DNA molecular lock based on IIS type enzyme recognition domain crossing and overlapping

Abstract

The invention discloses a DNA molecular lock based on IIS type enzyme recognition domain crossing and overlapping. A hierarchical verification framework of the molecular lock is constructed by differential enzyme digestion characteristics of IIS type FokI enzyme and BaeI enzyme; wherein the related DNA substrates are a four-chain compound S1 / S2 / S3 / S4 and a double-chain compound T1 / T2, when a corresponding IIS type enzyme is input, the substrate and a subsequently input single chain are subjected to a chain displacement reaction, and finally a report chain pops up to prove that unlocking is completed. According to the invention, the DNA molecular circuit can respond in a shorter time by adopting the specific recognition and cutting capability of the IIS type enzyme to DNA double strands, so that the authentication efficiency of the whole DNA molecular lock is improved.

Description

Technical Field

[0001] This invention relates to the field of DNA molecular circuit technology, and more specifically to a DNA molecular lock based on the cross-overlapping of IIS-type enzyme recognition domains. Background Technology

[0002] DNA molecular circuits have long been a research hotspot in the field of biocomputing due to their high-density information storage, efficient parallel processing capabilities, low energy consumption, and excellent biocompatibility. Utilizing the specificity of DNA molecules' base complementarity, programmability, and biocompatibility, they can perform complex information storage and processing operations. In recent years, artificially synthesized DNA molecular circuits have been widely applied in signal transduction, gene network regulation, and other fields, showing great potential, particularly in logic computing, molecular programming, biomolecular detection, information processing, drug delivery, and medical diagnostics. With their programmability and ease of operation, synthetic DNA molecular circuits have become an important tool for simulating and regulating DNA signaling networks, and have provided new avenues for complex molecular information processing.

[0003] The core of DNA encryption is to use DNA molecules as information carriers, leveraging their inherent characteristics of massive parallelism, high storage capacity, and low energy consumption, combined with traditional cryptographic principles, to achieve information encryption, authentication, and transmission at the molecular level. Because DNA cryptography is based on biological challenges, it can resist attacks with supercomputing power and possesses extremely high security. Compared to traditional electronic encryption technologies, it has unique advantages in molecular-level information density and biocompatibility, thus holding significant application value in the future field of information security.

[0004] DNA strand substitution technology is one of the core technologies for realizing DNA molecular locks. This technology designs competitive single strands to bind to the target DNA complex, triggering signal release and thus achieving controllable information processing. The DNA strand substitution reaction is based on the Watson-Crick complementary pairing principle, is a spontaneous reaction, and is characterized by its simplicity and high efficiency. However, to establish a multi-level verification mechanism, the participation of biological enzymes is required. Commonly used biological enzymes include DNA polymerases, RNA polymerases, restriction enzymes, and DNA / RNA ribozymes. Restriction endonucleases, in particular, are ideal tools for constructing genetic switches and feedback loops due to their ability to specifically recognize and cleave DNA sequences. Especially IIS-type restriction endonucleases, by introducing recognition sites at specific locations, can be used to specifically control the cleavage results, designing and implementing DNA molecular lock systems that respond to specific stimuli or signals, providing a powerful molecular biology tool for the development of information security protection devices.

[0005] In summary, DNA molecular circuits and encryption technology, with their unique advantages, have broad application prospects in the fields of biocomputing and information security. However, existing technologies still have shortcomings in areas such as the stability of complex circuits, the multi-verification capability of encryption systems, and the controllability of molecular locks. Therefore, there is an urgent need to develop a novel molecular lock system based on the synergistic effect of DNA strand substitution and enzymatic reactions, optimize signal response specificity and verification levels, improve system stability and application adaptability, provide technical support for high-security information protection and biological application scenarios, and meet the practical needs of fields such as medicine, finance, and biosensing. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a DNA molecular lock based on the cross-overlapping of IIS-type enzyme recognition domains.

