Information storage, encryption and decryption method based on DNA nano switch

Abstract

The invention discloses an information storage, encryption and decryption method based on a DNA nano switch, and belongs to the technical field of biomolecule self-assembly material information storage and encryption. According to the method, a key sequence is protected by means of an RNA chain, formation of an annular DNA nano-switch is prevented, and information storage and encryption are achieved, a target chain Tx chain is added in advance, then a secret key RNaseA nuclease is added to digest the RNA protection chain, the target chain Tx and a detection chain Dx form the annular DNA nano-switch based on base complementary pairing, RNaseA can digest all the RNA protection chains in a solution, and the RNA protection chains in the solution can be separated from the target chain Tx. The method has the advantages that the RNA protection chain Rx and the detection chain Dx are connected to form the annular DNA nano switch Sx, secondary connection of the residual RNA protection chain Rx and the detection chain Dx is avoided, the target chain Tx and the detection chain Dx are connected to form the annular DNA nano switch Sx after RNAseA nuclease digests the RNA protection chain, the agarose gel imaging analysis technology is combined, rapid decryption and accurate reading of molecular-level information can be achieved, and the accuracy of information encryption is improved. And a basic strategy is provided for promoting the development of a nano-scale biological information encryption technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of information storage and encryption of biomolecular self-assembly materials, and in particular relates to an information storage, encryption and decryption method based on a DNA nanoswitch. Background Art

[0002] DNA nanomaterials are two-dimensional or three-dimensional structures that are self-assembled using the complementary base pairing principle of deoxyribonucleic acid. Due to their precisely controllable shape, size and function, DNA nanostructures have shown great application prospects in the fields of drug delivery, gene therapy, bioimaging, disease diagnosis and biosensing. In recent years, DNA nanoswitches prepared by self-assembly using DNA origami by K. Halvorsen and WP Wong have gradually been developed into a new nanoscale qualitative and quantitative analysis experimental platform. Its biggest feature is that the detection signal can be output through agarose gel electrophoresis commonly used in laboratories. DNA nanoswitches are simple in design, stable in structure, easy to prepare and low in cost, easy to read signals, and the detection limit can be as high as picomolar. They also show great application potential in the fields of information storage and encryption. Since DNA nanoswitches of different configurations have different migration rates in agarose gel electrophoresis, K. Halvorsen et al. used a simple DNA nanoswitch to successfully arrange 26 letters in a specific gel electrophoresis diagram, input different information by combining different letters, and used RNase H endoribonuclease for specific enzyme digestion to store and "encrypt" the input information. Finally, DNA single strands were added to "decrypt" and read the transmitted information. Based on this strategy, the storage, encryption and transmission of information were successfully achieved. However, the information encryption-decryption process of this technology takes a long time, and the working temperature of RNase H nuclease is 37°C, which will specifically digest the RNA in the DNA-RNA hybrid chain, so there are still free RNA single strands in the solution. These free RNA single strands will form DNA-RNA hybrid chains again with DNA single strands at 25°C, affecting the accuracy of information encryption; and this method has great limitations in application and is not universal. Summary of the invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the present invention uses the principle of DNA molecule self-assembly and draws on the idea of ​​functional group protection in organic chemical synthesis to construct a new information control system, striving to achieve efficient and accurate storage, encryption and decryption of information.

[0004] In order to achieve the above purpose, the following technical solution is adopted: The present invention provides an information storage, encryption and decryption method based on DNA nanoswitch, comprising the following steps:

[0005] Step 1: Preparation of linear DNA nanoswitch: The information to be transmitted is stored through a combination of circular DNA nanoswitches with different configurations. The circular DNA nanoswitch is formed by connecting the detection chain Dx and the target chain Tx on the linear DNA nanoswitch through the principle of base complementary pairing, while the linear DNA nanoswitch is made by mixing the M13mp18 long single chain with a certain number of staple chains and detection chains using the principle of base complementary pairing, and self-assembly synthesized in a PCR instrument through the "annealing" program;

[0006] Step 2: Information encryption: Step 2 adopts one of the following strategies:

[0007] Strategy 1: Add the detection strand D 0 Paired RNA protection strand R 0 , incubate at room temperature to form DNA-RNA hybrid chains, and detect chain D 0 Implement protection and join the target chain T x Chain, T x Chain cannot be detected with chain D 0 Base complementary pairing occurs, so the linear DNA nanoswitch cannot be converted into a circular DNA nanoswitch, and the gel electrophoresis diagram does not show the circular DNA nanoswitch, thereby achieving the storage and encryption of the transmitted information;

