DNA origami structure with enhanced stability in DNA polymerase environment as well as preparation and application of DNA origami structure

Abstract

The invention relates to the technical field of molecular biology, and provides a DNA origami structure with enhanced stability in a DNA polymerase environment as well as preparation and application of the DNA origami structure. An optional number of conventional staple chains; one or more anti-enzymolysis staple chains, wherein the anti-enzymolysis staple chain comprises a staple section and a suspension section for preventing DNA polymerase degradation; wherein staple sections of the skeleton chain, the conventional staple chain and the anti-enzymolysis staple chain form a DNA origami body, and the DNA origami body comprises a plane or curved surface structure; in addition, the hanging section of the anti-enzymolysis staple chain is exposed out of the DNA origami body, and the hanging section is provided with a polyT block. The stability of the DNA origami structure in a DNA polymerase environment is effectively improved, characterization and analysis are facilitated, and the application range of the DNA origami structure in different enzyme systems is widened.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a DNA origami structure with enhanced stability in a DNA polymerase environment, and its preparation and application. Background Art

[0002] DNA origami technology is an important branch in the field of DNA nanostructures, which was proposed by Paul Rothemund of the California Institute of Technology in 2006. Rothemund's research is based on Seeman's theoretical framework and further expands the complexity and precision of DNA nanostructures. The core idea of DNA origami technology is to use a long single-stranded DNA (referred to as "scaffold") as the "skeleton" or "template", and bind a large number of short-stranded DNAs (referred to as "staples") through base complementary pairing to fold and stabilize this long-chain DNA, thereby forming a preset two-dimensional or three-dimensional shape. This technology can not only construct planar geometric figures such as stars and smiling faces, but also create more complex three-dimensional structures such as boxes and tubular structures. DNA origami technology has high structural precision, programmability, and biocompatibility, and can construct stable and multifunctional nanostructures. It has low cost, easily available materials, and can be dynamically regulated through external stimuli, and has broad application prospects in the fields of biomedicine, nanomanufacturing, etc.

[0003] DNA polymerase refers to enzymes that can catalyze the synthesis of DNA strands, and these enzymes play a key role in DNA replication, repair, and recombination processes. The discovery of DNA polymerase has brought revolutionary progress to the fields of molecular biology and genetic engineering. The basic function of DNA polymerase is to form a new DNA strand by adding deoxynucleotide triphosphates (dNTPs) under the guidance of an existing DNA strand (referred to as the template strand). This process is the core of semi-conservative replication, that is, each newly formed DNA double helix contains an old strand from the parent strand and a newly synthesized strand. DNA polymerase must rely on a short RNA primer to initiate the synthesis of the DNA strand and can only add nucleotides in the 5′ to 3′ direction. This directionality ensures the accuracy and efficiency of DNA replication.

[0004] In recent years, the combination of DNA origami technology and DNA polymerase has attracted extensive attention in the fields of molecular biology and nanotechnology. This combination utilizes the ability of DNA origami to precisely control spatial structures, providing a new platform for the application of DNA polymerase. Bryan Wei et al. studied how to drive the dynamic recombination and functional transformation of DNA origami structures through DNA-modifying enzymes to achieve programmable molecular switches. C.H. Fan et al. used atomic force microscopy (AFM) to directly observe the DNA replication activity of the Klenow fragment of DNA polymerase I at the single-molecule level. Thorsten L. Schmidt et al. utilized the DNA polymerase-assisted nick filling mechanism to achieve the transformation of wireframe DNA origami structures. With the continuous development of DNA origami technology, its application prospects in DNA polymerase are becoming increasingly broad. At this time, it is becoming increasingly important to study the stability of DNA origami in DNA polymerase. Summary of the Invention

[0005] The object of the present invention is to provide a preparation and application of a DNA origami structure with enhanced stability in a DNA polymerase environment, effectively improving the stability of the DNA origami structure in a DNA polymerase environment.

[0006] In the first aspect of the present invention, there is provided a DNA origami structure with enhanced stability in a DNA polymerase environment, comprising:

[0007] (a) one or more backbone strands;

[0008] (b) an optional number of conventional staple strands;

[0009] (c) one or more anti-enzyme digestion staple strands, wherein the anti-enzyme digestion staple strand comprises (i) a staple segment and (ii) a hanging segment that resists degradation by DNA polymerase;

[0010] Wherein, the backbone strand, the conventional staple strand, and the staple segment of the anti-enzyme digestion staple strand constitute the DNA origami body, and the DNA origami body comprises a planar or curved structure;

[0011] And, the hanging segment of the anti-enzyme digestion staple strand is exposed outside the DNA origami body, and the hanging segment has a polyT block.

[0012] In another preferred example, it comprises:

[0013] The length L of the polyT block 1 Each independently is 3 - 20 nt, preferably 4 - 15 nt, more preferably 5 - 10 nt; and / or

[0014] The length L of the hanging segment 2is 3 - 50 nt, preferably 4 - 20 nt, more preferably 5 - 10 nt.

[0015] In another preferred embodiment, it includes: the average length L of the polyT segment 1 is 5 nt.

