A method of modulating dna origami assembly cooperativity

Abstract

This invention discloses a method for regulating the synergistic assembly of DNA origami. The method involves cutting a backbone chain to obtain several segmented backbone chains of different lengths, each segmented backbone chain having several enzyme cleavage sites. When the segmented backbone chains self-assemble with a third staple chain according to the base complementarity principle, the binding rate decreases, thereby regulating the synergistic assembly of DNA origami. The segmented backbone chains include a first staple chain, a second staple chain, and a single-stranded DNA strand of the backbone chain forming a partial double helix, which is then cleaved along the partial double helix by a cleaving enzyme to form several fragments of different lengths. This invention achieves effective regulation of the synergistic assembly of the backbone chain and staple chain in DNA origami technology by cutting the backbone chain. This method has wide applicability, requires no significant adjustment of the assembly buffer or temperature conditions, is not limited to the backbone chain structure, and has no impact on the final DNA origami structure.

Description

Technical Field

[0001] This invention belongs to the field of DNA nanotechnology, specifically relating to a method for regulating the synergistic assembly of DNA origami. Background Technology

[0002] DNA nanotechnology is a technique that utilizes the complementarity of DNA molecules to construct complex nanostructures, offering high programmability and precision. It primarily uses DNA molecules as the building blocks for DNA nanostructures, forming specific nanoshapes through self-assembly. This leverages the unique base-pairing properties of DNA and the coding nature of its base sequences. Existing technologies use DNA self-assembly methods to design DNA structures, such as DNA origami, DNA bricks, and DNA tiles. DNA origami is currently one of the most important methods for constructing DNA nanostructures. Using DNA origami, a large number of two-dimensional and three-dimensional static DNA structures and dynamic DNA nanomachines of various shapes and sizes can be designed, leading to many important applications in biomedicine, sensing, drug delivery, and nanophotonics.

[0003] DNA origami is a technique that utilizes the complementary base pairing properties and self-assembly capabilities of DNA molecules to precisely fold long-chain DNA (backbone or scaffold chain) and short-chain DNA (staple or staple chain) into predetermined two-dimensional or three-dimensional nanostructures. Its core principle is to use a long single-stranded DNA as the "backbone," assisted by hundreds of short-chain DNA "staple chains" to fold and fix the backbone, thereby efficiently and accurately assembling it into a predetermined two-dimensional or three-dimensional nanostructure. This method boasts high yield, high programmability, and excellent site addressability. Under isothermal assembly conditions, it has been found that hundreds of staple chains can fold the backbone into the target structure within minutes. For example, Chinese invention patent CN107488661A discloses a nucleic acid nanostructure, which uses DNA origami technology to construct a hexagonal DNA nanostructure by assembling six triangular DNA origami structures. Specifically, this hexagonal DNA nanostructure is formed by hybridizing the scaffold chain with staple chains and capture chains, and then by connecting chains hybridizing with the scaffold chains of the six triangular DNA origami structures.

[0004] Traditional methods involve adding pre-designed structural monomers to the reaction system in a single step to obtain the desired DNA origami structure. While this method, based on base pairing principles, provides some synergy during assembly, the rapid self-assembly process, completed within minutes, is particularly challenging when hundreds of stapled strands are simultaneously assembled with the backbone strand. High specificity at each monomer connection point is required, making effective control of the assembly process and synergistic performance extremely difficult. Furthermore, chemical modifications to the DNA origami can only be performed pre-processed on the backbone strand, not during assembly. Chinese invention patent CN109477096A discloses a stepwise assembly method for DNA origami units, where pre-designed structural monomers are added in each step to achieve high-accuracy DNA origami assembly. However, this stepwise approach is cumbersome and unsuitable for mass production of DNA origami. Therefore, a simple, highly compatible method for controlling DNA origami synergy and final assembly yield is urgently needed.

[0005] To address the aforementioned issues, this invention provides a method for regulating the synergistic properties of DNA origami assembly. By constructing a segmented backbone chain to regulate the cleavage sites of the DNA backbone chain, the binding rate of double-stranded hybridization is effectively controlled, thereby achieving regulation of the synergistic properties of DNA origami. Summary of the Invention

[0006] The main objective of this invention is to provide a method and application for regulating the synergistic properties of DNA origami assembly, in order to overcome the shortcomings of the prior art.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] As the first objective of the invention, the present invention provides a method for regulating the synergistic performance of DNA origami assembly, comprising: cutting a backbone chain to obtain a number of segmented backbone chains of different lengths, such that the segmented backbone chains have a number of enzyme cleavage sites, and when the segmented backbone chains self-assemble with a second staple chain according to the base complementary pairing principle, the binding rate decreases, thereby achieving the regulation of the synergistic performance of DNA origami assembly.

[0009] Preferably, the first staple chain is a staple chain without enzyme cleavage sites.

[0010] Preferably, the second staple chain is a staple chain containing enzyme cleavage sites and serves as a primer.

[0011] Preferably, the number of the first staple chains is the same as the sum of the number of the second and third staple chains.

[0012] In a preferred embodiment, the segmented backbone chain comprises: forming a partial double strand by using a first staple chain, a second staple chain, and a single strand of DNA from the backbone chain; and then using a nicking enzyme to nick the backbone chain along the partial double strand to form several fragments of different lengths.

