostructure for capturing the label structure include information sites and check sites. The information sites representing different numbers are respectively arranged above and below the horizontal line of the cross structure, which is conducive to the observer to distinguish the information sites clearly at the nanoscale level, thereby facilitating the rapid reading of information. In addition, in order to meet the requirement that a single two-dimensional cross DNA nanostructure can represent a two-digit number, the information sites representing different digits are respectively arranged on the left and right sides of the cross structure as the high digit and the low digit, respectively. The distinction between the left and right sides is determined by the check site. The check digit is arranged on one side of the main body of the two-dimensional cross DNA nanostructure to determine the direction of information reading. When reading a number, the high digit is read first and then the low digit, thereby ensuring the accuracy of information reading. When reading, if all the information sites representing the same digit have no label structure or all the information sites representing the same digit have a label structure, then it is erroneous data. Only when only one of the information sites representing the same digit has a label structure is it correct data. Correspondingly, 5 capture sequences are arranged on the DNA nanostructure, and 1 capture sequence captures one label structure.

[0008] As a preferred embodiment, the size of the two-dimensional cross DNA nanostructure is set to be 100 nm in length and 100 nm in width; Figure 1 As shown, the preparation process of the logic element based on DNA nanostructure in the first aspect includes the following steps: step S1, mixing the DNA template chain, the functional DNA staple chain and the non-functional DNA staple chain in proportion in a buffer solution for PCR annealing to obtain a two-dimensional cross DNA nanostructure; step S2, ultrafiltration purification of the annealing product obtained in step S1 to replace the buffer solution to obtain the logic element based on DNA nanostructure.

[0009] Preferably, in step S1, the PCR annealing conditions are: the starting temperature is 95°C, maintained for 3 minutes, the end temperature is 25°C, each 1°C is used as a gradient, and each gradient stays for 100 seconds.

[0010] Preferably, in step S1, the molar ratio of the DNA template strand, the non-functional DNA staple strand and the functional DNA staple strand is 1:10:10.

[0011] Preferably, in step S2, the ultrafiltration purification is performed three times, and the ultrafiltration conditions for each time are 3000xg and 8 minutes.

[0012] Preferably, the buffer described in step S1 is 1×TAE / Mg 2+ ;

[0013] Preferably, the ultrafiltration step in step S2 is to anneal the obtained product with 1×TAE / Mg 2+ The buffer was mixed and added to a 100 kDa ultrafiltration tube and centrifuged.

[0014] In view of the basic characteristics of the DNA nanostructure-based logic element provided in the first aspect of the present invention, such as the site programmable, the stable connection relationship between the logic elements can be formed through the complementary sticky ends, and the connection relationship can be preserved, it is further applied to generate true random numbers, that is, in the second aspect, the present invention also provides the application of the DNA nanostructure-based logic element in the manufacture of a true random number generator, and the specific application includes the following steps:

[0015] Step S1, obtaining preset conditions according to the type of random numbers to be generated, and then making or selecting different logic elements based on DNA nanostructures according to the preset conditions; it should be noted that the differences here include different information represented by the sites and different sticky end group settings;

[0016] The method of obtaining the preset condition according to the type of random number to be generated as required refers to: determining the number of combinations of different numbers according to the number of bits of the random number to be generated, and then determining the number of logical elements with a connection relationship, that is, the value of N in the N-polymer, according to the number of combinations. Both dimers and polymers have good rigidity, which can ensure that the randomly generated results can be stored in the correct order and no errors will occur.

