A colloidal molecular system based on DNA circuit coding assembly and a preparation method thereof

Abstract

The application provides a colloidal molecule system based on DNA circuit coding assembly and a preparation method thereof. The application develops DNA circuit coding CMs with high precision and time dependence by using ssDNA with alternating polyA domains and functional domains and dynamic DNA circuit catalytic hairpin self-assembly (CHA) to code and assemble AuNPs, and the DNA circuit coding CMs can be used for adjusting peroxidase-like activity of AuNPs. The application can obtain DNA circuit coding CMs with dimeric, linear or reticular structures by changing the number and length of the polyA domains and the functional domains. The application can accurately adjust the peroxidase-like catalytic activity of the DNA circuit coding CMs system by blocking the catalytic active sites on the surface of the AuNPs or simply changing the concentration of the external DNA stimulant. The application provides a new idea for regulating the catalytic function of nanomaterials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of colloidal molecules, in particular to a colloidal molecule system based on DNA circuit coding assembly and a preparation method thereof. BACKGROUND

[0002] Proteins play an important role in physiology and pathology, and are spatiotemporally regulated by exogenous or endogenous factors such as small molecules, nucleic acids, etc. To respond to various regulatory factors, proteins change their structures to precisely regulate their activities, such as blocking catalytic sites or forming homodimers and multimers. In contrast, in materials science, the modulation of material catalytic function mainly relies on changing the synthesis conditions to form materials with different shapes, sizes and compositions. However, the regulation of catalytic activity after material synthesis is still a great challenge, especially under time-controllable conditions.

[0003] Dynamic control of the assembly of nanosynthetic particles (NPs) into small clusters, called colloidal molecules (CMs). Based on the interaction between the plasmonic, electronic and magnetic moments of individual NPs, CMs provide a promising strategy to modulate the catalytic function of NPs. As a classic nanosynthetic material, AuNPs have glucose oxidase and / or peroxidase-like activity, and have high application value in biosensing, tumor treatment and antibacterial, etc. To realize the self-assembly of NPs into highly ordered CMs, AuNPs are usually functionalized by DNA patches, polymer ligands or small molecules. In particular, due to the encodability, predictability, high specificity and DNA-DNA interaction, DNA-encoded nanoparticle assembly has attracted strong interest and made significant progress. In the early stage of this field, AuNPs were wrapped by dense thiol-modified double-stranded or single-stranded DNA (ssDNA) to mediate the assembly of CMs. However, due to the same chemical environment and shape isotropy of AuNPs, the surface anchoring of AuNPs with DNA is not region-selective, which leads to the generation of a mixture of CMs with different structures, making it difficult to explore the influence of CMs structure on the catalytic activity of AuNPs.

[0004] To realize precise control of CMs structure, additional modification steps are needed, such as region-specific surface coding of NPs with directional DNA patches, or merging of DNA-functionalized AuNPs into a DNA framework with unique strands. However, this process blocks the catalytic site, completely inhibiting the catalytic function of AuNPs, and cannot dynamically regulate the catalytic activity of synthesized NPs. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a colloidal molecule system based on DNA circuit coding assembly and a preparation method thereof. The present application integrates a dynamic DNA circuit, catalytic hairpin assembly (CHA), into AuNPs with ssDNA patterns containing alternating polyA and functional domains, for time-controllable assembly of AuNPs, and by changing the number and length of polyA structural domains and functional domains, CHA coding CMs with a dimeric, linear or reticular structure are obtained. Surprisingly, it is found that the peroxidase-like activity of AuNPs can be regulated by blocking different numbers of ssDNA containing polyA on the catalytic surface thereof. Interestingly, the inventors prove that the peroxidase-like activity of AuNPs can be inactivated by inter-particle interaction, so that in the CHA coding CMs system, the entire catalytic activity can be adjusted by changing the concentration of external DNA stimuli. The present application provides a new method for precisely regulating the catalytic function of AuNPs in a time-controllable manner.

[0006] In a first aspect, the present application provides a DNA circuit coding assembly CMs system, and the construction of DNA circuit coding CMs is achieved by integrating a dynamic DNA circuit, CHA, into AuNPs with ssDNA patterns containing alternating polyA and functional domains. The present application quickly and simply constructs a stimulus-responsive module based on a freeze-labeled strategy of adsorbing ssDNA containing polyA domains on the surface of AuNPs. By changing the number and length of polyA structural domains and functional domains, the construction module is automatically assembled and forms dimeric, linear or reticular CMs.

