Tetrahedral framework nucleic acid and preparation method therefor

Abstract

Provided are a tetrahedral framework nucleic acid and a preparation method therefor. Escherichia coli is used as a host to produce an M13 bacteriophage so as to obtain single-stranded DNA (ssDNA) in the M13 bacteriophage, ssDNA required for assembling the tetrahedral framework nucleic acid is obtained by cleavage with restriction endonuclease II, and then the tetrahedral framework nucleic acid is obtained by self-assembly of the ssDNA. The production costs of TDN are reduced while achieving large-scale industrial production of the TDN, and the present invention has good practicability.

Description

A tetrahedral framework nucleic acid and its preparation method Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a tetrahedral framework nucleic acid and a preparation method thereof. Background Art

[0002] Deoxyribonucleic acid (DNA) carries genetic information and is transcribed into mRNA, which in turn guides protein synthesis. It is an essential biological macromolecule for the maintenance of life and development. Deoxynucleotides are composed of deoxyribose, phosphate, and one of the four nucleobases: thymine (T), adenine (A), guanine (G), and cytosine (C). A, G, T, and C can selectively bind to each other through hydrogen bonds (AT, GC), forming the double-helical DNA structure. G and C interact through three hydrogen bonds, while A and T only have two. DNA's editability, predictability, and addressability make it an attractive material for applications in biology, physics, medicine, and engineering. DNA nanostructure technology has opened the door to new and emerging fields of DNA research. Based on the rules of complementary base pairing, DNA molecules can serve as universal building blocks for self-assembly to form more advanced, complex, and multidimensional nanostructures. Self-assembled DNA nanostructures, due to their suitable size and hydrophilic surface, can autonomously enter mammalian cells. In addition, compared with single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the ability of DNA nanostructures to resist nuclease degradation is also greatly improved.

[0003] DNA nanostructures can be functionalized in a precise and controllable manner by modifying various molecules on their surfaces, including nucleic acid aptamers, nanoparticles, drugs, proteins, and dye molecules. These excellent properties of DNA nanostructures have led to their widespread application in molecular diagnostics, bioimaging, biosensing, targeted drug delivery, and regenerative medicine. With the development of DNA nanostructures over the past 40 years, tetrahedral DNA nanostructures (TDNs) have stood out among various DNA nanomaterials due to their cell membrane and tissue permeability, high yield, structural stability, negligible immunogenicity, and multifunctional editability.

[0004] TDN consists of four equidistant single-stranded DNAs. According to the rules of Watson-Crick hybridization, each single-stranded DNA contains three blocks that can hybridize with three other strands to form a tetrahedral three-dimensional structure. Compared with other complex DNA structures, TDN is the simplest and most specialized DNA polyhedron. It can be easily synthesized with a yield of about 90%. In addition to its high yield, TDN also has the following outstanding features: (1) TDN can enter cells autonomously and in large quantities without any functional molecules; (2) TDN is non-toxic and has low immunogenicity; (3) The precise programmability and reversibility of TDN make it an ideal carrier. TDN can be functionalized by simply adding oligonucleotides or anti-oligonucleotides to the middle or end of ssDNA, inserting functional small molecules into double-stranded DNA through electrostatic adsorption, or chemically cross-linking between two ssDNAs through base complementary pairing. Based on the above characteristics, TDN has become a research hotspot in the biomedical field, including bioimaging, biosensing, molecular diagnosis, gene delivery, disease treatment and regenerative medicine.