[0017] The different settings of the sticky end groups refer to settings on the left side only, settings on the right side only, or settings on both sides; the two-dimensional cross DNA nanostructures are matched and connected through the complementary base relationship of the sticky end groups on both sides of the cross origami;

[0018] The sticky end groups are set to make the connection relationship between the logic elements representing the numbers random. In addition, all the logic elements are self-assembled under the same physical environment conditions, so the combination of numbers based on the logic elements is random; for example, in the random number generation based on dimers, when setting the sticky end groups, in order to ensure that the sticky end groups on the two-dimensional cross DNA nanostructure have an equal competitive relationship, such as Figure 3As shown, the sticky end group set on the left side of the logic element is the L group, and the six sticky end chains of the L group are L1, L2, L3, L4, L5, and L6 respectively; the sticky end group set on the right side of the logic element is the R group, and the six sticky end chains of the R group are R1, R2, R3, R4, R5, and R6 respectively; different logic elements have the sticky end groups on their left sides are all the six sticky end chains of the L group, and similarly, different logic elements have the sticky end groups on their right sides are all the six sticky end chains of the R group; the six sticky end chains of the L group correspond to the six sticky end chains of the R group one by one, and there is a base complementary relationship, and the base complementary relationship between the two represents the relationship of random number generation;

[0019] Step S2, mixing different logic elements based on DNA nanostructures in equal proportions, annealing and self-assembling in PCR to obtain assembly products, adding SA to the assembly products for connection, and forming a polymer with a labeled structure as a random number generator, and using an atomic force microscope AFM to directly observe the generation result; preferably, the annealing conditions are: starting temperature 45°C, slowly annealing in a water bath to an end temperature of 25°C; preferably, diluting the obtained annealing product to 1-2 nM; preferably, adding SA to the obtained dilution, according to the molar concentration of the site to SA is 1:50;

[0020] Further preferably, the application of the logic element based on DNA nanostructure in the production of a true random number generator also includes: step S3, detecting and judging whether the generation result of the random number generator is completely random, and the detection means mainly include: frequency test, run test and sequence test; specifically, the detection and judgment process is to characterize the sample morphology through an atomic force microscope AFM to obtain an AFM image, and the random number generation result is mainly statistically analyzed by objective observation, and all samples in the generated AFM image are statistically analyzed one by one. If there are some erroneous structures in the sample, they will be verified and excluded, and will not be counted in the generated result, and finally summarized and the proportion of each result will be calculated; wherein, the frequency test mainly looks at the proportion of 0 and 1 in the entire sample, and if the proportion is the same, the test is passed; the run test mainly looks at the total number of runs, and a run refers to a sequence of identical numbers without interruption, that is, a run such as "1111" or "0000" in a four-digit random number generator, by counting the proportion of consecutive 0 or 1 in the total number of these two samples. The two-bit test is to find out whether all results appear approximately the same number of times, that is, the proportion of all randomly generated possibilities in the total number of samples.

[0021] For example, in the production of a two-bit random number generator, such as Figure 4aAs shown, each logic element represents only one digit, and the encoded information is 0 or 1. The dimer is assembled into a dimer through the connecting chains on the left and right sides of the logic element. The dimer represents a random combination of two bits of information, that is, a two-bit random number. Through the randomly obtained two-bit random number AFM image, the samples in all the obtained AFM images are statistically analyzed, and then statistical tests are performed according to the requirements of frequency test, run test and sequence test.

[0022] For example, in the production of a four-digit random number generator, Figure 5a As shown, there are two bits of information encoded on the cross origami, 00, 01, 10, and 11, which are assembled into a dimer through the connecting chains on the left and right sides. At this time, the dimer represents four bits of information, that is, a four-bit random number. Through the randomly obtained four-bit random number AFM image, the samples in all the obtained AFM images are statistically analyzed, and then statistical tests are performed according to the requirements of frequency test, run test and sequence test.

[0023] For another example, in the production of an eight-bit random number generator, as shown in FIG6 , there are two bits of information encoded on the cross origami, 00, 01, 10, and 11, which are assembled into a tetramer through the connecting chains on the left and right sides. At this time, the tetramer represents eight bits of information, that is, an eight-bit random number. In the generation of eight-bit random numbers, its connecting chains have three groups, each corresponding to one another, to ensure the accuracy of the connection and to generate only tetramers.

