Nanogroove array and its manufacturing method and application

Abstract

The application relates to a nano-groove array and a manufacturing method and application thereof, the manufacturing method of the nano-groove array comprises the following steps: taking single-stranded DNA (ssDNA) as DNA bricks, and adopting the complementary pairing principle of the ssDNA to realize self-assembly, so that a nano-groove array with a base and a side wall is obtained; wherein the width and height of the nano-groove array are fixed, the groove number is determined, and the nano-groove array is elongated along a single direction; in the nano-groove array, the width of the side wall and the groove is equivalent to 2 double-helix DNA (dsDNA) diameters. Compared with the prior art, the groove number of the nano-groove array is controllable, the groove width is 4 nm, the period of the groove array is 8 nm, and self-assembly defects can be greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a nanogroove array, its manufacturing method, and its application. Background Technology

[0002] Nanogroove structures can construct parallel isolated regions within extremely small spaces, enabling the precise alignment of low-dimensional materials. They hold significant application value in semiconductor devices, nanophotonic devices, optoelectronics, and nanoquantum electrodynamics. In the experimental research phase, fabricating a single nanogroove with controllable morphology can generally meet most testing requirements. However, when the goal is upgraded to "a nanoscale array with regular morphology and consistent periodicity," the fabrication process places extremely high demands on the integrity of the design process, the stability of equipment precision, and the reliability of material properties.

[0003] To meet the demands of array fabrication, one of the current mainstream technologies is extreme ultraviolet (EUV) lithography, based on semiconductor chip manufacturing. While this technology can stably fabricate nanogroove arrays with a scale of 10 nm and above, it faces a key bottleneck in fabricating nanoarrays below 10 nm due to the post-Moore's Law era's demand for "smaller scales." ASML of the Netherlands announced the achievement of 8 nm dense trench printing in 2024, a technological breakthrough, but its high equipment cost and the fact that commercialization is not expected until 2033 make it difficult to meet the current needs of research and small-to-medium-scale industries for rapid fabrication of nanogroove arrays below 10 nm.

[0004] To overcome the scale and cost limitations of extreme ultraviolet lithography, DNA nanotechnology based on the self-assembly principle has emerged as a new research direction. This technology enables the self-assembly of DNA features in solution through complementary base pairing, achieving a resolution of up to 2 nm. It also supports user-designed 2D or 3D nanostructures: simply adding a specific sequence of single-stranded DNA (ssDNA) to the solution and annealing it yields the desired DNA structure. Depending on the design logic, the main template construction methods are DNA tiles, DNA origami, and DNA bricks. Among these, DNA brick nanotechnology allows for the design of uniformly spaced nanogrooves, enabling the spatial construction of groove arrays. Furthermore, the resulting DNA groove structures can serve as templates for the synthesis of various materials such as oxides, graphene, plasma materials, polymers, and carbon nanotubes, providing new pathways for the preparation of nanomaterials in multiple fields.

[0005] Specifically, DNA brick technology was originally used primarily for designing 3D structures with three mutually perpendicular extension axes. By designing recurrent chains along the perpendicular cross-sections of these axes, the extensions can be expanded. Current research has successfully constructed trench arrays with a spacing of 10.6 nm by designing two extension axes. However, this strategy has significant limitations in the fabrication of planar nanogrooved DNA templates: the biaxial extension approach easily leads to assembly defects, and the number of nanogrooves cannot be precisely controlled. This results in randomness in the length, width, and number of edge defects within the same batch of DNA templates, directly affecting the yield and structural consistency of planar nanogrooved arrays, making it unsuitable for applications with stringent precision requirements. Summary of the Invention

[0006] The purpose of this invention is to provide a nanogroove array, its manufacturing method and application, which utilizes the self-assembly of DNA bricks to form a nanogroove array with a groove spacing of less than 10 nm.

[0007] The objective of this invention can be achieved through the following technical solution: a method for manufacturing a nanogroove array, using ssDNA (single-stranded DNA) as DNA bricks, and employing the complementary pairing principle of ssDNA for self-assembly to obtain a nanogroove array with a base and sidewalls. The width and height of the nanogroove array are fixed, the number of grooves is determined, and it extends in a single direction.

[0008] Preferably, in the nanogroove array, the width of the sidewall and the groove are each 2 building units, that is, the width of the sidewall and the groove are equivalent to 2 double helix DNA (dsDNA) diameters.