[0008] Strategy 2: In the linear DNA nanoswitch solution obtained in step 1, add the detection chain D x Paired RNA protection strand R x , incubate at room temperature to form DNA-RNA hybrid chains, and detect chain D x RNA protected chain R x Protect it and join the target chain T at this time x The chain cannot form a circular DNA nanoswitch; the gel electrophoresis does not show the circular DNA nanoswitch, thus realizing the storage and encryption of the transmitted information;

[0009] Strategy 3: Add RNA protective chain R to the linear DNA nanoswitch solution obtained in step 1 1、 R 2、 R 3、 R 4 , incubated at room temperature to form a DNA-RNA hybrid chain, thereby making the detection chain D 1、 D 2、 D 3、 D 4 Protect all and join the target chain T at this time x The chain cannot form a circular DNA nanoswitch, and the gel electrophoresis does not show the circular DNA nanoswitch, thereby realizing the storage and encryption of the transmitted information;

[0010] Strategy 4: Add RNA protective chain R to the linear DNA nanoswitch solution obtained in step 1 0 and the RNA chain R required for encryption x , incubated at room temperature to form a DNA-RNA hybrid chain, thereby converting the detection chain D 0 and D that needs protection x Protect it and join the target chain T at this time x , a circular DNA nanoswitch cannot be formed, and the gel electrophoresis diagram does not show a circular DNA nanoswitch, thereby realizing the storage and encryption of the transmitted information;

[0011] Strategy 5: Add the detection strand D 0 , D 1 , D 2 , D 3 , D 4 Paired RNA protection strand R 0 , R 1、 R 2、 R 3、 R 4 , incubate at room temperature to form a DNA-RNA hybrid chain, and then x Protect it and join the target chain T at this time x , a circular DNA nanoswitch cannot be formed, and the gel electrophoresis diagram does not show a circular DNA nanoswitch, thereby realizing the storage and encryption of the transmitted information;

[0012] Step 3: Information decryption: Add RNase A enzyme to the mixed solution in step 2 and incubate for a period of time, perform agarose gel electrophoresis, and analyze with a gel imager to observe the bands at different positions on the gel image. The presence of a band is marked as 1, and the absence of a band is marked as 0. Then, based on the conversion relationship between binary and decimal, translate the information at different positions on the gel into the corresponding decimal 0-9 to achieve information decryption.

[0013] In the step 1, the long chain is selected as M13mp18 linear single chain, and the specific steps of its preparation process are as follows:

[0014] a. Form a double strand with a partial sequence in the M13mp18 circular single strand according to the principle of base complementary pairing. The partial sequence is: 5'-TGTATGATTTATTGGATGTTAATGCTACTACTATTAGTAG-3', and its paired sequence is named Oligo. The M13mp18 circular single strand is obtained by proliferating the M13mp18 phage in JM109 competent cells, purifying it by polyethylene glycol precipitation, and then extracting it by alkaline lysis method. The concentration is 250 ng / μL.

[0015] b. Mix M13mp18 circular single strand, Oligo, Cut Smart Buffer and ddH 2 O was added to a centrifuge tube and mixed;

[0016] c. Place the mixed solution into a PCR instrument, heat it at 95°C for 30 seconds, and then quickly cool it down to 50°C;

[0017] d. Add 1 μL of BtsCI enzyme to the mixed sample at 50°C, mix well, put into PCR instrument, digest at 50°C for 15 min, then heat to 80°C to inactivate the enzyme for 20 min, then cool to 25°C, and store the obtained M13mp18 linear single chain in a 4°C refrigerator for later use.

[0018] In step 1, the number of staple chains is 149 and the number of detection chains is 5, which are named as follows: D 0 , D 1 , D 2 , D 3 , D 4 Each detection chain consists of two parts, one of which is paired with the M13mp18 linear single chain; D 0 , D 1 , D 2 , D 3 , D 4 The other part can be combined with RNA protective chain R x Pairing, can also be paired with the target chain T x Chain pairing; each test chain D x The base length paired with the RNA protection chain is 12bp.

[0019] In step 1, D 0 The pairing position with the M13mp18 linear single strand was designed to be about the 2000th base from the 3' end of the M13mp18 linear single strand.