[0016] In another preferred embodiment, it includes: the average length L of the hanging segment 2 is 8 nt;

[0017] In another preferred embodiment, it includes:

[0018] each of the polyT segments independently includes X T bases, and the range of X is 3 - 20.

[0019] In another embodiment, it includes:

[0020] each of the polyT segments independently includes 5 T bases.

[0021] In another preferred embodiment, it includes:

[0022] The shape of the planar or curved surface structure is selected from the group consisting of: rectangle, triangle, circle, strip, sector, irregular shape or a combination thereof.

[0023] In another preferred embodiment, the DNA origami body is dense, loose, has a wireframe structure or has a grid structure.

[0024] In another preferred embodiment, it includes:

[0025] The anti - enzymatic stapling strand has the structure of formula I from 5′ - 3′:

[0026] Z 0 -R 1 -Z 1 -R 2 -Z 2 (I)

[0027] In the formula, Z 1 is a stapling segment, -R 1 -Z 0 and -R 2 -Z 2 are hanging segments;

[0028] Z 0 is none or a polyT segment;

[0029] R 1 is none or a first linking segment of 1 - 4 nt;

[0030] R 2 is none or a second linking segment of 1 - 4 nt;

[0031] Z 2 is none or a polyT segment;

[0032] Wherein, Z 1 and Z 2 at least one of them is not none.

[0033] In another preferred example, it includes:

[0034] Z 3 -R 3 -Z 4 -R 4 (II)

[0035] In the formula, Z 1 is a staple segment, -R 3 -Z 4 -R 4 is a hanging segment;

[0036] R 3 is none or a third linking segment of 1 to 4 nt;

[0037] Z 4 is a polyT segment, and one or more independent polyT segments are included in the polyT segment;

[0038] R 4 is none or an extension segment.

[0039] In another preferred example, the polyT segment is the most distal end of the hanging segment.

[0040] In another preferred example, the polyT segment is the distal end of the hanging segment.

[0041] In another preferred example, the polyT segment is the middle segment of the hanging segment.

[0042] In another preferred example, the polyT segment is the proximal end of the hanging segment.

[0043] In another preferred example, the polyT segment is the nearest proximal end of the hanging segment.

[0044] In another preferred example, the DNA origami structure has one or more features selected from the following group:

[0045] (a) In the anti-enzyme digestion staple strand, the length ratio L 2 / L 1 of the hanging segment and the polyT segment ranges from 1 to 2, preferably 1 to 1.5;

[0046] (b) The staple chain includes a conventional staple chain and the anti-enzyme digestion staple chain. In the DNA origami structure, the proportion of the anti-enzyme digestion staple chain in all the staple chains is 50% - 100%, preferably 80% - 100%.

[0047] (c) When the DNA origami body is a dense planar structure, the density of the hanging segments of the anti-enzyme digestion staple chain is 20 - 40 per 1000 nm 2 , preferably 35 - 40 per 1000 nm 2 .

[0048] In another preferred example, when the DNA origami body is a dense planar structure, the average density of the hanging segments of the anti-enzyme digestion staple chain is 37 per 1000 nm 2 .

[0049] In the second aspect of the present invention, a method for preparing the above DNA origami structure is provided, including:

[0050] S1. Provide a backbone chain and a staple chain, and one or more anti-enzyme digestion staple chains are included in the staple chain;

[0051] S2. The backbone chain and the staple chain are mixed and self-assembled to form the DNA origami structure as described above.

[0052] In another preferred example, step S1 further includes:

[0053] S1.1 Design the backbone chain and the conventional staple chain;

[0054] S1.2 The conventional staple chain is dissolved in ultrapure water and mixed with the backbone chain, nucleic acid electrophoresis buffer, and magnesium ion buffer;

[0055] S1.3 By adding the method of polyT, the anti-enzyme digestion staple chain is obtained.

[0056] In another preferred example, step S2 further includes:

[0057] The concentration ratio of the backbone chain to the staple chain is 1:10.

[0058] In the third aspect of the present invention, a reaction system is also disclosed, and the reaction system includes:

[0059] (a) The DNA origami structure as described above;

[0060] (b) DNA polymerase; and

[0061] (c) A buffer system for performing DNA polymerization reaction.

[0062] In another preferred example, the DNA polymerase is a polymerase with strand displacement activity.

[0063] In another preferred example, the DNA polymerase includes T4, Phi29, and the large fragment of Bst.

[0064] In another preferred example, the DNA polymerase includes the large fragments of Phi29 and Bst with strand displacement activity.

[0065] In another preferred example, performing the DNA polymerization reaction includes:

[0066] Incubating and characterizing the DNA origami structure in the solution where the DNA polymerase is located, and measuring the stability of the DNA origami structure before and after the DNA polymerization reaction.

[0067] In another preferred example, performing the DNA polymerization reaction includes:

[0068] Sa. Purifying the DNA origami structure, observing and recording the first characterization result;

[0069] Sb. Incubating the DNA origami structure in the solution where the DNA polymerase is located, setting the incubation time and incubation temperature;

[0070] Sc. When the incubation time is reached, measuring the reaction products in the buffer system, observing and recording the second characterization result;

[0071] Wherein, the methods used for characterization include performing stability tests using agarose gel electrophoresis and performing observations using atomic force microscopy.