[0007] The technical solution provided by this invention is: a DNA molecular lock based on the cross-overlapping recognition domains of IIS-type enzymes, wherein the hierarchical verification architecture of the molecular lock is constructed by the differential enzymatic digestion characteristics of IIS-type FokI enzymes and BaeI enzymes; wherein the DNA substrates involved are four-stranded complexes S1 / S2 / S3 / S4 and two-stranded complexes T1 / T2, and when the corresponding IIS-type enzyme is input, the substrate undergoes a strand substitution reaction with the subsequently input single strand, and finally a report strand is ejected to prove that the unlocking is completed.

[0008] Specifically, the recognition site sequence of the FokI enzyme is 5'-GGATG-3' : 5'-CATCC-3'. When it specifically recognizes and binds to the recognition site, it will perform double-strand cleavage 9-13 nucleotides downstream of the binding site to generate a DNA fragment with sticky ends. The recognition site sequence of BaeI enzyme is 5'-ACNNNNGTAYC-3' : 5'-GRTACNNNNGT-3', where the N site is any base, the Y site is a pyrimidine base, and the R site is a purine base; at the cleavage site, BaeI enzyme cuts on both sides of the recognition site. The recognition and cleavage domains of the FokI enzyme and the BaeI enzyme are designed to overlap. Based on the DNA substrates T1 / T2, the FokI restriction site was designed to be located at the BaeI enzyme recognition site, and the BaeI restriction site was located at the FokI enzyme recognition site.

[0009] Specifically, the DNA substrate S1 / S2 / S3 / S4 includes strands S1, S2, S3 and S4. Strand S1 hybridizes with strands S3 and S4 to form a DNA double helix structure, and overhangs are left on strands S1 and S3. Strand S2 hybridizes with S3 to form a DNA double helix structure, and overhangs are left on strand S3. Encrypted information is stored in strand S4. The DNA substrate T1 / T2 includes strands T1 and T2. Strains T1 and T2 hybridize to form a DNA double helix structure, and a dangling portion is left on the S3 strand. The portion K1 of strand T1, which is 11 nt long from the 3' end, stores encrypted information.

[0010] Specifically, the recognition and cleavage domains of the FokI enzyme and the BaeI enzyme are designed to overlap, including: DNA substrates T1 / T2 and FokI enzyme are placed in a reaction system that can maintain enzyme activity. FokI enzyme binds to the corresponding recognition sites on substrates T1 / T2 and cuts them at specific sites to generate enzyme digestion products A1 / A2 and K1 / K2. Based on the base arrangement of their overhangs, complementary DNA single strands AT and KT are designed as subsequent keys for the molecular lock. DNA substrates T1 / T2 and BaeI enzyme are placed in a reaction system that can maintain enzyme activity. BaeI enzyme binds to the corresponding recognition sites on substrates T1 / T2 and cuts them at specific sites to generate enzyme digestion products F1 / F2 and J1 / J2. Based on the base arrangement of their overhangs, it is ensured that the subsequently designed DNA single strands cannot have a complementary relationship with the base arrangement of their overhangs. DNA substrates S1 / S2 / S3 / S4, to which the recognition site of the FokI enzyme is embedded; DNA substrates S1 / S2 / S3 / S4 and FokI enzyme are placed in a reaction system that can maintain enzyme activity. FokI enzyme binds to the corresponding recognition sites on substrates S1 / S2 / S3 / S4 and cuts them at specific sites to generate enzyme digestion products S1 / N3 / S4. Based on the base arrangement of their overhangs, complementary DNA single strands P1, I1, and I2 are designed as subsequent keys for the molecular lock.

[0011] Specifically, when a corresponding IIS type enzyme is input, the substrate undergoes a chain substitution reaction with the subsequently input single strand, and a report chain is finally popped up to prove that the unlocking is complete: there are two correct keys corresponding to two ways of obtaining ciphertext information; one correct key is FokI-I1-I2, which obtains the single strand S4 containing the final ciphertext when input sequentially; the other correct key is BaeI-AT-KT, which obtains the single strand K1 containing the final ciphertext when input simultaneously.