[0013] As a preferred embodiment, the method for constructing the segmented skeleton chain includes:

[0014] S1. Construct pscaf phage particles;

[0015] DNA gene fragments were ligated into pscaf phage vectors using a double enzyme digestion method, and the pscaf phage particles were obtained after transformation and culture.

[0016] S2. Construct a backbone chain containing enzyme cleavage sites;

[0017] The pscaf phage particles and helper phages were co-transformed into competent Escherichia coli cells. After culturing and centrifugation, the target single-stranded DNA solution was obtained by lysis, which is the backbone chain containing enzyme cleavage sites.

[0018] S3. Construct a segmented skeleton chain;

[0019] After adding the first and second staple chains and annealing, they anneal with a specific region of the backbone chain containing the enzyme cleavage site to form a local double strand, guiding the cleavage enzyme to cleave the backbone DNA into fragments of different lengths, thus obtaining a segmented backbone chain.

[0020] Preferably, in S2, the sequence of the backbone chain containing the enzyme cleavage site is as shown in SEQ ID NO.1;

[0021] Preferably, the backbone chain containing enzyme cleavage sites contains 96 enzyme cleavage sites.

[0022] Preferably, the nicking enzyme is Nb.BtsI enzyme.

[0023] Preferably, in S3, the annealing conditions include: 85°C to 25°C, -1°C / 3 min.

[0024] Preferably, in S3, the sequence of the first staple chain is as shown in SEQ ID NO.3~SEQ ID NO.98.

[0025] Preferably, the molar ratio of the backbone chain containing the enzyme cleavage site to the first staple chain is 1:10.

[0026] In a preferred embodiment, the sequence of the second staple chain (primer containing restriction enzyme sites) is selected from at least two sequences of SEQ ID NO. 99 to SEQ ID NO. 194, but is not limited thereto.

[0027] Preferably, the molar ratio of the backbone chain containing the enzyme cleavage site to the second staple chain is 1:10.

[0028] When any two of the above-mentioned second staple strand sequences (primer sequences) containing enzyme cleavage sites are selected, the segmented backbone strand is cut into two segments; when any eight of the above-mentioned primer sequences containing enzyme cleavage sites are selected, the segmented backbone strand is cut into eight segments, and so on, to obtain cut fragments with sixteen, thirty-two, etc.

[0029] Preferably, the sequence of the third staple chain is selected from SEQ ID NO.99 to SEQ ID NO.194, and does not include the sequence of the second staple chain.

[0030] Preferably, the molar ratio of the backbone chain containing the enzyme cleavage site to the second staple chain is 1:10.

[0031] For example, when the second staple chain sequence is SEQ ID NO.144 and SEQ ID NO.149 respectively, the sequence of the third staple chain is selected from SEQ ID NO.99 to SEQ ID NO.194, but does not include the sequences SEQ ID NO.144 and SEQ ID NO.149; when the second staple chain sequence is SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.146, SEQ ID NO.147, SEQ ID NO.149, SEQ ID NO.154, SEQ ID NO.192, SEQ ID NO.193, the sequence of the third staple chain is selected from SEQ ID NO.99 to SEQ ID NO.194, but does not include the sequences SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.146, SEQ ID NO.147, SEQ ID NO.149, SEQ ID NO.154, SEQ ID NO.192, SEQ ID NO.193.

[0032] Preferably, the self-assembly includes incubating the third staple chain and the segmented skeleton chain together under constant temperature incubation conditions of 50~60℃ for 12 hours.

[0033] Preferably, the segmented skeleton chain is cut into 2 to 32 segments; more preferably, it is cut into 2 to 4 segments.

[0034] As another objective of the invention, the present invention also provides a DNA origami structure prepared by means of the method described above for regulating the synergistic assembly of DNA origami.

[0035] The DNA origami structure includes polygonal structures, including squares, rectangles, hexagons, etc., but is not limited to these.

[0036] This invention designs a single-stranded DNA backbone with reserved locations for enzyme cleavage sites. Then, staple strands are introduced at non-cleavage sites to form local double strands. Finally, the backbone is cut at the enzyme cleavage sites by primer DNA, resulting in segmented backbones of several different lengths.

[0037] In this invention, the designed pscaf7560 single-stranded DNA was cut into 2, 4, 8, 16 and 32 segments respectively, and finally formed a DNA origami structure.

[0038] For example, taking a two-segment cut as an example, a rectangular origami structure is designed based on pscaf7560 single-stranded DNA (sequence shown in SEQ ID NO.1), wherein the 56th and 3480th bases of the pscaf7560 single-stranded DNA are used as enzyme cleavage sites, and the primer sequences binding to the above two enzyme cleavage sites are shown in SEQ ID NO.144 and SEQ ID NO.149.

[0039] The backbone strand and staple strand were mixed in a 1:10 ratio. The pscaf7560 backbone strand and staple strand were combined and self-assembled to obtain a rectangular DNA origami structure.