[0024] As a preferred embodiment, in order to improve the connection yield, when the connection chain of the cross origami is 0.5 times more than the calculated addition amount, the connection yield is as high as 90%. The excess connection chains in the assembled cross origami will be removed by ultrafiltration, so as to solve its connection yield problem with more connection chains;

[0025] With the same technical concept as the second aspect, the third aspect of the present invention also provides a type of true random number generator, which is used to generate random numbers, and the true random number generator includes:

[0026] The encoding module is used to obtain preset conditions according to the type of random numbers to be generated, and then to make or select different logic elements based on DNA nanostructures according to the preset conditions;

[0027] A logic element database, used to store the above-mentioned different logic elements based on DNA nanostructures;

[0028] A reaction platform is connected to the logic element database and provides suitable reaction conditions for mixing the different logic elements based on DNA nanostructures under the same physical environment conditions to generate a self-assembly reaction to obtain a reaction product;

[0029] The result acquisition module is used to observe the reaction products and reaction results, and to detect and determine whether the generation results of the random number generator are completely random.

[0030] In a fourth aspect, the present invention further provides an application of the true random number generator described in the third aspect in information encryption. In view of the fact that the random number results generated by the above true random number generator have storage capabilities, they can be used in the field of information encryption.

[0031] Beneficial effects:

[0032] The molecular motion behavior of DNA molecules provides a pure physical noise source that can be used to generate high-quality true random values. Compared to traditional cryptography and quantum cryptography, the power of DNA computing lies in the incredible parallel processing ability of DNA molecules. Through a large number of DNA molecules, a large number of possibilities can be checked at the same time. This huge parallel computing along with the extraordinary density of data storage in DNA molecules will lead to many new achievements in different fields of computer science, such as its use to break modern cryptographic algorithms, none of which can resist DNA-based brute force cracking.

[0033] The first aspect of the present invention provides a type of logic element based on DNA nanostructure, which uses DNA nanostructure as a DNA computing element, and the designed DNA nanostructure has uniform size, recombined programmable sites provide precise site design, and abundant extensible side chains can provide stable and reliable physical connection relationships; the sticky end chains extending from the left and right sides or both sides of the two-dimensional cross DNA nanostructure of the present invention can make random connections between the cross origami; based on the above-mentioned logic element, the cross origami plates with a connection relationship are coaxial, and when applied to random number generation, it can be ensured that the polymer connected by multiple cross origami plates still has good rigidity, thereby ensuring that the generation results can be stored in the correct order without errors, so as to facilitate the observation of subsequent calculation results, and the above-mentioned logic element has encryption and storage capabilities.

[0034] The second aspect and other aspects of the present invention provide the application of the above-mentioned logic element based on DNA nanostructure in random number generation. Based on experimental verification, the researchers of this research group continuously improve and optimize the random number generation technology based on DNA origami. On the one hand, the design and assembly method of DNA origami are improved to increase the complexity and diversity of its structure, thereby increasing the entropy value of random numbers; on the other hand,