[0072] In another preferred example, performing the DNA polymerization reaction includes:

[0073] Dividing the DNA origami structure into two groups, one with polyT added and the other without polyT added, and conducting a control experiment.

[0074] In another preferred example, the incubation times in step Sb are 30 minutes, 60 minutes, and 90 minutes.

[0075] In another preferred example, the incubation temperature in step Sb is 37 °C, and the incubation experiment is carried out in a PCR instrument.

[0076] In the fourth aspect of the present invention, a kit is also disclosed, and the kit includes:

[0077] (a) A first container and the DNA origami structure described above located in the first container;

[0078] (b) A second container and the DNA polymerase located in the second container;

[0079] (c) Optional third container and a buffer system or buffer reagent for performing DNA polymerization reaction located in the third container.

[0080] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0081] Figure 1 Shows the atomic force microscopy characterization diagrams of rectangular DNA origami structures and fan-shaped DNA origami structures without adding polyT in an embodiment of the present application;

[0082] Figure 2 Shows the atomic force microscopy characterization diagrams of rectangular DNA origami structures and fan-shaped DNA origami structures with added polyT in an embodiment of the present application;

[0083] Figure 3 Shows a schematic diagram of a rectangular DNA origami structure with added polyT in an embodiment of the present application;

[0084] Figure 4 Shows the electrophoretic mobility diagrams of rectangular DNA origami structures and fan-shaped DNA origami structures in an embodiment of the present application;

[0085] Figure 5 Shows the electrophoretic mobility diagram of a rectangular DNA origami structure in a stability experiment with Bst large fragment DNA polymerase in an embodiment of the present application;

[0086] Figure 6 Shows the atomic force microscopy result diagram of a rectangular DNA origami structure in a stability experiment with Bst large fragment DNA polymerase in an embodiment of the present application;

[0087] Figure 7 Shows the electrophoretic mobility diagram of a rectangular DNA origami structure in a stability experiment with T4 and Phi29 DNA polymerases in an embodiment of the present application;

[0088] Figure 8 Shows the atomic force microscopy result diagram of a rectangular DNA origami structure in a stability experiment with Phi29 DNA polymerase in an embodiment of the present application;

[0089] Figure 9 Shows the electrophoretic mobility diagram of a fan-shaped DNA origami structure in a stability experiment with Bst large fragment and Phi29 DNA polymerases in an embodiment of the present application;

[0090] Figure 10 Shows the atomic force microscopy result diagram of the stability experiment of the sector-shaped DNA origami structure in the Bst large fragment DNA polymerase in one embodiment of the present application. Detailed implementation manners

[0091] Through extensive and in-depth research, the present inventors have for the first time provided the preparation and application of a DNA origami structure with enhanced stability in a DNA polymerase environment. Specifically, it includes a DNA origami body, and the DNA origami body includes a planar or curved structure composed of one or more backbone strands and one or more staple segments of anti-enzyme digestion staple strands; wherein, the anti-enzyme digestion staple strand includes a staple segment and a hanging segment that is exposed outside the planar or curved structure and resists degradation by DNA polymerase, and the hanging segment has a polyT block. The present application effectively improves the stability of the DNA origami structure in a DNA polymerase environment by adding polyT. Among them, the single strand formed by T bases has relatively high flexibility and is not easy to form stable base pairs with other strands. Its flexibility and low stability are the keys to preventing the binding and extension of DNA polymerase. Based on this, the present invention has been completed.

[0092] DNA origami structure

[0093] The present invention provides a DNA origami structure with enhanced stability in a DNA polymerase environment, as Figure 2 shown, including:

[0094] (a) One or more backbone strands;

[0095] (b) An optional number of conventional staple strands;

[0096] (c) One or more anti-enzyme digestion staple strands, wherein the anti-enzyme digestion staple strand includes (i) a staple segment and (ii) a hanging segment that resists degradation by DNA polymerase;

[0097] Among them, the staple segments of the backbone strand, the conventional staple strand, and the anti-enzyme digestion staple strand constitute the DNA origami body, and the DNA origami body includes a planar or curved structure;

[0098] And, the hanging segment of the anti-enzyme digestion staple strand is exposed outside the DNA origami body, and the hanging segment has a polyT block.

[0099] When there is no anti-enzyme digestion staple strand in the DNA origami structure, the DNA origami structure is as Figure 1 shown, Figure 1 and Figure 2 It shows that adding polyT has no obvious effect on the shape of the DNA origami structure.

[0100] In another embodiment, it includes:

[0101] The length L of the polyT segment 1 is independently 3 to 20 nt for each, preferably 4 to 15 nt, more preferably 5 to 10 nt; and / or

[0102] The length L of the hanging segment 2 is 3 to 50 nt, preferably 4 to 20 nt, more preferably 5 to 10 nt.

[0103] In another embodiment, it includes: The average length L of the polyT segment 1 is 5 nt.

[0104] In another embodiment, it includes: The average length L of the hanging segment 2 is 8 nt;

[0105] In another embodiment, it includes:

[0106] Each of the polyT segments independently includes X T bases, and the range of X is 3 to 20.

