A DNA information double-layer encryption method and system based on gradient fluorescent nucleotides and application thereof

Abstract

The application discloses a DNA information double-layer encryption method and system based on gradient fluorescent nucleotides and application thereof, and belongs to the field of DNA data storage and information security. The method encrypts original information through an algorithm to generate a digital key and ciphertext, encodes the ciphertext into a DNA sequence and clones the DNA sequence into a host bacterium for amplification and storage; a digital-fluorescence mapping table is constructed to map the digital key into an oligonucleotide probe containing gradient fluorescent nucleotides, and a primer extension reaction is performed to prepare a fluorescent key probe. When decryption is performed, the ciphertext is recovered through sequencing, the digital key is reconstructed by measuring the fluorescence intensity, and the original information is obtained through decryption. The application physically stores the key in the form of fluorescence intensity in the DNA molecule, realizes double-layer encryption of sequence information and fluorescence information, physically separates the key from the ciphertext, and significantly improves information security. The fluorescence intensity is linearly related to the number of gradient fluorescent nucleotides, multi-level gray code encoding can be realized, the storage density is expanded, and the application has a wide prospect in the fields of information security and anti-fake identification.

Description

Technical Field

[0001] This invention belongs to the field of DNA data storage and information security technology, specifically relating to a DNA information double-layer encryption method, system and application based on gradient fluorescent nucleotides, and particularly to a molecular encryption technology that utilizes the quantitative fluorescence characteristics of fluorescent nucleoside analog 3b to achieve physical storage of keys and visualization of information. Background Technology

[0002] DNA, as an information storage medium, possesses advantages such as ultra-high density, extremely low energy consumption, and extremely long storage period, and is widely recognized as one of the important directions for future information storage. With the rapid decline in the cost of DNA synthesis and sequencing, DNA storage technology is gradually moving from the laboratory to practical applications. However, the information security issues within DNA are also becoming increasingly prominent, becoming a key bottleneck restricting its large-scale application.

[0003] Existing DNA information encryption technologies can be mainly categorized as follows: physical encryption methods based on enzyme-cut hairpin structures, biological encryption methods based on CRISPR / Cas, algorithmic encryption methods based on DNA encoding and chaotic mapping, and information storage methods based on Z-DNA structures. Chinese patent application CN105119717A proposes a DNA encoding-based encryption system that uses primer sequences as keys to encode information into DNA base sequences, achieving information encryption and transmission. However, this type of method still belongs to single-dimensional sequence encryption; the key information still exists in sequence form. Once an attacker obtains the DNA sequence, they can recover all the information through sequencing, posing a risk of duplication or theft. Furthermore, existing technologies also propose the idea of ​​double-layer encryption. Chinese patent application CN120263395A uses a combination of AES and RSA to achieve double-layer encryption of computer files, improving the security of data transmission. However, this type of method still relies on mathematical algorithms for encryption, and the key exists in digital form, which cannot resist physical-level information theft and is difficult to directly apply to DNA storage systems.

[0004] In summary, current technologies rely excessively on DNA sequence encryption, failing to fully utilize the chemical modification properties of DNA molecules to construct multi-dimensional information encryption systems. There is a lack of encryption methods that can physically embed digital keys into DNA molecules and whose key information can be quantitatively read via fluorescence signals. While the fluorescence properties of nucleotide analogs have been extensively studied for nucleic acid detection, their application as "brightness-controllable display units" in the field of information encryption has not yet been reported. Summary of the Invention

[0005] To address the current technological limitations of relying excessively on DNA sequence encryption, failing to utilize the chemical modification properties of DNA molecules to construct multidimensional encryption systems, lacking methods for physically embedding digital keys into DNA and quantitatively reading them via fluorescence signals, and failing to apply the fluorescence properties of nucleotide analogs as "brightness-controllable display units" in the field of information encryption, this invention aims to provide a method, system, and application of dual-layer DNA information encryption based on gradient fluorescent nucleotides. This method physically stores digital keys in the form of fluorescence intensity within DNA molecules, achieving dual encryption of both sequence and fluorescence information.

[0006] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a method for double-layer encryption of DNA information based on gradient fluorescent nucleotides, comprising the following steps: S1, obtain the original information to be encrypted and the preset digital key, encrypt the original information using an encryption algorithm to generate ciphertext information, and encode the ciphertext information as the first DNA sequence; S2, Prepare a fluorescent key probe, wherein the fluorescent key probe is a DNA molecule incorporating gradient fluorescent nucleotides, and the number of gradient fluorescent nucleotides incorporated has a preset mapping relationship with the digital key; S3, construct the first DNA sequence into a biological vector to obtain a biological vector containing ciphertext; preserve the fluorescent key probe as an independent DNA molecule in combination with the biological vector containing ciphertext; S4, read the first DNA sequence from the biological vector containing the ciphertext to recover the ciphertext information, measure the fluorescence intensity of the fluorescent key probe to reconstruct the digital key, use the reconstructed digital key to decrypt the recovered ciphertext information, and output the original information.

[0007] In step S1, the encryption algorithm used in the encryption process is selected from one or a combination of the Blowfish algorithm, AES encryption algorithm, DES encryption algorithm, RSA encryption algorithm, or SHA-256 encryption algorithm; the encoding adopts a DNA coding table constructed based on tetra- or penta-base units, or a network-based encoding platform. This seamless integration of traditional computer encryption algorithms with biological DNA encoding technology broadens the application scope of the present invention.

[0008] In step S2, the steps for preparing the fluorescent key probe include: constructing a digit-fluorescence mapping table to map each digit of the digit key to an oligonucleotide containing gradient fluorescent nucleotide residues; and incorporating the number of gradient fluorescent nucleotides corresponding to the mapping relationship into the DNA template through a primer extension reaction to obtain the fluorescent key probe.

[0009] Precise conversion from digital keys to fluorescence signals was achieved: by constructing a digital-fluorescence mapping table, abstract numbers were transformed into physically measurable fluorescence signals; primer extension reactions enabled precise quantitative incorporation of gradient fluorescent nucleotides. For the first time, primer extension reactions were transformed from conventional PCR amplification into a "key encoding tool," enabling quantitative regulation of fluorescence signals.

[0010] Preferably, the gradient fluorescent nucleotide is 7-(thien-2-yl)-imidazo[4,5-b]pyridine-2′-deoxynucleotide (hereinafter referred to as 3b). A single incorporation can enhance the fluorescence intensity by about 5 times compared to the free monomer, and the enhancement of fluorescence intensity is approximately linearly proportional to the amount of incorporation.