[0035] Combined with other technologies, such as nanotechnology and optical technology, the efficiency and accuracy of random number generation can be improved. For example, light stimulation is used to control the structural changes of DNA origami, thereby achieving more accurate random number generation. As the technology continues to mature, random number generation technology based on DNA origami has begun to be applied in various fields. In the field of cryptography, DNA origami random numbers can be used in encryption algorithms to improve the security of information; compared with other algorithms, its advantages are: 1. Large key space: DNA origami encryption technology can use very long DNA chains as keys. For example, a skeleton chain of about 7,000 bases can achieve a theoretical key length of about 700 bits, which far exceeds the key strength of traditional encryption algorithms such as classic AES. Such a large key space makes brute force cracking almost impossible, greatly improving the security of encryption. 2. Encryption based on biomolecules is to perform the encryption process through thermodynamically controlled biomolecular reactions, which has high specificity. As a biomolecule, DNA has unique structure and reaction, which is completely different from the encryption system based on silicon-based computers. This makes it difficult for hackers to use conventional computer technology and algorithms to crack DNA origami encrypted information, avoiding attacks from computers. 3. DNA origami encryption can hide information in the structure and sequence of DNA, and this hiding method is very difficult to detect. Compared with traditional encryption methods, information is less likely to be discovered and stolen during transmission and storage, realizing the integration of encryption and steganography. 4. DNA itself has a huge information storage capacity, which is 5 million times that of current storage methods. 5. DNA molecules have high stability and have a certain resistance to interference from the physical and chemical environment. In contrast, some traditional encryption technologies may be affected by factors such as electromagnetic interference and temperature changes, resulting in loss or damage of information. DNA origami encryption technology can better protect the security of information in complex environments. In quantum physics research, it can be used as a random number source for simulation and verification of quantum experiments; in the biomedical field, it can also be used for randomized experimental design and data analysis. In addition, researchers are constantly exploring new application areas, such as applications in artificial intelligence, big data, etc., to provide more reliable random number support for the development of these fields.

[0036] The two-dimensional cross DNA nanostructure mainly used in the present invention has good biocompatibility, and other required chemical and biological materials are also non-toxic to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A schematic diagram of the synthesis of the two-dimensional cross DNA nano-encryption element of the present invention;

[0038] Figure 2a The schematic diagram of the arrangement of information sites and the information they represent in the specific implementation mode of the present invention is a statistical diagram of the yield of each information;

[0039] Figure 2b Schematic diagram and representative photos of logic elements corresponding to the two encoding information 0 and 1 in Example 1;

[0040] Figure 2c The logical elements and representative photos corresponding to the four coding information 00, 10, 01, and 11 in Example 2;

[0041] Figure 2d is the yield of logic elements corresponding to six encoding information of 0, 1, 00, 10, 01, and 11 in Example 1 and Example 2;

[0042] Figure 3 Schematic diagram of the distribution of sticky DNA chains on the logic element in Example 1;

[0043] Figure 4a This is a schematic diagram of the two-digit random number generation process in Example 1;

[0044] Figure 4b This is a characterization diagram of the dimer structure morphology obtained by the two-bit random number generation in Example 1;

[0045] Figure 4c This is a frequency test statistical chart of the two-digit random number generation result in Example 1;

[0046] Figure 4d A run test of the two-digit random number generation result in Example 1;

[0047] Figure 4e A statistical diagram of the sequence test of the two-digit random number generation result in Example 1;

[0048] Figure 5a This is a schematic diagram of the process of generating four random numbers in Example 2;

[0049] Figure 5b This is a characterization diagram of the dimer structure morphology obtained by generating four random numbers in Example 2;

[0050] Figure 5c The frequency test statistics of the four-digit random number generation results in Example 2;

[0051] Figure 5d It is a statistical graph of the run test of the four-digit random number generation result in Example 2;

[0052] Figure 5e A statistical diagram of the sequence test of the four-digit random number generation result in Example 2;

[0053] Figure 6a A schematic diagram of generating an eight-bit random number constructed in Example 3;

[0054] Figure 6b The tetramer structure morphology characterization diagram obtained by the eight-digit random number generation in Example 3;

[0055] Figure 7 The code book constructed for Example 3 is used for encryption and decryption schematic diagram. DETAILED DESCRIPTION

[0056] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0057] The following are descriptions of the raw materials and reagents used in the preparation process and examples:

[0058] Tris, disodium ethylenediaminetetraacetate (EDTA), magnesium acetate, glacial acetic acid, and boric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; 100 kDa ultrafiltration tubes were purchased from Pall China Company; and ultrapure water was used in the experiments.

