DNA (deoxyribonucleic acid) data storage molecular tag based on nanopore as well as preparation method and application of DNA data storage molecular tag

Abstract

The invention relates to a DNA data storage molecular tag based on nanopores and a preparation method and application thereof, and belongs to the technical field of DNA storage. The molecular tag is prepared by modifying azido polysaccharide onto an alkyne-containing DNA chain through a click chemical reaction. Hybridizing and fixing the molecular tag and a DNA bracket chain containing a specific notch structure, and scanning by using a glass nanopore; when the molecular tag passes through the nanopore, a characteristic ionic current blocking signal is generated, and accordingly data coding is achieved. The molecular tag disclosed by the invention is low in cost and simple and convenient to operate; polysaccharide with wide sources and efficient click chemistry are utilized, so that the preparation cost and complexity are remarkably reduced; the physicochemical properties of polysaccharide molecules are stable, the consistency of read signal amplitudes is ensured, the decoding accuracy is high, the label structure stability is good, and a guarantee is provided for long-term storage of data; based on a reversible hybridization storage structure, data updating can be realized without synthesizing a new DNA chain.

Description

Technical Field

[0001] This invention belongs to the field of DNA storage technology, and relates to a molecular tag for DNA data storage based on nanopores, its preparation method and application. Background Technology

[0002] DNA data storage methods fall into two categories: those based on DNA sequences and those based on DNA structures. Sequence-based data storage technology stores information on four deoxyribonucleotide sequences, writing the data through DNA synthesis and then reading it out through DNA sequencing. The advantages of this method are the ultra-high parallelism of DNA sequencing and the ultra-high information density of deoxyribonucleotide sequences. The disadvantages are that it relies on traditional large-scale de novo synthesis technology; the synthesis and sequencing of long-chain DNA requires high costs, and data storage requires a large number of unique DNA sequences. Each time data is written or rewritten, new DNA strands must be synthesized, resulting in high time costs and significant waste. Structure-based DNA information storage technology utilizes base pairing properties to create two-dimensional / three-dimensional nanostructures or adds molecular tags to scaffolds to achieve information storage. While data retrieval through sequencing technology is time-consuming, using DNA nanostructures for data storage eliminates the dependence on DNA sequencing. Multiple methods such as AFM, electron microscopy, and nanopore technology can be used for reading, saving retrieval time. In addition, whether through the self-assembly of DNA sequences or the addition of molecular tags to DNA strands, DNA nanostructures are inherently reconfigurable. This makes data erasure and rewriting unnecessary without further synthesis, reducing the number of DNA sequences that must be synthesized. Based on these advantages, data storage technologies relying on DNA nanostructures have received widespread attention and research. Summary of the Invention

[0003] In view of this, one objective of the present invention is to provide a nanopore-based DNA data storage molecular tag, another objective is to provide a method for preparing a nanopore-based DNA data storage molecular tag, and a third objective is to provide an application of a nanopore-based DNA data storage molecular tag.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a nanopore-based DNA data storage molecular tag, wherein the molecular tag is an azide polysaccharide, the polysaccharide being maltose or cyclodextrin, and the molecular tag is modified on alkyne DNA; The method for preparing the DNA data storage molecular tag involves mixing an alkyne-containing DNA solution with an azide polysaccharide solution, and then adding a catalyst system containing CuSO4, sodium ascorbate, and BTTAA to carry out a chemical reaction to prepare the tag. Preferably, the concentration of the alkyne-containing DNA solution is 50-150 μM, the concentration of the azide polysaccharide solution is 20-30 mM, the concentration of the CuSO4 solution is 15-25 mM, the concentration of the sodium ascorbate solution is 15-25 mM, and the concentration of the BTTAA solution is 5-7 mM. Preferably, the volume ratio of the alkyne-containing DNA solution: azide polysaccharide solution: CuSO4 solution: sodium ascorbate solution: BTTAA solution is 10:2:1:1:6; Preferably, the reaction temperature is 14-37℃ and the reaction time is 2-12 hours; Furthermore, the application of the nanopore-based DNA data storage molecular tag in DNA data storage; Preferably, its preparation method is as follows: S1: The molecular tag is immobilized onto the DNA scaffold strand through complementary base pairing; S2: The DNA scaffold strand is scanned using a nanopore reading device to detect the blocking signal of ion current generated when the molecular tag passes through the nanopore; S3: Data encoding is performed based on the changes in the ion current signal; Preferably, the DNA scaffold strand is a double-stranded DNA containing a gap structure; Furthermore, the present invention also provides a DNA data storage system, the DNA data storage system comprising the aforementioned nanopore-based DNA data storage molecular tag.