[0011] Preferably, in the primer extension reaction, the DNA template sequence is a preset sequence, and gradient fluorescent nucleotides are spaced apart on the oligonucleotides; the number of gradient fluorescent nucleotides incorporated is 1-10. The preset sequence ensures the reproducible preparation of the probe; the spaced distribution (8-10 bases) avoids mutual quenching between fluorescent groups, ensuring a linear relationship between fluorescence intensity and the number of incorporated nucleotides; the number of incorporated nucleotides (1-10) provides at least 10 levels of grayscale encoding capability, ensuring a linear relationship of fluorescence signal and encoding capacity.

[0012] More preferably, the DNA template uses a random sequence, which means that each position is independently selected from any one of the four bases A, T, C, and G, to ensure that the incorporation positions of the gradient fluorescent nucleotides have sequence diversity.

[0013] More preferably, the gradient fluorescent nucleotide has an incorporation site every 8 bases on the oligonucleotide, and the number of incorporations is 1-6.

[0014] Step S3 specifically includes: cloning the first DNA sequence into a plasmid vector, transforming it into a host bacterium for amplification culture, and obtaining a host bacterium containing encrypted information; and storing the fluorescent key probe in DNA molecule form together with the host bacterium containing the first DNA sequence or storing them separately.

[0015] Combining biological amplification technology with physical key storage resolves the conflict between information replication and key security in DNA storage.

[0016] Preferably, the plasmid vector is pUC57-Kan+, the host bacterium is Escherichia coli DH5α, and the amplification culture exceeds 100 generations to verify the long-term stability of the information.

[0017] In step S4, the fluorescence intensity of the fluorescent key probe is read by a fluorescence quantitative measurement device and converted into a heat map or pattern to realize the visualization of the key information.

[0018] Secondly, the present invention provides a DNA information double-layer encryption system for implementing the above method, characterized in that it comprises: The ciphertext processing module is used to process the original information to be encrypted into a digital key and ciphertext information, and to encode the ciphertext information into a first DNA sequence; A key fluorescence module, connected to the ciphertext processing module, is used to receive the digital key and incorporate a corresponding number of gradient fluorescent nucleotides into the DNA sequence based on the digital key to form a fluorescent key probe; wherein, the fluorescence intensity of the fluorescent key probe is proportional to the number of nucleotides incorporated, and the number of nucleotides incorporated has a preset mapping relationship with the digital key; The vector construction module is connected to the ciphertext processing module and the key fluorescence module, respectively, and is used to receive the first DNA sequence and construct it into a biological vector to obtain a biological vector containing ciphertext, and output the fluorescent key probe in the form of an independent DNA molecule combined with the biological vector containing ciphertext; The information decryption module, connected to the carrier construction module, is used to read the first DNA sequence from the biological carrier to recover the ciphertext information, measure the fluorescence intensity of the fluorescent key probe to reconstruct the digital key, and use the reconstructed digital key to decrypt the recovered ciphertext information and output the original information.

[0019] It also includes a standard curve establishment module, which is used to pre-establish a linear regression model of fluorescence intensity and dopant quantity, and to calibrate the detection sensitivity of different fluorescence channels.

[0020] Thirdly, the present invention provides a fluorescent key probe, wherein the fluorescent key probe is an oligonucleotide synthesized through a primer extension reaction, and the oligonucleotide incorporates a specific number of gradient fluorescent nucleotides; the number of gradient fluorescent nucleotides incorporated is uniquely determined by a digital key according to a preset digital-fluorescence mapping table, and the number of incorporations is proportional to the fluorescence intensity of the fluorescent key probe; the nucleotide sequence of the fluorescent key probe is a preset sequence, and the gradient fluorescent nucleotides are spaced apart on the oligonucleotide; the fluorescence intensity of the fluorescent key probe can be read by a fluorescence quantitative measurement device, and the reading of its fluorescence intensity does not depend on the determination of its DNA sequence.

[0021] Fourthly, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the program, when executed by a processor, implements the steps of the method described in the first aspect.

[0022] Fifthly, the present invention provides an application of a combined product comprising a fluorescent key probe and a host bacterium containing a ciphertext DNA sequence in secure information storage, anti-counterfeiting identification, or steganography.

[0023] The fluorescent key probe is the fluorescent key probe described above, and the host bacteria containing the encrypted DNA sequence is obtained by cloning the DNA sequence obtained by the method into a plasmid vector and transforming the host bacteria; the steganography information hiding uses the fluorescence intensity of the fluorescent key probe as the physical carrier of the hidden information to achieve the covert transmission and storage of information.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a double-layer encryption method for DNA information based on gradient fluorescent nucleotides, achieving double-layer encryption protection: the first layer is sequence information (ciphertext), and the second layer is fluorescent information (key). Attackers must simultaneously crack two different physical dimensions of information to obtain the original content, significantly improving information security. For the first time, the digital key is stored in the DNA molecule in a physicochemical modified form. The key is no longer a string of numbers or a sequence, but exists as a physical property of "the number of fluorescent molecules incorporated." The key can only be reconstructed by measuring fluorescence intensity, achieving a leap from "readable information" to "measurable physical quantity." Storing the digital key in physical form (fluorescence intensity) avoids the risk of traditional electronic keys being easily copied and stolen. The physical separation of the key and ciphertext (combined storage) increases the difficulty of cracking, achieving a secure mode of "separate storage and combined use." Decryption requires two completely different technical methods (sequencing + fluorescence), and even after thousands of generations, the ciphertext information can still be accurately read, with good stability of the fluorescent probe, enabling successful key reconstruction and decryption. This invention is the first to store digital keys in a physicochemical form, which is the amount of gradient fluorescent nucleotides incorporated. This achieves physical separation and protection of the key and ciphertext, and constructs a two-layer encryption system of sequence information and fluorescence information. This fundamentally solves the problems of easy key acquisition through sequencing and single security dimension in traditional DNA encryption technology.

[0025] Furthermore, the fluorescence signal is quantifiable, with a large encoding capacity: the number of gradient fluorescent nucleotides incorporated is highly linearly correlated with fluorescence intensity, enabling multi-level grayscale encoding. A single fluorescent site can encode multiple bits of information, expanding the information storage density. It exhibits good biocompatibility and high stability: experimental verification shows that plasmids containing encrypted information can still be accurately read after more than 100 generations of amplification in host bacteria; the fluorescent key probe maintains stable fluorescence characteristics under the same conditions, demonstrating good long-term preservation potential. It also boasts strong visualization capabilities and a wide range of applications: the fluorescence signal can be converted into a heatmap using a microplate reader, enabling the visualization of information and facilitating various applications such as anti-counterfeiting labels and steganography.

[0026] The system provided by this invention organically integrates biochemical operations (primer extension, cloning transformation) with an information processing module, forming a complete encryption system from input to output. The modular design allows each functional module to be independently optimized and upgraded (e.g., by replacing with a stronger encryption algorithm or using novel fluorescent nucleotides), exhibiting excellent scalability. The system interface is clearly defined, facilitating engineering implementation and commercial application; thus providing a technological foundation for automated, high-throughput DNA information encryption.