[0009] This invention discloses a method for manufacturing a trench array with an 8-nanometer spacing. The main building material of this trench array is single-stranded DNA (ssDNA), which is figuratively used as DNA bricks to construct the required trench array, i.e., the DNA template. This invention uses 8 bases of ssDNA as a binding domain and 8 base pairs (bp) of double-stranded DNA (dsDNA) as a building unit, wherein the elongation direction of the double-stranded DNA is parallel to the trench array.

[0010] Preferably, during self-assembly, the ssDNA having at least two binding domains is sequence-paired with at least two other ssDNAs having matching binding domains.

[0011] Preferably, the ssDNA has 2 to 6 binding domains, each binding domain having 8 bases.

[0012] More preferably, in accordance with structural stability requirements, the 20-25% ssDNA contains 2 binding domains, the 60-65% ssDNA contains 4 binding domains, and the ~15% ssDNA contains 6 binding domains.

[0013] In this invention, the complementary pairing principle of ssDNA is adopted. ssDNA generally has four binding domains: the number of binding domains in a single ssDNA is appropriately increased or decreased depending on its structural position to improve structural stability, with a minimum of two and a maximum of six; each binding domain has eight bases: the number of bases is reduced to minimize overall spin. During annealing, ssDNA with at least two binding domains pairs with at least two other ssDNA sequences with matching binding domains. As DNA "bricks" continuously pair and stack, a complex, desired structure is gradually formed.

[0014] Preferably, during self-assembly, a buffer solution to stabilize the solution environment and Mg2+ to adjust the charge of the ssDNA are added to the aqueous solution containing all the ssDNA. 2+ The solution pH was adjusted using Tris base (tris(hydroxymethyl)aminomethane). The solution was first heated to denature the ssDNA, and then annealed.

[0015] More preferably, the solution pH is adjusted to >8.5, the temperature is first raised to 80°C to denature the ssDNA, and then annealing is performed.

[0016] More preferably, after annealing, the temperature is maintained at 4°C.

[0017] In this invention, a buffer solution to stabilize the solution environment and Mg2+ to adjust the charge of ssDNA are also added to the aqueous solution containing all DNA bricks. 2+ 1. Tris base (tris(hydroxymethyl)aminomethane) to adjust the pH of the solution. The assembly solution can be temperature-controlled using a PCR instrument. First, heat to denature the ssDNA, then anneal using gradient cooling or isothermal methods. After annealing, maintain the solution at 4°C for subsequent purification, characterization, or other applications.

[0018] Preferably, the extension direction of the double helix DNA is defined as the z-axis, the nanogroove array extends along the z-axis, and corresponding periodic units are designed on the double helix DNA. By connecting the beginning and end of the periodic units, the double helix DNA is repeatedly spliced ​​in the z-axis direction.

[0019] In this invention, the extension direction of the double-helix DNA is parallel to the groove array, and this direction is defined as the z-axis. The x and y axes, which are perpendicular to the z-axis, allow the building blocks to be extended in six directions along the three extension axes. The nanogroove array in this design extends along the z-axis, therefore requiring the design of corresponding periodic units. By connecting the beginning and end of these periodic units, they are repeatedly spliced ​​along the z-axis, resulting in a long strip template with fixed x- and y-axis dimensions and a micrometer-scale z-axis.

[0020] Preferably, the method for manufacturing the nanotrench array includes the following steps: (1) Preliminary design and construction of DNA template with nanogroove array: Using building units, a geometric structure with a base and sidewalls is simulated and constructed in three-dimensional space to form a nanogroove array, wherein the width of the sidewalls and the grooves are 2 building units respectively. (2) Modify the DNA chain length in the design: use 32 nt (bases) as the main ssDNA chain length, fill and connect in the geometry described in step (1), wherein the DNA bricks located in the boundary region are modified to 48 nt ssDNA chain length, and the DNA bricks located in the side wall region are modified to ssDNA chain length less than 32 nt; (3) Adjusting template distortion: By deleting several bases from the DNA template, distortion compensation can be achieved over a long period of time; (4) Sequence assignment: After the spatial arrangement of ssDNA is completed, according to the principle of complementary base pairing, the base sequence is assigned to each ssDNA, namely A, T, C, G; (5) DNA strand annealing: Mix all ssDNA in buffer, adjust the pH, heat, and then anneal to obtain a DNA template solution with nanogroove array.