[0005] The beneficial effects of this invention are as follows: 1. Polysaccharide-based molecular tags are low-cost and easy to manufacture. Unlike traditional methods based on DNA sequences or complex DNA structures, this invention utilizes the inherent physical size and structure of polysaccharide molecules (such as cyclodextrin and maltose) as carriers of information encoding. This breaks away from the traditional approach of relying solely on nucleic acids for storage, providing a novel molecular option for high-density data storage. Widely available and readily accessible polysaccharide molecules are modified through click chemistry reactions to allow for base complementarity binding to long DNA chains as molecular tags, thus enabling data storage using DNA nanostructures.

[0006] 2. Polysaccharide-type molecular tags provide more stable signal output. Designing polysaccharide molecules as molecular tags for DNA data storage results in greater stability during repeated readings using glass nanopores. This reduces the risk of data readout errors caused by damage to the molecular tag during contact with the DNA or structural changes due to electric fields. Nanopore technology is used for reading. When the DNA strand with the polysaccharide tag is pulled through the nanopore, the polysaccharide molecule, due to its larger size than the DNA strand, generates a significant and characteristic ion current blocking signal. This signal has a significant and distinguishable amplitude difference from the pure DNA strand signal (encoding "0"), and can be clearly identified as encoding "1". This physical size-based "0 / 1" encoding mechanism is simple in principle and has strong anti-interference capabilities.

[0007] 3. Polysaccharide-type molecular tags have simple storage conditions and long shelf life. Because information is stored on a reversible DNA scaffold-molecular tag composite structure, erasing or rewriting data does not require resynthesizing the entire DNA strand; only the molecular tag needs to be replaced. This significantly reduces the cost of data updates and management compared to sequence storage methods that require the synthesis of new DNA strands. Polysaccharide molecules are structurally stable organic macromolecules, and their spatial conformation is not easily disrupted by electric fields or physical contact during nanopore scanning. Therefore, the amplitude of the current signal generated remains stable during repeated readings, reducing data readout errors and ensuring encoding accuracy. Compared to molecular tags formed by the self-assembly of DNA structures, polysaccharide molecular tags can maintain structural integrity under relatively broad conditions. This enables long-term data storage, providing a feasible solution for the ever-increasing data storage needs of today.

[0008] The polysaccharide molecular tagging technology provided by this invention combines the stability and low cost of polysaccharide chemistry with the high sensitivity and single-molecule resolution of nanopore detection technology. It exhibits significant comprehensive advantages in terms of cost, ease of operation, signal stability, and storage lifespan, offering a highly promising and feasible solution to address the challenges of high cost, complex operation, and poor long-term stability currently faced by DNA data storage technologies.

[0009] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0010] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1Electron microscopy image of the pore size of the nanopore; Figure 2 This is a schematic diagram illustrating the complementary base pairing between long DNA and short segments of modified polysaccharide molecules. Figure 3 This is a signal diagram of a long DNA chain containing two gaps. Figure 4 Electrophoresis diagram to verify whether short DNA fragments successfully bind to β-maltose azide; Figure 5 Electrophoresis diagram to verify whether the purification of short DNA fragments with β-maltose azide probe was successful; Figure 6 Electrophoresis diagram to verify whether short DNA fragments successfully bind to β-cyclodextrin; Figure 7 Electrophoresis diagram to verify whether the purification of short DNA fragments with β-cyclodextrin probes was successful; Figure 8 Image of β-cyclodextrin molecular tag signal read from nanopores; Figure 9 Image of maltose molecular tag signal read from nanopores Figure 10 This is a graph showing the relationship between the amplitude of the cyclodextrin molecular tag signal and the voltage. Detailed Implementation

[0011] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0012] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0013] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0014] Example 1: Preparation and Validation of Polysaccharide Molecular Tags Azide polysaccharides modify alkyne-containing DNA: 1. Combine 10 μl DNA solution (100 μM, 1×TE), 2 μl azide polysaccharide solution (25 mM), 1 μl CuSO4 solution (20 mM), 1 μl sodium ascorbate solution (20 mM), and 6 μl BTTAA solution (6 mM). Incubate overnight at 24°C with shaking (14-37°C is acceptable; if 14, 24, or 37°C is used, the incubation time should be shortened to 2 hours). No pH buffer was added to the system.