[0027] The fluorescent key probe product provided by this invention creates a completely new information carrier. It is no longer a "sequence" that stores information, but a "physical probe" that stores a key, thus expanding the functional boundaries of DNA molecules. This probe features: the number of elements incorporated is uniquely determined by a digital key; fluorescence intensity is proportional to the number of elements incorporated; the sequence is pre-defined, and the fluorescent groups are spaced apart; fluorescence reading is independent of sequence determination. These characteristics make it an ideal carrier for realizing the function of a "physical key." As a standalone product, the fluorescent key probe can be manufactured, sold, and applied independently. "Independent of sequence determination" means that even if the probe sequence is determined, the key information cannot be obtained, which is a fundamental difference from traditional DNA encryption.

[0028] The applications provided by this invention combine fluorescent key probes with host bacteria containing encrypted text, applying them to specific scenarios such as secure information storage (long-term preservation of confidential data), anti-counterfeiting identification (anti-counterfeiting of luxury goods and important documents), and steganography (information hiding). This advances biological encryption technology from theoretical research to practical application, covering multiple commercially valuable fields such as information security, brand protection, and covert communication. Attached Figure Description

[0029] Figure 1 A schematic diagram of the synthetic steps of fluorescent nucleosides 3a and 3b and their corresponding active forms; Figure 2 For fluorescent nucleoside 3b 1 H NMR characterization image; Figure 3 For fluorescent nucleoside 3b 13 C10 NMR characterization image; Figure 4 This is a high-resolution mass spectrometry (HR-MS) characterization of fluorescent nucleoside 3b; Figure 5 This is a schematic diagram of the DNA information double-layer encryption method based on gradient fluorescent nucleotides according to the present invention; Figure 6The diagram shows the properties of 3b incorporation into DNA using gradient fluorescent nucleotides. (A) shows the workflow of 3b-incorporated oligonucleotides in data visualization; (B) shows the protocol for 3b incorporation using Deepvent DNA polymerase and FAM-labeled primers for primer extension reactions; (C) shows PAGE analysis of primer extension reaction products containing different numbers of 3b residues, with lane 1 showing the primers and lanes 2-7 showing primer extension reaction products containing 1-6 3b residues, visualized in the FAM channel; (D) shows the protocol for 3b incorporation using Deepvent DNA polymerase and non-fluorescent primers for primer extension reactions; (E) shows the relative fluorescence intensity of primer extension reaction products containing 1-6 3b residues relative to free 3b monomers; (F) shows the "2025" pattern displayed by 3b-incorporated oligonucleotides in a 96-well plate; and (G) shows the visual effect of the "face" pattern. Figure 7 The image shows the sequencing results of a plasmid containing DNA sequence 1, extracted after 2000 generations of transmission of an ancient Chinese poem. Figure 8 The image shows the sequencing results of a plasmid containing DNA sequence 2, extracted after 2000 generations of transmission of an ancient Chinese poem. Figure 9 The image shows the sequencing results of a plasmid containing DNA sequence 3, extracted after 2000 generations of transmission of an ancient Chinese poem. Figure 10 This is a schematic diagram of the module structure of the DNA information double-layer encryption system of the present invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] The chemical name of gradient fluorescent nucleotide 3b is 7-(thiophen-2-yl)-imidazo[4,5-b]pyridine-2′-deoxynucleotide.

[0032] The Deep vent DNA polymerase used in this embodiment of the invention was purchased from New England Biolabs, catalog number M0258S; the KOD XL DNA polymerase was purchased from Sigma-Aldrich, catalog number 71087; and the plasmid extraction kit was purchased from Guangzhou Meiji Biotechnology Co., Ltd., catalog number P1112-03.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1: Preparation and characterization of gradient fluorescent nucleotide 3b Figure 1 This paper presents a complete synthetic route for the synthesis of fluorescent nucleosides 3a and 3b from starting materials through multiple reactions, and their further conversion into their phosphorus amide monomers (5a and 5b) and triphosphate active forms (6a and 6b). Compound 1 is a key intermediate, a 5-cyano-2'-deoxyuridine derivative; compound 2 is a 5-thioformamide-substituted nucleoside intermediate; compounds 3a and 3b are fluorescent nucleoside core molecules modified with different aromatic ketone structures; compounds 4a and 4b are 5'-O-dimethoxytriphenylmethyl protected forms; compounds 5a and 5b are phosphorus amide active monomers used for the solid-phase synthesis of oligonucleotides; and compounds 6a and 6b are the corresponding triphosphate derivatives. In the diagram, the R group represents different aromatic substitution structures (a and b represent different fluorescent parent nuclei). This example focuses on the preparation and characterization of the target molecule 3b, its phosphorus amide monomer 5b, and triphosphate compound 6b.

[0034] This embodiment provides a method for synthesizing gradient fluorescent nucleotide 3b and confirming its structure. Compound 3b is a core modified nucleoside with photophysical properties. To incorporate it into the DNA strand for data encryption and storage, it must first be converted into the corresponding phosphoramidite active monomer. The complete process of synthesizing 3b and its derivatives from precursor molecule 1 is described in detail below.

[0035] 1. Synthesis of intermediates and target compounds (i) Synthesis of compound 1 2'-Deoxyuridine (5.0 g, 21.9 mmol), 4-dimethylaminopyridine (268 mg, 0.1 eq), and triethylamine (6.7 g, 65.7 mmol, 3.0 eq) were dissolved in acetonitrile (80 mL), and acetic anhydride (5.6 g, 54.8 mmol, 2.5 eq) was added. After stirring for 30 min, methanol (10 mL) was added to quench the reaction, and stirring was continued for 10 min. The mixture was concentrated under reduced pressure, and the residue was extracted with dichloromethane (2 × 80 mL). The organic phases were combined, washed with saturated brine (100 mL × 3), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give a colorless foamy substance (6.5 g, 20.8 mmol, 95%).

[0036] The product was dissolved in acetonitrile (70 mL), and iodine (3.2 g, 12.5 mmol, 0.6 eq) and cerium ammonium nitrate (5.5 g, 10.4 mmol, 0.5 eq) were added. The mixture was stirred at 80 °C for 1 h. After cooling, the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (150 mL), washed successively with sodium thiosulfate aqueous solution (100 mL) and saturated sodium chloride solution (150 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give a white solid 5-iodinated product (8.7 g, 19.9 mmol, 95%).