[0007] The present application can regulate the peroxidase-like activity of AuNPs by blocking the surface catalytic sites. In the CHA coding CM system, the entire catalytic activity can be adjusted by changing the concentration of external DNA stimuli. With the increase of the concentration of DNA stimuli from 0 to 1 μM, the absorption intensity at 652 nm gradually decreases, and the catalytic activity of the system gradually weakens with the increase of the number of dimeric structures.

[0008] The present application labels ssDNA containing polyA on AuNPs by a freeze-labeled strategy, the functional domains on the ssDNA start the CHA circuit to achieve the construction of the DNA circuit coding CMs system, and the peroxidase-like catalytic activity of AuNPs is regulated by blocking the surface catalytic sites or changing the external DNA stimuli.

[0009] Preparation of dimeric CMs system

[0010] In the dimer CMs system, under external DNA stimulation, A and B effectors form stable hybrid double strands through a CHA reaction, thereby synthesizing a dimer structure. The design strategy of this invention is applicable to AuNPs of different sizes, such as 13 nm and 5 nm.

[0011] The preparation steps of the above-mentioned dimer CMs system are as follows: the dual-domain encoder is added to effector A or effector B and annealed to form encoder-A or encoder-B dual complex; then the AuNPs solution is mixed with the encoder-A or encoder-B dual complex, frozen at -30 to -15℃ for 10 to 60 min, thawed and centrifuged at 3 to 6℃ at 10,000 to 15,000 rpm / min for 20 to 40 min with buffer A, and the precipitate is washed; the precipitate is resuspended in buffer A, TNak buffer for dissolving external DNA stimuli is added, and incubated at 35 to 40℃ for 2 to 4 h to form the dimer CMs system.

[0012] Dimeric CMs system dual-domain encoder sequence (SEQ ID NO:1):

[0013] (5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAA AAA AAA AAA AAA AAA AAAAAA-3');

[0014] The A sequence of the effector in the dimer CMs system (SEQ ID NO:2):

[0015] (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATGTGT AGA TAT TGC ACA TTA CT-3');

[0016] The effector B sequence of the dimer CMs system (SEQ ID NO:3):

[0017] (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT ATC TAC ACA TGG TAT TGC ACATTA GAC CAT GTG TAGA-3');

[0018] DNA stimulator sequence (SEQ ID NO:4): (5'-TATTGCACATTACTAAGTTGCA).

[0019] Further, the construction method of the above-mentioned dimer CMs system is as follows: the double-domain encoder is added into effector A or effector B for annealing to form an encoder-A or an encoder-B double complex; based on the freeze labeling method, the prepared 13 nm AuNPs or 5 nm AuNPs solution is mixed with the encoder-A or the encoder-B, and then frozen at-20℃; after thawing, the particles are collected and resuspended in buffer A at pH 7.4 after centrifugation at 4℃ and at a speed of 12,000 rpm / min for 30 min, and the obtained stimulus-responsive construction encoder-A coated building block and the encoder-B coated building block are stored in a dark environment at 4℃; the external DNA stimulus is added into a mixture containing the encoder-A coated building block, the encoder-B coated building block and TNak buffer, and then the mixture is incubated at 37℃ for 3 h to form a dimer structure; the buffer A is a buffer containing 0.1M NaCl and 0.01M phosphate.

[0020] The preparation method of the 13 nm AuNPs solution is as follows: all glassware is immersed in chromic acid overnight, and then washed with ddH2O; then sodium citrate is added into a boiling HAuCl4 solution, and the heating is stopped after the color changes from light yellow to wine red, and the solution is cooled to room temperature under stirring. The obtained AuNPs solution is stored in a dark environment at 4℃.

[0021] The preparation of the 5 nm AuNPs solution is as follows: ddH2O and trisodium citrate are sequentially added into a round-bottom flask under magnetic stirring, and then HAuCl4 solution is added, and the stirring is continued in an ice bath for 3-6 min, and then NaBH4 is added, and the stirring is continued until the color changes to orange red. The prepared AuNPs solution is stored at 4℃ in a dark environment for standby use.