[0059] M13mp18 phage genomic DNA was purchased from Biobio Biotechnology Co., Ltd. with a product number and specification of B3003-50 pmol; non-functional DNA stapler chain, functional DNA stapler chain, biotin-ssDNA chain and streptavidin were all purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0060] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0061] Taking the preparation of two-digit, four-digit and eight-digit random number generators as examples, the preparation process of the DNA nano encryption element of the present invention and its application in the preparation of random number generators are specifically described;

[0062] Example 1: Production of a two-digit random number generator

[0063] As a preferred embodiment of the method for generating a two-digit random number of the present invention, the specific steps of Embodiment 1 include:

[0064] According to the requirement of generating two-bit random numbers, a preset condition is obtained, and according to the preset condition, a DNA sequence is encoded on the sticky end of the two-dimensional cross DNA nanostructure and a marking structure is used to mark the two-dimensional cross DNA nanostructure to construct a variety of different logic elements; the preset conditions of this embodiment include the number represented by each logic element and the connection relationship between each cross;

[0065] The two-dimensional cross DNA nanostructure is marked with a marking structure to represent different numbers. Specifically, in this embodiment, five DNA capture chains are anchored on the surface of the DNA nanostructure, and one chain captures one SA. The two-dimensional cross DNA nanostructure is self-assembled by the M13mp18 template chain and the staple chain. The DNA sequence of the M13mp18 template chain is provided in GenBank: X02513.1, and the non-functional DNA staple chain can be found in the literature "Crystalline two-dimensional DNA-origami arrays. Angew Chem Int Ed Engl. 2011Jan 3; 50(1): 264-7." The stapler chain used in the two-dimensional cross DNA nanostructure in the above document, the DNA capture chain is a capture sequence modified with biotin added to the end of some non-functional DNA stapler chains in the above document, and different information sites are encoded by controlling whether the biotin is modified or not. The serial number of this part of the non-functional DNA stapler chain in the above document and its corresponding information site number in the present application are as follows: site 1 corresponds to the position of CO-M-95 in the document, site 2 corresponds to the position of CO-M-90 in the document, site 3 corresponds to the position of CO-M-166 in the document, site 4 corresponds to the position of CO-M-171 in the document, and site 5 corresponds to the position of CO-M-154 in the document; the nucleotide sequences of the five capture sequences are shown in SEQ ID NO: 1 to 5.

[0066] The sticky DNA chain is obtained by adding corresponding sticky ends to the ends of some non-functional DNA staple chains in the above literature. The sticky ends here are the connecting chains in Table 1, which are connected to the sequences of C0-A-L1 to C0-A-L6 and C0-A-R1 to C0-A-R6 in the above literature as sticky DNA chains, such as Figure 3 After a large number of experimental verifications, the connection yield of 6 connecting chains is the best. Each group of sticky ends extends from the left and right sides of the cross origami DNA nanostructure. It should be noted that in order to simplify the schematic diagram, Figure 3 Each sticky die forging chain of the same sticky end group is represented by a thick line, and the thick lines with the same linear mark on the same side belong to a sticky end group. In order to form this sticky end structure, the specific design of the two-bit random number generator is shown in Table 1 below:

[0067] Six connecting chains Left connecting chain Right connecting chain First link chain L1: SEQ ID NO: 6 R1: SEQ ID NO: 12 Second link chain L2: SEQ ID NO: 7 R2: SEQ ID NO: 13 The third link L3: SEQ ID NO: 8 R3: SEQ ID NO: 14 The fourth link L4: SEQ ID NO: 9 R4: SEQ ID NO: 15 Fifth link L5: SEQ ID NO: 10 R5: SEQ ID NO: 16 The sixth link L6: SEQ ID NO: 11 R6: SEQ ID NO: 17

[0068] like Figure 2aAs shown, information position 1 and information position 2 represent information 0, and information position 1 is a low position, and information position 2 is a high position; information position 3 and information position 4 represent information 1, and position 3 is a low position, and position 4 is a high position; position 5 represents the information check position Marker, which is used to mark the direction of the cross origami and determine whether the origami is in the correct position;

[0069] like Figure 4a As shown, there is only one digit on the cross origami, and the information is 0 or 1. The dimer is assembled through the connecting chains on the left and right sides. The dimer represents two bits of information, that is, a two-bit random number.