[0005] Currently, TDN production relies on industrial synthesis of ssDNA, using the principle of base complementary pairing to self-assemble in a specific buffer. This synthesis method is expensive, difficult to mass-produce, and poses safety risks. While TDN research has shown significant potential in the medical field, cost and production constraints have hindered further clinical use of this raw material, limiting its application in basic medical research and industrialization. Therefore, a new method for constructing TDN is needed. Summary of the Invention

[0006] In response to some deficiencies in the prior art, the present invention provides a tetrahedral framework nucleic acid and a preparation method thereof. The present invention utilizes Escherichia coli as a host to produce M13 bacteriophage, thereby obtaining ssDNA in the M13 phage, and obtains single-stranded DNA required for assembling the tetrahedral framework nucleic acid by cleavage with restriction endonuclease II. The single-stranded DNA is then self-assembled to obtain the tetrahedral framework nucleic acid. The tetrahedral framework nucleic acid realizes large-scale industrial production of TDN while reducing the production cost of TDN, and has excellent practicality.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The present invention first provides a method for preparing a tetrahedral framework nucleic acid, the preparation method comprising:

[0009] S1. Design a nucleic acid fragment containing single-stranded DNA, referred to as the target sequence fragment; the structure of the nucleic acid fragment containing single-stranded DNA is [L1-L2]n-L3, where L2 is single-stranded DNA, and L1 and L3 are specific DNA sequence fragments that can form a hairpin structure and contain a restriction endonuclease II cleavage site; n is 4; and L1 and L3 cannot complement each other to form a double strand.

[0010] S2. The target sequence fragment is connected to the phage display plasmid and transferred into E. coli competent cells. After culturing the E. coli cells into which the target sequence fragment has been transferred for a period of time, M13 helper phage is added for co-cultivation;

[0011] S3. Extracting phage from the cultured product to obtain a target sequence fragment with a vector backbone;

[0012] S4. After annealing the obtained target sequence fragment with the vector backbone, restriction endonuclease II is added to cut it to obtain four single-stranded DNA fragments;

[0013] S5. The four single-stranded DNAs obtained are placed in a TM buffer solution, treated at 90-98° C., and then gradually cooled to complete DNA annealing to obtain the tetrahedral framework nucleic acid.

[0014] Preferably, the sequences of L1 and L3 are the same or different; each L1 is the same or different, and each L2 is the same or different.

[0015] Preferably, the L1 or L3 is selected from any one of the nucleotide sequences shown in SEQ ID No: 5 to 9.

[0016] Preferably, in step S2, the phage display plasmid is preferably pBluescript II-KS(+), and the Escherichia coli competent cells are preferably JM109;

[0017] The Escherichia coli transformed with the target sequence fragment is cultured until the OD value is 0.01-0.1, and then a helper phage is added for co-culture.

[0018] Preferably, in step S2, there is no particular limitation on the specific type of M13 helper phage, and any M13 helper phage commonly used in the art can be used. In the embodiment of the present invention, it is specifically M13K07 helper phage.

[0019] Preferably, in step S2, the amount of helper phage added is 1×10 11The co-culture conditions after adding 0.5-2 μL of pfu / mL helper phage are as follows: a shaking speed of 150-250 rpm, a temperature of 28-37°C, kanamycin is added after 1-2 hours, and the culture is continued for 1-2 hours before fermentation culture; further, the fermentation culture conditions are a stirring speed of 250-600 rpm, a temperature of 28-42°C, a pH of 6.5-7.5, and a culture of 8-14 hours.

[0020] The fermentation culture medium is 2×YT medium containing 5 mM MgCl 2 .

[0021] Preferably, in step S1, the single-stranded DNA is selected from any one of the sequences shown in SEQ ID Nos: 1 to 4.

[0022] Preferably, in step S5, the mixture is treated at 90-98° C. for 1 min and then gradually cooled to room temperature.

[0023] The present invention also provides the tetrahedral framework nucleic acid prepared by the above method.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention utilizes Escherichia coli (E. coli) as a host to produce M13 phage, thereby obtaining ssDNA within the M13 phage. Restriction endonuclease II is then used to cleave the four single strands required for TDN assembly, and TDN is then self-assembled. This invention utilizes synthetic biology techniques to achieve large-scale industrial production of TDN while reducing its production cost, demonstrating excellent practicality.