[0021] In this invention, the DNA template design structure has fixed dimensions in the x-axis and y-axis directions in three-dimensional space, and extends infinitely in the z-axis direction. During self-assembly, the design structure is connected end-to-end along the z-axis, causing the target synthesized DNA template to extend along the z-axis. The x, y, and z-axis directions mentioned here refer only to relative positions.

[0022] In this invention, DNA bricks are used to construct a base and sidewalls with specific x and y axis dimensions. The base and multiple sidewalls are interconnected. The overall width of the nanogroove array is determined by the width of the base. The area accommodating the sidewalls is changed by altering the width. The height of the nanogroove array is determined by both the base and the sidewalls. The number of nanogrooves is determined by the number of sidewalls on the base. The spacing between the nanogrooves is determined by the spacing between the sidewalls.

[0023] In this invention, the width of the designed base can be appropriately adjusted according to the application scenario, serving as a width limit to accommodate a controllable number of sidewalls.

[0024] In this invention, the trench design requirements include: ① a minimum trench spacing of 4 nm; ② reducing the overall chiral spin of the template; ③ accommodating 6 nanogrooves on a single DNA template; ④ the designed trench units having unidirectional extensibility; and ⑤ adjusting the length of ssDNA at the structural edge to improve stability.

[0025] More preferably, the DNA template has four or more base layers.

[0026] More preferably, the DNA template has four layers as its sidewalls.

[0027] More preferably, when designing the base and sidewalls in the x and y axis directions, double helix DNA is used as the building unit, and its minimum feature size in the x and y axes is 2 nm. The designed sidewalls are composed of double helix DNA with 2 layers wide and 4 layers high, so the sidewalls are 4 nm wide and 8 nm high. The designed base is 4 layers of double helix DNA, and the sidewalls are superimposed on the base, so the height of the nanogroove array is 16 nm.

[0028] More preferably, in step (2), 50% of the ssDNA bricks located in the sidewall region are modified to an ssDNA chain length of 16 nt.

[0029] More preferably, in step (3), distortion compensation over a long period is achieved by deleting one base pair from the end of the DNA template.

[0030] In this invention, the overall twisting design requires that no overall twisting occurs within a length range of approximately 500 nm.

[0031] More preferably, the design length of the DNA template is set to 63 bp (base pairs) or a multiple thereof.

[0032] More preferably, the design length of the DNA template is set to 63 bp.

[0033] More preferably, in step (4), the ssDNA cannot contain consecutive bases or consecutive CG.

[0034] More preferably, in step (4), consecutive bases or consecutive CGs refer to a number of consecutive bases or CGs of 5 or more. For example, AAAAA, TTTTTTT, CGCGCGCGCG.

[0035] More preferably, in step (5), the buffer solution contains Mg 2+ TE buffer.

[0036] More preferably, in step (5), Tris base is added to the buffer solution, and the pH is controlled to be >8.5 by adjusting the concentration of Tris base.

[0037] More preferably, in step (5), the concentration of each ssDNA in the final annealing solution is not less than 100 nM.

[0038] More preferably, in step (5), the temperature is first raised to 80°C, and then annealed. The temperature is first gradually lowered to 60°C, and then gradually lowered to 25°C. The entire annealing time is 40~200 h.

[0039] More preferably, when the temperature is gradually reduced to 60°C, each temperature increment is maintained for more than 2 minutes.

[0040] More preferably, when the temperature is gradually reduced to 25°C, the temperature is maintained at each temperature for more than 1 hour.

[0041] More preferably, after obtaining the DNA template solution with nanogroove array in step (5), excess ssDNA is removed using a centrifugal filter.

[0042] More preferably, the centrifugal filter has a pore size range of 10 kD to 300 kD, uses a relative centrifugal force of 2400×g, centrifuges for 2 to 5 minutes, and centrifuges at least 4 times.

[0043] More preferably, after centrifugation, a solution containing Mg is used. 2+ Rinse with buffer solutions of TE and Tris.

[0044] More preferably, the bottom of the remaining solution after centrifugation is rinsed with buffer each time to further disperse the DNA template, and each rinse is performed at least 10 times.

[0045] Preferably, the method for manufacturing the nanotrench array includes the following steps: 1) Preliminary design and construction of a DNA template with a nanogroove array: A geometric structure with a base and sidewalls was simulated and constructed in three-dimensional space using building units to form a groove array. The width of the sidewalls and grooves are each 2 building units, approximately 4 nm.