[0037] The solid was dissolved in pyridine (70 mL), and hexamethyldisilazane (25 mL, 119.4 mmol, 6.0 eq) was added. The mixture was stirred at room temperature for 12 h under argon protection. Pyridine was removed by vacuum distillation, and the resulting colorless oil was redissolved in anhydrous pyridine (100 mL). Cuprous cyanide (10.7 g, 119.4 mmol, 6.0 eq) was added, and the mixture was heated to 120 °C and stirred for 6 h under argon protection. The mixture was cooled to room temperature, and pyridine was removed by vacuum distillation. The residue was dissolved in ethyl acetate (200 mL), filtered through a diatomaceous earth filter, and washed thoroughly with ethyl acetate. The filtrate and washings were combined, washed with saturated brine (125 mL × 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a light gray foamy substance (6.4 g, 87% overall yield of the three steps). This substance was used directly in the next reaction without purification.

[0038] (ii) Synthesis of compound 2 Compound 1 (2.0 g, 6.0 mmol) was dissolved in DMF (16 mL), and 70% sodium hydrosulfide aqueous solution (1.44 g, 18 mmol, 3.0 eq), diethylamine hydrochloride (2.97 g, 27 mmol, 4.5 eq), pyridine (1.5 mL, 18 mmol, 3.0 eq), and water (4 mL) were added. The mixture was stirred at 80 °C for 3 h under argon protection. The reaction solution was poured into brine (70 mL, 2 M) and extracted with ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (50 mL × 1), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting yellow crude solid was slurried with ethyl acetate (15 mL) to give a yellow solid protected nucleoside 2 (1.6 g, 73%). 1 H NMR (400 MHz, DMSO-) d6 Confirmed by high-resolution mass spectrometry (HRMS-ESI): 1 H NMR (400 MHz, DMSO-) d6 ): δ 8.21 (s, 1H, H-6), 6.18 (t, J= 6.4 Hz, 1H, H-1'), 5.82 (s, 2H, NH2), 4.32-4.28 (m, 1H, H-3'), 3.98-3.94 (m, 1H, H-4'), 3.71-3.65 (m, 2H, H-5'), 2.31-2.24 (m, 2H, H-2'); HRMS-ESI (m / z): Theoretical value [M+H] + 306.0752, measured value 306.0756.

[0039] (iii) Synthesis of compounds 3a and 3b Compound 2 (1 g, 2.7 mmol) was dissolved in DMF (10 mL), and 2-bromo-1-indanone (815 mg, 3.8 mmol, 1.4 eq) was added. The mixture was heated in an oil bath to 105 °C and stirred for 1 h. After cooling, the reaction solution was poured into water (50 mL), and a yellow precipitate formed. The precipitate was filtered, washed thoroughly with water, and dried. The solid was dissolved in an ethanol / water mixture (4:1, v / v; 20 mL), and sodium hydroxide (216 mg, 5.4 mmol, 2.0 eq) was added. The mixture was stirred at room temperature for 30 min. The reaction solution was carefully neutralized with 10% acetic acid aqueous solution and concentrated under reduced pressure. The resulting yellow crude solid was slurried with water (15 mL), filtered, and washed with ethyl acetate (10 mL) to give a white solid nucleoside 3a (880 mg, 82%). The structure of compound 3a was determined by 1H NMR spectroscopy (1H NMR spectroscopy). 1 H NMR, carbon spectrum ( 13 Confirmed by C NMR and high-resolution mass spectrometry (HRMS-ESI): 1 H NMR (400 MHz, DMSO- d6 ): δ 12.32 (s, 1H, NH), 8.17 (s, 1H, H-2), 7.86-7.83 (m, 1H, Ar-H), 7.75-7.72 (m, 1H, Ar-H), 7.58-7.54 (m, 2H, Ar-H), 6.21 (t,J= 6.6 Hz, 1H, H-1'), 5.35 (s, 1H, OH), 5.16 (s, 1H, OH), 4.35-4.32 (m, 1H, H-3'), 3.87-3.85 (m, 1H, H-4'), 3.71-3.60 (m, 2H, H-5'), 2.75-2.66 (m, 1H, H-2'), 2.24-2.18 (m, 1H, H-2'). 13 C NMR (101 MHz, DMSO-) d6): δ 160.8, 152.1, 146.2, 142.3, 137.5, 133.0, 128.6, 128.0, 126.4, 113.4, 87.2, 83.4, 70.5, 61.4, 39.3. HRMS-ESI (m / z): Theoretical value [M+H] + 390.1089, measured value 390.1093.

[0040] Following the preparation method of 3a, using 5-bromo-5,6-dihydro-4H-cyclopentano[b]thiophene-4-one as the starting material, 3b was obtained as a yellow solid. The identification results of compound 3b are shown in [link to documentation]. Figures 2-4 The structure of compound 3b was determined by 1H NMR spectroscopy (NMR spectroscopy). 1 HNMR, carbon spectroscopy ( 13 The results were confirmed by C NMR and high-resolution mass spectrometry (HRMS-ESI). HRMS-ESI verification showed the theoretical value [M+H]+ 404.0943 and the measured value 404.0948.

[0041] (iv) Synthesis of compounds 4a and 4b Compound 3a (250 mg, 0.63 mmol) was co-evaporated with pyridine (2 × 2.5 mL) to dryness. The residue was dissolved in anhydrous pyridine (8 mL), and 4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl) (278 mg, 0.82 mmol, 1.3 eq) was added. The mixture was stirred at room temperature for 18 h. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (gradient elution, dichloromethane / methanol = 50:1 v / v) to give compound 4a (290 mg, 66%) as a white solid. The structure of compound 4a was determined by proton NMR spectroscopy (1H NMR spectroscopy). 1 Confirmed by 1H NMR and high-resolution mass spectrometry (HRMS-ESI): 1 H NMR (400 MHz, CDCl3): δ 8.15 (s, 1H), 7.82-7.78 (m, 1H), 7.72-7.68 (m, 1H), 7.52-7.48 (m, 2H), 7.40-7.20 (m, 9H), 6.85-6.80 (m, 4H), 6.35 (t, J = 6.4 Hz, 1H), 4.52-4.48 (m, 1H), 4.15-4.12 (m, 1H), 3.78 (s, 6H), 3.55-3.48 (m, 2H), 3.35-3.28 (m, 1H), 2.65-2.58 (m, 1H), 2.40-2.35 (m, 1H). HRMS-ESI (m / z): Theoretical value [M+H] +692.2395, measured value 692.2401.

[0042] Following the preparation method of 4a, a white solid 4b (300 mg, 69%) was prepared from nucleoside 3b (250 mg, 0.62 mmol). The structure of compound 4b was determined by proton nuclear magnetic resonance spectroscopy (1H NMR). 1 Confirmed by 1H NMR and high-resolution mass spectrometry (HRMS-ESI): 1 HNMR (400 MHz, CDCl3): δ 8.18 (s, 1H), 7.76-7.72 (m, 2H), 7.40-7.20 (m, 9H), 7.20-7.18 (m, 1H), 6.85-6.80 (m, 4H), 6.36 (t, J = 6.5 Hz, 1H), 4.53-4.49 (m, 1H), 4.16-4.13 (m, 1H), 3.78 (s, 6H), 3.56-3.49 (m, 2H), 3.36-3.29 (m, 1H), 2.66-2.58 (m, 1H), 2.41-2.36 (m, 1H). HRMS-ESI (m / z): Theoretical value [M+H]+706.2248, measured value 706.2253.