[0026] Furthermore, the present invention adds an additional DNA fragment to the target sequence fragment in the design, allowing a palindromic hairpin double-stranded DNA structure to form at the pre-cleavage site of the single-stranded DNA. This structure also contains a recognition site for restriction endonuclease II. After annealing, the produced single-stranded DNA can be cleaved using the corresponding restriction endonuclease II, ultimately obtaining pure single-stranded DNA. The single-stranded DNA obtained by the present method is pure and free of impurities, and does not contain vector backbone DNA fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 shows the structure formed after annealing of ssDNA in Example 1.

[0028] FIG2 is an electrophoretic band diagram of the single-stranded DNA of the target vector before and after enzyme digestion.

[0029] FIG3 shows the structure formed after ssDNA annealing in Example 2.

[0030] Figure 4 shows gel electrophoresis verification of single-stranded DNA obtained after enzyme digestion.

[0031] FIG5 is an electrophoretic band diagram of four single-stranded DNAs assembled into TDN.

[0032] FIG6 is an AFM characterization image of a tetrahedral framework nucleic acid. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, all that is not explained in detail is accomplished using conventional experimental methods, and the processes involved in the embodiments are understood and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the art, and therefore will not be described in detail.

[0034] Unless otherwise specified, all culture media or other reagent materials involved in the present invention are conventionally prepared or purchased.

[0035] Example 1:

[0036] In this example, a nucleic acid fragment [L1-L2]n-L3 comprising single-stranded DNA was first prepared. In [L1-L2]n-L3, n = 4; the four L1s and L3s were all different, with the nucleotide sequences of the four L1s and L3s being SEQ ID Nos: 5 to 9, respectively; and the four single-stranded DNA L2s were all different, with the nucleotide sequences of SEQ ID Nos: 1 to 4, respectively.

[0037] The specific steps are as follows:

[0038] (1) Selection of bacterial strains and culture medium:

[0039] The helper phage M13K07 used in this example was purchased from NEB with a titer of 1×10 11 pfu / mL; pBluescript II-KS(+) phage display plasmid 1 μg (20 μL, 50 ng / μL), purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.; JM109 Escherichia coli genotype: recA1, endA1, gyrA96, thi-1, hsdR17 (rk - mk + ),e14 - (mcrA - ),supE44,relA1,Δ(lac-proAB) / F'[traD36,proA B + ,lacIq,lacZΔM15] were purchased from Takara.

[0040] 2×YT medium: tryptone 1.6% (W / V), yeast extract 1% (W / V), NaCl 0.5% (W / V), pH 7.0.

[0041] (Other embodiments are the same)

[0042] (2) Vector construction and identification:

[0043] In this step, four single-stranded DNAs with nucleotide sequences as shown in SEQ ID Nos: 1 to 4 are inserted into the same pBluescriptII-KS(+) vector, wherein:

[0044] SEQ ID No: 1 Sequence:

[0045]

[0046] SEQ ID No: 2 Sequence:

[0047]

[0048] SEQ ID No: 3 Sequence:

[0049]

[0050] SEQ ID No: 4 Sequence:

[0051]

[0052] Then, a hairpin structure sequence with a recognition site for the restriction endonuclease EcoRI is added between each two fragments of the four single-stranded DNAs and at both ends of the four single-stranded DNAs after being connected, to obtain nucleic acid fragments [L1-L2]n-L3 containing single-stranded DNA; the nucleotide sequences of the hairpin structure sequences L1 and L3 with the recognition site for the restriction endonuclease EcoRI are shown in SEQ ID Nos: 5 to 9, respectively.

[0053] SEQ ID No: 5:

[0054]

[0055] SEQ ID No: 6:

[0056]

[0057] SEQ ID No: 7:

[0058]

[0059] SEQ ID No: 8:

[0060]

[0061] SEQ ID No: 9:

[0062]

[0063] The nucleic acid fragment containing single-stranded DNA obtained by combination is shown in SEQ ID No: 10.