[0046] 2) Modify the DNA strand length in the design: Use 32 nt (bases) as the main ssDNA strand length for filling and ligation within the above geometry. Additionally, the DNA bricks located in the boundary regions need to be modified to 48 nt ssDNA strands to ensure structural stability. Note that ssDNA strands shorter than 32 nt should be used in the sidewall regions.

[0047] 3) Adjusting template distortion: An 8 bp building block can cause a systematic overall distortion in the DNA template. This distortion can be compensated for over a long period by artificially deleting a few bases.

[0048] 4) Sequence assignment: After the spatial arrangement of ssDNA is completed, according to the principle of complementary base pairing, each ssDNA is assigned a base sequence, namely A, T, C, G.

[0049] 5) DNA strand annealing: All ssDNA strands are mixed in a specific buffer solution and adjusted to a specific pH. Annealing is then performed to obtain a DNA template solution with a specific nanogroove array.

[0050] 6) Use a centrifugal filter to remove excess ssDNA.

[0051] More preferably, in step 1), the DNA template expands periodically along the z-axis, which is visually represented by the extension of nanogrooves along the z-axis. During DNA self-assembly, the expansion speed along the z-axis is faster than that along the x and y axes. Adopting a multi-directional expansion method (simultaneously expanding along the x and z axes) can easily lead to various assembly defects caused by assembly misalignment, while periodic expansion in a single direction can avoid the occurrence of such defects.

[0052] More preferably, the DNA template base layer should have four or more layers to improve the overall structural rigidity and help resist systemic overall distortion. The sidewall height is adjusted according to the assembly materials, and generally also uses four layers.

[0053] More preferably, in step 2), 50% of the ssDNA bricks of the sidewalls use 16 nt ssDNA strands to prevent the sidewalls from forming prematurely at higher temperatures during the annealing step, which would cause the sidewall structure to become free and result in the loss of the DNA template sidewalls.

[0054] More preferably, in step 3), the helical rotation period of double-stranded DNA (dsDNA) in nature is 10.5 bp. Therefore, the design length of the DNA template needs to be set to 63 bp or a multiple thereof, so that under the design of 8 bp building units, removing 1 bp from 8 building units yields a length of 63 bp, which is exactly equal to 6 rotation periods (6 × 10.5 bp = 63 bp), thereby reducing the overall spin effect of double-stranded DNA.

[0055] More preferably, in step 4), excessive consecutive bases or consecutive CGs should be avoided, such as AAAAA, TTTTTTT, CGCGCGCGCG, etc.

[0056] More preferably, in step 5), the buffer system contains Mg 2+ The TE buffer was used, and the pH was controlled to be >8.5 by adjusting the Tris base concentration. The concentration of each ssDNA in the final annealing solution was not less than 100 nM. During the annealing process, the temperature was first heated to 80℃, then gradually cooled to 60℃, holding for >2 min at each temperature, and then gradually cooled to 25℃, holding for >1 h at each temperature. The entire annealing time was 40~200 h.

[0057] More preferably, in step 6), removing excess unassembled ssDNA requires using a centrifugal filter with a pore size ranging from 10 kD to 300 kD, centrifuging at a relative centrifugal force of 2400 × g for 2–5 min, repeating at least 4 times, and using a filter containing Mg. 2+ Rinse with buffer solutions of TE and Tris.

[0058] More preferably, the bottom of the remaining solution after centrifugation is rinsed with buffer each time to further disperse the DNA template, and each rinse is performed at least 10 times.

[0059] A nanogroove array, fabricated using the above-described method, has a period of 8 nm and a trench width of 4 nm.

[0060] One application of the above-mentioned nanogroove array is to use the nanogroove array in the fields of semiconductor devices, nanophotonic devices, optoelectronics, or nanoquantum electrodynamics.

[0061] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a method for manufacturing a trench array with an 8-nanometer period and a 4-nanometer width, which utilizes the self-assembly of DNA bricks to form a nano-trench array with a trench spacing of less than 10 nm.

[0062] 2. The DNA template design of this invention has the following significant features: the number of trenches in the nanogroove array is controllable; the trench width is 4 nm; the trench period is 8 nm; repeating units are designed and extended in a single extension axis direction; self-assembly defects can be significantly reduced.

[0063] 3. By controlling the size and shape of the nanogrooves, this invention can achieve an 8 nm array spacing, a 4 nm trench width, and a 4 nm sidewall width.