[0107] In another embodiment, it includes:

[0108] Each of the polyT segments independently includes 5 T bases.

[0109] In another embodiment, it includes:

[0110] The shape of the planar or curved surface structure is selected from the group consisting of: rectangle, triangle, circle, strip, sector, irregular shape or a combination thereof.

[0111] In another embodiment, the DNA origami body is dense, loose, has a wireframe structure or has a grid structure.

[0112] In another embodiment, it includes:

[0113] The anti-enzyme digestion staple chain has a structure of formula I from 5'-3':

[0114] Z 0 -R 1 -Z 1 -R 2 -Z 2 (I)

[0115] In the formula, Z 1 is a staple segment, -R 1 -Z 0 and -R 2 -Z 2 are hanging segments;

[0116] Z 0 is none or a polyT segment;

[0117] R 1 is a first linker segment of 0 or 1 to 4 nt;

[0118] R 2 is a second linker segment of 0 or 1 to 4 nt;

[0119] Z 2 is a polyT segment of 0 or polyT blocks;

[0120] wherein at least one of Z 1 and Z 2 is not 0.

[0121] In another embodiment, it includes:

[0122] Z 3 -R 3 -Z 4 -R 4 (II)

[0123] In the formula, Z l is a staple segment, -R 3 -Z 4 -R 4 is a hanging segment;

[0124] R 3 is a third linker segment of 0 or 1 to 4 nt;

[0125] Z 4 is a polyT segment, and the polyT segment includes one or more independent polyT blocks;

[0126] R 4 is a 0 or extension segment.

[0127] In another embodiment, the polyT block is the distal most end of the hanging segment.

[0128] In another embodiment, the polyT block is the distal end of the hanging segment.

[0129] In another embodiment, the polyT block is the middle segment of the hanging segment.

[0130] In another embodiment, the polyT block is the proximal end of the hanging segment.

[0131] In another preferred example, the polyT block is the proximal most end of the hanging segment.

[0132] In another embodiment, the DNA origami structure has one or more features selected from the group consisting of:

[0133] (a) In the anti - enzymatic staple strand, the length ratio L 2 / L1 ranges from 1 to 2, preferably from 1 to 1.5;

[0134] (b) The staple chain includes a conventional staple chain and an anti-enzyme-degradation staple chain. In the DNA origami structure, the proportion of the anti-enzyme-degradation staple chain in all the staple chains ranges from 50% to 100%, preferably from 80% to 100%;

[0135] (c) When the DNA origami body is a dense planar structure, the density of the hanging segments of the anti-enzyme-degradation staple chain is 20 - 40 per 1000 nm 2 , preferably 35 - 40 per 1000 nm 2 .

[0136] In another embodiment, when the DNA origami body is a dense planar structure, the average density of the hanging segments of the anti-enzyme-degradation staple chain is 37 per 1000 nm 2 .

[0137] Figure 3 shows an embodiment in which the origami structure is a single-layer rectangular plane, that is, a single layer, and 216 staples are all on it. All polyT segments face only one side, that is, all the added 5 T bases face one direction. The length of the rectangular origami structure is about 96.56 nm, the width is about 60.19 nm, and the area is about 5811.9 nm 2 , and at this time the average density is about 37 per 1000 nm 2 .

[0138] The single strand formed by T bases has relatively high flexibility and is not easily paired with other strands to form stable base pairs. Its flexibility and low stability are the keys to preventing DNA polymerase from binding and extending. G and C bases are prone to form GC pairs, especially under high concentration and low temperature conditions, which may lead to the formation of stable secondary structures, such as hairpin structures, by themselves or with other strands. These stable structures may be recognized by DNA polymerase, instead increasing the possibility of strand displacement or extension. Therefore, the effect may be inferior to that of T bases. Therefore, T bases are more suitable for preventing DNA origami from being damaged.

[0139] Preparation method of DNA origami structure

[0140] The present invention provides a preparation method of the above DNA origami structure, including:

[0141] S1. Providing a backbone chain and a staple chain, and the staple chain includes one or more anti-enzyme-degradation staple chains;

[0142] S2. Mixing and self-assembling the backbone chain and the staple chain to form the DNA origami structure as described above.

[0143] In another embodiment, step S1 further includes:

[0144] S1.1 Design a backbone chain and a conventional staple chain;

[0145] S1.2 Dissolve the conventional staple chain in ultrapure water and mix it with the backbone chain, nucleic acid electrophoresis buffer, and magnesium ion buffer;

[0146] S1.3 Obtain an enzyme-resistant staple chain by adding polyT.

[0147] In another embodiment, step S2 further includes:

[0148] The concentration ratio of the backbone chain to the staple chain is 1:10.

[0149] Application of DNA origami structure

[0150] The present invention also discloses a reaction system, which includes:

[0151] (a) The DNA origami structure described above;

[0152] (b) DNA polymerase; and

[0153] (c) A buffer system for performing DNA polymerization reaction.

[0154] In another embodiment, the DNA polymerase is a polymerase with strand displacement activity.