[0015] 2. Verification of the click reaction by polyacrylamide gel electrophoresis 3. Prepare 15% polyacrylamide gel. Add a mixed solution containing azide polysaccharide to pores α1, α2 and α3 (normal reaction system), add a mixed solution without azide polysaccharide to pores β1, β2 and β3, and add DNA solution (without other components in the reaction system) to pores γ1, γ2 and γ3 (as shown in Table 1).

[0016] Table 1:

[0017] 4. Electrophoretic banding confirms that the azide polysaccharide was successfully modified onto alkyne-containing DNA. The polysaccharide-modified DNA has a larger molecular weight and migrates slower relative to unmodified DNA during electrophoresis.

[0018] 5. Purify the probe by extracting M-DNA (DNA successfully modified with azide polysaccharide) from polyacrylamide using the QIAEX II gel extraction kit.

[0019] 6. Verify the success of purification using polyacrylamide gel electrophoresis (as shown in Table 2).

[0020] Table 2:

[0021] Example 2: Glass substrate modification and double-stranded DNA scaffold fixation 1. Modification of the glass substrate The specific experimental procedures are as follows: (1) Place the glass slide into the slot, add Decon90 solution, and soak the glass slide in it overnight. Decon90 is an alkaline cleaning solution that can remove organic residues, inorganic salt deposits and biological contaminants from the surface of the glass slide. (2) Sonicate in a staining tank containing Decon90 for 10 minutes, clean the glass slide with ddH2O, and then sonicate in ddH2O twice for 10 minutes each time, and rinse with water; (3) Acetone has the effect of removing organic pollutants. Use acetone to clean the surface of the glass slide again. Since acetone is volatile, it is necessary to add enough acetone to cover the glass slide, sonicate for 30 minutes, and then rinse with water. (4) Add methanol to the staining tank and submerge the glass slide, sonicate for 30 minutes, rinse with water, drain as much water as possible, and put it in a 90°C oven to evaporate all the moisture on the surface of the glass slide. (5) Place the glass slide in the piranha solution (98% concentrated sulfuric acid: 30% hydrogen peroxide = 3:1), incubate in a 90°C oven for 2 hours, rinse with water, drain as much water as possible, remove the glass slide and dry it with nitrogen gas; (6) Place the glass slide in sodium ethoxide (35 mL anhydrous ethanol, 15 mL ddH2O, 2 g NaOH, dissolved by ultrasonication) and sonicate for 15 minutes. Then sonicate in ddH2O for 15 minutes. Rinse with water, dry the glass slide with high-purity nitrogen, and place it in a staining jar. Sodium ethoxide is a strong alkaline reagent that can not only clean the glass but also activate the glass surface. The main component of glass is silicon dioxide, and its surface contains a large number of silanol groups (-Si-OH). The alkalinity of sodium ethoxide can promote the deprotonation of silanol groups, forming more -Si-O. - This enhances the surface reactivity and provides active sites for APTES silanization; (7) Treatment with 3-aminopropyltriethoxysilane (APTES): Mix 29 mL of methanol, 1.5 mL of acetic acid, and 0.3 mL of APTES, add the mixture to a staining tank, seal with Parafilm, incubate at room temperature for 1 hour, sonicate with ddH2O for 5 minutes, remove the glass slide, and dry with high-purity nitrogen. APTES is a silane coupling agent that can introduce amino groups (-NH2) onto the surface of a glass substrate. The ethoxy group (-OCH2CH3) of APTES undergoes a hydrolytic condensation reaction with the silanol groups on the glass surface to form Si-O-Si bonds, thereby fixing the amino group onto the glass surface. The glass surface treated with sodium ethoxide has more activated hydroxyl groups, which is more conducive to the binding of APTES. (8) PEG treatment: Ten glass slides were treated at once. 30 mg of methoxy polyethylene glycol succinimide ester (mPEG-SVA) and 1 mg of Biotin-PEG-NHS were dissolved in 300 μL of 100 mM NaHCO3 (sterilized at high temperature). PEG-SVA and Biotin-PEG-NHS formed covalent bonds through the reaction of NHS ester and amino groups, thus immobilizing PEG and biotin on the glass surface. Two glass slides were placed in a container filled with ddH2O, sandwiching 50 µL of the mixed PEG solution between them. The mixture was incubated in a clean bench at room temperature in the dark for 2 hours. This completed the modification of the glass substrate, facilitating the subsequent immobilization of double-stranded DNA scaffolds on the glass slides.