[0012] This invention provides a DNA molecular lock based on the overlapping recognition domains of IIS-type enzymes. It utilizes the differentiated enzymatic digestion characteristics of IIS-type FokI and BaeI enzymes to construct a hierarchical verification architecture for this molecular lock. IIS-type enzymes can recognize non-palindromic DNA sequences and cleave them at a specific distance beyond the DNA double-strand recognition site sequence to generate customized sticky ends. This enables more complex orthogonal designs and provides flexible control capabilities for dynamic molecular lock systems. The DNA substrates involved are four-stranded complexes S1 / S2 / S3 / S4 and double-stranded complexes T1 / T2. Only when the corresponding IIS-type enzyme is input can the substrate undergo a strand displacement reaction with the subsequently input single strand, ultimately resulting in a report strand indicating successful unlocking.

[0013] This invention, based on the asymmetric cleavage characteristics of IIS-type FokI enzymes, utilizes the spacing between the recognition and cleavage sites of FokI enzymes, combined with Toehold-mediated DNA strand substitution, to provide a hierarchical manipulation method for DNA molecular cascade circuits. Based on the differentiated cleavage characteristics of IIS-type FokI and BaeI enzymes, and through the interpenetration of the recognition and cleavage sites of the two IIS-type enzymes, a flexibly configurable DNA molecular lock is designed and implemented via the modular assembly of DNA molecular circuits.

[0014] This invention utilizes the specific recognition and cleavage capabilities of IIS-type enzymes on double-stranded DNA, enabling DNA molecular circuits to respond in a shorter time, thereby improving the overall authentication efficiency of DNA molecular locks. The cleavage site of the IIS-type enzyme is located outside its recognition site, and the recognition site can be a non-palindromic symmetric sequence. This characteristic allows for precise cleavage without damaging the original DNA sequence, providing a high-precision foundation for the construction of DNA logic gates and logic circuits. By designing different recognition sequences for IIS-type enzymes, various different cleavage results can be achieved, and primers and DNA fragments suitable for specific research needs can also be designed. This programmability allows DNA molecular circuits to be flexibly adjusted according to different requirements, thus meeting the needs of various application scenarios and enabling the construction of more complex molecular lock systems. Attached Figure Description

[0015] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A schematic diagram of a DNA molecular lock based on FokI and BaeI enzymes provided by this invention; Figure 2 PAGE gel electrophoresis image of the molecular lock FokI-I1-I2 provided by this invention; Figure 3 PAGE gel electrophoresis image of the molecular lock BaeI-AT-KT provided by this invention; Figure 4 PAGE gel electrophoresis image of the molecular lock for the input key FokI-I2-I1 provided by this invention; Figure 5 PAGE gel electrophoresis image of the molecular lock for the input key FokI-P1-I1 / I2 provided by this invention; Figure 6 PAGE gel electrophoresis image of the molecular lock for the input key FokI-I1-P1 provided by this invention; Figure 7 PAGE gel electrophoresis image of the molecular lock for the input key BaeI-I1-I2 provided by this invention; Figure 8 PAGE gel electrophoresis image of the molecular lock for the input key FokI-AT-KT provided by this invention; Figure 9 The fluorescence results of DNA molecular locking based on FokI and BaeI enzymes provided by this invention; The lane numbers in the diagram are labeled with the names of the reactants within each lane, and a "+" sign indicates a mixture of reactants. In the diagram, the arrows correspond to the 3' end of the DNA sequence, and the blunt ends correspond to the 5' end. Detailed Implementation

[0018] The present invention will be further explained below with reference to specific implementation schemes, but this explanation does not limit the scope of the invention.

[0019] To address the challenges posed by supercomputers to traditional information security protection, this invention provides a novel DNA molecular lock based on the cross-overlap design of IIS-type enzyme recognition domains. It utilizes the differentiated enzymatic digestion characteristics of IIS-type FokI and BaeI enzymes to construct a hierarchical verification architecture of competitive inhibition and synergistic activation, thereby realizing a highly complex and highly secure dynamic molecular lock system.

[0020] This invention relates to the following specific technical solutions: A novel DNA molecular lock based on the cross-overlap design of IIS-type enzyme recognition domains was first designed. The DNA substrates were designed in which the four-stranded complexes S1 / S2 / S3 / S4 contain the recognition sequence of FokI enzyme 5'-GGATG-3' : 5'-CATCC-3'; the two-stranded complexes T1 / T2 contain both the recognition sequence of FokI enzyme 5'-GGATG-3' : 5'-CATCC-3' and the recognition sequence of BaeI enzyme 5'-ACNNNNGTAYC-3' : 5'-GRTACNNNNGT-3'.