[0043] (v) Synthesis of compounds 5a and 5b (phosphamide monomers) Compound 4a (205 mg, 0.29 mmol) was co-evaporated with toluene (2 × 3 mL) to dryness. The residue was dissolved in anhydrous dichloromethane (5 mL) under inert gas protection, and N,N-diisopropylethylamine (DIPEA; 113 mg, 0.15 mL, 0.88 mmol, 3.0 eq) and 2-cyanoethyl N,N-diisopropylchlorophosphine (89 mg, 0.38 mmol, 1.3 eq) were added sequentially with stirring at room temperature for 20 min. The reaction solution was diluted with dichloromethane (30 mL), washed with 5% sodium bicarbonate aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1:1 v / v) to give a white solid phosphorus amide monomer 5a (223 mg, 85%).

[0044] Following the preparation method of 5a, yellow foamy compound 5b (280 mg, 88%) was prepared from 4b. The structure of compound 5b was determined by phosphorus nuclear magnetic resonance spectroscopy (NMR spectroscopy). 31 Confirmed by P NMR 31 P NMR (162 MHz, CDCl3) verification: δ 149.2, 148.7.

[0045] (vi) Synthesis of compounds 6a and 6b (triphosphates) Compound 3a (30 mg, 0.075 mmol) was co-distilled with anhydrous pyridine (2 × 2 mL) to dryness. The residue was dissolved in dry trimethyl phosphate (1.5 mL) under nitrogen protection, and freshly distilled phosphorus oxychloride (11.2 μL, 0.12 mmol, 1.6 eq) was added at 0 °C with stirring, and stirring was continued for 2 h. A DMF solution of tributylammonium pyrophosphate (0.5 M, 1.5 mL, 0.75 mmol, 10 eq) and tributylamine (168 μL, 0.75 mmol, 10 eq) were added at once, the ice bath was removed, and stirring was continued at room temperature for 30 min. The reaction solution was quenched with 2 M triethylamine-carbonate buffer (pH 8.0, 15 mL) and extracted with dichloromethane (4 × 5 mL). The aqueous phase was concentrated under reduced pressure to about 5 mL and purified by reversed-phase flash column chromatography (C18 column; linear gradient: 0→40% acetonitrile in 50 mM triethylamine-carbonate buffer (pH 8.0), elution for 30 min). The fraction containing the product was collected, lyophilized, and the tetratriethylammonium salt 6a of the 3a triphosphate compound was obtained (white foamy substance, 30 mg, 43%).

[0046] Following the preparation method of 6a, a yellow foamy compound 6b (45 mg, 64%) was prepared from 3b. The structure of compound 6b was determined by phosphorus nuclear magnetic resonance spectroscopy (NMR spectroscopy). 31 Confirmed by P NMR, 31 P NMR (162 MHz, D2O) verification: δ-5.8 (d, 1P), -11.2 (d, 1P), -21.5 (t, 1P), confirming the triphosphate structure.

[0047] 2. Oligonucleotide synthesis and purification Oligonucleotides were synthesized using an ABI 392-08 synthesizer. During synthesis, the modifying structural unit 5b (i.e., the phosphoridamide monomer of 3b) was incorporated, strictly adhering to the manufacturer's protocol for 3'-cyanoethyl phosphoridamide. After synthesis, the oligonucleotides were cleaved from the solid-phase support and the protecting groups were removed. The synthesized product was purified by high-performance liquid chromatography (HPLC) under the following elution conditions: 0.1 M triethylamine acetate buffer (pH 7.0) and acetonitrile gradient elution to obtain high-purity target oligonucleotides. The molecular weight of the purified oligonucleotides was verified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry in linear negative ion mode using 3-hydroxypyridinecarboxylic acid as the matrix. The results showed that the measured molecular weight was consistent with the theoretical molecular weight, with an error within 0.1%, confirming the successful synthesis of the target product.

[0048] Example 2: Quantitative Verification of Fluorescence Properties This embodiment verifies the positive correlation between the amount of 3b doping and the fluorescence intensity.

[0049] (1) Template design and synthesis To ensure broad applicability of 3b incorporation, randomly generated DNA template sequences were used. The adenosine nucleotides in the templates were flanked by different types of base pairs, including GC, TT, and GT, to avoid sequence bias. DNA templates of varying lengths were designed, incorporating 1 to 6 3b residues evenly distributed, with an incorporation site spaced every 8 bases. Specific template sequences are as follows: Template-1 (with 1 3b incorporated): 5'-XXXX A XXXX-3' (where position A is the 3b incorporation site, with 5 "random" bases on each side, and X represents any one of the four bases A, T, C, and G, with each position chosen independently) Template-2 (with two 3b elements): 5'-XXXX A XXXXXXX A XXXX-3' Template-6 (with 6 3b atoms): 5'-A X8 A X8 A X8 A X8 A X8 A-3' (X8 represents 8 random bases, each position independently selected from A, T, C, G) (2) Primer extension reaction To clearly distinguish between the template and the product, FAM-labeled primers were used for primer extension.

[0050] The primer extension reaction system consisted of 50 μL of DNA template (100 μM, 1.35 μL), primers (300 μM, 0.5 μL), native dNTPs (1 mM, 1.0 μL), 3b-triphosphate (120 μM, 1.15 μL), Deep vent or KOD XL DNA polymerase (2 U / μL, 1.2 μL), and 1× polymerase buffer. The reaction was incubated at 60°C for 60 minutes, followed by heating at 95°C for 5 minutes to terminate the reaction.

[0051] (3) PAGE analysis The product was analyzed by denaturing polyacrylamide gel electrophoresis (PAG) and observed in the FAM channel. The results showed that 3b was efficiently incorporated into the DNA strand. With increasing 3b incorporation, the product band shifted upwards (see [link to analysis]). Figure 6 The presence of C indicates that oligonucleotide products containing 1-6 3b residues were successfully prepared.

[0052] (4) Fluorescence intensity measurement To eliminate fluorescence interference from FAM, the above experiment was repeated using unlabeled primers. The primer extension reaction products were purified and transferred to 96-well black plates. Fluorescence signals were measured using a microplate reader (excitation wavelength 375 nm, emission wavelength 510 nm). Relative fluorescence intensity was calculated based on the fluorescence intensity of the free 3b monomer. The results showed that a single incorporation of 3b increased the fluorescence intensity by approximately 5 times compared to the free nucleoside monomer, and the increase in fluorescence intensity was approximately linearly proportional to the number of 3b residues incorporated (R0). 2 >0.99), indicating that 3b has excellent gradient fluorescence characteristics (see Figure 6 (E).