[0064] 4. This invention enables precise control over the number of nanogrooves by setting addressable connecting chains on the base to connect the sidewall structure; and by deleting bases at specific locations to reduce overall distortion, it constructs neatly arranged nanogrooves.

[0065] 5. The present invention utilizes periodically repeating units and adopts a single expansion direction to effectively control assembly defects.

[0066] 6. The long axis dimension of the array of the present invention reaches the micrometer scale, and the array has a large effective area.

[0067] 7. The synthesis process of this invention does not involve any volatile organic reagents or high-energy-consuming equipment, making it environmentally friendly.

[0068] 8. The present invention has high synthesis efficiency, simple method, and is easy to mass-produce. Attached Figure Description

[0069] Figure 1 This is a schematic diagram of the interaction and binding domains between ssDNA bricks in this invention.

[0070] Figure 2 This is a schematic diagram of the connection method of ssDNA bricks in three-dimensional space.

[0071] Figure 3 This is a schematic diagram of the morphology of DNA nanogrooves.

[0072] Figure 4 This is a transmission electron microscope (TEM) image of DNA nanogrooves.

[0073] Figure 5 The spacing of DNA nanogrooves is measured based on electron microscopy images.

[0074] Figure 6 The finite element modeling framework simulation is performed before and after correcting the template element length.

[0075] Figure 7 Transmission electron microscopy characterization of the DNA nanogroove template after the removal of three sidewalls. Detailed Implementation

[0076] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0077] A method for designing and fabricating a DNA nanogroove array includes the following steps: (1) Design DNA template with nanogroove array: simulate and build a geometric structure with base and sidewall in three-dimensional space to form nanogroove array; (2) Modify the DNA chain length in the design: use 32 nt as the main ssDNA chain length, fill and connect in the geometry described in step (1), wherein the DNA bricks located in the boundary region are modified to 48 nt ssDNA chain length, and the DNA bricks located in the side wall region are modified to ssDNA chain length less than 32 nt. (3) Adjusting template distortion: By deleting several bases from the DNA template, distortion compensation can be achieved over a long period of time; (4) Sequence assignment: After the spatial arrangement of ssDNA is completed, according to the principle of complementary base pairing, the base sequence is assigned to each ssDNA, namely A, T, C, G; (5) DNA strand annealing: Mix all ssDNA in buffer, adjust the pH, heat, and then anneal to obtain a DNA template solution with nanogroove array.

[0078] As a preferred technical solution, when it is necessary to design and fabricate a DNA nanogroove array with an 8-nanometer period and a 4-nanometer width, when designing the base and sidewalls in the x and y axes, double-helical DNA is used as the building unit, and its minimum feature size in the x and y axes is 2 nm. The designed sidewalls are composed of double-helical DNA with a width of 2 layers and a height of 4 layers, so the sidewalls are 4 nm wide and 8 nm high. The designed base is composed of 4 layers of double-helical DNA, and the sidewalls are stacked on the base. Thus, the height of the nanogroove array is 16 nm, and the designed groove size is equivalent to double-helical DNA with a width of 2 layers, so the groove width is 4 nm.

[0079] The following detailed description is based on specific embodiments.

[0080] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0081] Example 1 Design, fabrication, and characterization of a DNA nanogroove array with an 8-nanometer period and a 4-nanometer width. Step 1: like Figure 1 , Figure 2As shown, the target structure was designed using the DNA brick design method and the nanobricks web software (https: / / yin.hms.harvard.edu / bricks / try / ), subsequently generating a DNA template with nanogrooves. The internal sequence of the DNA structure was edited using the cadnano open-source software to improve overall stability, and the design length of the DNA template was modified to 63 bp. While meeting the sequence pairing requirements of the structure, cadnano was used to randomly assign values ​​to the ssDNA within it.

[0082] The length of local ssDNA was changed: the boundary strand was changed to 48 nt, and 50% of the sidewall strands were changed to 16 nt; the overall distortion of the designed structure was improved by deleting one base pair at the end of each layer along the template z-axis. This stabilized the edge structure and made it more conducive to obtaining the desired assembly structure, such as... Figure 3 As shown.

[0083] The obtained ssDNA was dispersed in a low-concentration TE buffer, and the concentration was adjusted to 100 μM. Two μL of each ssDNA sample was taken, mixed, and dispersed in TE / Mg buffer. 2+ The buffer solution contains a final concentration of 100 nM for each ssDNA. The mixed solution contains 1×TE and 40 mM Mg. 2+ The pH was adjusted to 9.0 using Tris base.