[0040] More specifically, pscaf7560 single-stranded DNA was mixed with a first staple strand (SEQ ID NO.3~SEQ ID NO.98) and a second staple strand containing restriction enzyme sites (such as SEQ ID NO.144 and SEQ ID NO.149) at a ratio of 1:10 and then annealed. The annealing conditions were: temperature decreasing from 85℃ to 25℃, -1℃ / 3min, for 200 minutes, to form a local double-stranded structure. Then, Nb.BtsI nicking enzyme was added to cut the backbone strand into two segments. Finally, the remaining staple strands (SEQ ID NO.99~SEQ ID NO.194, excluding SEQ ID NO.144 and SEQ ID NO.149) were added and mixed at a ratio of 1:10 and incubated at a constant temperature of 50~60℃ for 12 hours. The pscaf7560 backbone strand bound to the staple strand and self-assembled to obtain a rectangular DNA origami structure.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] 1. This invention achieves effective control of the assembly synergy between the backbone chain and staple chain in DNA origami technology by cutting the backbone chain; this method has wide applicability, does not require significant adjustment of assembly buffer or temperature conditions, and is not limited to the backbone chain structure, nor does it affect the final DNA origami structure; and the obtained DNA origami structure product has comparable stability performance to products obtained by other methods in the prior art.

[0043] 2. This invention allows for the modification of the segmented backbone chain obtained after cutting the backbone chain before further assembly with the staple chain. This enables the simple and effective design of DNA origami product structures as needed, simplifies the chemical modification process of DNA origami, and is suitable for large-scale promotion and application. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 This is a schematic diagram simulating the principle of regulating the synergistic assembly of DNA origami using existing technologies and the present invention.

[0046] Figure 2 This is a schematic diagram of a DNA origami structure designed in this invention.

[0047] Figure 3 This is an atomic force microscope image of the DNA origami structure obtained by assembling the DNA backbone strands cut into two segments in Example 1 of this invention.

[0048] Figure 4 This is a comparison diagram of the assembly kinetics of DNA origami obtained in Examples 1-3 of the present invention as the DNA backbone chain is cut into different numbers of fragments.

[0049] Figure 5 This is a comparison graph of the gel yield of the DNA origami structures of Examples 1-5 of the present invention with the gel yield of the DNA backbone strand cut into different numbers of fragments and the gel yield of the uncut DNA backbone strand. The vertical axis of the graph is the gel yield.

[0050] Figure 6 The images show a comparison of atomic force microscopy images of DNA backbone strands obtained in Example 1 of this invention, one uncut and the other cut into two segments, after being assembled into DNA origami and incubated.

[0051] Figure 7 The images are atomic force microscopy comparisons of DNA origami assembled from DNA backbone strands cut into 2, 4, and 8 segments respectively, according to Examples 1-3 of the present invention.

[0052] Figure 8 The figures show a comparison of the yields of complete DNA origami products assembled from DNA backbone strands cut into 2 and 4 segments, respectively, obtained in Examples 1-3 of this invention. Detailed Implementation

[0053] Together with Figure 1 Reading the following detailed description will provide a more complete understanding of the invention. Detailed embodiments of the invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary examples of specific implementations of the invention, which may be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as intended to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0054] This invention provides a method for regulating the synergistic properties of DNA origami assembly, see reference. Figure 1 Figure A presents a comparative simulation of the principle of regulating the synergistic assembly of DNA origami using existing technologies and the present invention. Figure A shows a simulation diagram of the conventional assembly of the backbone chain and staple chain in existing technologies. In conventional assembly, the binding of the staple chain (gray and yellow) and the backbone chain (blue) exhibits a positive synergistic effect, meaning that the precursor binding promotes subsequent binding. Figures B and C show simulation diagrams of regulating the synergistic assembly of DNA origami by introducing cuts at different sites on the backbone chain. In B, cuts are introduced at different sites on the backbone chain to first reduce the rate of precursor binding and then promote subsequent binding; in C, the binding rate of both chains is reduced simultaneously. Based on the above principles, in practical applications, the assembly rate of DNA origami structures is extremely fast, usually completing the general assembly of the structure within a few minutes. Therefore, fluorescence detection of the assembly process requires high operational skills. The present invention significantly reduces the assembly rate by cutting the backbone chain. On the one hand, this makes fluorescence detection more accurate; on the other hand, the cut backbone chain can be chemically modified (e.g., phosphorylation, methylation) to achieve regulation of the DNA origami structure.

[0055] One specific embodiment provides a method for regulating the coordination of DNA origami assembly, specifically including:

[0056] 1. Construct a segmentable DNA backbone chain system

[0057] (1) Construct the pscaf7560 plasmid; Phage particles are hybrid vectors that combine the characteristics of plasmids and bacteriophages. They can stably replicate in E. coli as double-stranded plasmids, and can also initiate single-stranded DNA synthesis and package it into phage particles after infection by helper phages. Since phage particles themselves do not encode phage proteins and rely on helper phages to provide replicase and packaging proteins, they are widely used in the field of single-stranded DNA preparation.

[0058] The pscaf phage vector contains two restriction enzyme sites: KpnI and BamHI.

[0059] This invention first designed a gene fragment 7560 with a length of 7179 bp, which was synthesized by Suzhou Genewiz Company, and its sequence is shown in SEQ ID NO.2.

[0060] The gene fragment 7560 was ligated into the pscaf phage vector using a double enzyme digestion method. After transformation, culture and identification, pscaf7560 phage particles rich in the target DNA single strand were constructed. The final backbone chain produced was 7560 nt in length and contained 96 Nb.BtsI restriction sites, as shown in SEQ ID NO.1.

[0061] (2) Prepare a backbone DNA strand containing restriction enzyme sites;

[0062] The pscaf7560 phage particle constructed in step (1) and the helper phage pSB4423 (plasmid HP17_KO7, addgene#120346) were co-transformed into XL1-Blue Escherichia coli competent cells (purchased from Beyotime Biotechnology Co., Ltd.).

