CN117316264A - Method and equipment for realizing calculable structure based on DNA paper folding - Google Patents

Abstract

The embodiments of this specification provide a method and equipment for realizing a computable structure based on DNA origami, including: designing a DNA origami monomer containing multiple programmable edges; and preparing a DNA origami monomer according to the design results of the DNA origami monomer. The DNA origami monomers are combined and characterized; the DNA sequences of the programmable edges in the DNA origami monomers are encoded, and corresponding DNA nanostructures are assembled according to different connection rules between origami sheets. The DNA origami structure of the present invention provides more codable points, has greater information capacity, and improves the application capabilities of DNA origami in computing scenarios.

Description

A method and device for realizing computable structures based on DNA origami

Technical field

This document relates to the fields of DNA nanomanufacturing and DNA computing technology, and in particular to a method and equipment for realizing a computable structure based on DNA origami.

Background technique

The concept of DNA origami was first proposed in 2006. This technology is based on the Watson-Crick base pairing principle and designs hundreds of short ssDNA (called staples) through artificial coding to transform a single strand usually about 7000nt in length. Circular DNA backbones (called scaffolds) fold and bind into preset shapes, demonstrating the technology's powerful ability to encode DNA sequences. At the same time, Rothemund demonstrated the high addressability of DNA origami by adding hairpin-like modified structures to the staples at designated sites to arrange specific patterns on the surface of DNA origami. The emergence of this programmable, addressable and easy-to-characterize nanotechnology has improved the problem of DNA tile self-assembly systems involving a large number of short chains, and is a milestone in the history of nanofabrication.

Self-assembly nanotechnology represented by DNA tiles and DNA origami provides a programmable molecular platform for DNA computing. However, DNA origami monomers whose size is limited by fixed-length scaffolds are difficult to meet increasingly complex application requirements. A feasible method is to divide codable connection regions on DNA origami. According to the shape, size, biochemical properties and other characteristics of DNA origami monomers, multiple DNA origami can be ordered by defining connection rules on the codable regions. self-assemble into larger-scale structural arrays. Finite self-assembly structures are the cornerstone of constructing complex nanodevices and molecular arrays. The current method to expand the scale of DNA origami takes advantage of the reliability of complementary DNA base pairing. By encoding the DNA sequence of the connecting region, small-sized DNA origami can be connected with the corresponding The strategy is scaled up to large-size structures, effectively controlling the self-assembly behavior between DNA origami. However, related research based on programmable self-assembly of two-dimensional DNA origami has some limitations: first, the assembly directions of the DNA origami monomers used are limited to the four directions of U, D, L, and R; second, programmable self-assembly The generated large-scale DNA origami arrays are often applied to molecular canvases. As a highly programmable nanotool, in the field of DNA computing, DNA origami has more advantages than small molecular structures such as DNA tiles - allowing more coded sites, It is easier to characterize, but there are few studies on developing DNA origami components that can be used for computing based on the pasting model in DNA computing.

Contents of the invention

One or more embodiments of this specification provide a method and device for realizing a computable structure based on DNA origami, including:

S1. Design DNA origami monomers containing multiple programmable edges;

S2. Prepare DNA origami monomers according to the design results of the DNA origami monomers and characterize them;

S3. Encode the DNA sequence of the programmable edge in the DNA origami monomer, and assemble to form the corresponding DNA nanostructure according to different connection rules between the origami.

Further, the DNA origami monomer includes a main body region and a coding connection region, the main body region is used to provide structural stability support, and the coding connection region is used to connect different DNA origami monomers according to the coding; The main area includes an origami main body, a central bridging area and an edge bridging area;

The DNA origami monomer contains 8 programmable edges, each of which contains 6 double helices.

Further, step S1 is specifically:

The DNA origami monomer is formed by hybridizing the scaffold chain scaffold and the staple chain staple based on the principle of complementary base pairing to complete self-assembly, wherein scaffold is the M13 phage plasmid circular DNA sequence;

Use staple to create crossovers between helices. Adjacent double crossovers are spaced 32nt apart. One base pair is deleted between every three pairs of double crossovers to balance plane twisting;

Introduce a single-stranded scaffolding bridge between non-adjacent double helix structures and design the length of the single-stranded scaffolding bridge in DNA origami.

Further, the length of the single-stranded scaffolding bridge in the DNA origami is specifically:

The lengths of the single-stranded scaffolding bridges in the central bridging region are 7, 2, 7, 7, and 2nt respectively from the center to the outside. The lengths of the single-stranded scaffolding bridges in the edge bridging region are all 8nt. The flexible single strands of the bridging sequence are composed of thymidine-rich nucleosides. Composed of acid (T).