[0084] The mixed solution was transferred to PCR-specific centrifuge tubes and annealed and assembled in PCR equipment, with a solution volume of 70 μL per PCR tube. A two-stage, segmented cooling method was used for annealing. First, the temperature was heated from room temperature to 80°C at a rate of 6 °C / s and held for 15 min. Then, a first rapid cooling phase (80°C → 60°C) was performed: the temperature was decreased in integer increments of 1°C at a rate of 0.1 °C / s, held for 6 min at each temperature. This was followed by a second slow cooling phase (59°C → 25°C): the temperature was decreased in integer increments of 1°C at a rate of 0.1 °C / s, held for 3 h at each temperature. After annealing, the temperature was rapidly reduced from 25°C to 4°C at a rate of 6 °C / s. At this point, the DNA nanogroove array was annealed and assembled, and the assembled solution could be stored long-term at 4°C.

[0085] To reduce excess ssDNA in the DNA template solution, the assembly solution was purified using a 30kD filter-concentrated centrifuge tube. 1×TE and 40 mM Mg were used. 2+ The DNA template in the centrifuge tube was washed and centrifuged multiple times with a washing solution of pH 9.0, repeated 4 times. The purified DNA template solution can be stored at 4°C for a long time.

[0086] DNA templates were characterized and analyzed using transmission electron microscopy (TEM).

[0087] During TEM imaging, the carbon-supported copper mesh needs to be treated with glow discharge to obtain hydrophilic properties. 4 μL of DNA template solution is added to the front surface of the copper mesh and deposited for 3 min. Excess solution is then blotted away with clean filter paper near the edge of the copper mesh. Negative staining is then performed using 1% wt uranyl acetate solution, typically 4 μL added and deposited for 7 s. Excess solution is then blotted away with clean filter paper near the edge of the copper mesh. The copper mesh is then placed on a sample holder and inserted into the TEM vacuum chamber to acquire morphological images.

[0088] See Figure 4 This is a TEM image of the assembled DNA nanogroove array. The black areas between the array segments represent the groove regions highlighted after negative staining.

[0089] See Figure 5 This is a schematic diagram of the dimensions of the DNA nanogroove array. The width of the DNA template remains essentially constant at 53 nm, extending along the z-axis. The widths of the grooves and sidewalls are labeled: average sidewall width 4.01 nm; average groove width 4.21 nm; average array period 8.22 nm.

[0090] Example 2 A finite element modeling framework for DNA nanogroove arrays.

[0091] The overall distortion correction effect of DNA nanogrooves was simulated using the open-source web platform software Cando (https: / / cando-dna-origami.org / ). Cando uses a mechanical model of DNA to predict the 3D shape and flexibility of programmed DNA. This model assumes that the double helix structure is a uniform elastic rod with axial tensile, torsional, and bending stiffness. In the design, the length of a single template unit without distortion correction was 64 bp, equivalent to 24 nm. To visually understand the distortion of the DNA nanogroove array, three template units were selected, connected end-to-end, as the simulated DNA structure. Following the Cando operating manual, the exposed free strands at both ends were removed. Figure 6 As shown, comparing the thermal fluctuation three-view (RMSF) of the initial group (origin) with a length of 64 bp * 3 and the modified group (modified) with a length of 63 bp * 3, the results show that the initial group exhibits significant distortion, while the distortion characteristics of the modified group are reduced, and no overall twisting occurs. The comparison between this framework simulation and experimental characterization confirms that the designed DNA template structure can effectively prevent overall distortion and can be used as a planar nanogroove array.

[0092] Example 3 Design, prepare, and characterize DNA groove array templates containing 4 sidewalls (3 grooves).

[0093] The target structure in Example 1 was modified using the open-source software scadnano: three sidewalls were removed to give it three wider nanogrooves, while the remaining base sequence remained unchanged.

[0094] Based on the new structure, 2 μL of the remaining ssDNA was taken, mixed, and dispersed in TE / Mg. 2+ The buffer solution was adjusted to a final concentration of 100 nM for each ssDNA. The mixed solution contained 1×TE and 40 mM Mg. 2+ The pH was adjusted to 9.0 using Tris base.