[0063] With the assistance of helper phages, pscaf7560 phage particles replicate, producing a large amount of target single-stranded DNA with a length of 7560 nt, namely the pscaf7560 backbone chain.

[0064] After culturing, a phage particle suspension rich in target single-stranded DNA was obtained. The phage precipitate was collected by centrifugation and resuspended in 4 mL of 1 M Tris (tris(hydroxymethyl)aminomethane, purchased from Sigma-Aldrich). The phage shell was lysed using a protein lysis agent (0.2 M sodium hydroxide and 1% sodium dodecyl sulfate, both purchased from Sigma-Aldrich). The precipitate was removed by centrifugation, and a supernatant containing the target single-stranded DNA was obtained. Subsequently, ethanol was added to precipitate the DNA. After centrifugation, the supernatant was discarded, and the solution was resuspended in pure water to obtain a pure target single-stranded DNA solution.

[0065] 2. DNA origami assembly with segmented, customizable sequence backbone strands.

[0066] Based on the fact that the pscaf7560 skeleton chain prepared above can be assembled normally, this embodiment adopts the method of cutting it into different numbers of segments to control the assembly synergy.

[0067] The specific steps include: first, mixing the pscaf7560 backbone chain with staple chains without restriction enzyme sites at a ratio of 1:10, annealing in buffer, reducing the temperature from 85℃ to 25℃, -1℃ / 3min (i.e., holding for 3min for every 1℃ decrease), adding staple chains containing restriction enzyme sites as primers (sequences are SEQ ID NO.99 and SEQ ID NO.194), allowing them to bind to specific regions of the backbone chain; according to the number of staple chains containing restriction enzyme sites added, using Nb.BtsI restriction enzyme to cleave the backbone chain into several segments n (n≥2); after inactivating the enzyme at 80℃ for 20min, adding the remaining staple chains containing restriction enzyme sites (sequences are shown as SEQ ID NO.99~SEQ ID NO.194, except for the primer sequences), and incubating at 58.3℃ for 12 hours.

[0068] SYBR Green fluorescent dye (which specifically binds to double-stranded DNA) was added to the buffer, and the changes in fluorescence signal during the assembly process were monitored using a real-time quantitative PCR instrument to assess synergy.

[0069] As a preferred embodiment, the DNA origami structure is a rectangular origami structure of 97.92 nm × 72 nm.

[0070] It should be noted that in some specific embodiments of the present invention, the selected staple chains include staple chains containing restriction enzyme sites and staple chains without restriction enzyme sites. The sequences of staple chains without restriction enzyme sites are shown in SEQ ID NO. 3 to SEQ ID NO. 98; the sequences of staple chains containing restriction enzyme sites are shown in SEQ ID NO. 99 to SEQ ID NO. 194, all of which contain GCAGTG. The remaining sequences vary depending on the binding region between the staple chain and the backbone chain. The staple chains containing restriction enzyme sites include a first staple chain containing restriction enzyme sites and a second staple chain containing restriction enzyme sites. First, according to the number of fragments cut and the sequence of the sites, the first staple chain containing restriction enzyme sites is added as a primer to form a segmented backbone chain. Then, the second staple chain containing restriction enzyme sites is added. During self-assembly based on the base complementary pairing principle, the binding rate decreases, thereby regulating the synergistic assembly of DNA origami and obtaining a DNA origami structure.

[0071] In a preferred embodiment, the ratio of the backbone chain to the staple chain without enzyme cleavage sites is 1:10.

[0072] As a preferred embodiment, gradient annealing is used, with the temperature decreasing from 85°C to 25°C, -1°C / 3min (holding for 3min for each 1°C decrease), for a total of 200 minutes.

[0073] Example 1

[0074] This embodiment constructs a method for origami assembly of a segmentable custom sequence DNA backbone chain, specifically including:

[0075] I. Preparation process of pscaf7560 backbone DNA

[0076] (1) Construction of pscaf7560 phage particles: BamHI and KpnI restriction sites were modified at both ends of the 7560 gene fragment, respectively. The original pscaf vector and the 7560 gene fragment (as shown in SEQ ID NO.2) were subjected to double enzyme digestion with BamHI and KpnI, respectively. Then, the 7560 gene fragment and the original pscaf vector were ligated using T4 DNA ligase to obtain pscaf 7560 phage particles. The construction of pscaf7560 phage particles was commissioned to Suzhou Genewiz Biotechnology Co., Ltd.

[0077] (2) Co-conversion: Prepare 10 mL YT medium, 25 mL LB solid medium, and 200 mL YT medium in advance. After sterilizing at 121℃ for 15 minutes, add carbenicillin and chloramphenicol (purchased from APExBIO) to 25 mL LB solid medium and 200 mL YT medium so that the concentration of carbenicillin in the medium is 50 mg / mL and the concentration of chloramphenicol is 20 mg / mL. After mixing thoroughly, pour the LB solid medium into the culture dish.

[0078] 50 ng of pscaf7560 phage prepared using the above method and 50 ng of helper phage pSB4423 (plasmid HP17_KO7, addgene#120346) were added together to 20 μL of XL1-Blue competent cells (purchased from Beyotime Biotechnology Co., Ltd.). The mixture was gently pipetted and incubated on ice for 30 minutes, followed by heat shock at 42°C for 40 seconds. Then, 200 μL of antibiotic-free YT medium was added for recovery culture. After incubation at 30°C for 1 hour, the bacterial culture was evenly spread on LB agar plates containing chloramphenicol and carbenicillin and incubated at 30°C for 48 hours.