Further, the specific method for preparing DNA origami monomers based on the design results of the DNA origami monomers is:

Add a sequence of closed staples to 8 programmable edges, specifically:

Mix staples and scaffolds in 1×TAE buffer with 12.5mM Mg ²⁺ at a concentration ratio of 5:1 to obtain a solution with a concentration of 2.5nM;

The DNA origami structure is obtained through PCR annealing reaction. The specific requirements of the PCR annealing reaction are as follows: heating at 95°C for 5 minutes, then cooling at a constant rate of -0.1°C/6s, and annealing from 95°C to 4°C.

Further, the characterization of DNA origami monomers is specifically:

Preparing a characterization sample of the DNA origami monomer, and performing atomic force microscopy (AFM) characterization on the characterization sample;

The preparation method of the characterization sample is as follows: pipet 5 ul of DNA origami monomer sample onto the surface of a smooth mica sheet, and leave it for 3 minutes for adsorption.

Further, the different connection logic includes deterministic connection and non-deterministic connection:

The deterministic connection is: when a pair of programmable edges simultaneously add elongated and shortened connecting DNA sequences in the solution, and the sticky end of the elongated connecting DNA sequence is complementary to the scaffold of the shortened connecting DNA sequence, this Logically connect deterministic connections between programmable edges;

The non-deterministic connection is: when there is a pair of programmable edges in the solution and a blunt-end connecting DNA sequence that is completely complementary to the scaffold is added, the pair of programmable edges are logically connected with a non-deterministic connection;

There is no division of positive and negative edges in the non-deterministic connection, and there is a division of positive and negative edges in the deterministic connection.

Further, the DNA origami monomer assembly method is:

All the DNA origami monomers were annealed at a constant speed of -0.1°C/8min in the temperature range of 35°C-20°C, incubated at 20°C for 1 hour, and then annealed at a constant speed of -0.1°C/6s to 4°C for storage.

One or more embodiments of the present specification provide an electronic device, including: a processor; and a memory arranged to store computer-executable instructions. When executed, the computer-executable instructions cause the processor to implement the above-mentioned based on Steps of the method to realize the computable structure of DNA origami.

One or more embodiments of this specification provide a storage medium for storing computer-executable instructions that, when executed, implement the steps of the above method for implementing a computable structure based on DNA origami.

Using the embodiments of the present invention, a DNA origami with eight mutually independent codable edges was designed and realized. By calculating the single-strand length of the bridging sequence, an origami structure that complies with thermodynamic stability was designed, and was finally prepared and characterized to meet the application requirements. The required computable DNA origami structure; the DNA origami of the present invention provides more coded sites through 8 programmable edges, the coding area is denser, and the information capacity is larger; the programmable edges can be based on different molecular assembly characteristics, respectively Realizing three different connection states: closed, deterministic, and non-deterministic, it improves the application capabilities of DNA origami in computing scenarios.

The above description is only an overview of the technical solution of the present invention. In order to have a clearer understanding of the technical means of the present invention, it can be implemented according to the content of the description, and in order to make the above and other objects, features and advantages of the present invention more obvious and understandable. , the specific embodiments of the present invention are listed below.

Description of drawings

In order to more clearly illustrate one or more embodiments of this specification or technical solutions in the prior art, the drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, in the following description The drawings are only some of the embodiments recorded in this specification. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is a flow chart of a method for implementing a computable structure based on DNA origami provided by one or more embodiments of this specification;

Figure 2 is a schematic diagram of the scaffold shape of the 8-edge DNA origami structure provided by one or more embodiments of this specification;

Figure 3 is a schematic diagram of three connection states of the programmable edges of DNA origami provided by one or more embodiments of this specification;

Figure 4 is a schematic diagram of the computable DNA origami structure region division provided by one or more embodiments of this specification;

Figure 5 is a schematic diagram of the double helix arrangement in the 8-edge DNA origami design provided by one or more embodiments of this specification;

Figure 6 is the AFM characterization result of preparing the 8-edge DNA origami of the present invention provided by one or more embodiments of this specification;

Figure 7 is a schematic diagram and implementation results of the deterministic connection logic provided by one or more embodiments of this specification;

Figure 8 is a schematic diagram and implementation results of the non-deterministic connection logic provided by one or more embodiments of this specification;

Figure 9 is a schematic structural diagram of an electronic device provided by one or more embodiments of this specification.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in one or more embodiments of this specification, the following will describe the technical solutions in one or more embodiments of this specification in conjunction with the drawings in one or more embodiments of this specification. The technical solution is described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of this specification, rather than all the embodiments. Based on one or more embodiments of this specification, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the protection scope of this document.