[0070] According to the above preset conditions, for the generation of two-bit random numbers, and considering the site information of the logic element and the setting of the sticky end, four logic elements need to be prepared, and each logic element is operated according to the following steps:

[0071] (1) Mix the M13mp18 template strand, non-functional DNA staple strand, and DNA functional strand. The final concentrations of the template strand and all DNA staple strands are 5uM and 50nM, respectively.

[0072] (2) Using a gradient PCR instrument, the mixture was slowly annealed. The annealing conditions were as follows: the starting temperature was 95°C, maintained for 3 minutes; the end temperature was 25°C, with each 1°C as a gradient, and each gradient was kept for 100 seconds, thereby obtaining a two-dimensional cross DNA nanostructure;

[0073] (3) After the annealing process is completed, the two-dimensional cross DNA nanostructure sample is taken out and centrifuged using a 100 kDa centrifuge tube to remove excess DNA short chains. The centrifugation conditions are: add 300 μL of 1×TAE-Mg2+ buffer to 100 μL of sample, centrifuge at 2800 rcf / min for 8 minutes, and repeat the centrifugation operation three times.

[0074] Logic element characterization: In order to clearly characterize the logic element used in this Example 1, the two-dimensional cross DNA nanostructures prepared above were added with a labeling structure SA. The specific operation was as follows: the prepared two-dimensional cross DNA nanostructure was mixed with the information site and SA at a molar ratio of 1:50, and then annealed and incubated for 24 hours. The annealing conditions were: the starting temperature was 37°C, the end temperature was 25°C, and each 1°C was used as a gradient. Each gradient stayed for 10 minutes, and the annealing time was maintained for 2 hours. The annealed product was ultrafiltration purified and replaced with a buffer of 1×TAE / Mg 2+ The ultrafiltration step is to anneal the obtained product with 1×TAE / Mg 2+The buffer solution was mixed and added to a 100 kDa ultrafiltration tube for ultrafiltration to purify excess SA, and centrifuged at 3000 x g for 8 minutes to obtain a purified cross-plate DNA nano-encryption element encoding site information; the final collected sample was observed using an atomic force microscope (AFM) to observe the morphology of the sheet structure, such as Figure 2b The figure shows the logic elements corresponding to the two coding information 0 and 1. The constructed DNA nanostructure presents a cross-layer structure. The AFM characterization results show that the rectangular two-dimensional DNA nanostructure is about 100nm long and 100nm wide, presenting a regular cross structure.

[0075] The four logic elements prepared above are mixed evenly in an equimolar ratio, the concentration of each logic element is 3nM, the mixed solution system is 80uL, annealed in PCR for 14 hours, annealed in a water bath, and slowly cooled from 45°C to 25°C. After the annealing program is completed, the obtained annealing product is diluted to 1-2nM; SA is added to the obtained dilution, that is, the structure SA is marked on the dimer structure formed by the above logic element, and the specific operation is the same as the specific operation of adding the marking structure SA when characterizing the above logic element;

[0076] The dimer structure was characterized by AFM. Figure 4b The following is a partial observation view of the obtained dimer structure; by counting multiple AFM characterization images, the statistics of the generated results are randomly tested to determine whether the random number generation results are completely random. The frequency test results are shown in Figure 4c As shown, the run test results are as follows Figure 4d The sequence test results are shown in Figure 4e shown.

[0077] Example 2: Preparation of a four-digit random number generator

[0078] The difference between Example 2 and Example 1 is that, in the generation of four-digit random numbers, Figure 5a As shown, each cross origami has two digits of coded information, namely 00, 01, 10, and 11. The dimers are assembled into a dimer through the connecting chains on the left and right sides. At this time, the dimer represents four digits, that is, four random numbers.