[0155] In another embodiment, the DNA polymerase includes T4, Phi29, and Bst large fragment.

[0156] In another embodiment, the DNA polymerase includes Phi29 and Bst large fragment with strand displacement activity.

[0157] In another embodiment, performing the DNA polymerization reaction includes:

[0158] Incubate and characterize the DNA origami structure in the solution where the DNA polymerase is located, and measure the stability of the DNA origami structure before and after the DNA polymerization reaction.

[0159] In another embodiment, performing the DNA polymerization reaction includes:

[0160] Sa. Purify the DNA origami structure, observe and record the first characterization result;

[0161] Sb. Incubate the DNA origami structure in the solution where the DNA polymerase is located, and set the incubation time and incubation temperature;

[0162] Sc. When the incubation time reaches, measure the reaction products in the buffer system, observe and record the second characterization result;

[0163] Among them, the methods for characterization include performing stability tests using agarose gel electrophoresis (AGE) and observation using atomic force microscopy (AFM).

[0164] In another embodiment, performing the DNA polymerization reaction includes:

[0165] Dividing the DNA origami structures into two groups, one with polyT added and the other without polyT added, and conducting a control experiment.

[0166] In another embodiment, the incubation times in step Sb are 30 minutes, 60 minutes, and 90 minutes.

[0167] In another embodiment, the incubation temperature in step Sb is 37 °C, and the incubation experiment is carried out in a PCR instrument.

[0168] The present invention also discloses a kit, which includes:

[0169] (a) A first container and the aforementioned DNA origami structure located in the first container;

[0170] (b) A second container and DNA polymerase located in the second container;

[0171] (c) Optionally, a third container and a buffer system or buffer reagent for performing the DNA polymerization reaction located in the third container.

[0172] The main advantages of the present invention include:

[0173] (a) Effectively improves the stability of the DNA origami structure in a DNA polymerase environment, enables the DNA origami structure to maintain structural integrity in various enzyme reactions, and improves its tolerance and stability.

[0174] (b) The method in this application is convenient for characterization and analysis, can be characterized by methods such as agarose gel electrophoresis and atomic force microscopy, can directly observe the changes in the DNA origami structure, and is convenient for the analysis and verification of experimental results.

[0175] (c) This application improves the applicability of the DNA origami structure to various DNA polymerases, can conduct experiments with various DNA polymerases, and broadens the application scope of the DNA origami structure in different enzyme systems.

[0176] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0177] Example 1

[0178] Design and self-assemble two DNA origami monomers using caDNAno and PERDIX-Win-MATLAB.

[0179] Classify the DNA origami according to the structure, which is classified into: dense structure and wireframe structure. The dense structure is designed as a rectangular DNA origami, and the wireframe structure is designed as a fan-shaped DNA origami, as Figure 1 shown.

[0180] 1.1 Synthesis of rectangular DNA origami: Dissolve 216 staple strands equally in ddH2O so that the final concentration of each strand is 400 nM. Mix 100 nM of single-stranded DNA of P7249 (M13mp18) with the staple strands (mixed at a molar concentration ratio of 1:10), and add 10x TAE, 100 mM magnesium acetate, and ddH2O, where the final concentration of P7249 single-stranded DNA is 20 nM and the final concentration of the staple strands is 200 nM. Dilute TAE to 1x and magnesium acetate to 10 mM; put the mixed solution into a PCR instrument, and lower the temperature from 90 °C to 4 °C, with a cooling rate of 0.123 °C every 10 seconds.

[0181] 1.2 Synthesis of fan-shaped DNA origami: Dissolve 158 staple strands equally in ddH2O so that the final concentration of each strand is 400 nM. Mix 100 nM of single-stranded DNA of P7249 (M13mp18) with the staple strands (mixed at a molar concentration ratio of 1:10), and add 10x TAE, 100 mM magnesium acetate, and ddH2O, where the final concentration of P7249 single-stranded DNA is 20 nM and the final concentration of the staple strands is 200 nM. Dilute TAE to 1x and magnesium acetate to 10 mM; place the mixed solution in a PCR instrument and cool it according to the following temperature program:

[0182] First, lower the temperature from 90 °C to 86 °C at a rate of 4 °C every 5 minutes;

[0183] Then lower the temperature to 70 °C at a rate of 1 °C every 5 minutes;

[0184] Next, lower the temperature to 40 °C at a rate of 1 °C every 15 minutes;

[0185] Finally, lower the temperature to 25 °C at a rate of 1 °C every 10 minutes;

[0186] Finally, maintain the temperature at 4 °C.

[0187] 1.3 Method for adding polyT to stabilize DNA origami: Add 5 T bases to the 3′ ends of 216 staple strands of the rectangular DNA origami and resynthesize according to the aforementioned step 1.1; add 5 T bases to the 3′ ends of 158 staple strands of the fan-shaped DNA origami and resynthesize according to the aforementioned step 1.2.

[0188] Purification: Use PEG centrifugation technology to purify and concentrate the self-assembled DNA origami and remove the excess staple strands. The centrifugation conditions are 16000 rcf, 25 °C, and 25 minutes.