[0064] SEQ ID No: 10:

[0065]

[0066] The above sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The target sequence fragment and the pBluescript II-KS(+) plasmid were then linearized using restriction endonucleases Sac I and Kpn I. The target sequence fragment was then ligated into the pBluescript II-KS(+) plasmid using T4 DNA ligase. The plasmid vector containing the target sequence fragment was then heat-shocked and transformed into JM109 competent E. coli cells. Single colonies were selected on 2×YT solid medium plates containing 50 μg / mL ampicillin. Successful vector construction was subsequently verified by colony PCR, digestion with the endonucleases Sac I and Kpn I, and DNA sequencing.

[0067] Colony PCR was performed using T3 and T7 primer sequences:

[0068] The sequence of the T3 primer is: 5′-AATTAACCCTCACTAAAGGGA-3′ (SEQ ID No: 11);

[0069] The sequence of the T7 primer is: 5'-TAATACGACTCACTATAG-3' (SEQ ID No: 12).

[0070] (3) Phage fermentation

[0071] 100 mL of the JM109 Escherichia coli obtained in step (2) was cultured. When the OD value reached 0.05, 1 μL of the M13K07 helper phage was added and the culture was placed in a shaker for infection culture at 200 rpm and 37°C. After 2 h, 100 μL of 30 μg / mL kanamycin was added and the culture was continued for 1.5 h to prepare the original seed bacteria.

[0072] Use a 5L fermentor with 3L of 2×YT medium containing 5mM MgCl2 and sterilize by autoclaving at 121°C for 20 minutes. Add the prepared seed culture to the fermentor, followed by 3mL each of 50μg / mL ampicillin and 30μg / mL kanamycin. Fermentation conditions are 300rpm, 37°C, and pH 7 for 16 hours.

[0073] (4) Extraction of single-stranded DNA:

[0074] The obtained fermentation broth was centrifuged at 5000 g and 4° C. for 20 minutes, and the supernatant was collected. 240 g of PEG8000 and 180 g of NaCl were added, dissolved, and placed in a 4° C. refrigerator for 2 hours.

[0075] Then centrifuge at 8000 g and 4°C for 20 min to collect the phage precipitate.

[0076] Add 40 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH = 8) solution to resuspend the precipitate, then add 80 mL of PPB2 (200 mM NaOH, 1% (w / v) SDS) solution and mix gently. React for 5 minutes. Then add 60 mL of PPB3 (3 M KOAc, adjust pH to 5.5 with acetic acid) solution and mix. React for 10 minutes. Centrifuge at 10,000 g and 4°C for 20 minutes, and collect the supernatant.

[0077] An equal volume of -20°C pre-cooled ethanol was added, mixed thoroughly, and placed in a -20°C refrigerator for 1 hour. Centrifuge at 10,000 g and 4°C for 20 minutes to obtain a precipitate.

[0078] 100 mL of 70% alcohol was added to wash the sample, and the sample was centrifuged at 10,000 g and 4° C. for 20 minutes to obtain a single-stranded DNA precipitate.

[0079] Centrifuge at 10,000 g for 2 minutes at 4°C, and remove the remaining supernatant.

[0080] After drying, add 20 mL of TE solution and allow to dissolve at room temperature for 5 hours. Measure the DNA concentration using a Onedrop micro-spectrophotometer and identify it by gel electrophoresis to obtain single-stranded DNA with a vector backbone.

[0081] (5) Enzymatic cleavage of single-stranded DNA and self-assembly of tetrahedral framework nucleic acids:

[0082] After obtaining single-stranded DNA, 20 μL of the sample was placed at 95°C for 1 minute, then gradually cooled at 1°C / min until it reached 25°C to complete DNA annealing. The sequences of SEQ ID No: 3 and 4 at both ends of the single-stranded DNA formed a local double-stranded DNA through base pairing. This double-stranded DNA contained the recognition site 5'-GAATTC-3' for the EcoRI restriction endonuclease.

[0083] EcoRIIII endonuclease and buffer (purchased from Takara) were added, mixed, and then placed at 37°C for enzyme digestion to obtain single-stranded DNA.

[0084] The single-stranded DNA after enzyme digestion was placed in TM buffer (20mM Tris+50mM MgCl2) at 95°C for 1 minute, and then gradually cooled to room temperature at a rate of 1°C / min to complete DNA annealing and obtain tetrahedral nucleic acid.