Method Example

According to an embodiment of the present invention, a method for realizing a computable structure based on DNA origami is provided. Figure 1 is a flow chart of a method for realizing a computable structure based on DNA origami provided by one or more embodiments of this specification. As shown in Figure 1, the implementation method of a computable structure based on DNA origami according to an embodiment of the present invention specifically includes:

S1. Design DNA origami monomers containing multiple programmable edges.

Design the origami structure according to the application purpose, including dividing functional areas, designing geometric shapes, and designing scaffold trends. In this example, we choose to construct a two-dimensional origami structure. The skeleton chain for preparing DNA origami is the M13mp18 phage plasmid circular DNA sequence. The skeleton chain design is a closed loop. First, the DNA origami monomer is divided into a main body area and a coding connection area based on the connection type probe machine PM. The main area is used to provide structural stability support, including the origami main body, central bridging area and edge bridging area. Region, as shown in Figure 4; the coded connection region is used to connect different DNA origami monomers according to the coding and participate in the assembly behavior between origami. The schematic diagram of the edge DNA origami structure of Example 8 is shown in Figure 2, and the connection schematic diagram is shown in Figure 3.

Step S1 is specifically as follows:

The DNA origami monomer is self-assembled through the hybridization of the scaffold chain scaffold and the staple chain staple based on the principle of complementary base pairing. Among them, scaffold is the M13 phage plasmid circular DNA sequence;

Use staple to create crossovers between helices. Adjacent double crossovers are spaced 32nt apart. One base pair is deleted between every three pairs of double crossovers to balance plane twisting;

Introduce a single-stranded scaffolding bridge between non-adjacent double helix structures and design the length of the single-stranded scaffolding bridge in DNA origami.

In order to make the constructed DNA origami unit have high rigidity and programmability, the length of the single-strand bridge in the DNA origami needs to be designed to prevent structural distortion caused by excessive traction or relaxation. In this embodiment, the DNA origami is comprehensively considered. The scaffold design drawing and the geometric distance between the double strands determine that the lengths of the single-stranded bridges in the central bridging region are 7, 2, 7, 7, and 2nt from the center to the outside respectively. The lengths of the single-stranded scaffolding bridges in the edge bridging region are all 8nt. The bridge sequence The flexible single strand is composed of thymine-rich nucleotides T.

According to the sequence length, a semi-manual method is used to generate the DNA sequence in the software caDNAno2. According to the designed distribution strategy, select the square stacking mode in the software and draw the double helix arrangement of the side view of the DNA origami in the double helix stacking window. The drawing interface and results are shown in Figure 5. Then fill in the double cross arrangement window with staples and draw the main view of the DNA origami.

In order to stabilize the structure of the main region of the designed DNA origami self-assembly structure, the connection region meets the following requirements: the codable sites of different elements can contact each other; the number is as large as possible. This embodiment designs 8 connection edges. Each edge contains 6 double helices; the DNA sequence is independent of the main region.

S2. Prepare DNA origami monomers based on the design results of the DNA origami monomers, and characterize the DNA origami monomers.

The specific method for preparing DNA origami monomers according to the design results of the DNA origami monomers is:

Add a sequence of hairpin-like closing staples to 8 programmable edges, specifically:

Mix staples and scaffolds in 1×TAE buffer with 12.5mM Mg ²⁺ at a concentration ratio of 5:1 to obtain a solution with a concentration of 2.5nM;

The DNA origami structure is obtained through PCR annealing reaction. The specific requirements of the PCR annealing reaction are as follows: heating at 95°C for 5 minutes, then cooling at a constant rate of -0.1°C/6s, and annealing from 95°C to 4°C.

The prepared DNA origami structure was characterized by Atomic Force Microscope (AFM). First, a characterization sample of the DNA origami monomer was prepared. The preparation method of the characterization sample was as follows: Pipette 5 ul of the DNA origami monomer sample onto smooth mica. Place on the surface of the tablet and leave it for 3 minutes for adsorption.

The prepared characterization samples were characterized by atomic force microscopy (AFM). The characterization process was performed in Cypher ES liquid phase mode, using BL-AC40TS probe scanning, and software Asylum Research 16.33.234 for image processing. The scan results are shown in Figure 6.

S3. Encode the DNA sequence of the programmable edge in the DNA origami monomer, and assemble to form the corresponding DNA nanostructure according to different connection rules between the origami.