[0189] 1.4 Atomic force microscopy characterization of the synthesized rectangular DNA origami and fan-shaped DNA origami: Fix the mica sheet on the iron sheet, drop 10 μL of the prepared and diluted DNA origami solution onto the surface of the clean mica sheet, let it stand for 3 min, wash away the salts with ddH2O and dry with nitrogen, then drop 50 μL of 1xTAE 10 mM Mg2+ on the prepared and dried sample and observe its morphology under a scanning probe microscope, as Figure 1 and Figure 2 shown.

[0190] 1.5 Agarose gel electrophoresis characterization of the synthesized rectangular DNA origami and fan-shaped DNA origami: First, according to the standard operating procedure, mix the sample containing DNA origami (5 μL) with an appropriate 6X DNA Loading buffer (1 μL), and then load it onto a pre-prepared and stained 1.5% agarose gel. Use 1XTBE 10 mM Mg2+ buffer as the electrophoresis medium, apply a constant voltage (70 V) for electrophoresis until the samples are fully separated in the gel (3 hours). After completing the agarose gel electrophoresis, use a gel imaging system to image the agarose gel and observe the migration status of each band in the lane, as Figure 4 shown.

[0191] Figure 4 shows the electrophoresis migration diagrams of the rectangular DNA origami structure and the fan-shaped DNA origami structure in an embodiment of the present application; where L represents 1 kb DNA ladder, that is, a DNA ladder with 1000 base pairs, which is a DNA molecular weight standard used for estimating the DNA length; S represents 20 nM P7249, that is, the backbone chain sample; 1 represents the sample of the fan-shaped DNA origami structure without adding polyT; 2 represents the sample of the fan-shaped DNA origami structure with added polyT; 3 represents the sample of the rectangular DNA origami structure without adding polyT; 4 represents the sample of the rectangular DNA origami structure with added polyT; 1-4 are all purified samples, and the concentration of each sample is 5 nM. The samples here are all samples before the DNA polymerase reaction.

[0192] In nucleic acid electrophoresis, generally, a longer migration distance indicates a smaller and more regular nucleic acid molecule, and a darker color indicates a larger amount of nucleic acid. From Figure 4 the comparison between lanes S and 1 in Figure 4 it can be seen that the single backbone chain migrates farther than the fan-shaped origami structure. Because the backbone chain is more regular and compact, the resistance coefficient it receives is relatively small. While the fan-shaped origami structure has a large molecular weight and a relatively loose structure, and may have more holes and unfolded regions, so the overall folding density is lower, which makes the effective electrophoretic migration speed of the fan-shaped origami slower, manifested as a trailing migration band; from Figure 4 the comparison between lanes 1 and 3 in

[0193] Example 2

[0194] A method for applying a DNA origami structure with enhanced stability in a DNA polymerase environment, comprising the following steps:

[0195] Explore the stability of rectangular DNA origami in Bst large fragment, T4, and Phi29 DNA polymerases.

[0196] 2.1 Set up an experimental group with polyT added and a control group without polyT for the rectangular DNA origami.

[0197] 2.2 Mix the rectangular DNA origami with the corresponding DNA polymerase buffer, DNA polymerase, dNTP, and water in a PCR tube. After shaking and mixing evenly, place it in a PCR instrument, set the incubation temperature to 37 °C, and the incubation times to 30 minutes, 60 minutes, and 90 minutes. Every 30 minutes, take out the experimental group and control group samples from the PCR instrument, take a part of the solution and place it on ice for atomic force microscopy imaging and agarose gel electrophoresis, and continue to incubate the remaining samples in the PCR instrument.

[0198] 2.3 Agarose gel electrophoresis characterization was performed on the experimental and control group samples incubated for 30 minutes, 60 minutes, and 90 minutes: First, according to the standard operating procedure, the sample containing DNA origami (10 μL) was mixed with an appropriate 6X DNA Loading buffer (2 μL), and then loaded onto a pre-prepared and stained 1.5% agarose gel. 1XTBE 10 mM Mg2+ buffer was used as the electrophoresis medium, and a constant voltage (70 V) was applied for electrophoresis until the samples were fully separated in the gel (3 hours). After completing the agarose gel electrophoresis, the agarose gel was imaged using a gel imaging system to observe the migration status of each band in the lane, such as Figure 5 and Figure 7 shown.

[0199] Figure 5 shows the electrophoretic migration map of the rectangular DNA origami structure in the Bst large fragment DNA polymerase in an embodiment of the present application. Among them, 37 °C is when the enzyme activity is very good; from Figure 5 it can be seen that the original contents of lanes 1 and 2 were quite equal. Then, in the case of adding Bst large fragment DNA polymerase, it can be seen from the darker colors of lanes 3 and 4, 5 and 6, 7 and 8 that under the condition of incubating for different durations, the band migration was slightly backward because the enzyme bound to the origami, so the rate decreased; there are two horizontal lines in lanes 3, 5, and 7. The migration rate of the band is affected by the combined effects of molecular weight, structural shape, and the charge carried. The upper band represents the DNA origami bound to the DNA polymerase, and the lower band is the normal structure without bound enzyme, indicating that the DNA origami structure with added polyT exists in a bound form after the reaction, showing that it has not been degraded; while lanes 4, 6, and 8 show that the DNA origami structure without added polyT has become a diffuse form. The amount of the rectangular DNA origami structure with added polyT is significantly more and more stable than that without added polyT. Therefore, the method of adding polyT can enhance the stability of the rectangular DNA origami structure in the Bst large fragment DNA polymerase.