[0085] FIG1 shows the structure formed after annealing of ssDNA in this embodiment. As can be seen from the figure, a hairpin structure of a local double-stranded DNA is generated after annealing of ssDNA, and the double-stranded DNA sequence contains a restriction endonuclease recognition site.

[0086] After digestion with EcoRI restriction endonuclease and annealing, the sample was added to 10*Loading buffer and detected using a 1% agarose gel. 40 μL of sample was added to each gel well. The results are shown in Figure 2.

[0087] Figure 2 shows the electrophoretic banding of the single-stranded DNA of the target vector before and after enzyme digestion. As can be seen from the figure, the molecular weight of the main band after digestion is lower than that before digestion, indicating that digestion has occurred. Two new bands were generated. Molecular weight analysis revealed that one is a tetrahedral nucleic acid and the other is a hairpin structure removed by digestion. This indicates that the tetrahedral framework nucleic acid can be obtained through self-assembly after digestion.

[0088] Example 2:

[0089] In this example, a nucleic acid fragment [L1-L2]n-L3 comprising single-stranded DNA was first prepared. In [L1-L2]n-L3, n = 4; the four L1s and L3s were all different, with the nucleotide sequences of the four L1s and L3s being SEQ ID Nos: 5 to 9, respectively; the four single-stranded DNA fragments L2 were identical, with the nucleotide sequences of SEQ ID Nos: 1 to 4 being replicated four times on the pBluescript II-KS(+) phage display plasmid.

[0090] The specific steps are as follows:

[0091] (1) Selection of bacterial strains and culture medium:

[0092] The helper phage M13K07 used in this example was purchased from NEB with a titer of 1×10 11 pfu / mL; pBluescript II-KS(+) phage display plasmid 1 μg (20 μL, 50 ng / μL), purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.; JM109 Escherichia coli genotype: recA1, endA1, gyrA96, thi-1, hsdR17 (rk - mk + ),e14 - (mcrA - ),supE44,relA1,Δ(lac-proAB) / F'[traD36,proA B + ,lacIq,lacZΔM15] were purchased from Takara.

[0093] (2) Vector construction and identification:

[0094] Referring to step (2) in Example 1, the nucleotide sequences in this example, such as the four single-stranded DNAs shown in SEQ ID Nos: 1 to 4, were repeated four times and then inserted into the pBluescriptII-KS(+) vector respectively. To increase the final yield, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8 and SEQ ID No: 9 were added at both ends and between each single-stranded DNA sequence, respectively, to obtain nucleic acid fragments containing single-stranded DNAs, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15 and SEQ ID No: 16, respectively.

[0095] SEQ ID No: 13:

[0096]

[0097] SEQ ID No: 14:

[0098]

[0099] SEQ ID No: 15:

[0100]

[0101] SEQ ID No: 16:

[0102]

[0103] The above sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The target sequence fragment and the pBluescript II-KS(+) plasmid were then linearized using restriction endonucleases Sac I and Kpn I. The target sequence fragment was then ligated into the pBluescript II-KS(+) plasmid using T4 DNA ligase. The plasmid vector containing the target sequence fragment was then heat-shocked and transformed into JM109 competent E. coli cells. Single colonies were selected on 2×YT solid medium plates containing 50 μg / mL ampicillin. Successful vector construction was subsequently verified by colony PCR, digestion with the endonucleases Sac I and Kpn I, and DNA sequencing.

[0104] Colony PCR was performed using T3 and T7 primer sequences:

[0105] The sequence of the T3 primer is: 5′-AATTAACCCTCACTAAAGGGA-3′ (SEQ ID No: 11);

[0106] The sequence of the T7 primer is: 5'-TAATACGACTCACTATAG-3' (SEQ ID No: 12).