[0022] II. DNA Fixation: After modifying the glass substrate, streptavidin was applied to the glass slide. The DNA scaffold was then immobilized on the glass substrate through the high affinity and strong specific binding of biotin at the 5' end of the double-stranded DNA scaffold to streptavidin. The interaction between biotin and streptavidin is the strongest known non-covalent interaction, exhibiting excellent stability and highly specific recognition capabilities. It is not easily affected by fluctuations in reagent concentration, changes in external environmental conditions, pH gradients, chemical denaturants, or organic solvents. Streptavidin has four biotin-binding sites. The double-stranded DNA was immobilized on the glass slide through this specific binding.

[0023] This embodiment uses a glass slide with a PEG:Biotin ratio of 30:1 (mPEG-SVA:Biotin-PEG-NHS = 30:1). The specific experimental procedures are as follows: (1) Clean the PEG-modified glass slides with fresh deionized water; (2) Drop 10 μL of streptavidin with a concentration of 10 μg / mL onto a glass slide, cover the glass slide from one side edge with another glass slide modified with PEG, and try to avoid and remove air bubbles as much as possible. Incubate at room temperature for 15 minutes. (3) Rinse the glass slide multiple times with deionized water to remove excess streptavidin. (4) Sample preparation for nanomanipulation detection of DNA gap structures: Take 10 μL of 100 pM B4-Gap (or 4B-Gap) solution and drop it onto a glass slide. Gently lower the coverslip from one edge of the glass slide to ensure that the DNA solution fully covers the surface of the glass slide. Incubate at room temperature for 1.5 hours. (5) Wash away excess unbound DNA on the glass slide with 1xPBS solution; (6) Place the glass slide into a culture dish containing buffer solution for subsequent nanomanipulation detection.

[0024] Example 3: Reading Azide Cyclodextrin Molecular Tag Data Based on Glass Nanopores Pretreatment and preparation of glass nanopores: The glass nanopores in this embodiment were fabricated using QF100-70-7.5 glass capillaries from Sutter Chemical Company, USA. These capillaries are made of quartz glass, 7.5 cm long, with an outer diameter of 1 mm and an inner diameter of 0.7 mm, and contain a flow guide wire (Filament). The flow guide wire helps the electrolyte solution better fill the tip of the glass nanopore, connecting it to the sample cell to form a circuit.

[0025] Before drawing glass capillaries into glass nanopores, pretreatment is required to thoroughly clean the glass tube and minimize interference during signal detection. The specific experimental procedures are as follows: (1) Immerse the quartz glass capillary in acetone solution. Since acetone is volatile, it is necessary to add a sufficient amount of acetone solution and sonicate it in an ultrasonic cleaner for 20 minutes. (2) Transfer the glass capillary tube in acetone solution to deionized water at 18.2 MΩ·cm, and sonicate it twice in an ultrasonic cleaner for 20 minutes each time. After the first ultrasonic cleaning, put the glass capillary tube into clean deionized water for a second ultrasonic treatment; (3) Immerse the glass capillary in piranha solution (98% concentrated sulfuric acid: 30% hydrogen peroxide = 3:1) for 3 hours. Piranha solution has strong oxidizing properties, which can remove organic pollutants on the glass surface. Moreover, piranha solution can generate a large number of silanol groups (-Si-OH) on the glass surface, which increases the hydrophilicity of the glass surface and is beneficial to improving the wettability of glass nanopores with electrolyte solution. (4) In order to remove the piranha solution, the glass capillary was immersed in deionized water and ultrasonically cleaned 5 times, 10 minutes each time; (5) Immerse the glass capillary in anhydrous ethanol and ultrasonically clean it for 20 minutes; (6) Soak the glass capillary tube in deionized water and ultrasonically clean it twice, 10 minutes each time; (7) Place the ultrasonically sonicated glass capillary in a 90 ℃ oven for 4 hours to dry the moisture on the glass capillary; (8) After the dried glass capillary is cooled to room temperature, it can be used to draw glass nanopores.