[0053] Example 3: Thermal Stability Analysis This embodiment examines the thermal stability of DNA double strands containing 3b to verify that they meet the requirements for subsequent biological operations.

[0054] (1) Sample preparation Oligonucleotides (primer extension products) containing different numbers (1 to 6) of 3b residues prepared in Example 2 were used as test strands. Natural DNA strands (without any modifications) completely complementary to each test strand were synthesized. The test strands and complementary strands were mixed in an annealing buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl) at a 1:1 molar ratio. The mixture was heated to 95°C and held for 5 minutes, then slowly cooled to 25°C to form a complete DNA double-stranded structure. Simultaneously, a DNA double-stranded structure with the same sequence composed entirely of natural nucleotides was prepared as a control group.

[0055] (2) Determination of melting temperature Melting temperature analysis was performed using a Cary 3500 UV-Vis spectrometer in a buffer solution containing 0.1 M sodium chloride, 10 mM magnesium chloride, and 10 mM sodium diarsinate (pH 7.0). The temperature was cycled between 20 °C and 80 °C at a rate of 0.6 °C / min, with absorbance measured at 260 nm every 0.6 °C. Each sample was measured three times. The temperature corresponding to the first derivative of absorbance versus temperature (dA / dT) was determined as the melting temperature (Tm) of the DNA double strand by plotting the first derivative of the absorbance versus temperature.

[0056] Table 1: Melting temperatures of DNA double strands with different 3b incorporation amounts

[0057] The results showed that the Tm values ​​of DNA double strands containing 1 to 6 3b residues were very close to those of the control group DNA double strands composed entirely of natural nucleotides. The Tm value of DNA double strands containing up to 6 3b residues decreased by only about 1.2 °C compared to the control group. The Tm value variations for all 3b-containing double strands were within ±1.5 °C. This small difference indicates that the incorporation of gradient fluorescent nucleotide 3b did not significantly disrupt the stability of the DNA double helix structure, and its thermodynamic properties are highly similar to those of natural DNA, meeting the requirements for subsequent biological manipulation.

[0058] Example 4: Construction of Digital-Fluorescence Map and Preparation of Key Probes This example illustrates how to convert a digital key into a fluorescent key probe.

[0059] (1) Construction of digital-fluorescence mapping table Numbers 0-9 are mapped to oligonucleotides containing a specific number of 3b residues. Since the number of 3b incorporations is linearly proportional to fluorescence intensity, and a single incorporation can enhance fluorescence intensity by approximately 5 times, for ease of differentiation, numbers 1-6 are mapped using 1-6 3b incorporations. Number 0 uses an oligonucleotide without 3b residues (fluorescence intensity is background value). Numbers 7-9 can be achieved through combinations or by extending the linear range. The mapping relationships established in this example are as follows: Number 0 → 0 3b residues (background fluorescence); Number 1 → 1 3b residue; Number 2 → 2 3b residues; Number 3 → 3 3b residues; Number 4 → 4 3b residues; Number 5 → 5 3b residues; Number 6 → 6 3b residues; Number 7 → Contains 3 and 4 3b residues (two probes mixed in a 1:1 molar ratio); Number 8 → Contains 3 and 5 3b residues (two probes mixed in a 1:1 molar ratio); Number 9 → Contains 4 and 5 3b residues (two probes mixed in a 1:1 molar ratio).

[0060] (2) Key probe preparation Taking the digital key "13452" as an example, each digit corresponds to the number of 3b residues that need to be incorporated. Using the primer extension reaction of Example 2, five sets of oligonucleotide probes containing 1, 3, 4, 5, and 2 3b residues were prepared, respectively. After the reaction, the products were purified by HPLC, quantified, and stored separately to form independent key probe sets.

[0061] (3) Fluorescence verification The fluorescence intensity of each probe was measured (excitation 375 nm, emission 510 nm). The results showed that the fluorescence intensity of each probe matched the preset mapping value, and the relative standard deviation was less than 5%, indicating that the key could be accurately reconstructed.

[0062] Example 5: Application of a 3b-based DNA information double-layer encryption method Combination Figure 5 , Figure 6 and Figure 7 This embodiment will be described. This embodiment demonstrates the complete process from encrypting the original information to finally decrypting and recovering it. The overall process is as follows: Figure 5 As shown.

[0063] (1) Encryption of original information and algorithm The original information is selected from the full text of "Qing Yu An • Yuan Xi" by Xin Qiji, a poet of the Song Dynasty: "In the east, a thousand trees burst into bloom at night, and stars fall like rain. Fragrant carriages and jeweled horses fill the roads, the sound of phoenix flutes stirs, jade dragons gleam, and fish and dragons dance all night. Moths, willow branches, and golden threads adorn the scene, laughter and fragrance linger. I searched for him a thousand times in the crowd, then suddenly turning around, I found him where the lights were dim." The widely used Blowfish algorithm is employed to encrypt the poem with a pre-configured digital key "13452," generating ciphertext (in hexadecimal representation).

[0064] (2) Encryption of DNA The encrypted information was encoded using DNA according to the following rules: the encrypted information was first converted into binary form, and each 2 bits of the binary encrypted information was mapped to one base (00→A, 01→T, 10→C, 11→G). Necessary primer binding sequences were then added to obtain DNA molecules suitable for PCR amplification (see Table 2). Alternatively, a DNA coding table based on tetra- or penta-base units could be used for encoding. In this embodiment, a published coding platform (http: / / storage.dailab.xyz:16666 / codec) was used to convert the encrypted information into three 500-nucleotide DNA sequences, and corresponding PCR amplification primers were designed. The three DNA sequences were named Seq-1, Seq-2, and Seq-3 (see Table 2).

[0065] Table 2: Sequences generated by the encoding algorithm

[0066] (3) Preparation of fluorescent key probes Following the method in Example 4, fluorescent key probes corresponding to each digit of the digital key "13452" were prepared based on the digital-fluorescence mapping table. Specifically, oligonucleotide probes containing 1, 3, 4, 5, and 2 3b residues were prepared and stored independently as physical keys.

[0067] (4) Construction and amplification of biological vectors Three DNA sequences, Seq-1, Seq-2, and Seq-3, were cloned into the multiple cloning site of the pUC57-Kan+ plasmid vector and transformed into *E. coli* DH5α competent cells. The cells were plated on LB agar containing 50 μg / mL kanamycin and incubated overnight at 37°C. Positive clones were picked for colony PCR verification to confirm the correct insertion fragments. Positive clones were then inoculated into LB liquid agar containing kanamycin and cultured at 37°C with shaking for 12 hours to obtain a seed culture. The seed culture was inoculated at a 1:100 ratio into fresh agar and passaged continuously, once every 12 hours, for a total of 200 passages (each passage involves approximately 10 generations of bacterial division, totaling approximately 2000 generations), to achieve multi-generational replication and long-term preservation of information.