[0107] (3) Phage fermentation:

[0108] 100 mL of the JM109 Escherichia coli obtained in step (2) was cultured. When the OD value reached 0.05, 1 μL of the M13K07 helper phage was added and the culture was placed in a shaker for infection culture at 200 rpm and 37°C. After 2 h, 100 μL of 30 μg / mL kanamycin was added and the culture was continued for 1.5 h to prepare the original seed bacteria.

[0109] Use a 5L fermentor with 3L of 2×YT medium containing 5mM MgCl2 and sterilize by autoclaving at 121°C for 20 minutes. Add the prepared seed culture to the fermentor, followed by 3mL each of 50μg / mL ampicillin and 30μg / mL kanamycin. Fermentation conditions are 300rpm, 37°C, and pH 7 for 16 hours.

[0110] (4) Extraction of single-stranded DNA:

[0111] The obtained fermentation broth was centrifuged at 5000 g and 4° C. for 20 minutes, and the supernatant was collected. 240 g of PEG8000 and 180 g of NaCl were added, dissolved, and placed in a 4° C. refrigerator for 2 hours.

[0112] Then centrifuge at 8000 g and 4°C for 20 min to collect the phage precipitate.

[0113] Add 40 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH = 8) solution to resuspend the precipitate, then add 80 mL of PPB2 (200 mM NaOH, 1% (w / v) SDS) solution and mix gently. React for 5 minutes. Then add 60 mL of PPB3 (3 M KOAc, adjust pH to 5.5 with acetic acid) solution and mix. React for 10 minutes. Centrifuge at 10,000 g and 4°C for 20 minutes, and collect the supernatant.

[0114] An equal volume of -20°C pre-cooled ethanol was added, mixed thoroughly, and placed in a -20°C refrigerator for 1 hour. Centrifuge at 10,000 g and 4°C for 20 minutes to obtain a precipitate.

[0115] 100 mL of 70% alcohol was added to wash the sample, and the sample was centrifuged at 10,000 g and 4° C. for 20 minutes to obtain a single-stranded DNA precipitate.

[0116] Centrifuge at 10,000 g for 2 minutes at 4°C, and remove the remaining supernatant.

[0117] After drying, add 20 mL of TE solution and allow to dissolve at room temperature for 5 hours. Measure the DNA concentration using a Onedrop micro-spectrophotometer and identify it by gel electrophoresis to obtain single-stranded DNA with a vector backbone.

[0118] (5) Enzymatic cleavage of single-stranded DNA and self-assembly of tetrahedral framework nucleic acids:

[0119] After obtaining single-stranded DNA, take 20 μL of the sample and place it at 95°C for 1 minute, then perform a gradient cooling at 1°C / min until it reaches 25°C to complete DNA annealing. The sequences of SEQ ID No: 3 and 4 at both ends of the single-stranded DNA form a local double-stranded DNA through base pairing. This double-stranded DNA contains the HindⅢ restriction endonuclease recognition site 5'-AAGCTT-3'.

[0120] Add HindⅢ endonuclease and buffer, mix well, and incubate at 37℃ for enzyme digestion to obtain single-stranded DNA.

[0121] The single-stranded DNA after enzyme digestion was placed in a TM buffer (20mM Tris+50mM MgCl2) buffer solution at 95°C for 1 minute, and then gradually cooled to room temperature at a rate of 1°C / min to complete the annealing of the DNA to obtain a tetrahedral framework nucleic acid.

[0122] Figure 3 shows the structure formed after annealing of the ssDNA in Example 2. As can be seen from the figure, a partially double-stranded DNA hairpin structure is generated after annealing of the ssDNA. This double-stranded DNA sequence contains restriction endonuclease recognition sites, which combine to form the four chains of SEQ ID Nos. 1-4 required for tetrahedral nucleic acid. This is repeated four times on different vectors to increase yield.

[0123] The sample, digested and annealed with HindIII restriction endonuclease, was added to 10× Loading buffer and examined on a 1% agarose gel. 40 μL of sample was added to each well. The results are shown in Figure 4. Figure 4 shows gel electrophoresis verifying the single-stranded DNA obtained after enzyme digestion. As can be seen, two new bands were generated after enzyme digestion. Molecular weight analysis revealed that one band was the target single-stranded DNA, and the other was the hairpin structure removed by enzyme digestion.