[0022] Preparation of linear CMs system

[0023] The linear CMs system, the linear structure length of the linear CMs system is different, and contains different number (n) of AuNPs (n = 3, 4, 5, 6, 7 and 11), which may be that after the last CHA reaction, the released DNA stimulus needs to find the next construction module, resulting in different interactions of these stimulus-responsive construction modules, which indicates that the CHA reaction can push the stimulus-responsive construction modules into an ordered linear structure with different number of AuNPs.

[0024] The preparation steps of the linear CMs system are as follows: adding a three-domain encoder to a mixed solution containing effector A and effector A1, annealing to form an A-encoder-A1 triple-stranded complex; preparing a B-encoder-B1 complex in the same way; then mixing the A-encoder-A1 and B-encoder-B1 complexes, and then mixing with an AuNPs solution, freezing and coordinating at -30 to -15°C for 10 to 60 min, centrifuging at 3 to 6°C at 10,000 to 15,000 rpm / min for 20 to 40 min after thawing, and washing the precipitate; resuspending the precipitate in buffer A, adding TNak buffer for dissolving external DNA stimulants, incubating at 35 to 40°C for 2 to 4 h, and forming a linear CMs system.

[0025] Effector A (SEQ ID NO: 5) of the linear CMs system: (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CTA TCT ACC CAT ACA TAA CTC AT-3');

[0026] Effector A1 (SEQ ID NO: 6) of the linear CMs system: (5'-TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CTA TCT ACC CAT ACA TAA CTC AT-3');

[0027] Three-domain encoder (SEQ ID NO: 7) of the linear CMs system: (5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAA AAA AAA AAA AAA AAA AAA ATG AGT TAT G TAT GG GTAGAT-3');

[0028] Effector B (SEQ ID NO: 8) of the linear CMs system: (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT ATC TAC ACA TGG TAT TGC ACA TTA GAC CAT GTG TAG A-3');

[0029] Encoder-B1 (SEQ ID NO: 9) of the linear CMs system: (5'-TGC AAT ATC TAC ACA TGG TAT TGC ACA TTA GAC CAT GTG TAG AAT CTA CCC ATA CAT AAC TCAT-3');

[0030] DNA stimulator sequence (SEQ ID NO: 4): (5'-TATTGCACATTACTAAGTTGCA).

[0031] Further, the construction method of the linear CMs structure system is as follows: a three-domain encoder is added to the effector A and the effector A1, and annealing is performed to form an A-encoder-A1 triple-stranded complex in 1xTA buffer; an B-encoder-B1 complex is prepared in the same way; based on the freeze-label method, AuNPs solution is mixed with the A-encoder-A1 triple-stranded complex or the B-encoder-B1 complex, and then frozen at -20°C; after thawing, centrifugation is performed at 4°C at a speed of 12000 rpm / min for 30 min, and the collected particles are resuspended in buffer A at pH 7.4, to obtain the stimulus-responsive construction A-encoder-A1 coated construction module and the B-encoder-B1 coated construction module, which are stored in a dark environment at 4°C; an external DNA stimulator is added to a mixture containing the A-encoder-A1 coated construction module, the B-encoder-B1 coated construction module and TNak buffer, and then the mixture is incubated at 37°C for 3 h to form a linear structure; the buffer A is a buffer containing 0.1M NaCl and 0.01M phosphate.

[0032] Preparation of the reticular CMs system

[0033] The reticular CMs system is designed with a double-domain encoder, which contains a polyA domain (poly15A) for anchoring AuNPs and a functional domain for hybridization with effectors. Under the external DNA stimulus, A and B effectors on different stimulus-responsive construction modules are hybridized with each other through the CHA reaction, causing multiple AuNPs to approach and form a grid-like structure.

[0034] The preparation steps of the reticular CMs system are as follows: a double-domain encoder is added to the effector A or the effector B, and annealing is performed to form an encoder(mini)-A or an encoder(mini)-B; then the encoder(mini)-A and the encoder(mini)-B are mixed, and then mixed with AuNPs solution, and then frozen at -30 to -15°C for 10 to 60 min; after thawing, centrifugation is performed at 3 to 6°C at a speed of 10000 to 15000 rpm / min for 20 to 40 min, and the precipitate is washed; the collected precipitate particles are resuspended in buffer A at pH 7.4, to obtain the stimulus-responsive construction Encoder (mini) A coating construction module and Encoder (mini)B-coating building block, stored in dark environment at 4°C; then, TNak buffer solution dissolving external DNA stimuli was added, incubated at 35-40°C for 2-4h, forming reticular CMs system.