[0068] (5) Plasmid extraction and sequencing Plasmid DNA was extracted from the passaged bacterial culture using a plasmid extraction kit. Using the extracted plasmid as a template, PCR amplification was performed using universal primers M13F / M13R. The amplified products were purified by agarose gel electrophoresis and then sequenced by Sanger sequencing. Sequencing results showed that the three DNA sequences remained intact after 200 passages, without any mutations or deletions (see...). Figures 7-9 This indicates that the information is stably preserved in the host bacteria.

[0069] (6) Fluorescent key reading Take the fluorescent key probe mixture prepared in step (3) (stored separately), dilute it to a concentration of 100 nM, add it to a 96-well black plate, and measure the fluorescence intensity (excitation 375 nm, emission 510 nm) in a microplate reader. Based on the measured fluorescence intensity value, reconstruct the digital key according to the digital-fluorescence mapping table. The reconstruction results of three parallel measurements were all "13452", consistent with the original key.

[0070] (7) Decryption and Recovery The DNA sequence obtained from sequencing was converted into ciphertext using the Wukong decoding platform (corresponding to the encoding platform). The ciphertext was then decrypted using the Blowfish algorithm with the reconstructed digital key "13452," successfully recovering the original ancient poem: "In the east, a thousand trees burst into bloom at night, and stars fall like rain. Fragrant carriages and jeweled horses fill the road, the sound of phoenix flutes stirs, jade dragons gleam, and fish and dragons dance all night. Moths, willow branches, and golden threads adorn the scene, laughter and fragrance linger. I searched for him a thousand times in the crowd, and suddenly, turning around, I found him where the lights were dim."

[0071] Example 6: Visualization of Fluorescent Keys Combination Figure 6 This embodiment will be described. This embodiment illustrates how fluorescent key probes are arranged in a pattern in a 96-well plate to achieve the visualization of information.

[0072] (1) Probe array fabrication Primer extension products containing 1, 3, and 6 3b residues were prepared according to the method in Example 2, representing low, medium, and high fluorescence intensities, respectively. The products were then diluted to the same concentration (50 nM) with sodium dimethylarsinate buffer (pH 7.0).

[0073] (2) Pattern arrangement Drawing inspiration from the brightness control principle of electronic displays, fluorescence intensity was modulated using different numbers of 3b residues to achieve information visualization. In a 96-well plate, PEX products containing different numbers of 3b residues were arranged in a preset pattern to create a face pattern and a "2025" number pattern. To enhance contrast, products with significant intensity differences were selected: the lowest intensity (1 3b) was assigned to the darkest hue, medium intensity (3 3b) to the medium brightness, and the highest intensity (6 3b) to the brightest hue. The PEX products were aliquoted into the designated wells and diluted with sodium dimethylarsinate buffer (pH 7.0). (3) Fluorescence imaging and thermogram conversion A 96-well plate was placed in a microplate reader, with the excitation wavelength set to 375 nm and the emission wavelength to 510 nm. The fluorescence intensity of each well was read. The collected intensity data was imported into image processing software and converted into a heatmap according to a preset grayscale mapping rule. The results showed that the heatmap accurately reproduced the expected "2025" pattern and face pattern (see...). Figure 6 China F and Figure 6 (G), which verified the potential of 3b as a brightness-controllable visualization material.

[0074] By introducing the fluorescent nucleoside analog 3b, this invention expands the molecular alphabet, significantly increasing the coding space and encryption complexity, providing additional degrees of freedom for information security. 3b triphosphate can be efficiently incorporated into DNA polymerase, making it suitable for fluorescence detection; its fluorescence activation effect exhibits a template-dependent gradient response. This tunable fluorescence characteristic allows different intensity levels to represent different data states, similar to how an oscilloscope uses electron density to render an image. The dual-layer encryption system constructed in this invention encodes the original data within the DNA sequence, while the decryption key is embedded in the fluorescence response generated by 3b, which can be read using a microplate reader. This method demonstrates how fluorescence-based readout can supplement sequence-based information, adding a new dimension to molecular encryption strategies and showing broad application prospects in areas such as secure information storage, anti-counterfeiting, and steganography.

[0075] Example 7: DNA Information Double-Layer Encryption System This embodiment provides a DNA information double-layer encryption system for implementing the above method (see...). Figure 10 ),include: The ciphertext processing module is used to process the original information to be encrypted into a digital key and ciphertext information through an encryption algorithm, and to convert the ciphertext information into a first DNA sequence according to a preset encoding rule. The encryption algorithm is preferably a symmetric encryption algorithm or an asymmetric encryption algorithm, and the encoding rule preferably uses quaternary encoding to map the binary ciphertext into four base combinations of A, T, C, and G.

[0076] A key fluorescence module, connected to the ciphertext processing module, receives the digital key and incorporates a corresponding number of gradient fluorescent nucleotides into the DNA sequence based on the digital key to form a fluorescent key probe. The fluorescence intensity of the fluorescent key probe is linearly proportional to the number of nucleotides incorporated, and the number of nucleotides incorporated has a preset mapping relationship with the digital key. Preferably, the gradient fluorescent nucleotides are fluorescently labeled dNTP analogs, incorporated via primer extension or terminal transferase; the mapping relationship is that each bit of the digital key corresponds to a specific number of nucleotides incorporated, or the entire key is mapped to fluorescence intensity levels through quantization encoding.

[0077] The vector construction module, connected to both the encrypted text processing module and the key fluorescence module, receives the first DNA sequence and constructs it into a biological vector to obtain a biological vector containing the encrypted text. It then outputs the fluorescent key probe as an independent DNA molecule combined with the biological vector containing the encrypted text. The biological vector is preferably a plasmid vector, a phage vector, or a bacterial artificial chromosome, and the host bacterium is preferably *Escherichia coli*. The output method includes either encapsulating the fluorescent key probe and the biological vector containing the encrypted text together in the same storage container or storing them separately.

[0078] The information decryption module, connected to the vector construction module, is used to read the first DNA sequence from the biological vector to recover the encrypted information, measure the fluorescence intensity of the fluorescent key probe to reconstruct the digital key, and use the reconstructed digital key to decrypt the recovered encrypted information and output the original information. The sequencing method is preferably second-generation sequencing or third-generation single-molecule sequencing, and the fluorescence intensity measurement is preferably performed using a fluorescence spectrophotometer or fluorescence microscope in conjunction with image analysis software.

[0079] Furthermore, the system also includes a standard curve establishment module, which is used to pre-establish a linear regression model of fluorescence intensity and dopant quantity, and to calibrate the detection sensitivity of different fluorescence channels to ensure the accuracy of key reconstruction.