[0079] After the colonies have grown, single colonies are picked and added to 3 mL of YT medium containing carbenicillin and chloramphenicol for expansion culture at 30°C and 280 rpm for 24 hours. Then the bacterial culture is transferred to 200 mL of YT medium containing carbenicillin and chloramphenicol and cultured at 30°C and 280 rpm for 48 hours.

[0080] (3) Extraction of pscaf7560 backbone DNA: Collect the bacterial culture, centrifuge at 4℃ and 8000 RCF for 20 minutes, discard the precipitate, retain the supernatant (containing phage particles), add 3% sodium chloride and 4% PEG-8000 (both purchased from Sigma-Aldrich), gently shake until completely dissolved, and incubate in an ice water bath for 30 minutes.

[0081] After the ice bath, the sample was centrifuged at 4°C and 8000 RCF for 20 minutes. The supernatant was discarded, and the precipitate was resuspended in 4 mL of 1M Tris (tris(hydroxymethyl)aminomethane, purchased from Sigma-Aldrich). 8 mL of protein lysis buffer (0.2 M sodium hydroxide and 1% sodium dodecyl sulfate, both purchased from Sigma-Aldrich) was added for lysis for 3 minutes, followed immediately by 6 mL of 3M potassium acetate (purchased from Sigma-Aldrich) for neutralization. The sample was then centrifuged at 4°C and 8000 RCF for 15 minutes, and the precipitate was discarded. At this point, the single-stranded DNA was dissolved in the supernatant.

[0082] Add 10 mL of pure ethanol (purchased from Sigma-Aldrich) to the supernatant and freeze at -20°C for 8 hours. Then centrifuge at 8000 RCF for 15 minutes at 4°C, discard the supernatant, add 15 mL of 75% ice-cold ethanol (prepared in advance and stored at -20°C), centrifuge at 8000 RCF for 15 minutes at 4°C, discard the supernatant, resuspend the precipitate with ultrapure water to obtain the pscaf7560 backbone DNA, the sequence of which is shown in SEQ ID NO.1.

[0083] II. Construction of Segmentable Customizable Sequence Skeleton Chains

[0084] This embodiment verifies the DNA origami assembly performance of the pscaf7560 backbone strand constructed in step one, specifically including:

[0085] See Figure 2A rectangular origami structure with a theoretical size of 97.92 nm × 72 nm was designed, along with 192 staple chains, including 96 staple chains without enzyme cleavage sites, the sequences of which are shown in SEQ ID NO.3~SEQ ID NO.98, and 96 staple chains with enzyme cleavage sites, the sequences of which are shown in SEQ ID NO.99~SEQ ID NO.194.

[0086] A rectangular origami structure with a theoretical size of 97.92 nm × 72 nm was designed, along with 192 staple chains (SEQ ID NO.3~SEQ ID NO.194). The backbone chain and staple chains were mixed at a 1:10 ratio and annealed in a buffer solution containing 10 mM Tris and 1 mM EDTA (purchased from Sigma-Aldrich). Annealing was performed using a PCR instrument, with the temperature decreasing from 85℃ to 25℃ at -1℃ / 3 min for 200 minutes. This step yielded a DNA origami structure assembled from uncut pscaf7560 backbone chains. The product was analyzed by separating the target band by agarose gel electrophoresis and recovering the product. The prepared DNA origami structure was characterized using atomic force microscopy.

[0087] Further, the pscaf7560 single-stranded DNA prepared above was mixed with 96 staple strands without restriction enzyme sites (sequences shown in SEQ ID NO.3 to SEQ ID NO.98) and two staple strands with restriction enzyme sites as primers (sequences shown in SEQ ID NO.144 and SEQ ID NO.149). The molar ratio of pscaf7560 single-stranded DNA to staple strands without restriction enzyme sites was 1:10, and the molar ratio of pscaf7560 single-stranded DNA to primers was 1:10. The mixture was annealed in a buffer solution containing 10 mM Tris and 1 mM EDTA (purchased from Sigma-Aldrich). The annealing temperature was controlled using a PCR instrument, and the annealing temperature was reduced from 85℃ to 25℃, -1℃ / 3min, for a total of 200 minutes.

[0088] The staple strand and primer, lacking enzyme cleavage sites, anneal to a specific region of the 7560 backbone strand to form a local double strand. The Nb.BtsI cleavage enzyme recognizes and cleaves at the double-stranded position containing the sequence 5'-NN↓CACTGC-3', where ↓ indicates the cleavage site. In this embodiment, the cleavage positions on the backbone strand are the 56th and 3480th bases, corresponding to... Figure 2 The coordinates [x,y] in the diagram are [4,23] and [5,0], where x represents the horizontal coordinate and y represents the vertical coordinate. The skeleton chain is cut into two segments with lengths of 3424 nt and 4136 nt, respectively, to obtain a segmented skeleton chain.