DNA sequences encoding programmable edges realize different connection logics between origami. Different connection logics specifically include deterministic connections and non-deterministic connections:

The deterministic connection is: when a pair of programmable edges simultaneously add elongated and shortened connecting DNA sequences in the solution, and the sticky end of the elongated connecting DNA sequence is complementary to the scaffold of the shortened connecting DNA sequence, this Logically connect deterministic connections between programmable edges;

The non-deterministic connection is: when there is a pair of programmable edges in the solution and a blunt-end connecting DNA sequence that is completely complementary to the scaffold is added, the pair of programmable edges are logically connected with a non-deterministic connection;

There is no division of positive and negative edges in the non-deterministic connection, and there is a division of positive and negative edges in the deterministic connection.

When realizing a specific array through deterministic connection, elongated and shortened DNA sequences are added to a pair of programmable edges that exist simultaneously in the solution. The sticky end of the elongated sequence is complementary to the scaffold of the shortened sequence. At this time, the relationship between the pair of edges is are logically connected with deterministic connections. As shown in Figure 7, taking the first array as an example, the 8 edges E ₁ -E ₈ of origami B ₁ are encoded as OFF in sequence, OFF, OFF, OFF, OFF; the 8 edges E ₁ -E ₈ of origami B ₂ are encoded as OFF, OFF, OFF, OFF, OFF,/> Among them, OFF means closed, D means deterministic connection, the subscript means a pair of connected positive and negative edges, and the superscript means that the current edge is used as a positive edge (+) or a negative edge (-). B ₁ B ₂ realizes the connection arrays of E ₂ & E ₈ , E ₃ & E ₇ , E ₄ & E ₆ through deterministic connection logic. The connection sequences of the other two sets of connection arrays in Figure 7 can be obtained in the same way. In the experiment of this embodiment, biotin-streptavidin modified sequences were added to different positions of B ₁ and B ₂ to distinguish them. The AFM scanning image of the experimental results is shown in the lower figure of Figure 7. The three arrays are from top to bottom. The following correspond to three scanned images from left to right, that is, a deterministic connection encoding between origami can realize a corresponding self-assembly array.

When realizing a specific array through non-deterministic connection, a blunt-end connecting DNA sequence that is completely complementary to the scaffold is added to a pair of programmable edges that exist simultaneously in the solution. At this time, the pair of edges are logically connected with a non-deterministic connection. As shown in Figure 8, the eight edges E ₁ -E ₈ of the origami B ₁ are sequentially encoded as OFF, N, N, N, OFF, OFF, OFF, OFF, and N represents a non-deterministic connection.

When performing self-assembly of DNA origami monomers, first encode and prepare each origami monomer through the above method, add corresponding sequences to the 8 edges according to the encoding, and then assemble the prepared monomers. Specifically: mix all the components involved in the assembly reaction. Origami monomers were annealed at a constant speed of -0.1°C/8min in the temperature range of 35°C-20°C, incubated at 20°C for 1 hour, and then annealed at a constant speed of -0.1°C/6s to 4°C for storage.

The beneficial effects of the present invention are as follows:

The present invention designs and implements a DNA origami with 8 mutually independent codable edges. By calculating the single-strand length of the bridging sequence, an origami structure that meets the thermodynamic stability is designed, and finally a computable origami that meets the application requirements is prepared and characterized. DNA origami structure; the DNA origami of the present invention provides more coded sites through 8 programmable edges, with denser coding areas and greater information capacity; the programmable edges can achieve closure and determination respectively based on different molecular assembly characteristics , non-deterministic three different connection states, which improves the application capabilities of DNA origami in computing scenarios.

Device Embodiment 1

An embodiment of the present invention provides an electronic device, as shown in Figure 9, including: a memory 90, a processor 92, and a computer program stored on the memory 90 and executable on the processor 92. The computer program When executed by the processor 92, the following method steps are implemented:

S1. Design DNA origami monomers containing multiple programmable edges;

S2. Prepare DNA origami monomers according to the design results of the DNA origami monomers and characterize them;

S3. Encode the DNA sequence of the programmable edge in the DNA origami monomer, and assemble to form the corresponding DNA nanostructure according to different connection rules between the origami.

Device Embodiment 2

Embodiments of the present invention provide a computer-readable storage medium. The computer-readable storage medium stores a program for implementing information transmission. When the program is executed by the processor 92, the following method steps are implemented:

S1. Design DNA origami monomers containing multiple programmable edges;

S2. Prepare DNA origami monomers according to the design results of the DNA origami monomers and characterize them;

S3. Encode the DNA sequence of the programmable edge in the DNA origami monomer, and assemble to form the corresponding DNA nanostructure according to different connection rules between the origami.