[0200] Figure 7 shows the electrophoretic migration map of the rectangular DNA origami structure in the T4 and Phi29 DNA polymerases in an embodiment of the present application; from Figure 7It can be seen that the original contents of the samples in lanes 1-4 were quite equivalent. Then, in the case of adding T4 DNA polymerase, the color differences between 5 and 6, 9 and 10, and 13 and 14 were not significant. This is because T4 DNA polymerase does not have strand displacement activity, so it does not destroy the structure of the DNA origami. However, in the case of adding Phi29 DNA polymerase, the color differences between lanes 7 and 8, 11 and 12, and 15 and 16 became increasingly obvious over time. Lanes 8, 12, and 16 showed that the rectangular DNA origami structures without adding polyT were increasingly severely damaged, but lanes 7, 11, and 15 showed that the rectangular DNA origami structures with added polyT could stably exist. Therefore, the method of adding polyT can enhance the stability of the rectangular DNA origami structure in Phi29 DNA polymerase without reducing its stability in T4 DNA polymerase.

[0201] 2.4 Atomic force microscopy characterization was performed on the experimental group and control group samples incubated for 30 minutes and 90 minutes: Fix the mica sheet on the iron sheet, drop 10 μL of the prepared and diluted DNA origami solution onto the surface of the clean mica sheet, let it stand for 3 min, wash away the salts with ddH2O and dry it with nitrogen. Drop 50 μL of 1xTAE 10 mM Mg2+ onto the prepared and dried sample, and observe its morphology under a scanning probe microscope, as Figure 6 and Figure 8 shown.

[0202] Figure 6 shows the atomic force microscopy result diagram of the stability experiment of the rectangular DNA origami structure in a Bst large fragment DNA polymerase in an embodiment of the present application; it can be visually seen from Figure 6 that the rectangular DNA origami structure without adding polyT was significantly decomposed after being incubated in Bst large fragment DNA polymerase for 30 minutes, while the rectangular DNA origami structure with added polyT still showed a relatively complete state after being incubated in Bst large fragment DNA polymerase for 90 minutes. Therefore, the method of adding polyT can enhance the stability of the rectangular DNA origami structure in Bst large fragment DNA polymerase.

[0203] Figure 8 shows the atomic force microscopy result diagram of the stability experiment of the rectangular DNA origami structure in Phi29 DNA polymerase in an embodiment of the present application; it can be seen from Figure 8It can be intuitively seen that in the Phi29 DNA polymerase, for the rectangular DNA origami structure without adding polyT, as the time increases from 30 minutes to 90 minutes, the proportion of the intact structure in the total structure decreases from 16.9% to 12%, showing an obvious decline. While for the rectangular DNA origami structure with added polyT in the Phi29 DNA polymerase, as the time increases from 30 minutes to 90 minutes, the ratio of the intact structure in the total structure decreases from 36% to 34%, quantitatively demonstrating that the method of adding polyT can enhance the stability of the rectangular DNA origami structure in the Phi29 DNA polymerase.

[0204] Example 3

[0205] A method for applying a DNA origami structure with enhanced stability in a DNA polymerase environment, comprising the following steps:

[0206] Explore the stability of the fan-shaped DNA origami in Bst large fragment and Phi29 DNA polymerase.

[0207] 3.1 Set the fan-shaped DNA origami into an experimental group with added polyT and a control group without added polyT.

[0208] 3.2 Mix the fan-shaped DNA origami with the corresponding DNA polymerase buffer, DNA polymerase, dNTP, and water in a PCR tube. After shaking and mixing evenly, place it in a PCR instrument, set the incubation temperature to 37°C, and the incubation times to 30 minutes, 60 minutes, and 90 minutes. Take out the samples of the experimental group and the control group from the PCR instrument every 30 minutes. Take a part of the solution and place it on ice for atomic force microscopy imaging and agarose gel electrophoresis, and continue to incubate the remaining samples in the PCR instrument.

[0209] 3.3 Characterize the samples of the experimental group and the control group incubated for 30 minutes, 60 minutes, and 90 minutes by agarose gel electrophoresis again according to the method in step 2.3 above. As Figure 7 shown.

[0210] Figure 9 shows the electrophoretic migration diagram of the stability experiment of the fan-shaped DNA origami structure in the Bst large fragment and Phi29 DNA polymerase in an embodiment of the present application; from Figure 9It can be seen that the original contents of lanes 3-6 were quite the same. Then, in the case of adding Bst large fragment DNA polymerase, the color contrast differences in lanes 7 and 8, 11 and 12, 15 and 16 were obvious. For the sector DNA origami structures with polyT added, the amounts were significantly more and more stable than those without polyT added; in the case of adding Phi29 DNA polymerase, the color contrast differences in lanes 9 and 10, 13 and 14, 17 and 18 were not large. Because the sector origami structure had more space in contact with the enzyme and was relatively easier to be damaged compared to the rectangle, its stability was slightly weaker; therefore, the method of adding polyT could enhance the stability of the sector DNA origami structure in Bst large fragment DNA polymerase and did not reduce its stability in Phi29 DNA polymerase.