[0035] Reticular CMs system dual-domain encoder (SEQ ID NO: 10):

[0036] (5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAA AAA AAA-3')

[0037] Reticular CMs system effector A (SEQ ID NO: 11):

[0038] (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATGTGT AGA TAT TGCACATTACT-3')

[0039] Reticular CMs system effector B (SEQ ID NO: 12):

[0040] (5'-ATAAGCTACGGTACGCTACGGTGCAATATCTACACATGGTATTGCACATTAGACCATGTGTAGA-3')

[0041] DNA stimuli sequence (SEQ ID NO: 4): (5'-TATTGCACATTACTAAGTTGCA).

[0042] Further, the construction of the above-mentioned reticular CMs structure system: the dual-domain encoder containing 15 consecutive adenines was added to effector A or effector B, annealed in 1xTA buffer to form Encoder (mini) -A or Encoder (mini) -B double complex; based on the freeze-labeling method, the AuNPs solution was mixed with Encoder (mini) -A or Encoder (mini) -B double complex, then frozen at-20°C; after thawing, centrifuged at 4°C with buffer A at a speed of 12000rpm / min for 30min, washed three times, and the collected particles were resuspended in buffer A at pH 7.4, obtaining the stimuli-responsive constructed Encoder (mini) A-coating building block and Encoder (mini) B-coating building block, stored in dark environment at 4°C.

[0043] In particular, the DNA circuit-encoded CMs system was prepared according to the following steps:

[0044] (1) Preparation of 13 nm AuNPs: all glassware was soaked in chromic acid overnight, then rinsed with deionized water; then, 10.1 mL of sodium citrate (34 mM) was quickly added to a boiling solution of HAuCl4 (88.2 mL, 1 mM), and when the color changed from light yellow to wine red, the heating solution was stopped, and the solution was continuously stirred to cool to room temperature. The prepared AuNPs solution was stored in a dark environment at 4°C;

[0045] (2) Preparation of 5 nm AuNPs: first, 18.5 mL of deionized water and 0.5 mL of trisodium citrate (10 mM) were sequentially added to a clean round-bottom flask, and stirred uniformly on a magnetic stirrer, then 0.5 mL of HAuCl4 solution (10 mM) was added, stirred in an ice bath for 5 minutes, then 0.5 mL of freshly prepared frozen NaBH4 (10 mM) was slowly added, and the stirring was continued until the color changed to orange red. The prepared AuNPs solution was stored in a dark environment at 4°C;

[0046] (3) Construction of the dimer CMs system: the dual-domain encoder (50 μM) was added to 50 μL of effector A (50 μM) or effector B (50 μM) to anneal and form an encoder-A or encoder-B double complex. Based on the freeze-labeling method, 1 μL of 10 μM of the encoder-A or encoder-B was mixed with the prepared 13 nm or 5 nm AuNPs solution, then frozen at -20°C, and after thawing, centrifuged at 12,000 rpm / min for 30 min at 4°C with buffer A (0.1 M NaCl, 0.01 M phosphate buffer, pH 7.4), washed three times, and the collected particles were resuspended in 100 μL of buffer A. The obtained stimulus-responsive building blocks were stored in a dark environment at 4°C. In order to realize the CHA-induced dimer structure, 1 μL of external DNA stimulus was added to a mixture containing 100 μL of encoder-A coated building blocks, 100 μL of encoder-B coated building blocks, and 100 uL of TNak buffer, and then the mixture was incubated at 37°C for 3 h to form a dimer structure;

[0047] (4) Linear CMs structure system construction: 20 μL of three-domain encoder (60 μM) is added into effect body A (30 μM) and effect body A1 (30 μM), and annealing is carried out in 1*TA buffer to form A-encoder-A1 triple-stranded complex, in addition, B-encoder-B1 complex is prepared in the same way, and the construction module is prepared by using the above-mentioned freeze labeling method, 4 μL of external DNA stimulator (10 μM) is added into a mixture containing 100 μL of A-encoder-A1 coated construction module, 100 μL of B-encoder-B1 coated construction module and 100 uL of TNak buffer, so as to realize CHA-induced linear structure, and the mixture is incubated at 37°C for 3h to form a linear structure;