[0026] (9) Fix the pretreated glass capillary tube on the guide rail of MODEL P-2000, ensuring that both ends of the glass tube are in the groove on the guide rail; (10) Cover the protective cover, enter the corresponding program, set the corresponding parameters, heat to 630, filament value to 4, velocity value to 61, delay value to 150, pull value to 155; (11) After setting the parameters, press the PULL key on the keyboard. At this time, the indicator light inside the protective cover turns red. After a short delay of a few seconds, the glass capillary is drawn into two identical glass nanopores with an inner diameter of about 10-17 nm. (12) Inject 100 μL of electrolyte solution into a 1.5 mL centrifuge tube beforehand, insert the drawn glass nanopore into the centrifuge tube, and ensure that the tip of the glass nanopore enters the liquid surface; (13) Fix the glass nanopores using a special device and inject an electrolyte solution into the inner wall of the glass nanopores using a liquid injector; (14) Centrifuge the glass nanopores after the solution has been injected at 5000 rpm for 10 minutes to remove air bubbles at the tip of the glass nanopores and ensure that the electrolyte solution fully fills the tip of the quartz glass nanopores.

[0027] DNA data storage detection: The detection system comprises three main modules: a probe module, a displacement control module, and a signal acquisition module. The probe module mainly includes a glass nanopore, a scaffold for fixing the glass nanopore, a glass slide modified with a DNA double-stranded scaffold, and a stage containing an electrolyte buffer solution. The quartz glass nanopore serves as a probe for capturing the double-stranded DNA scaffold and for nanomanipulation. Its tip is filled with electrolyte buffer solution and mounted on a pre-designed scaffold, allowing the glass nanopore to be fixed and aligned precisely with the center of the stage. The double-stranded DNA scaffold is bound to the surface of a polyethylene glycol-modified quartz glass substrate through a sandwich reaction involving biotin-streptavidin-biotin binding. Polyethylene glycol effectively prevents DNA molecules from being adsorbed onto the glass slide surface due to electrostatic interactions. The displacement control module includes a triaxial motorized stage and a piezoelectric system. The triaxial motorized stage provides coarse micron-level control of the relative position between the glass slide and the glass nanopore, while the piezoelectric system provides nanon-level precision control of the capture and manipulation of DNA molecules by the glass nanopore. The signal acquisition system consists of a patch-clamp signal amplifier and a digital-to-analog converter. These components are connected to the glass nanopores and the electrolyte buffer solution containing the glass slide via Ag / AgCl electrodes, and external electromagnetic noise is shielded by a Faraday cage. All equipment is placed on a vibration-damping platform to minimize interference from physical vibrations during nanomanipulation of the glass nanopores.

[0028] I. Sample Preparation for Testing: Mix 3 μL of 10 nM long-chain DNA solution, 1 μL of 2 μM molecular tag solution modified with β-cyclodextrin, 1 μL of 10 mM EDTA, and 4 μL of 10% PBS thoroughly. Incubate at 37°C for 2 hours. Take the solution of the double-stranded DNA scaffold and drop it evenly onto a glass slide. Cover the glass slide with Parafilm sealing film to avoid and remove air bubbles as much as possible. Incubate at room temperature for 1.5 hours. II. Nanopore Capture: Data was recorded using Clampex software in "gap-free" mode. The culture dish with a glass slide was fixed on the sample stage, and the glass nanopore (14.7 nm) was fixed directly above the glass slide. The patch-clamp system was connected via an Ag / AgCl electrode, with a 300 mV bias voltage output at the trans end. The sampling frequency was set to 25 kHz, the low-pass filter frequency to 1 kHz, the step size of the piezoelectric ceramic motor was set to 1 nm / ms, the delay to 100 ms, and the actual motor movement speed to 10 nm / s. The displacement of the glass nanopore was controlled by the detection platform and gradually moved closer to the glass slide. When the glass nanopore entered the 2 M LiCl, 10 mM Tris, pH 7.4 buffer solution above the glass slide, the patch-clamp collected a stable ion current. The distance between the nanopore and the glass slide was controlled to reach the critical point. Then, the nanomanipulation system was used to scan the 5 μm × 5 μm plane around the critical point until double-stranded DNA was successfully captured.