[0020] In step 1, D 1 With D 0 The distance between them is about 600 bases, D 2 With D 0 The distance between them is about 1200 bases, D 3 With D 0 The distance between them is about 1800 bases, D 4 With D 0 The distance between them is about 2400 bases.

[0021] In step 1, D 0 There are four detection chains, namely D 1 , D 2 , D 3 , D 4, when the target chain T x Connection detection chain D 0 and D x A circular DNA nanoswitch will be formed. In the gel image, four circular DNA nanoswitches with different configurations are S 4 , S 3 , S 2 , S 1 .

[0022] The specific steps of the preparation process of the linear DNA nanoswitch in step 1 are as follows:

[0023] S1. Mix 149 staple chains at a concentration ratio of 1:1 into a centrifuge tube to form a staple chain mixture M1;

[0024] S2. Mix the detection strands at a concentration ratio of 1:1:1:1:1 to form a detection strand mixture M2;

[0025] S3. Mixing the staple chain mixture M1 and the detection chain mixture M2 into a staple chain-detection chain mixture M3;

[0026] S4. Mixing the above M13mp18 linear single strand with the staple strand-detection strand mixture M3 to form a mixture M4;

[0027] S5. Place the mixture M4 into a PCR instrument for an "annealing" procedure, first keep it at 95°C for 5 min, then cool it down to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch;

[0028] S6. Add 190 μL 1X PBS to the above linear DNA nanoswitch for dilution and set aside.

[0029] In step 2, there are 5 RNA protection chains, each of which is 12 bases long and is connected to D 0 , D 1 , D 2 , D 3 , D 4 The chains are complementary to each other.

[0030] In step 2, there are 4 target chains T, each of which is 24 bases long and consists of two parts, one of which is connected to D 0 The other part is connected to D 1 , D 2 , D 3 , D 4 connected.

[0031] The specific steps of the information encryption process in step 2 are as follows:

[0032] I. Add RNA protection strand to the diluted linear DNA nanoswitch and incubate at room temperature for 30 min.

[0033] II. After incubation, add target chain T according to different encryption strategies x Mix well and place in a 4℃ refrigerator for later use.

[0034] The enzyme used in step 3 is RNase A nuclease, which has a working temperature of 37°C and a working time of 10 min. After enzyme digestion, the mixture is incubated at 25°C for 30 min. x With detection chain D 0 and D x They are connected to form circular DNA nanoswitches with different configurations, which will appear as bands at different positions of the gel due to different mobility during gel electrophoresis.

[0035] In the step 3, the concentration of agarose gel is 1.2%, the buffer is 0.5X TBE, the gel electrophoresis condition is 80V voltage, and the running time is 2 hours.

[0036] The agarose gel electrophoresis image decryption method in step 3 is that when the four positions correspond to the presence of a circular DNA nanoswitch, it is represented as 1, and when there is no circular DNA nanoswitch, it is represented as 0. From top to bottom, observe whether there is a circular DNA nanoswitch at the four positions in each swimming band, and represent them with 1 and 0 respectively to obtain a binary representation.

[0037] In step 3, from top to bottom, the circular DNA nanoswitch S n The position is translated into the number 2 n-1 , and then sum them up to translate them into 1 of the decimal numbers 0-9 to decrypt the encrypted information.

[0038] The beneficial effects of the present invention are as follows: the present invention utilizes the DNA nanoswitch formed by self-assembly of DNA molecules, and combines the functional group protection concept in the organic chemical synthesis process to propose a new, fast, and more accurate information encryption and decryption method. In the present invention, the key sequence is protected by the RNA chain to prevent the formation of a circular DNA nanoswitch, thereby realizing information storage and encryption. In the present invention, by adding the target chain T in advance, the target chain T x The target strand is then digested by adding the "key" RNase A nuclease to the RNA protection strand. x With detection chain D x Based on the formation of a circular DNA nanoswitch based on complementary base pairing, RNase A can digest all RNA protective chains in the solution, avoiding the residual RNA protective chain R x With detection chain D xSecondary connection affects the accuracy of information encryption. After RNase A nuclease digests the RNA protection chain, the target chain T x With detection chain D x Connect to form a circular DNA nanoswitch x Combined with agarose gel imaging analysis technology, it can achieve rapid decryption and accurate reading of molecular-level information, providing a basic strategy for promoting the development of nanoscale bioinformation encryption technology. This method can not only be used for digital encryption-decryption, but can also be expanded to other directions such as letter encryption. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the principle of a method for information storage, encryption and decryption based on a DNA nanoswitch of the present invention;

[0040] Figure 2 The decrypted target information of the agarose gel electrophoresis image of the first embodiment of the present invention;

[0041] Figure 3 The decrypted target information of the agarose gel electrophoresis image of the second embodiment of the present invention;

[0042] Figure 4 The decrypted target information of the agarose gel electrophoresis image of the third embodiment of the present invention;

[0043] Figure 5 The decrypted target information of the agarose gel electrophoresis image of the fourth embodiment of the present invention;

[0044] Figure 6 This is the decrypted target information of the agarose gel electrophoresis image of Example 5 of the present invention.