[0048] (5) Network CMs structure system construction: 50 μL of double-domain encoder containing 15 consecutive adenines (50 μM) is added into 50 μL of effect body A (50 μM) or effect body B (50 μM), and annealing is carried out in 1*TA buffer to form Encoder (mini) -A or Encoder (mini) -B double complex, and the Encoder (mini) A coated construction module and Encoder (mini) B coated construction module are prepared by using the above-mentioned freeze labeling method. 1 μL of external DNA stimulator (10 μM) is added into a mixture containing 100 μL of Encoder (mini) a coated construction module, 100 μL of Encoder (mini) A coated construction module and 100 μL of TNak buffer, so as to operate polyA-induced network structure, and the mixture is incubated at 37°C for 3h to form a network structure.

[0049] The catalytic activity of the CMs system is from 0 to 555 nM, and the absorption intensity gradually decreases with the increase of the concentration of the encoder and the effector, which corresponds to the change of the color of the oxidized TMB from dark to light, indicating that the peroxidase-like activity of the AuNPs gradually decreases.

[0050] The beneficial effects of the present application are:

[0051] (1) The present application develops a CMs based on DNA circuit coding assembly, and different CMs systems such as dimer CMs system, linear CMs system and network CMs system can be constructed by adjusting the number of PloyA in the CMs structure system.

[0052] (2) The present application develops a CMs based on DNA circuit coding assembly, and the peroxidase-like catalytic activity of the CHA coding CMs system can be accurately adjusted by blocking the surface catalytic site or simply changing the concentration of the external DNA stimulator.

[0053] (3)At the same time, the application develops a CM based on DNA circuit coding assembly, which is simple in preparation process, and the construction strategy is suitable for AuNPs of different sizes.

[0054] (4)In summary, the DNA circuit coding assembly CM system is successfully constructed, the CM system with high precision and time-dependent CHA control according to the application can adjust the AuNPs peroxidase-like catalytic activity by blocking the surface catalytic site or changing the external DNA stimulus, which provides a new idea for regulating the catalytic function of nanomaterials. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is a DNA circuit coding assembly CM system construction principle diagram.

[0056] Figure 2 is a representative TEM image of the dimer CM structure, scale, 50nm; wherein 2A is a dimer CM system with 13nm AuNPs; 2B is a dimer structure CM system with 5nm AuNPs.

[0057] Figure 3 is a statistical distribution of the center-to-center distance of the dimer CM system particles assembled by AuNPs with different particle sizes (TEM image measurement); N≈50; wherein 3A is the statistical distribution of the center-to-center distance of the dimer structure particles assembled by 13nm AuNPs; 3B is the statistical distribution of the center-to-center distance of the dimer structure particles assembled by 5nm AuNPs.

[0058] Figure 4 is a CHA coded linear structure CM; A-E are schematic diagrams and TEM images of linear structures of AuNPs with different numbers (n) (n=3, 4, 5, 6, 7 and 11); from left to right); scale, 20nm. The size of AuNPs is 13nm.

[0059] Figure 5 is a CHA coded mesh structure CM, wherein, Figure 5 A is a representative TEM image of the mesh structure; scale, 100nm; Figure 5 B is a DLS measurement of the mesh structure. The size of AuNPs is 13nm.

[0060] Figure 6 is the time required to complete the freeze-based labeling process at -20℃.

[0061] Figure 7Figure 7A is the peroxidase-like activity of AuNPs blocked by two-domain encoders containing polyA domain (15A) and different concentrations of A effector, where 7A is the AuNPs-mediated chemical reaction related to TMB oxidation; 7B-F are the concentrations of encoders and effector used to anchor AuNPs, respectively, 0 nM, 4.4 nM, 22 nM, 111 nM, 555 nM.

[0062] Figure 8 Figure 7A is the peroxidase-like activity of AuNPs blocked by two-domain encoders containing polyA domain (15A) and different concentrations of A effector, where 7A is the AuNPs-mediated chemical reaction related to TMB oxidation; 7B-F are the concentrations of encoders and effector used to anchor AuNPs, respectively, 0 nM, 4.4 nM, 22 nM, 111 nM, 555 nM.

[0063] Figure 9 Figure 8A is the UV-Vis absorption spectra and color change of TMB in the CHA-encoded dimeric structure system; curve a is the dimeric structure; curve b is the building block + non-specific DNA stimulus; curve c is the building block.