[0079] For example, if the encoded information is 01, the corresponding information sites 1 and 4 and the check site 5 capture the marked structure; if the encoded information is 10, the corresponding information sites 2 and 3 and the check site 5 capture the marked structure; when 01 and 10 appear at the same time, it is difficult to distinguish them without the mark of the check site 5. With the check site 5, the direction of the origami can be adjusted according to the direction of the check site 5 to determine whether the origami represents information.

[0080] like Figure 2cFIG. 2 shows logic elements corresponding to four coding information, 00, 10, 01, and 11, respectively, manufactured according to the manufacturing method of the logic element in Example 1; Figure 2d The figure shows the yield of logic elements corresponding to six types of coding information, 0, 1, 00, 10, 01, and 11, in Example 1 and Example 2.

[0081] In the generation of four-digit random numbers, cross DNA nano-encryption elements with connecting chains and encoding information of 00, 01, 10, and 11 were mixed evenly in an equal molar ratio. They were placed in a water bath for annealing, and the temperature was slowly reduced from 45°C to 25°C. After the annealing process was completed, the annealed product was diluted to 1-2 nM and characterized by AFM. Figure 5b The figure shows the correct result in the cross-plate polymer. By statistically analyzing multiple AFM characterization images, frequency test, run test, and sequence test are performed to determine whether the random number generation result is completely random. The frequency test results are shown in Figure 5c As shown, the run test results are as follows Figure 5d The sequence test results are shown in Figure 5e shown.

[0082] Example 3: Production of an eight-digit random number generator

[0083] The difference between Example 3 and Example 2 is that there are three groups of connecting chains, and the connecting chain sequences are as follows:

[0084] As shown in SEQ ID NOs: 6 to 41, SEQ ID NOs: 6 to 17 are one group, and SEQ ID NOs: 18 to 29 are the second group,

[0085] SEQ ID NOs: 30 to 41 are the third group, and each group of connecting chains is connected to the sequences of C0-A-L1 to C0-A-L6 and C0-A-R1 to C0-A-R6 in the above literature as sticky DNA chains;

[0086] In the generation of eight-digit random numbers, the same information encoding method is used as in the four-digit random number generation method in Example 2, such as Figure 6a As shown, there are two bits of information encoded on the cross origami, 00, 01, 10, and 11 respectively. The difference is that in this embodiment 3, the tetramer is assembled into a tetramer through the connecting chains on the left and right sides. At this time, the tetramer represents eight bits of information, that is, an eight-bit random number. In the generation of the eight-bit random number, its connecting chain has three groups, each group corresponds to one another, to ensure the accuracy of the connection and to generate only tetramers.

[0087] In the generation of eight-digit random numbers, cross DNA nano-encryption elements with connecting chains and encoding information of 00, 01, 10, and 11 were mixed evenly in an equal molar ratio, placed in a water bath for annealing, and slowly cooled from 45°C to 25°C. After the annealing process, the annealed product was diluted to 1-2 nM and characterized by AFM. Figure 6b As shown, the eight-bit random number generator can be used as a code book for encryption and decryption.

[0088] Example 4: Eight-bit random number generator for encryption and decryption

[0089] By listing the read information of the tetramer in the AFM image in the eight-bit random number generator one by one into the code book, Figure 7 As shown, the binary information represented by the tetramer generated by the eight-bit random number generator can be used as a key for encryption and decryption, and the key can be randomly selected for encryption during the encryption process.