[0045] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0047] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0049] Embodiment 1

[0050] Example DNA information encryption and decryption method using strategy 1:

[0051] (1) Convert the target information, i.e. the decimal numbers “0, 1, 2, 3, 4, 5, 6, 7, 8, 9”, into the corresponding binary codes: 0000, 0001, 0010, 0011, 0100, 0101, 0110, 0111, 1000, 1001.

[0052] (2) The 1 and 0 values ​​on the above four binary digits respectively indicate whether the corresponding circular DNA nanoswitch (S1, S2, S3, S4) at each position is ringed. For example, the 1 on the first digit of the binary digit indicates that S4 is ringed, and 0 indicates that it is not ringed. Therefore, the addition scheme of the protection chain and the target chain corresponding to strategy 1 is shown in Table 1 below.

[0053] Table 1 Adding scheme of protection chain and target chain corresponding to strategy 1

[0054]

[0055] (3) The mixture M4 was placed in a PCR instrument and subjected to an “annealing” procedure, first maintained at 95°C for 5 min, and then cooled to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch, which was then diluted and stored.

[0056] (4) As shown in Table 1, RNA protection strand R0 and corresponding target strand Tx were added to the diluted linear DNA nanoswitch. After incubation for a period of time, due to the presence of R0, the detection strand D0 could not be connected to the target strand Tx, thereby failing to form a circular DNA nanoswitch, i.e., the corresponding band could not be displayed on the agarose gel image, thus achieving information storage and encryption.

[0057] (5) RNase A is added to the reaction solution and incubated for a period of time. When R0 in the reaction solution is digested, the sequence originally protected by R0 in the detection chain D0 is exposed and paired with the target chain Tx to form a circular DNA nanoswitch. The corresponding band is displayed on the agarose gel image, thereby decrypting the encrypted information.

[0058] (6) The strips appearing from top to bottom in each swimming band are represented by 2n-1, and then all the strips are summed up to convert the binary code into a decimal number and decrypt the target information. The result is as follows: Figure 2 shown.

[0059] Embodiment 2

[0060] Example DNA information encryption and decryption method using strategy 2:

[0061] (1) Convert the target information, i.e. the decimal numbers “0, 1, 2, 3, 4, 5, 6, 7, 8, 9”, into the corresponding binary codes: 0000, 0001, 0010, 0011, 0100, 0101, 0110, 0111, 1000, 1001.

[0062] (2) The 1 and 0 values ​​on the above four binary digits respectively indicate whether the corresponding circular DNA nanoswitch (S1, S2, S3, S4) at each position is ring-formed. For example, the 1 on the first digit of the binary digit indicates that S4 is ring-formed, and 0 indicates that it is not ring-formed. Therefore, the addition scheme of the protection chain and the target chain corresponding to strategy 2 is shown in Table 2 below.

[0063] (3) The mixture M4 was placed in a PCR instrument and subjected to an “annealing” procedure, first maintained at 95°C for 5 min, and then cooled to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch.

[0064] (4) As shown in Table 2, RNA protection chains R x After incubation for a period of time, the corresponding target chain T is added x Because R x The existence of makes the detection chain D x Unable to link with target T x Connection, so that a circular DNA nanoswitch cannot be formed, that is, the corresponding band cannot be displayed on the agarose gel image, thus realizing the storage and encryption of information.

[0065] (5) Add RNase A to the above reaction solution and incubate for a period of time. x After digestion, the detection chain D x The original R x The protected sequence is exposed to the target strand T x After pairing, a circular DNA nanoswitch is formed, which displays corresponding bands on the agarose gel image, thus decrypting the encrypted information.

[0066] (6) Use 2 n-1 Then sum all the strips to convert the binary code into decimal numbers and decrypt the target information. The result is as follows Figure 3 shown.