[0064] Figure 10 Figure 8A is the UV-Vis absorption spectra and color change of TMB in the CHA-encoded dimeric structure system; curve a is the dimeric structure; curve b is the building block + non-specific DNA stimulus; curve c is the building block.

[0065] Figure 11 Figure 8A is the UV-Vis absorption spectra and color change of TMB in the CHA-encoded dimeric structure system; curve a is the dimeric structure; curve b is the building block + non-specific DNA stimulus; curve c is the building block.

[0066] Figure 12 Figure 9A is a typical TMB catalytic curve in response to external DNA stimulus concentrations of 0-1 μΜ in the CHA program GMs with dimeric structure; curve a is the external DNA stimulus concentration of 0 nM; curve b is the external DNA stimulus concentration of 10 nM; curve c is the external DNA stimulus concentration of 100 nM; curve d is the external DNA stimulus concentration of 1 μΜ. DETAILED DESCRIPTION

[0067] The present application is herein described, by way of example only, with reference to embodiments thereof. It is construed that persons skilled in the art might easily appreciate other advantages and functionalities of the present application from this disclosure. The present application can be implemented or applied in other different embodiments, and each detail in the present disclosure can be modified or changed based on different viewpoints and applications without departing from the spirit of the present application.

[0068] This work was supported by the National Natural Science Foundation of China (82202649), the Chongqing Science and Technology Joint Project (2023MSXM035), the Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0190), and the Second Affiliated Hospital of Chongqing Medical University (kryc-yq-2117).

[0069] It is understood that the process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art; all pressure values and ranges refer to absolute pressure.

[0070] In addition, it should be understood that the one or more method steps mentioned in the present application do not exclude the presence of other method steps before and after the combination steps or the insertion of other method steps between the explicitly mentioned steps, unless otherwise stated; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present application does not exclude the presence of other devices / apparatuses before and after the combination devices / apparatuses or the insertion of other devices / apparatuses between the two explicitly mentioned devices / apparatuses, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool to identify each method step, and is not intended to limit the arrangement order of each method step or to limit the scope of the present application, and changes or adjustments of the relative relationship are also considered as the scope of the present application can be implemented without substantial changes in technical content.

[0071] Example 1 Preparation of DNA circuit encoded assembled colloidal molecules

[0072] 1. Materials and methods

[0073] 1.1 Materials

[0074] HPLC purified DNA was synthesized by Shanghai Shengong. HAuCl4·4H2O was purchased from Sinopharm Chemical Holding Co., Ltd. (China, Shanghai). 30% H2O2 was purchased from Chuantung Chemical Co., Ltd. (China, Chongqing). 3,3,5,5-tetramethylbenzidine was purchased from Beyotime (TMB, China, Shanghai). Trisodium citrate and sodium borohydride (NaBH4) were purchased from Shengong Biotechnology (China, Shanghai).

[0075] The phosphate buffer solution referred to in the present application refers to the PBS buffer solution, which is the most widely used buffer solution in biochemical research and is well known in the art. The TA buffer solution contains 40 mM Tris-Ac, 10 mM MgAc2, and pH 8.0. The TNaK buffer solution contains: 20 mM Tris, 5 mM KCl, 140 mM NaCl, and pH 7.5. Both the TA buffer solution and the TNaK buffer solution are commonly used buffer solutions known in the art.

[0076] 1.2 Characterization instruments

[0077] UV-Vis spectrophotometer (Japan, Shimadzu). JEM-1200EX Transmission Electron Microscope (Japan, JEOL Ltd). Nano ZS90 Dynamic Light Scattering (UK, Worcestershire).

[0078] 1.3 Principles of CMs system construction

[0079] A single valence AuNP is encoded by a dual-domain encoder containing a polyA domain and a functional domain, a bivalency AuNP is encoded by a triple-domain encoder containing two functional domains separated by a polyA domain, and a multivalency AuNP is encoded by multiple dual-domain encoders containing a short polyA domain (15A) and a functional domain.

[0080] 2. Preparation of AuNPs

[0081] 2.1 Preparation of 13 nm AuNPs

[0082] All glassware was chromated with ddH2O overnight. Then, sodium citrate (34 mM, 10.1 mL) was quickly added into the boiling solution of HAuCl4(34 mM, 88.2 mL). The heating was stopped after the color changed from light yellow to wine red, and the solution was cooled to room temperature with stirring. The obtained AuNPs solution was stored at 4 °C in the dark.