[0090] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A logic element based on DNA nanostructure, characterized in that: The logic element is a two-dimensional cross DNA nanostructure, which is assembled from a DNA template chain and a DNA staple chain by DNA origami. The DNA template chain serves as a main chain, and an excess of DNA staple chains serves as auxiliary chains. The main chain and the auxiliary chains are hybridized and complemented at specific positions to assemble to form the two-dimensional cross DNA nanostructure. Part of the DNA staple chains are programmable auxiliary chains, i.e., functional DNA staple chains, and the remaining DNA staple chains are non-functional DNA staple chains. A part of the functional DNA staple chains is used to generate sticky end chains after subsequent assembly. This part of the functional DNA staple chains is a non-functional DNA staple chain. The NA stapler chain is recorded as a sticky DNA chain. After assembly, multiple free sticky terminal chains extend on one side or both sides of the edge of the main body of the two-dimensional cross DNA nanostructure. These sticky terminal chains constitute a sticky terminal chain group, and the base sequences of the sticky terminal chains in the sticky terminal chain group are all different from each other; the other part of the functional DNA stapler chain is a DNA capture chain. The DNA capture chain includes two blocks, namely, one part of the block is used as a stapler chain for the assembly of the two-dimensional cross DNA nanostructure main body, and the other part is used to extend at least one capture sequence for capturing the label structure on the surface of the two-dimensional cross DNA nanostructure main body after assembly.

2. A logic element based on DNA nanostructure as claimed in claim 1, characterized in that: There are 6 sticky end chains in the sticky end group and they are evenly distributed on one side edge of the main body of the two-dimensional cross DNA nanostructure.

3. A logic element based on DNA nanostructure as claimed in claim 1, characterized in that: The capture sequence is a nucleotide sequence added to the 5' end of the DNA staple strand.

4. A logic element based on DNA nanostructure as claimed in claim 1, characterized in that: The labeling structure is selected from any one of nano-gold-thiolated oligonucleotide, streptavidin SA and fluorescent group.

5. The logic element based on DNA nanostructure according to claim 1, characterized in that: The label structure is SA, the capture sequence is a capture sequence modified with biotin, and the DNA capture chain is a DNA capture chain modified with biotin.

6. A logic element based on DNA nanostructure as claimed in claim 1, characterized in that: The specific sites on the two-dimensional cross DNA nanostructure used to capture the marking structure include information sites and check sites. The information sites representing different numbers are respectively arranged above and below the horizontal line of the cross structure, and the information sites representing different positions of numbers are respectively arranged on the left and right sides of the cross structure as high positions and low positions, respectively. The check bit is arranged on a single side of the two-dimensional cross DNA nanostructure body to determine the direction of information reading; correspondingly, 5 capture sequences are arranged on the DNA nanostructure, and 1 capture sequence captures one marking structure.

7. A logic element based on DNA nanostructure as claimed in claim 1, characterized in that: The DNA template chain is the M13mp18 phage genome DNA sequence provided in GenBank: X02513.1; the size of the two-dimensional cross DNA nanostructure is set to be 100 nm in length and 100 nm in width.

8. Use of the logic element based on DNA nanostructure as claimed in claim 1 in making a true random number generator.

9. The use according to claim 8, characterized in that The application comprises the following steps: Step S1, obtaining preset conditions according to the type of random numbers to be generated, and then making or selecting different logic elements based on DNA nanostructures according to the preset conditions; These include different information represented by the sites and different sticky end group settings; The obtaining of the preset condition according to the type of random number to be generated means: determining the number of combinations of different numbers according to the number of bits of the random number to be generated, and then determining the number of logical elements having a connection relationship, i.e., the value of N in the N-mer, according to the number of combinations; The different settings of the sticky end groups refer to settings on the left side only, settings on the right side only, or settings on both sides; the two-dimensional cross DNA nanostructures are matched and connected through the complementary base relationship of the sticky end groups on both sides of the cross origami; Step S2, different logic elements based on DNA nanostructures are mixed in equal proportions, annealed and self-assembled in PCR to obtain assembly products, SA is added to the assembly products for connection, and the formed polymer with a labeled structure is used as a random number generator.

10. The use according to claim 9, characterized in that: The annealing conditions are as follows: starting temperature 45°C, slow annealing in a water bath to an end temperature of 25°C; diluting the obtained annealing product to 1-2 nM; adding SA to the obtained dilution, and the molar concentration of the site to SA is 1:50.

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