[0080] The system constructed in this embodiment achieves the physical separation and independent storage of the key and ciphertext. The key information is embedded in the DNA molecule in the form of fluorescence intensity. The complete information cannot be obtained by sequence sequencing alone, which significantly improves the security of encrypted data. At the same time, the multi-level incorporation of gradient fluorescent nucleotides can achieve more than 8 levels of grayscale encoding, expanding the information storage density per unit DNA molecule.

[0081] Example 8: Computer-readable storage medium This embodiment provides a computer-readable storage medium storing a computer program thereon. When the program is executed by a processor, it implements steps S1-S4 of the method described in the first aspect of the present invention. Specifically, the computer-readable storage medium includes, but is not limited to, non-volatile storage media such as read-only memory (ROM), random access memory (RAM), hard disk, solid-state drive (SSD), USB flash drive, and optical disc (CD / DVD). When the program is executed, it performs the following functions: processing the original information into a digital key and ciphertext information and encoding it into a first DNA sequence (S1); controlling the preparation process of the fluorescent key probe (S2); recording the construction and combination preservation information of the biological vector (S3); and controlling the reading of sequencing data, fluorescence intensity analysis, and decryption calculation during the decryption process (S4). Those skilled in the art will understand that the above steps can be automated by combining software programs with conventional computer hardware.

[0082] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A double-layer encryption method for DNA information based on gradient fluorescent nucleotides, characterized in that, Includes the following steps: S1, obtain the original information to be encrypted and the preset digital key, encrypt the original information using an encryption algorithm to generate ciphertext information, and encode the ciphertext information as the first DNA sequence; S2, Prepare a fluorescent key probe, wherein the fluorescent key probe is a DNA molecule incorporating gradient fluorescent nucleotides, and the number of gradient fluorescent nucleotides incorporated has a preset mapping relationship with the digital key; S3, construct the first DNA sequence into a biological vector to obtain a biological vector containing ciphertext; preserve the fluorescent key probe as an independent DNA molecule in combination with the biological vector containing ciphertext; S4, read the first DNA sequence from the biological vector containing the ciphertext to recover the ciphertext information, measure the fluorescence intensity of the fluorescent key probe to reconstruct the digital key, use the reconstructed digital key to decrypt the recovered ciphertext information, and output the original information.

2. The DNA information double-layer encryption method based on gradient fluorescent nucleotides according to claim 1, characterized in that, In step S1, the encryption algorithm used in the encryption process is selected from one or a combination of the Blowfish algorithm, AES encryption algorithm, DES encryption algorithm, RSA encryption algorithm or SHA-256 encryption algorithm; the encoding adopts a DNA coding table based on tetra-base units or penta-base units, or a network-based encoding platform.

3. The DNA information double-layer encryption method based on gradient fluorescent nucleotides according to claim 1, characterized in that, In step S2, the steps for preparing the fluorescent key probe include: constructing a digit-fluorescence mapping table to map each digit of the digit key to an oligonucleotide containing gradient fluorescent nucleotide residues; and incorporating the number of gradient fluorescent nucleotides corresponding to the mapping relationship into the DNA template through a primer extension reaction to obtain the fluorescent key probe.

4. The DNA information double-layer encryption method based on gradient fluorescent nucleotides according to claim 3, characterized in that, In the primer extension reaction, the DNA template sequence is a preset sequence, and gradient fluorescent nucleotides are spaced apart on the oligonucleotides; the number of gradient fluorescent nucleotides incorporated is 1-10.

5. The DNA information double-layer encryption method based on gradient fluorescent nucleotides according to claim 1, characterized in that, Step S3 specifically includes: cloning the first DNA sequence into a plasmid vector, transforming it into a host bacterium for amplification culture, and obtaining a host bacterium containing encrypted information; and storing the fluorescent key probe in DNA molecule form together with the host bacterium containing the first DNA sequence or storing them separately.

6. The DNA information double-layer encryption method based on gradient fluorescent nucleotides according to claim 1, characterized in that, In step S4, the fluorescence intensity of the fluorescent key probe is read by a fluorescence quantitative measurement device and converted into a heat map or pattern to visualize the key information.

7. A DNA information double-layer encryption system for implementing the DNA information double-layer encryption method based on gradient fluorescent nucleotides as described in any one of claims 1-6, characterized in that, include: The ciphertext processing module is used to process the original information to be encrypted into a digital key and ciphertext information, and to encode the ciphertext information into a first DNA sequence; A key fluorescence module, connected to the ciphertext processing module, is used to receive the digital key and incorporate a corresponding number of gradient fluorescent nucleotides into the DNA sequence based on the digital key to form a fluorescent key probe; wherein, the fluorescence intensity of the fluorescent key probe is proportional to the number of nucleotides incorporated, and the number of nucleotides incorporated has a preset mapping relationship with the digital key; The vector construction module is connected to the ciphertext processing module and the key fluorescence module, respectively, and is used to receive the first DNA sequence and construct it into a biological vector to obtain a biological vector containing ciphertext, and output the fluorescent key probe in the form of an independent DNA molecule combined with the biological vector containing ciphertext; The information decryption module, connected to the carrier construction module, is used to read the first DNA sequence from the biological carrier to recover the ciphertext information, measure the fluorescence intensity of the fluorescent key probe to reconstruct the digital key, and use the reconstructed digital key to decrypt the recovered ciphertext information and output the original information.

8. A fluorescent key probe, characterized in that, The fluorescent key probe is an oligonucleotide synthesized via a primer extension reaction, incorporating a specific number of gradient fluorescent nucleotides. The number of gradient fluorescent nucleotides incorporated is uniquely determined by a digital key according to a preset digital-fluorescence mapping table, and the number of incorporations is proportional to the fluorescence intensity of the fluorescent key probe. The nucleotide sequence of the fluorescent key probe is a preset sequence, with the gradient fluorescent nucleotides spaced apart on the oligonucleotide. The fluorescence intensity of the fluorescent key probe can be read by a fluorescence quantitative measurement device, and the reading of its fluorescence intensity is independent of the determination of its DNA sequence.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the program is executed by the processor, it implements the steps of the DNA information double-layer encryption method based on gradient fluorescent nucleotides as described in any one of claims 1 to 6.

10. The application of a combined product comprising a fluorescent key probe and a host bacterium containing a ciphertext DNA sequence in secure information storage, anti-counterfeiting identification, or steganalysis, characterized in that, The fluorescent key probe is the fluorescent key probe of claim 8, and the host bacterium containing the encrypted DNA sequence is obtained by cloning the DNA sequence obtained by the method of any one of claims 1 to 6 into a plasmid vector and transforming the host bacterium; the steganography information hiding is achieved by using the fluorescence intensity of the fluorescent key probe as the physical carrier of the hidden information, thereby realizing the covert transmission and storage of information.

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