[0021] Taking the schematic diagram of a DNA molecule lock based on FokI and BaeI enzymes as an example, the specific steps include: Step 1: The DNA substrate S1 / S2 / S3 / S4 includes strands S1, S2, S3 and S4. Strand S1 hybridizes with strands S3 and S4 to form a DNA double helix structure, and hangover portions are left on strands S1 and S3; strand S2 hybridizes with S3 to form a DNA double helix structure, and hangover portions are left on strand S3, wherein ciphertext information is stored in strand S4; Step 2: The DNA substrate T1 / T2 includes strands T1 and T2. Strains T1 and T2 hybridize to form a DNA double helix structure, and a hanging portion is left on the S3 strand. The portion (K1) of strand T1 from the 3' end, which is 11nt in length, stores the encrypted information. Step 3: The FokI enzyme recognition site sequences embedded in substrates S1 / S2 / S3 / S4 have been marked in the box. The cleavage sites are 9 nt and 13 nt downstream of the recognition site, indicated by blue scissors. Step 4: The FokI enzyme recognition site sequence embedded in substrate T1 / T2 has been marked in the box, with the cleavage sites at 9nt and 13nt downstream of the recognition site, indicated by blue scissors; the BaeI enzyme recognition site sequence embedded in the substrate has also been marked in the box, with the cleavage sites at 10nt and 15nt upstream of the recognition site and 7nt and 12nt downstream of the recognition site, indicated by orange scissors. Step 5: Based on the DNA substrates T1 / T2 in Step 4, design the specific recognition sequences of FokI and BaeI enzymes on the double strand to ensure that the FokI restriction site is located on the BaeI enzyme recognition site, and the BaeI restriction site is also located on the FokI enzyme recognition site, so as to achieve the competitive inhibition function of the two. Step 6: When FokI is introduced into the substrate, it will first bind to and cleave the corresponding recognition sites on substrates S1 / S2 / S3 / S4, generating enzyme digestion products SI / N3 / S4 and R1 / R2, and bind to and cleave the corresponding recognition sites on substrates T1 / T2, generating enzyme digestion products F1 / F2 and J1 / J2.

[0022] Step 7: According to Step 6, if the correct key FokI-I1-I2 is input at this time, the pre-designed DNA domains a* and b* on the single strand I1 are input. Through complementary base pairing with the bases of the bases a and b on the enzyme digestion products S1 / N3 / S4, a DNA strand substitution reaction is triggered. Under the action of branching migration, the domains c* and d* bind to the domains c and d of strand N3 in S1 / N3 / S4 in turn, displacing the single strand N3 and generating the intermediate product S1 / S4. The pre-designed bases d on the single strand I2 can form a base complementary relationship with the bases d* on the intermediate product S1 / S4. The domain e binds to the domain e* of strand S1 in S1 / S4, displacing the single strand S4. Finally, the report chain generates an output signal, proving that the molecular lock has been unlocked and the encrypted information stored in the single strand S4 has been obtained.

[0023] Step 8: When BaeI is introduced into the substrate, it will first bind to the corresponding recognition site on substrate T1 / T2 and cleave it to generate enzyme digestion products H1 / H2, A1 / A2 and K1 / K2.

[0024] Step 9: According to Step 8, if the input is the correct key BaeI-AT-KT, the pre-designed domain f* on the single-strand AT forms a complementary relationship with the base domain f on the intermediate product A1 / A2. The domain g* on AT combines with the domain g of strand A1 in A1 / A2 under the action of branch migration, popping single-strand A2. The pre-designed h* on the single-strand KT triggers the Toehold-mediated DNA strand displacement reaction by base complementary pairing with the base domain h on the intermediate product K1 / K2. Under the action of branch migration, the domain i* on KT combines with the domain i of strand K2 in K1 / K2, displacing single-strand K1. Finally, the report chain generates an output signal, proving that the molecular lock has been unlocked and the ciphertext information stored in single-strand K1 has been obtained.