[0124] Figure 5 shows the electrophoretic band diagram of four single-stranded DNAs assembled into TDN. As can be seen from the figure, different numbers of single-stranded DNAs are combined together through the principle of base complementary pairing. As the number increases, the molecular weight also increases. When the four single strands are mixed in equal proportions and annealed, a tetrahedral nucleic acid can be obtained.

[0125] Atomic force microscopy was used to detect tetrahedral framework nucleic acids. The detection results are shown in FIG6 . From FIG6 , it can be observed that the successfully assembled tetrahedral nucleic acid molecules have a tetrahedral spatial structure, which is consistent with the theoretical size of tetrahedral nucleic acids in combination with the measured size.

[0126] In summary, the present invention utilizes Escherichia coli (E. coli) as a host to produce M13 phage, thereby obtaining ssDNA within the M13 phage. Restriction endonuclease II cleaves the four single strands required for TDN assembly, and TDN is self-assembled. This invention utilizes synthetic biology techniques to achieve large-scale industrial production of TDN while reducing its production cost, demonstrating excellent practicality.

[0127] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a tetrahedral framework nucleic acid, characterized in that: The preparation method comprises: S1. Design a nucleic acid fragment containing single-stranded DNA, referred to as the target sequence fragment; the structure of the nucleic acid fragment containing single-stranded DNA is [L1-L2]n-L3, where L2 is single-stranded DNA, and L1 and L3 are specific DNA sequence fragments that can form a hairpin structure and contain a restriction endonuclease II cleavage site; n is 4; and L1 and L3 cannot complement each other to form a double strand. S2. The target sequence fragment is connected to the phage display plasmid and transferred into E. coli competent cells. After culturing the E. coli cells into which the target sequence fragment has been transferred for a period of time, M13 helper phage is added for co-cultivation; S3. Extracting phage from the cultured product to obtain a target sequence fragment with a vector backbone; S4. After annealing the obtained target sequence fragment with the vector backbone, restriction endonuclease II is added to cut it to obtain four single-stranded DNAs; S5. The four single-stranded DNAs obtained are placed in a TM buffer solution, treated at 90-98° C., and then gradually cooled to complete DNA annealing to obtain the tetrahedral framework nucleic acid.

2. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein The sequences of L1 and L3 are the same or different; each L1 is the same or different, and each L2 is the same or different.

3. The method for preparing a tetrahedral framework nucleic acid according to claim 2, wherein: The L1 or L3 is selected from any one of the nucleotide sequences shown in SEQ ID No: 5 to 9.

4. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein In step S2, the phage display plasmid is pBluescript II-KS(+), the E. coli competent cells are JM109; and the helper phage is M13K07 helper phage.

5. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein In step S2, the Escherichia coli transformed with the target sequence fragment is cultured until the OD value is 0.01-0.1, and then a helper phage is added for co-culture.

6. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein In step S2, the amount of helper phage added is: 1×10 11 pfu / mL helper phage 0.5-2 μL; The co-culture conditions after addition are: shaker speed 150-250 rpm, temperature 28-37°C, kanamycin is added after 1-2 hours, and the culture is continued for 1-2 hours before fermentation culture.

7. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein In step S2, the fermentation culture conditions are as follows: a stirring speed of 250-600 rpm, a temperature of 28-42° C., a pH of 6.5-7.5, and a culture time of 8-14 hours; and the fermentation culture medium is a 2×YT medium containing 5 mM MgCl 2 .

8. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein In step S1, the single-stranded DNA is selected from any one of the sequences shown in SEQ ID Nos: 1 to 4.

9. The method for preparing a tetrahedral framework nucleic acid according to claim 1, wherein In step S5, the mixture is treated at 90-98° C. for 1 min and then gradually cooled to room temperature.

10. The tetrahedral framework nucleic acid prepared by the method according to any one of claims 1 to 9.

Images