[0067] Table 2 Adding scheme of protection chain and target chain corresponding to strategy 2

[0068]

[0069] Embodiment 3

[0070] Example DNA information encryption and decryption method using strategy 3:

[0071] (1) Convert the target information, i.e. the decimal numbers “0, 1, 2, 3, 4, 5, 6, 7, 8, 9”, into the corresponding binary codes: 0000, 0001, 0010, 0011, 0100, 0101, 0110, 0111, 1000, 1001.

[0072] (2) The 1 and 0 values ​​on the above four binary digits represent the corresponding circular DNA nanoswitch (S 1 , S 2 , S 3 , S 4 ) is a ring. Example: The first digit of a binary number is 1, indicating that S 4 Therefore, the protection chain and target chain adding scheme corresponding to strategy 3 are shown in Table 3 below.

[0073] (3) The mixture M4 was placed in a PCR instrument and subjected to an “annealing” procedure, first maintained at 95°C for 5 min, and then cooled to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch, which was then diluted and stored.

[0074] (4) As shown in Table 3, RNA protective chain R was added to the diluted linear DNA nanoswitch. 1 , R 2 , R 3 , R 4 and the corresponding target chain T x After incubation for a period of time, because R x The existence of detection chain D x Unable to link with target T x Connection, so that a circular DNA nanoswitch cannot be formed, that is, the corresponding band cannot be displayed on the agarose gel image, thus realizing the storage and encryption of information.

[0075] (5) Add RNase A to the above reaction solution and incubate for a period of time. x After digestion, the detection chain D x The original R x The protected sequence is exposed and then binds to the target strand T xThe pairing forms a circular DNA nanoswitch, which displays corresponding bands on the agarose gel image, thereby decrypting the encrypted information.

[0076] (6) Use 2 n-1 Then sum all the strips to convert the binary code into decimal numbers and decrypt the target information. The result is as follows Figure 4 shown.

[0077] Table 3 Adding scheme of protection chain and target chain corresponding to strategy 3

[0078]

[0079] Embodiment 4

[0080] Example DNA information encryption and decryption method using strategy 4:

[0081] (1) Convert the target information, i.e. the decimal numbers “0, 1, 2, 3, 4, 5, 6, 7, 8, 9”, into the corresponding binary codes: 0000, 0001, 0010, 0011, 0100, 0101, 0110, 0111, 1000, 1001.

[0082] (2) The 1 and 0 values ​​on the above four binary digits represent the corresponding circular DNA nanoswitch (S 1 , S 2 , S 3 , S 4 ) is a ring. Example: The first digit of a binary number is 1, indicating that S 4 Therefore, the protection chain and target chain adding scheme corresponding to strategy 4 are shown in Table 4 below:

[0083] Table 4 Addition scheme of protection chain and target chain corresponding to strategy 4

[0084]

[0085] (3) The mixture M4 was placed in a PCR instrument and subjected to an “annealing” procedure, first maintained at 95°C for 5 min, and then cooled to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch, which was then diluted and stored.

[0086] (4) As shown in Table 3, RNA protective chain R was added to the diluted linear DNA nanoswitch. 0 , R x and the corresponding target chain T x After incubation for a period of time, because R 0 , R x The existence of makes the detection chain Dx Unable to link with target T x Connection, so that a circular DNA nanoswitch cannot be formed, that is, the corresponding band cannot be displayed on the agarose gel image, thus realizing the storage and encryption of information.

[0087] (5) Add RNase A to the above reaction solution and incubate for a period of time. x After digestion, the detection chain D x The original R x The protected sequence is exposed to the target strand T x After pairing, a circular DNA nanoswitch is formed, which displays corresponding bands on the agarose gel image, thus decrypting the encrypted information.

[0088] (6) Use 2 n-1 Then sum all the strips to convert the binary code into decimal numbers and decrypt the target information. The result is as follows Figure 5 shown.

[0089] Embodiment 5

[0090] Example DNA information encryption and decryption method using strategy 5:

[0091] (1) Convert the target information, i.e. the decimal numbers “0, 1, 2, 3, 4, 5, 6, 7, 8, 9”, into the corresponding binary codes: 0000, 0001, 0010, 0011, 0100, 0101, 0110, 0111, 1000, 1001.

[0092] (2) The 1 and 0 values ​​on the above four binary digits represent the corresponding circular DNA nanoswitch (S 1 , S 2 , S 3 , S 4 ) is a ring. Example: The first digit of a binary number is 1, indicating that S 4 Therefore, the protection chain and target chain adding scheme corresponding to strategy 5 are shown in Table 5 below.