[0025] The following is in conjunction with the appendix Figures 1-9 The embodiments and examples will be described in further detail the implementation of the present invention.

[0026] The DNA sequences used in the examples were purchased from Sangon Biotech Co., Ltd., and were purified by PAGE. The DNA molecular sequences in the examples are shown in Table 1. The reagents used in the examples were: EDTA Na2 (disodium ethylenediaminetetraacetate), TRIS (tris(hydroxymethyl)aminomethane), glacial acetic acid, magnesium acetate, ammonium persulfate, polyacrylamide, N,N'-methylenebisacrylamide, tetramethylethylenediamine, and Stains-all. 1×TAE / Mg 2+Buffer solution: 40 mmol / L TRIS, 20 mmol / L acetic acid, 1 mmol / L EDTA Na2, 12.5 mmol / L magnesium acetate, pH=8.0. 40% acrylamide stock solution: 190 g acrylamide and 10 g N,N'-methylenebisacrylamide, dissolved in water at 37°C, then diluted to 500 mL with deionized water. The concentration of all DNA strands was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc. USA). Fluorescence signals were detected using a real-time fluorescence PCR instrument (AgiLent, G8830A), with a maximum absorption wavelength of 550 nm and a maximum emission wavelength of 564 nm. The FokI enzyme, BaeI enzyme, and rCutSmart Buffer used in this example are also described. TM All buffer solutions were purchased from New England Biolabs. A standardized reaction system was maintained: a 50 µL total reaction volume containing 5 µL of 10×rCutSmartBuffer, 1 µL of FokI or BaeI enzyme, 1 µg of DNA substrate, and sterile deionized water to bring the total reaction volume to 50 µL. In this standard reaction system, at 37°C, 1 µL of FokI or BaeI enzyme completely digested 1 µg of DNA substrate within 1 hour. The DNA substrates S1 / S2 / S3 / S4 and T1 / T2 in the examples were prepared by annealing (95°C for 4 min, 65°C for 30 min, 50°C for 30 min, 37°C for 30 min, 25°C for 30 min, and stored at 25°C). Enzymatic digestion was performed at 37°C, and strand displacement was performed at 25°C.

[0027] The results of the examples were detected using PAGE gel electrophoresis.

[0028] Table 1. DNA sequences used in the examples Example 1 Correct key FokI-I1-I2 input for DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 2 Give the PAGE detection results when the correct key FokI-I1-I2 is input. Figure 2In lane 8 of the diagram, after adding FokI, I1, and I2 to the substrate reaction system composed of complexes S1 / S2 / S3 / S4 and T1 / T2, a comparison with the substrate complex bands represented by lanes 2 and 4 shows that the substrate bands have all disappeared, and a band at the same molecular level as the output single-chain S4 shown in lane 1 has been generated. This indicates that the input FokI enzyme has completed its enzymatic digestion reaction, and the intermediate product generated by digestion reacts with single-chain I1 and I2 through a chain substitution reaction to generate the final output chain S4. The fluorescence experimental results are as follows... Figure 9 As shown, there is no obvious increase in fluorescence without any input (curve 1); when the key FokI-I1-I2 is input, a significant increase in fluorescence output signal can be observed (curve 9), which is the same as the results of the PAGE experiment.

[0029] Example 2 Correct key BaeI-AT-KT input for DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 3 Provide the PAGE detection results when the correct key BaeI-AT-KT is input. Figure 3 In lane 7 of the diagram, when BaeI enzyme, single-chain AT, and KT are added to the substrate reaction system composed of complexes S1 / S2 / S3 / S4 and T1 / T2, a significant change in the lane bands can be observed. Compared with the bands represented by substrates S1 / S2 / S3 / S4 in lane 2, it can be seen that substrates S1 / S2 / S3 / S4 are still present in the solution, while the gel band corresponding to substrate T1 / T2 in lane 3 has disappeared, and a gel band appears at the same molecular level as the complex K2 / KT in lane 6. This indicates that the added BaeI enzyme successfully carried out the enzymatic digestion reaction, and its digestion product K1 / K2 underwent a chain substitution reaction with single-chain KT to generate the output complex K2 / KT. Finally, comparing lane 7 with the single-chain K1 band in lane 1, it can be observed that a band exists at the same position as K1, which is the final output chain K1 of the molecular lock. The fluorescence experimental results are as follows. Figure 9 As shown, there is no obvious increase in fluorescence without any input (curve 1); when the key BaeI-AT-KT is input, a significant increase in fluorescence output signal can be observed (curve 8), which is the same as the results of the PAGE experiment.