The computer-readable storage medium in this embodiment includes but is not limited to: ROM, RAM, magnetic disk or optical disk, etc.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. scope.

Claims (10)

1. A method for realizing a computable structure based on DNA origami, which is characterized by including: S1. Design DNA origami monomers containing multiple programmable edges; S2. Prepare DNA origami monomers according to the design results of the DNA origami monomers, and characterize the DNA origami monomers; S3. Encode the DNA sequence of the programmable edge in the DNA origami monomer, and assemble to form the corresponding DNA nanostructure according to different connection rules between the origami. 2. The method according to claim 1, characterized in that, The DNA origami monomer includes a main body region and a coding connection region. The main body region is used to provide structural stability support, and the coding connection region is used to connect different DNA origami monomers according to coding; the main body region includes Origami body, central bridge area and edge bridge area; The DNA origami monomer contains 8 programmable edges, each of which contains 6 double helices. 3. The method according to claim 2, characterized in that step S1 is specifically: The DNA origami monomer is formed by hybridizing the scaffold chain scaffold and the staple chain staple based on the principle of complementary base pairing to complete self-assembly, wherein scaffold is the M13 phage plasmid circular DNA sequence; Use staple to create crossovers between helices. Adjacent double crossovers are spaced 32nt apart. One base pair is deleted between every three pairs of double crossovers to balance plane twisting; Introduce a single-stranded scaffolding bridge between non-adjacent double helix structures and design the length of the single-stranded scaffolding bridge in DNA origami. 4. The method according to claim 3, characterized in that the length of the single-stranded scaffold bridge in the DNA origami is specifically: The lengths of the single-stranded scaffolding bridges in the central bridging region are 7, 2, 7, 7, and 2nt respectively from the center to the outside. The lengths of the single-stranded scaffolding bridges in the edge bridging region are all 8nt. The flexible single strands of the bridging sequence are composed of thymidine-rich nucleosides. Acid T composition. 5. The method according to claim 3, characterized in that the specific method for preparing DNA origami monomers according to the design results of the DNA origami monomers is: Add a sequence of hairpin-like closing staples to 8 programmable edges, specifically: Mix staples and scaffolds in 1×TAE buffer with 12.5mM Mg ²⁺ at a concentration ratio of 5:1 to obtain a solution with a concentration of 2.5nM; The DNA origami structure is obtained through PCR annealing reaction. The specific requirements of the PCR annealing reaction are as follows: heating at 95°C for 5 minutes, then cooling at a constant rate of -0.1°C/6s, and annealing from 95°C to 4°C. 6. The method according to claim 5, characterized in that the characterization of the DNA origami monomer is specifically: Preparing a characterization sample of the DNA origami monomer, and performing atomic force microscopy (AFM) characterization on the characterization sample; The preparation method of the characterization sample is as follows: pipet 5 ul of DNA origami monomer sample onto the surface of a smooth mica sheet, and leave it for 3 minutes for adsorption. 7. The method according to claim 3, characterized in that the different connection rules include deterministic connections and non-deterministic connections: The deterministic connection is: when a pair of programmable edges simultaneously add elongated and shortened connecting DNA sequences in the solution, and the sticky end of the elongated connecting DNA sequence is complementary to the scaffold of the shortened connecting DNA sequence, this Logically connect deterministic connections between programmable edges; The non-deterministic connection is: when there is a pair of programmable edges in the solution and a blunt-end connecting DNA sequence that is completely complementary to the scaffold is added, the pair of programmable edges are logically connected with a non-deterministic connection; There is no division of positive and negative edges in the non-deterministic connection, and there is a division of positive and negative edges in the deterministic connection. 8. The method according to claim 5, characterized in that the assembly method of DNA origami monomers is: All the DNA origami monomers were annealed at a constant speed of -0.1°C/8min in the temperature range of 35°C-20°C, incubated at 20°C for 1 hour, and then annealed at a constant speed of -0.1°C/6s to 4°C for storage. DNA origami monomer assembly. 9. An electronic device, characterized in that it includes: processor; and, A memory arranged to store computer-executable instructions that, when executed, cause the processor to implement the steps of the method for realizing a computable structure based on DNA origami according to any one of claims 1 to 8 . 10. A storage medium, characterized in that it is used to store computer-executable instructions. When executed, the computer-executable instructions realize the realization of the computable structure based on DNA origami as claimed in any one of claims 1 to 8. Method steps.