[0211] 3.4 The experimental group and control group samples incubated for 30 minutes and 90 minutes were subjected to atomic force microscopy characterization again according to the method in step 2.4 described above, as Figure 10 shown.

[0212] Figure 10 The atomic force microscopy result diagram of the stability experiment of the sector DNA origami structure in Bst large fragment DNA polymerase in an embodiment of the present application is shown. It can be intuitively seen from Figure 10 that the sector DNA origami structure without polyT added had significantly decomposed after being incubated in Bst large fragment DNA polymerase for 30 minutes, while the sector DNA origami structure with polyT added still presented a relatively complete state after being incubated in Bst large fragment DNA polymerase for 90 minutes. Therefore, the method of adding polyT could enhance the stability of the sector DNA origami structure in Bst large fragment DNA polymerase.

[0213] In summary, this embodiment successfully demonstrated an efficient and stable DNA origami structure, providing new possibilities for future biomedical research and applications. We encourage those skilled in the art to implement the present invention according to this specification and explore its application potential in other fields.

[0214] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A DNA origami structure with enhanced stability in a DNA polymerase environment, characterized in that: include: (a) one or more backbone chains; (b) a conventional staple chain of any number; (c) one or more enzymatically resistant staple strands, wherein the enzymatically resistant staple strands comprise (i) staple segments and (ii) hanging segments resistant to DNA polymerase degradation; Wherein, the backbone chain, the conventional staple chain, and the staple segments of the anti-enzymatic staple chain constitute a DNA origami body, and the DNA origami body includes a planar or curved surface structure; Furthermore, the hanging segment of the enzymatically resistant staple chain is exposed from the DNA origami body, and the hanging segment has a polyT block.

2. The DNA origami structure according to claim 1, characterized in that: include: The length L1 of the polyT blocks is independently 3 to 20 nt, preferably 4 to 15 nt, more preferably 5 to 10 nt; and / or The length L2 of the hanging segment is 3 to 40 nt, preferably 4 to 20 nt, and more preferably 5 to 10 nt.

3. The DNA origami structure according to claim 1, characterized in that: include: The polyT blocks each independently include X T bases, where X ranges from 3 to 20.

4. The DNA origami structure according to claim 1, characterized in that: include: The shape of the plane or curved structure is selected from the following group: rectangle, triangle, circle, strip, fan, irregular shape or a combination thereof.

5. The DNA origami structure according to claim 1, characterized in that: include: The enzymatically resistant staple chain has a structure of Formula I from 5′ to 3′: Z0-R1-Z1-R2-Z2 (I) Where Z1 is the staple section, -R1-Z0 and -R2-Z2 are the hanging sections; Z0 is none or polyT block; R1 is the first linker segment of none or 1 to 4 nt; R2 is the second linker segment of none or 1 to 4 nt; Z2 is none or polyT block; Among them, at least one of Z1 and Z2 is not zero.

6. The DNA origami structure according to claim 1, characterized in that: include: The enzymatically resistant staple chain has a structure of Formula II from 5′ to 3′: Z3-R3-Z4-R4 (II) In the formula, Z l is the staple section, -R3-Z4-R4 is the hanging section; R3 is the third linker segment of none or 1 to 4 nt; Z4 is a polyT segment, wherein the polyT segment includes one or more independent polyT blocks; R4 is none or an extended segment.

7. The DNA origami structure according to claim 1, characterized in that: The DNA origami structure has one or more features selected from the following group: (a) In the enzymatically resistant staple chain, the length ratio of the dangling segment to the polyT block is L2 / L l The range is 1 to 2, preferably 1 to 1.5; (b) The staple chains include conventional staple chains and the enzymatically resistant staple chains. In the DNA origami structure, the number of the enzymatically resistant staple chains accounts for 50% to 100%, preferably 80% to 100% of all staple chains. (c) When the DNA origami body is a compact planar structure, the density of the hanging segments of the anti-enzymatic staple chains is 20 to 40 per 1000 nm. 2 , preferably 35 to 40 / 1000nm 2 .

8. A method for preparing a DNA origami structure according to any one of claims 1 to 7, characterized in that: include: S1. Providing a skeleton chain and a staple chain, wherein the staple chain includes one or more anti-enzymatic staple chains; S2. The backbone chain and the staple chain are mixed and self-assembled to form a DNA origami structure as described in any one of claims 1 to 7.

9. A reaction system, characterized in that: The reaction system comprises: (a) The DNA origami structure according to any one of claims 1 to 7; (b) DNA polymerase; and (c) Buffer system for DNA polymerization reaction.

10. A kit, characterized in that: The kit comprises: (a) a first container and a DNA origami structure according to any one of claims 1 to 7 located in the first container; (b) a second container and a DNA polymerase in the second container; (c) an optional third container and a buffer system or buffer reagent for performing DNA polymerization reaction in the third container.

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