[0093] Table 5 Addition scheme of protection chain and target chain corresponding to strategy 5

[0094]

[0095] (3) The mixture M4 was placed in a PCR instrument and subjected to an “annealing” procedure, first maintained at 95°C for 5 min, and then cooled to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch, which was then diluted and stored.

[0096] (4) As shown in Table 5, RNA protection chain R was added to the diluted linear DNA nanoswitch. 0 , R 1 , R 2 , R 3 , R 4 and the corresponding target chain T x After incubation for a period of time, because R x The existence of makes the detection chain D x Unable to link with target T x Connection, so that a circular DNA nanoswitch cannot be formed, that is, the corresponding band cannot be displayed on the agarose gel image, thus realizing the storage and encryption of information.

[0097] (5) Add RNase A to the above reaction solution and incubate for a period of time. x After digestion, the detection chain D x The original R x The protected sequence is exposed and then binds to the target strand T x The chains pair to form a circular DNA nanoswitch, which displays corresponding bands on the agarose gel image, thereby decrypting the encrypted information.

[0098] (6) Use 2 n-1 Then sum all the strips to convert the binary code into decimal numbers and decrypt the target information. The result is as follows Figure 6 shown.

[0099] The encryption link in step (4) of the above embodiments 1-5 is to detect the chain D x Design of a portion of the linear single-stranded connection position with M13mp18 and detection of strand D x Another part of the sequence protection design, such as Figure 1 As shown, the following steps are included:

[0100] a. Design detection chain D x Sequence, detection chain D x It consists of two parts, one part is connected to the M13mp18 linear single strand, and the other part is connected to the RNA protection strand and the target strand T strand.

[0101] b. Determine the detection chain D 0 The paired position with the M13mp18 linear single strand, D 0 The pairing position with the M13mp18 linear single strand was designed to be about 2000 bases away from the 3' end of the M13mp18 linear single strand.

[0102] c. Determine the detection chain D 1 , D 2 , D3 , D 4 The paired position with the linear single strand of M13mp18. 1 With D 0 The distance between them is about 600 bases. 2 With D 0 The distance between them is about 1200 bases. 3 With D 0 The distance between them is about 1800 bases. 4 With D 0 The distance between them is about 2400 bases.

[0103] d. Design of RNA protection chain, RNA protection chain R x Designed to be compatible with the detection chain D x Partial sequence pairing.

[0104] In the above embodiments 1-5, the target chain T is added in advance in step (4). x However, due to the existence of the RNA protective chain, the circular DNA nanoswitch cannot be formed at this time.

[0105] In the above embodiments 1-5, the target chain T in step (4) x There are 4 chains, each consisting of two parts, one of which can be connected to D 0 The other part can be connected with D 1 , D 2 , D 3 , D 4 connected.

[0106] In the above embodiments 1-5, the decryption link in step (5) is that the target chain T in step (5) x Addition and selection of RNA digestion enzyme.

[0107] In the above examples 1-5, the RNase A nuclease in step (5) can digest all RNA in the reaction solution with high efficiency and high speed. The addition of this enzyme makes it possible to x Connected protection chain R x is digested, and the target chain T x Chain will detect chain D 0 and detection chain D 1 , D 2 , D 3 , D 4 connected.

[0108] In the above embodiments 1-5, in step (6), due to the target chain T x The special design of the chain makes part of it contact with the detection chain D 0The other part can be connected to the detection chain D 1 , D 2 , D 3 , D 4 Connected, in the target chain T x A circular DNA nanoswitch is formed under the action of the chain, which is connected with different detection chains to obtain circular DNA nanoswitches with different configurations, and the positions presented on the agarose gel are also different.

[0109] In the above Examples 1-5, in step (6), according to the different positions of the circular DNA nanoswitch on the agarose gel electrophoresis image, the position with the circular DNA nanoswitch is represented as 1, and the position without the circular DNA nanoswitch is represented as 0. Observing the four positions in each swimming band from top to bottom, the presence or absence of the circular DNA nanoswitch is represented by 1 and 0, respectively, and then it is decrypted and read according to the conversion relationship between binary and decimal.