[0030] Example 3 Typical error key FokI-I2-I1 input DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 4The PAGE detection results are given when the erroneous key FokI-I2-I1 is input. This erroneous key is obtained by swapping the single-chain input order, which verifies the sensitivity of the molecular lock to the timing of operations. Figure 4 In lane 5, after adding FokI enzyme to the substrate reaction system composed of complexes S1 / S2 / S3 / S4 and T1 / T2, comparison with the bands of substrate S1 / S2 / S3 / S4 and intermediate product S1 / S4 / N3 shown in lanes 2 and 3 indicates that FokI digestion is complete, meaning the initial molecular lock recognition is complete. In lane 6, when the input chain I2 is mixed with the solution after the digestion reaction of the previous band, no band corresponding to the expected intermediate product S1 / S4 in lane 7 appears. In lane 8, when the input chain I1 is mixed with the solution after the chain substitution reaction in lane 8, a band corresponding to intermediate product S1 / S4 shown in lane 7 appears, but the output chain S4 band corresponding to lane 1 after correct unlocking does not appear, indicating that molecular lock unlocking has failed. The fluorescence experimental results are as follows. Figure 9 As shown, there is no significant increase in fluorescence without any input (curve 1); when the key FokI-I2-I1 is input, the fluorescence output signal still shows no significant change (curve 5), which is the same as the results of the PAGE experiment.

[0031] Example 4 Typical error key FokI-P1-I1 / I2 input DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 5 The PAGE detection results when the erroneous key FokI-P1-I1 / I2 is input are given. This erroneous key blocks the subsequent cascade reaction by adding a single-chain P1 with a preferential substitution rate before the input single-chain I1 / I2, which utilizes its faster chain substitution kinetics to cover the Toehold region exposed by the enzyme digestion product. Figure 5 In lane 5, after adding FokI enzyme to the substrate reaction system composed of complexes S1 / S2 / S3 / S4 and T1 / T2, comparison with the bands of substrate S1 / S2 / S3 / S4 and intermediate product S1 / S4 / N3 represented in lanes 2 and 3 shows that FokI digestion is complete, indicating that the molecular lock has been initially recognized. In lane 6, after mixing the solution of input chain P1 with the digestion reaction of the previous band, a band at the same position as S1 / S4 / P1 in lane 4 can be observed. In lane 8, after mixing the solution of the two input chains I1 and I2 with the chain displacement reaction of the previous band, the band corresponding to the intermediate product S1 / S4 / P1 present in lanes 4 and 6 does not disappear, and the output chain S4 band corresponding to lane 1 after correct unlocking does not appear, indicating that the molecular lock unlocking has failed. The fluorescence experiment results are as follows. Figure 9As shown, there was no significant increase in fluorescence without any input (curve 1); when the key FokI-P1-I1 / I2 was input, the fluorescence output signal still showed no significant change (curves 3 and 4), which is the same as the results of the PAGE experiment.

[0032] Example 5 Typical error key FokI-I1-P1 input DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 6 The PAGE detection results are given when the erroneous key FokI-I1-P1 is input. This erroneous key is intended to test for interference from non-temporally sequential intermediate products. Figure 6 In lane 5, after adding FokI enzyme to the substrate reaction system composed of complexes S1 / S2 / S3 / S4 and T1 / T2, the band corresponding to substrate S1 / S2 / S3 / S4 in lane 2 disappeared, and the band corresponding to the enzyme digestion product S1 / S4 / N3 in lane 3 appeared, indicating that FokI digestion was complete and the molecular lock was initially correctly identified. In lane 6, after mixing the input chain I1 with the solution after the digestion reaction of the previous band, a band corresponding to the intermediate product S1 / S4 in lane 7 was observed. However, in lane 8, after mixing the input chain P1 with the solution after the substitution reaction of the previous band, the band corresponding to the intermediate product S1 / S4 in lane 7 remained almost unchanged, and the output chain S4 band corresponding to lane 1 after correct unlocking did not appear, indicating that the molecular lock unlocking failed. The fluorescence experimental results are as follows. Figure 9 As shown, there is no significant increase in fluorescence without any input (curve 1); when the key FokI-I1-P1 is input, the fluorescence output signal still shows no significant change (curve 2), which is the same as the PAGE experimental results.