[0089] III. Regulation of Coordination in DNA Origami Assembly with Segmentable Customizable Sequence Backbone Strands

[0090] The segmented backbone obtained in the above steps of this embodiment is subjected to synergistic regulatory operations to achieve DNA origami assembly. The specific steps include:

[0091] The segmented backbone strands obtained in step two were added to the remaining 94 staple strands containing restriction enzyme sites, with sequences SEQ ID NO.99~SEQ ID NO.194 (excluding sequences SEQ ID NO.144 and SEQ ID NO.149). The mixture was incubated at 58.3℃ for 12 hours to complete the origami assembly, thereby achieving the coordinated regulation of DNA origami assembly. This process can be observed by adding specific fluorescent molecules. Specifically, SYBR Green fluorescent dye is added to the buffer to specifically bind to double-stranded DNA, and the changes in fluorescence signal during the assembly process are monitored using a real-time quantitative PCR instrument to assess the coordinated nature of the assembly.

[0092] The DNA origami product obtained in the above steps was used to separate and recover the target band by agarose gel electrophoresis, and the prepared DNA origami structure was characterized by atomic force microscopy.

[0093] See Figure 3 The image shows an atomic force microscope image of a rectangular DNA origami structure, with an image size of 2 μm × 2 μm. The measured size of the rectangle in the image is 97.94 nm × 75.49 nm, which is close to the theoretical size of 97.92 nm × 72 nm, proving that the structure meets expectations. In particular, there is no significant difference in the microstructure of the DNA origami products obtained by uncutting and two-segment cutting.

[0094] See Figure 2 The diagram shows a simulated schematic of the pscaf7560 DNA backbone chain restriction enzyme sites of the DNA origami structure designed in this embodiment. The blue lines in the diagram represent the backbone chain, the breaks in the blue lines are the Nb.BtsI restriction sites, which are marked in red, and the gray lines represent the staple chain.

[0095] Example 2

[0096] The only difference between this embodiment and Example 1 is the number of staple chains containing enzyme cleavage sites added as primers, resulting in a different number of segments in the obtained segmented backbone chain.

[0097] In step two, four primers are added, with sequences shown in SEQ ID NO.144, SEQ ID NO.149, SEQ ID NO.192, and SEQ ID NO.193. The 96 staple chains without restriction enzyme sites are the same as in Example 1, with cleavage positions on the backbone chain at positions 56, 3480, 1688, and 5208, corresponding to the bases at positions 56, 3480, 1688, and 5208, respectively. Figure 2 The coordinates [x,y] are shown in red circles [4,23], [5,0], [4,11], and [5,12], where x represents the horizontal coordinate and y represents the vertical coordinate. The DNA backbone is cut into 4 segments.

[0098] In step three, the remaining 92 staple chains containing enzyme cleavage sites are added, with sequences as shown in SEQ ID NO.99~SEQ ID NO.194 (excluding sequences SEQ ID NO.144, SEQ ID NO.149, SEQ ID NO.192, and SEQ ID NO.193), to complete the origami assembly and thus achieve synergistic regulation.

[0099] Example 3

[0100] The only difference between this embodiment and Example 1 is the number of staple chains containing enzyme cleavage sites added as primers, resulting in a different number of segments in the obtained segmented backbone chain.

[0101] In step two of this embodiment, eight staple chains containing enzyme cleavage sites are added as primers, with sequences as shown in SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 147, SEQ ID NO. 149, SEQ ID NO. 154, SEQ ID NO. 192, and SEQ ID NO. 193. The 96 staple chains without enzyme cleavage sites are the same as in Example 1, with cleavage positions on the backbone chain at positions 120, 6480, 3352, 1272, 56, 3480, 1688, and 5208, corresponding to the bases at positions 120, 6480, 3352, 1272, 56, 3480, 1688, and 5208, respectively. Figure 2 The coordinates [x,y] in the figure are [2,23], [4,23], [8,23], [1,0], [5,0], [7,2], [4,11], and [5,12], respectively, where x represents the horizontal coordinate and y represents the vertical coordinate. The DNA backbone is cut into 8 segments.

[0102] In step three, the remaining 88 staple chains containing enzyme cleavage sites are added, with sequences SEQ ID NO.99~SEQ ID NO.194 (excluding sequences SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.146, SEQ ID NO.147, SEQ ID NO.149, SEQ ID NO.154, SEQ ID NO.192, and SEQ ID NO.193), to complete the origami assembly and thus achieve synergistic regulation.

[0103] See Figure 4 The figure shows the assembly kinetics of DNA origami in Examples 1-3 as the DNA backbone strand was cut into different numbers of segments (the vertical axis is the normalized fluorescence intensity). The DNA backbone strands included no cutting, 2-segment cutting, 4-segment cutting, and 8-segment cutting, respectively. As can be seen from the figure, the assembly rate decreased significantly as the number of segments cut into the backbone DNA increased during the assembly of the origami structure.

[0104] Example 4

[0105] The only difference between this embodiment and Example 1 is the number of staple chains containing enzyme cleavage sites added as primers, resulting in a different number of segments in the obtained segmented backbone chain.

[0106] In step two of this embodiment, sixteen staple chains containing enzyme cleavage sites are added as primers (sequences shown in SEQ ID NO. 115, SEQ ID NO. 118, SEQ ID NO. 135, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 147, SEQ ID NO. 149, SEQ ID NO. 154, SEQ ID NO. 155, SEQ ID NO. 158, SEQ ID NO. 166, SEQ ID NO. 171, SEQ ID NO. 192, and SEQ ID NO. 193) and 96 staple chains without enzyme cleavage sites (sequences shown in SEQ ID NO. 3 to SEQ ID NO. 98). The corresponding enzyme cleavage sites are... Figure 2 The coordinates [x,y] in the diagram are [2,15], [8,15], [1,20], [8,20], [2,23], [4,23], [8,23], [1,0], [7,2], [2,23], [4,11], [8,1], [8,3], [1,6], [5,12], and [2,1], where cutting occurs at the red circles. Here, x represents the horizontal coordinate and y represents the vertical coordinate, and the DNA backbone is cut into 16 segments.