[0110] The information encryption and decryption method based on the DNA nanoswitch structure of the present invention comprises a linear DNA nanoswitch, 5 detection chains D x , 5 RNA protection chains R x , 4 target chains T x , and an RNA endoribonuclease (RNase A). In order to facilitate the encryption and decryption of information, the protection chain in the present invention is selected as RNA, the target chain is selected as DNA, and the digestion enzyme is selected as RNase A nuclease. This enzyme has high digestion efficiency and fast action speed, and will not digest the DNA sequence in the reaction solution, so it will not destroy the DNA nanoswitch, ensuring the stability of the nanostructure. 1 With D 0 The distance between them is about 600 bases, D 2 With D 0 The distance between them is about 1200 bases, D 3 With D 0 The distance between them is about 1800 bases, D 4 With D 0 The distance between them is about 2400 bases, which makes the four circular DNA nanoswitches presented in the end have different migration rates on the gel. It can be clearly observed that the positions of circular DNA nanoswitches with different configurations on the gel are different, ensuring the accuracy of reading information.

[0111] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0112] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for information storage, encryption and decryption based on DNA nanoswitch, characterized in that: The following steps are involved: Step 1: Preparation of linear DNA nanoswitch: The information to be transmitted is stored through a combination of circular DNA nanoswitches with different configurations. The circular DNA nanoswitch is formed by connecting the detection chain Dx and the target chain Tx on the linear DNA nanoswitch through the principle of base complementary pairing, while the linear DNA nanoswitch is made by mixing the M13mp18 long single chain with a certain number of staple chains and detection chains using the principle of base complementary pairing, and self-assembly synthesized in a PCR instrument through the "annealing" program; Step 2: Information encryption: Step 2 adopts one of the following strategies: Strategy 1: Add the RNA protection chain R0 paired with the detection chain D0 to the linear DNA nanoswitch solution obtained in step 1, incubate at room temperature to form a DNA-RNA hybrid chain to protect the detection chain D0, and then add the target chain T x Chain, to achieve the storage and encryption of transmitted information; Strategy 2: In the linear DNA nanoswitch solution obtained in step 1, add the detection chain D x Paired RNA protection strand R x , incubate at room temperature to form DNA-RNA hybrid chains, and detect chain D x RNA protected chain R x Protect it and join the target chain T at this time x Chain, to achieve the storage and encryption of transmitted information; Strategy 3: Add RNA protection chains R1, R2, R3, and R4 to the linear DNA nanoswitch solution obtained in step 1 at the same time, and incubate at room temperature to form DNA-RNA hybrid chains, so that the detection chains D1, D2, D3, and D4 are all protected. At this time, add the target chain T x Chain, to achieve the storage and encryption of transmitted information; Strategy 4: Add RNA protection chain R0 and RNA chain R required for encryption to the linear DNA nanoswitch solution obtained in step 1. x , incubate at room temperature to form a DNA-RNA hybrid chain, thereby separating the detection chain D0 and the D x Protect it and join the target chain T at this time x , to achieve the storage and encryption of transmitted information; Strategy 5: Add RNA protection chains R0, R1, R2, R3, and R4 paired with detection chains D0, D1, D2, D3, and D4 to the linear DNA nanoswitch solution obtained in step 1. 1、 R 2、 R 3、 R4, incubate at room temperature to form a DNA-RNA hybrid chain, and then transfer the detection chain D x Protect it and join the target chain T at this time x , to achieve the storage and encryption of transmitted information; Step 3: Information decryption: Add RNase A enzyme to the mixed solution in step 2 and incubate for a period of time, perform agarose gel electrophoresis, and analyze with a gel imager to observe the bands at different positions on the gel image. The presence of a band is marked as 1, and the absence of a band is marked as 0. Then, based on the conversion relationship between binary and decimal, translate the information at different positions on the gel into the corresponding decimal 0-9 to achieve information decryption.

2. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 1 is characterized in that: In the step 1, the long chain is selected as M13mp18 linear single chain, and the specific steps of its preparation process are as follows: a. Form a double strand with a partial sequence in the M13mp18 circular single strand according to the principle of base complementary pairing. The partial sequence is: 5'-TGTATGATTTATTGGATGTTAATGCTACTACTATTAGTAG-3', and its paired sequence is named Oligo. The M13mp18 circular single strand is obtained by proliferating the M13mp18 phage in JM109 competent cells, purifying it by polyethylene glycol precipitation, and then extracting it by alkaline lysis method. The concentration is 250 ng / μL. b. Add M13mp18 circular single strand, Oligo, Cut Smart Buffer and ddH2O into a centrifuge tube and mix well; c. Place the mixed solution into a PCR instrument, heat it at 95°C for 30 seconds, and then quickly cool it down to 50°C; d. Add 1 μL of BtsCI enzyme to the mixed sample at 50°C, mix well, put into PCR instrument, digest at 50°C for 15 min, then heat to 80°C to inactivate the enzyme for 20 min, then cool to 25°C, and store the obtained M13mp18 linear single chain in a 4°C refrigerator for later use.

3. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 2 is characterized in that: In the step 1, there are 149 staple chains and 5 detection chains, which are named as D0, D1, D2, D3, and D4. Each detection chain consists of two parts, one of which is paired with the M13mp18 linear single chain; the other part of D0, D1, D2, D3, and D4 can be paired with the RNA protection chain R x Pairing, can also be paired with the target chain T x Chain pairing; each test chain D x The base length paired with the RNA protection chain is 12bp.

4. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 3 is characterized in that: In the step 1, the pairing position of D0 and the M13mp18 linear single strand is designed to be about the 2000th base from the 3' end of the M13mp18 linear single strand; In the step 1, the distance between D1 and D0 is about 600 bases, the distance between D2 and D0 is about 1200 bases, the distance between D3 and D0 is about 1800 bases, and the distance between D4 and D0 is about 2400 bases.

5. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 4 is characterized in that: In step 1, there are four detection chains except D0, namely D1, D2, D3, and D4. x Connect detection chains D0 and D x A circular DNA nanoswitch will be formed. In the gel image, four circular DNA nanoswitches with different configurations are S4, S3, S2, and S1 from top to bottom.

6. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 5 is characterized in that: The specific steps of the preparation process of the linear DNA nanoswitch in step 1 are as follows: S1. Mix 149 staple chains at a concentration ratio of 1:1 into a centrifuge tube to form a staple chain mixture M1; S2. Mix the detection strands at a concentration ratio of 1:1:1:1:1 to form a detection strand mixture M2; S3. Mixing the staple chain mixture M1 and the detection chain mixture M2 into a staple chain-detection chain mixture M3; S4. Mixing the above M13mp18 linear single strand with the staple strand-detection strand mixture M3 to form a mixture M4; S5. Place the mixture M4 in a PCR instrument and perform an "annealing" program, first keep it at 95°C for 5 minutes, then cool it down to 25°C at a rate of 1°C / min to obtain a linear DNA nanoswitch; S6. Add 190 μL 1X PBS to the above linear DNA nanoswitch for dilution and set aside.

7. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 6 is characterized in that: In step 2, there are 5 RNA protection chains, each of which is 12 bases long and complementary to the D0, D1, D2, D3, and D4 chains respectively; In the step 2, there are 4 target chain T chains, each of which is 24 bases long and consists of two parts, one of which is connected to the D0 chain, and the other is connected to D1, D2, D3, and D4 respectively.

8. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 7 is characterized in that: The specific steps of the information encryption process in step 2 are as follows: I. Add RNA protection strand to the diluted linear DNA nanoswitch and incubate at room temperature for 30 min. II. After incubation, add target chain T according to different encryption strategies x Mix well and place in a 4℃ refrigerator for later use.

9. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 8, characterized in that: The enzyme used in step 3 is RNase A nuclease, which has a working temperature of 37°C and a working time of 10 min. After enzyme digestion, the mixture is incubated at 25°C for 30 min. x With detection chains D0 and D x Connected to form circular DNA nanoswitches with different configurations, which will show bands at different positions of the gel due to different mobility during gel electrophoresis; In the step 3, the concentration of agarose gel is 1.2%, the buffer is 0.5X TBE, the gel electrophoresis condition is 80V voltage, and the running time is 2 hours.

10. The information storage, encryption and decryption method based on DNA nanoswitch according to claim 9, characterized in that: The agarose gel electrophoresis image decryption method in step 3 is that when the four positions correspond to the presence of a circular DNA nanoswitch, it is represented as 1, and when there is no circular DNA nanoswitch, it is represented as 0. From top to bottom, observe whether there is a circular DNA nanoswitch at the four positions in each swimming band, and represent it with 1 and 0 respectively, to obtain a binary representation; In step 3, from top to bottom, the circular DNA nanoswitch S n The position is translated into the number 2 n-1 , and then sum them up to translate them into 1 of the decimal numbers 0-9 to decrypt the encrypted information.

Images