[0033] Example 6 Typical error key BaeI-I1-I2 input DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 7 The PAGE detection results are given when the erroneous key BaeI-I1-I2 is input. This erroneous key is intended to detect interference between the input enzyme and the single-stranded non-matching key. Figure 7In lane 6, after adding BaeI enzyme to the substrate reaction system consisting of complexes S1 / S2 / S3 / S4 and T1 / T2, no significant change in the band pattern can be observed. Comparison with the bands corresponding to substrates S1 / S2 / S3 / S4 and T1 / T2 in lanes 2 and 4 shows that the original substrate bands did not disappear, indicating that the input of BaeI did not trigger the enzymatic cleavage reaction, and the initial recognition of the molecular lock failed. Subsequent lanes 7 and 9 did not show the output strand S4 band corresponding to lane 1 after correct unlocking, further confirming the failure of molecular lock unlocking. The fluorescence experiment results are as follows... Figure 9 As shown, there is no significant increase in fluorescence without any input (curve 1); when the key BaeI-I1-I2 is input, the fluorescence output signal still shows no significant change (curve 7), which is the same as the results of the PAGE experiment.

[0034] Example 7 Typical error key FokI-AT-KT input DNA molecular lock verification according to Figure 1 The design diagram of the DNA molecular lock, Figure 8 The PAGE detection results are given when the erroneous key FokI-AT-KT is input. This erroneous key is intended to detect interference between the input enzyme and the single-stranded non-matching key. Figure 8 In lane 8, after adding FokI enzyme, single-chain AT, and KT to the substrate reaction system composed of complexes S1 / S2 / S3 / S4 and T1 / T2, comparison with the bands corresponding to the intermediate products S1 / N3 / S4 in lane 3 shows that the input of FokI enzyme triggered the enzymatic cleavage reaction, indicating initial successful recognition of the molecular lock. However, comparison with the output chain K1 band in lane 2 revealed no corresponding gel band after correct unlocking, proving that the molecular lock unlocking failed. Fluorescence experimental results are as follows... Figure 9 As shown, there is no significant increase in fluorescence without any input (curve 1); when the key BaeI-I1-I2 is input, the fluorescence output signal still shows no significant change (curve 6), which is the same as the results of the PAGE experiment.

[0035] In summary, this invention constructs a DNA molecular lock based on the differentiated enzymatic digestion characteristics of IIS-type FokI and BaeI enzymes through the modular assembly of DNA molecular circuits. This DNA molecular lock can achieve modular assembly and flexible configuration through the interpenetration of recognition and cleavage sites of the two IIS-type enzymes and the encoding of DNA functional domains. Furthermore, combined with the permutation and combination of DNA sequence key inputs and logical control, it can achieve information protection at the molecular level: unlocking and releasing the ciphertext when the correct enzyme and sequence key combination is input, and maintaining a locked state when there is an incorrect input or no input. By transforming the specific regulatory capabilities of proteases into the core logic of security authentication, and through the modular design of DNA molecular circuits, the molecular lock system can flexibly adapt to different security level requirements, providing a new technical method for the subsequent construction of information security protection molecular devices with high sensitivity and multiple verification mechanisms.

[0036] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0037] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A DNA molecule lock based on the cross-over of IIS type enzyme recognition domains, characterized in that, The hierarchical verification architecture of the molecular lock is constructed by the differential enzyme cutting characteristics of the IIS type FokI enzyme and the BaeI enzyme; the DNA substrates involved are a four-strand complex S1 / S2 / S3 / S4 and a double-strand complex T1 / T2, and when the corresponding IIS type enzyme is input, the substrate and the subsequently input single strand undergo strand displacement reaction, and finally the report strand is popped out to prove that the unlocking is completed.