[0095] The mixed solution was transferred to PCR-specific centrifuge tubes and annealed for assembly in a PCR instrument, with a solution volume of 70 μL per PCR tube. Annealing was performed using the same two-stage, stepped cooling method as in Example 1. The assembly solution was purified using a 30 kDa filter-concentrating centrifuge tube. Purification was performed using 1×TE and 40 mM MgSO4. 2+ The DNA template in the centrifuge tube was washed and centrifuged multiple times with a washing solution of pH 9.0, repeated 4 times. The purified DNA template solution can be stored at 4°C for a long time.

[0096] Characterization analysis of DNA templates was performed using transmission electron microscopy (TEM), such as... Figure 7 As shown in the figure. The results show that the width of the base is the same as that of the original template, and the base has 4 sidewalls that are evenly spaced and separated by 3 wider nanogrooves.

[0097] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for manufacturing a nanogroove array, characterized in that, Using ssDNA as DNA bricks, self-assembly was performed according to the complementary pairing principle of ssDNA to obtain a nanogroove array with a base and sidewalls. The width and height of the nanogroove array are fixed, the number of grooves is determined, and it extends in a single direction. The method for manufacturing the nanogroove array includes the following steps: (1) Design DNA template with nanogroove array: simulate and build a geometric structure with base and sidewall in three-dimensional space to form nanogroove array; (2) Modify the DNA chain length in the design: use 32 nt as the main ssDNA chain length, fill and connect in the geometry described in step (1), wherein the DNA bricks located in the boundary region are modified to 48 nt ssDNA chain length, and the DNA bricks located in the side wall region are modified to ssDNA chain length less than 32 nt. (3) Adjusting template distortion: By deleting several bases from the DNA template, distortion compensation can be achieved over a long period of time; (4) Sequence assignment: After the spatial arrangement of ssDNA is completed, according to the principle of complementary base pairing, the base sequence is assigned to each ssDNA, namely A, T, C, G; (5) DNA strand annealing: Mix all ssDNA in buffer, adjust the pH, heat, and then anneal to obtain a DNA template solution with nanogroove array.

2. The method for manufacturing a nanotrench array according to claim 1, characterized in that, In three-dimensional space, the design structure of the DNA template has fixed dimensions in the x-axis and y-axis directions and extends infinitely in the z-axis direction. During self-assembly, the design structure is connected end to end in the z-axis, so that the target synthesized DNA template extends along the z-axis.

3. The method for manufacturing a nanotrench array according to claim 2, characterized in that, DNA bricks are used to construct a base and sidewalls with specific x and y axis dimensions. The base is interconnected with multiple sidewalls. The overall width of the nanogroove array is determined by the width of the base. The area that accommodates the sidewalls is changed by changing the width. The height of the nanogroove array is determined by both the base and the sidewalls. The number of nanogrooves is determined by the number of sidewalls on the base. The spacing of the nanogrooves is determined by the spacing of the sidewalls.

4. The method for manufacturing a nanotrench array according to claim 1, characterized in that, During self-assembly, a buffer solution to stabilize the solution environment and Mg2+ to adjust the charge of the ssDNA are added to an aqueous solution containing all ssDNA. 2+ First, use Tris base to adjust the pH of the solution, then heat to denature the ssDNA, and finally anneal it.

5. The method for manufacturing a nanotrench array according to claim 4, characterized in that, Adjust the solution pH to >8.5, first heat to 80℃ to denature the ssDNA, and then anneal.

6. The method for manufacturing a nanotrench array according to claim 1, characterized in that, When designing the base and sidewalls in the x and y axes, double helical DNA is used as the building unit. Its minimum feature size in the x and y axes is 2 nm. The designed sidewalls are composed of double helical DNA with 2 layers wide and 4 layers high. Therefore, the sidewalls are 4 nm wide and 8 nm high. The designed base is composed of 4 layers of double helical DNA. The sidewalls are superimposed on the base, so the height of the nanogroove array is 16 nm.

7. The method for manufacturing a nanotrench array according to claim 1, characterized in that, In step (2), 50% of the ssDNA bricks located in the sidewall region are modified to an ssDNA chain length of 16 nt; And / or, set the design length of the DNA template to 63 bp or a multiple thereof.

8. A nanogroove array, characterized in that, The trench is manufactured using the manufacturing method described in any one of claims 1 to 7, wherein the trench width is 4 nm and the trench period is 8 nm.

9. An application of the nanogroove array according to claim 8, characterized in that, The nanogroove array can be used in semiconductor devices, nanophotonic devices, optoelectronics, or nanoquantum electrodynamics.

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