[0029] III. Structure Recognition: A nanomanipulation system was used to slowly move a glass nanopore upwards from the bottom of the double-stranded DNA to identify the DNA structure. After detecting the cyclodextrin molecular tag structure, the data was processed. Data analysis showed that cyclodextrin, as a molecular tag, has excellent signal discrimination and is significantly different from the current change value formed by the transient pore blockage of long-chain DNA. Furthermore, the signal amplitude is stable, exhibiting good "1" characteristics suitable for DNA data storage and encoding.

[0030] Example 4 Mix 1 μL of 2 μM long-chain DNA solution, 1 μL of 2 μM maltose molecular tag solution, 1 μL of 10 mM EDTA solution, and 4 μL of 1 PBS solution thoroughly. Incubate at 37 °C for 2 hours. Take the solution of the double-stranded DNA scaffold and drop it evenly onto a glass slide. Cover the glass slide with Parafilm sealing film, avoiding and removing air bubbles as much as possible. Incubate at room temperature for 1.5 hours. Nanopore capture: Data was recorded using Clampex software in "gap-free" mode. The culture dish with a glass slide was fixed on the sample stage, and the glass nanopore was fixed directly above the glass slide. The patch-clamp system was connected via an Ag / AgCl electrode, with a 300 mV bias voltage output at the trans end. The sampling frequency was set to 25 kHz, the low-pass filter frequency to 1 kHz, the step size of the piezoelectric ceramic motor was set to 1 nm / ms, the delay to 100 ms, and the actual motor movement speed to 10 nm / s. The displacement of the glass nanopore was controlled using nanomanipulation techniques, gradually bringing it closer to the glass slide. When the glass nanopore entered the 2 M LiCl, 10 mM Tris, pH 7.4 buffer solution above the glass slide, the patch-clamp collected a stable ionic current. The distance between the nanopore and the glass slide was controlled to reach a critical point. Then, the nanomanipulation system was used to scan a 5 μm × 5 μm plane around the critical point until double-stranded DNA was successfully captured.

[0031] Structure recognition: A nanomanipulation system was used to slowly move a glass nanopore upwards from the bottom of the double-stranded DNA to identify the DNA structure. After detecting the maltose molecular tag structure, data processing revealed that because maltose is a trisaccharide with a smaller diameter, the signal amplitude read from the nanopore was also smaller. Furthermore, due to the inherent limitations of the glass nanopore itself, the background noise was around 10 pA, while the signal amplitude generated by maltose was smaller than the background noise, requiring fitting with a large amount of signal data.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nanopore-based DNA data storage molecular tag, characterized in that: The molecular tag is an azide polysaccharide, the polysaccharide being maltose or cyclodextrin, and the molecular tag is modified on alkyne DNA.

2. The method of claim 1, wherein: The product is prepared by mixing an alkyne-containing DNA solution with an azide polysaccharide solution, and then adding a catalyst system containing CuSO4, sodium ascorbate, and BTTAA to carry out a chemical reaction.

3. The method of claim 2, wherein: The concentration of the alkyne-containing DNA solution is 50-150 μM, the concentration of the azide polysaccharide solution is 20-30 mM, the concentration of the CuSO4 solution is 15-25 mM, the concentration of the sodium ascorbate solution is 15-25 mM, and the concentration of the BTTAA solution is 5-7 mM.

4. The method of claim 2, wherein: The volume ratio of the alkyne-containing DNA solution: azide polysaccharide solution: CuSO4 solution: sodium ascorbate solution: BTTAA solution is 10:2:1:1:

6.

5. The method of claim 2, wherein: The reaction temperature is 14-37℃, and the reaction time is 2-12 hours.

6. The application of the nanopore-based DNA data storage molecular tag according to claim 1 in DNA data storage.

7. Use according to claim 6, characterized in that, Its preparation method is as follows: S1: The molecular tag is immobilized onto the DNA scaffold strand through complementary base pairing; S2: The DNA scaffold strand is scanned using a nanopore reading device to detect the blocking signal of ion current generated when the molecular tag passes through the nanopore; S3: Data encoding is performed based on the changes in the ion current signal.

8. Use according to claim 7, characterized in that: The DNA scaffold strand is a double-stranded DNA with a gap structure.

9. A DNA data storage system characterized by: The DNA data storage system includes the nanopore-based DNA data storage molecular tag as described in claim 1.

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