2. The DNA molecular lock based on the cross-over of IIS type enzyme recognition domain according to claim 1, characterized in that, The recognition site sequence of the FokI enzyme is 5'-GGATG-3' : 5'-CATCC-3', when it specifically recognizes and binds to the recognition site, it will perform double-strand cutting at 9-13 nucleotides downstream of the binding site, generating a DNA fragment with a sticky end; The recognition site sequence of the BaeI enzyme is 5'-ACNNNNGTAYC-3' : 5'-GRTACNNNNGT-3', wherein the N site is any base, the Y site is a pyrimidine base, and the R site is a purine base; at the enzyme cutting position, the BaeI enzyme cuts on both sides of the recognition site; The recognition domain and the cutting domain of the FokI enzyme and the BaeI enzyme are cross-overlapping designed; According to the DNA substrate T1 / T2, the FokI enzyme cutting site is designed to be on the recognition site of the BaeI enzyme, and the BaeI enzyme cutting site is designed to be on the recognition site of the FokI enzyme.

3. The DNA molecular lock based on the cross-over of IIS type enzyme recognition domain according to claim 1, characterized in that, The DNA substrate S1 / S2 / S3 / S4 includes strands S1, S2, S3 and S4, the strand S1 is hybridized with the strands S3 and S4 to form a DNA double helix structure, and a overhang is left on the strands S1 and S3; the strand S2 is hybridized with the strand S3 to form a DNA double helix structure, and a overhang is left on the strand S3, wherein the ciphertext information is stored in the strand S4; The DNA substrate T1 / T2 includes strands T1 and T2, which are hybridized to form a DNA double helix structure, and a overhang is left on the S3 strand, wherein the ciphertext information is stored in the 11nt length part K1 of the strand T1 from the 3' end.

4. The DNA molecular lock based on the cross-over of IIS type enzyme recognition domain according to claim 1, characterized in that, The recognition domain and the cutting domain of the FokI enzyme and the BaeI enzyme are cross-overlapping designed, including: The DNA substrate T1 / T2 and the FokI enzyme are placed in a reaction system capable of maintaining enzyme activity, the FokI enzyme binds to the corresponding recognition site on the substrate T1 / T2 and cuts at a specific site to generate enzyme cutting products A1 / A2 and K1 / K2, according to the base arrangement of the overhang, the complementary DNA single strands AT and KT are designed as the subsequent key of the molecular lock; The DNA substrate T1 / T2 and the BaeI enzyme are placed in a reaction system capable of maintaining enzyme activity, the BaeI enzyme binds to the corresponding recognition site on the substrate T1 / T2 and cuts at a specific site to generate enzyme cutting products F1 / F2 and J1 / J2, according to the base arrangement of the overhang, it is ensured that the subsequently designed DNA single strands cannot have complementary relationship with the base arrangement of the overhang; The DNA substrate S1 / S2 / S3 / S4 has the recognition site of the FokI enzyme embedded thereon; DNA substrate S1 / S2 / S3 / S4 and FokI enzyme are placed in a reaction system capable of maintaining enzyme activity, the corresponding recognition site on the substrate S1 / S2 / S3 / S4 is combined by FokI enzyme and cut at a specific site to generate enzyme digestion product S1 / N3 / S4, according to the base arrangement of the overhang, the complementary DNA single strand P1, I1 and I2 are designed as the subsequent key of the molecular lock.

5. The DNA molecular lock based on the cross-over of IIS type enzyme recognition domain according to claim 1, characterized in that, The substrate and the subsequently input single strand undergo strand displacement reaction when the corresponding IIS type enzyme is input, and the final report chain is popped out to prove that the unlocking is completed: there are two correct keys corresponding to two ways of obtaining the ciphertext information; one correct key is FokI-I1-I2, which obtains the single strand S4 where the final ciphertext is located when inputting in sequence; the other correct key is BaeI-AT-KT, which obtains the single strand K1 where the final ciphertext is located when inputting simultaneously.

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