[0107] In step three, the remaining 80 staple chains containing enzyme cleavage sites are added, with sequences SEQ ID NO.99~SEQ ID NO.194 (excluding sequences SEQ ID NO.115, SEQ ID NO.118, SEQ ID NO.135, SEQ ID NO.142, SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.146, SEQ ID NO.147, SEQ ID NO.149, SEQ ID NO.154, SEQ ID NO.155, SEQ ID NO.158, SEQ ID NO.166, SEQ ID NO.171, SEQ ID NO.192, and SEQ ID NO.193), to complete the origami assembly, thereby achieving synergistic regulation.

[0108] Example 5

[0109] The only difference between this embodiment and Example 1 is the number of staple chains containing enzyme cleavage sites added as primers, resulting in a different number of segments in the obtained segmented backbone chain.

[0110] In this embodiment, thirty-two staple chains containing restriction enzyme sites were added as primers, with sequences such as SEQ ID NO. 99, SEQ ID NO. 101, SEQ ID NO. 103, SEQ ID NO. 105, SEQ ID NO. 107, SEQ ID NO. 109, SEQ ID NO. 111, SEQ ID NO. 113, SEQ ID NO. 115, SEQ ID NO. 118, SEQ ID NO. 135, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 147, SEQ ID NO. 149, SEQ ID NO. 154, SEQ ID NO. 155, SEQ ID NO. 158, SEQ ID NO. 166, SEQ ID NO. 171, SEQ ID NO. 175, SEQ ID NO. 177, SEQ ID NO. 179, SEQ ID NO. 181, SEQ ID NO. 183, SEQ ID NO. 185, SEQ ID NO. 186 ...86, SEQ ID NO. 175, SEQ ID NO. 177, SEQ ID NO. 179, SEQ ID NO. 181, SEQ ID NO. 183 As shown in SEQ ID NO. 187, SEQ ID NO. 189, SEQ ID NO. 192, and SEQ ID NO. 193, the sequences of 96 staple chains without restriction enzyme sites are shown in SEQ ID NO. 3 to SEQ ID NO. 98, and the corresponding restriction enzyme sites are... Figure 2The coordinates [x,y] are [2,15], [6,15], [1,14], [5,14], [2,13], [6,13], [1,16], [5,16], [2,15], [8,15], [1,20], [8,20], [2,23], [4,23], [8,23], [1,0], [7,2], [2,23], [4,11], [8,1], [8,3] respectively. Cuttings occurred at the red circles [1,6], [1,8], [5,8], [2,7], [6,7], [1,10], [5,10], [2,9], [6,9], [5,7], and [2,1], where x represents the horizontal axis and y represents the vertical axis, indicating that the DNA backbone was cut into 32 segments.

[0111] In step three, the remaining 64 staple chains containing restriction enzyme sites are added, with sequences SEQ ID NO.99~SEQ ID NO.194 (excluding sequences SEQ ID NO.99, SEQ ID NO.101, SEQ ID NO.103, SEQ ID NO.105, SEQ ID NO.107, SEQ ID NO.109, SEQ ID NO.111, SEQ ID NO.113, SEQ ID NO.115, SEQ ID NO.118, SEQ ID NO.135, SEQ ID NO.142, SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.146, SEQ ID NO.147, SEQ ID NO.149, SEQ ID NO.154, SEQ ID NO.155, SEQ ID NO.158, SEQ ID NO.166, SEQ ID NO.171, SEQ ID NO.175, SEQ ID NO.177, SEQ ID NO.179, SEQ ID NO.174 ...4, SEQ ID NO.175, SEQ ID NO.174, SEQ ID NO.175, SEQ ID NO.174, SEQ ID NO.175, SEQ ID NO.174, SEQ ID NO.175, SEQ ID NO.174, SEQ ID NO.175, SEQ ID NO.174, SEQ ID NO.175, SEQ ID NO.174, SEQ ID NO.1 (SEQ ID NO.181, SEQ ID NO.183, SEQ ID NO.185, SEQ ID NO.187, SEQ ID NO.189, SEQ ID NO.192, SEQ ID NO.193) to complete the origami assembly, thereby achieving synergistic control.

[0112] See Figure 5 The figure shows a comparison of the complete yield under different cutting numbers. As can be seen from the figure, the gel yield analysis of the DNA origami structures of Examples 1-5 as the DNA backbone strand was cut into different numbers of segments (the vertical axis is the normalized gel yield). As can be seen from the figure, the yield decreases as the number of backbone strand segments increases, but the rate of yield decrease gradually slows down after 4 segments.

[0113] See Figure 6 These are atomic force microscopy images, showing the uncut DNA backbone and the DNA backbone cut into two segments for assembly into DNA origami, after incubation. The image size is 2 μm × 2 μm, and the scale bar is 200 nm. Figure 6 Figures A and B shown are comparison images of DNA origami formed by uncut DNA backbone strands before and after incubation at 45°C for 2 hours. Figure 6 Figures C and D shown are comparison images of DNA origami formed after cutting two segments of the DNA backbone strand and incubating at 45°C for 2 hours. The comparison shows that there is no significant difference between the DNA origami formed after cutting two segments of the DNA backbone strand and the DNA origami formed without cutting the DNA backbone strand before and after incubation, and both can maintain the integrity of the structure.

[0114] See Figure 7 The images are atomic force microscopy (AFM) images of DNA origami assembled from DNA backbone strands cut into 2, 4, and 8 segments, respectively. The image size is 2 μm × 2 μm, and the scale bar is 200 nm. Images A, B, and C in the figure correspond to the DNA origami assembled from 2, 4, and 8 segments, respectively. The results show that all three types of origami assembled into the target structure. As the number of segments increases, cutting into 2 and 4 segments ensures the integrity of the DNA origami, while the structural integrity decreases significantly when cutting into 8 segments.

[0115] See Figure 8 The figure shows a comparison of the intact product yield under different cutting numbers. As can be seen from the figure, when the DNA backbone is cut into 2 or 4 segments, the yield of the intact product is above 60%, while when it is cut into 8 segments, the yield of the intact product is below 50%. Obviously, the higher the number of segments, the lower the yield of the intact product. Therefore, cutting the DNA backbone into 2 to 4 segments can ensure the integrity of the product.

[0116] In summary, the DNA origami product structure obtained by adopting the technical solution of this invention is not significantly different from the DNA origami product structure obtained by the uncut method. Moreover, this invention can simplify the preparation steps of DNA origami, and while ensuring the integrity of the origami structure product, it can also control the assembly speed of the origami structure. On the other hand, by selectively cutting the DNA backbone chain sites to form segmented DNA backbone chains and then chemically modifying them, the applications of DNA origami structure products can be expanded, making them suitable for large-scale promotion and application.

[0117] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A method for regulating the coordination of DNA origami assembly, comprising: The backbone chain is cut to obtain a segmented backbone chain of several different lengths, and the segmented backbone chain has several enzyme cutting sites. When the segmented backbone chain and the third staple chain self-assemble through the base complementary pairing principle, the binding rate decreases, thereby achieving the regulation of the synergistic performance of DNA origami assembly. The segmented backbone strand comprises forming a partial double strand with the DNA single strand of the backbone strand using a first staple strand, a second staple strand, and a backbone strand, and then using a nicking enzyme to nick the backbone strand along the partial double strand to form several fragments of different lengths. The first staple chain is a staple chain without enzyme cleavage sites; The second staple chain is a staple chain containing enzyme cleavage sites and serves as a primer; The number of the first staple chain is the same as the sum of the number of the second and third staple chains.

2. The method for regulating the synergistic assembly of DNA origami according to claim 1, characterized in that: The method for constructing the segmented skeleton chain includes: S1. Construct pscaf phage particles; DNA gene fragments were ligated into pscaf phage vectors using a double enzyme digestion method, and the pscaf phage particles were obtained after transformation and culture. S2. Construct a backbone chain containing enzyme cleavage sites; The pscaf phage particles and helper phages were co-transformed into competent Escherichia coli cells. After culturing and centrifugation, the target single-stranded DNA solution was obtained by lysis, which is the backbone chain containing enzyme cleavage sites. S3. Construct a segmented skeleton chain; After adding the first and second staple chains and annealing, they form a local double strand with the backbone chain containing the enzyme cleavage site, guiding the cleavage enzyme to cleave the backbone DNA into fragments of different lengths, thus obtaining a segmented backbone chain.

3. The method for regulating the synergistic assembly of DNA origami according to claim 2, characterized in that: In S2, the sequence of the backbone chain containing the enzyme cleavage site is shown in SEQ ID NO.1; The backbone chain containing enzyme cleavage sites contains 96 enzyme cleavage sites.

4. The method for regulating the synergistic assembly of DNA origami according to claim 2, characterized in that: In S3, the annealing conditions include: 85°C to 25°C, -1°C / 3min; And / or, the nicking enzyme is Nb.BtsI enzyme.

5. The method for regulating the synergistic assembly of DNA origami according to claim 2, characterized in that: In S3, the sequence of the first staple chain is shown as SEQ ID NO.3~SEQ ID NO.98; And / or, the molar ratio of the backbone chain containing the enzyme cleavage site to the first staple chain is 1:

10.

6. The method for regulating the synergistic assembly of DNA origami according to claim 2, characterized in that: The sequence of the second staple chain is selected from at least two sequences as shown in SEQ ID NO. 99 to SEQ ID NO. 194; And / or, the molar ratio of the backbone chain containing the enzyme cleavage site to the second staple chain is 1:

10.

7. The method for regulating the synergistic properties of DNA origami assembly according to claim 2, characterized in that: The sequence of the third staple chain is SEQ ID NO.99~SEQ ID NO.194, and does not include the sequence of the second staple chain; And / or, the molar ratio of the backbone chain containing the enzyme cleavage site to the third staple chain is 1:

10.

8. The method for regulating the synergistic assembly of DNA origami according to claim 2, characterized in that: The self-assembly includes incubating the third staple chain and the segmented skeleton chain together under constant temperature incubation conditions of 50~60℃ for 12 hours.

9. The method for regulating the coordinating properties of DNA origami assembly according to any one of claims 1-8, characterized in that: The segmented skeleton chain is cut into 2 to 32 segments.

10. A DNA origami structure, prepared using the method for regulating the synergistic assembly of DNA origami as described in any one of claims 1-9.

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