[0083] 2.2 Preparation of 5 nm AuNPs

[0084] ddH2O (18.5 mL) and trisodium citrate (10 mM, 0.5 mL) were added into a round bottom flask in sequence with magnetic stirring. Then, HAuCl4solution (10 mM, 0.5 mL) was added, and the solution was stirred in an ice bath for 5 min before the addition of freshly frozen NaBH4(10 mM, 0.5 mL) slowly. The stirring was continued until the color changed to orange red. The prepared AuNPs solution was stored at 4 °C in the dark.

[0085] 3. Preparation of dimeric CMs system

[0086] 3.1 Preparation of dual complex of encoder-A or encoder-B

[0087] The dual-domain encoder (SEQ ID NO: 1: 5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAAAAA AAA AAA AAA AAA AAA AAA-3'), Effector A (SEQ ID NO: 2: 5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CT-3') or Effector B (SEQ ID NO: 3: 5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT ATC TAC ACA TGG TAT TGC ACA TTA GAC CAT GTG TAGA-3') were denatured at 95 °C, slowly recovered to room temperature, and stored at 4 °C for standby.

[0088] 3.2 Preparation of dimeric CMs system

[0089] Based on the freeze-labeling method, the AuNPs (13 or 5 nm) solution was mixed with the encoder-A or encoder-B (10 mM, 1 pL) and frozen at -20 °C. After thawing, the mixture was centrifuged (12,000 rpm / min, 30 min) at 4 °C with buffer A (0.1 M NaCl, 0.01 M PBS, pH 7.4) and washed three times. The precipitate was resuspended in 100 pL of buffer A, and the resulting stimuli-responsive building blocks were stored in a dark environment at 4 °C. To achieve CHA-induced dimeric structures, an external DNA stimulus (SEQ ID NO: 4: 5'-TATTGCACATTACTAAGTTGCA, 10 pM, 1 pL) was added to a mixture containing encoder-A (100 pL), encoder-B (100 pL) building blocks, and 100 pL of TNak buffer, and the mixture was incubated at 37 °C for 3 h to form the dimeric CMs system.

[0090] 4. Preparation of linear CMs system

[0091] 4.1 Preparation of A-encoder-A1, B-encoder-B1 complex

[0092] In 40 μΐ, of a mixture containing Effector A (SEQ ID NO: 5: 5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CT-3', 30 μΜ), Effector A1 (SEQ ID NO: 6: 5'-TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CT ATC TC C AT C AT A CTA CTC AT-3', 30 μΜ) was added with three-domain encoder (SEQ ID NO: 7: 5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA ATG AGT TAT GTA TGG GTA GAT-3', 60 μΜ, 20 μΐ,), denatured at 95 °C, slowly recovered to room temperature, and stored at 4 °C for standby. In addition, B (SEQ ID NO: 8: 5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CT-3')-encoder-B1 (SEQ ID NO: 9: 5'-TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CT ATC TC C AT C AT A CTA CTC AT-3') complex was prepared in the same way.

[0093] 4.2 Preparation of linear CMs system

[0094] Based on the freeze-labeling method, external DNA stimuli (10 μΜ, 4 μΐ,) were added in a mixture containing 100 μΐ, of A-encoder-A1, 100 μΐ, of B-encoder-B1 coated building blocks and 100 uL of TNak buffer to achieve CHA-induced linear structure. The mixture was incubated at 37 °C for 3 h to form linear structure.

[0095] 5. Preparation of reticular CMs system

[0096] 5.1 Encoder (mini) -A, encoder (mini) Preparation of B complex

[0097] A dual-domain encoder (SEQ ID NO:10: 5'-CCG TAG CGT ACC GTA GCT TAT AAAAAA AAAAAA AAA-3', 50μM, 50μL) is added to a 50μL effector A (SEQ ID NO:11: 5'-ATA AGC TAC GGT ACG CTACGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGCACAT TACT-3', 50μM) or effector B (SEQ ID NO:12: 5'-ATAAGCTACGGTACGCTACGGTGCAA TATCTACACATGGTATTGCACATTAGACCATGTGTAGA-3', 50μM), and then annealed to form encoder (mini)-A or encoder (mini)-B.

[0098] 5.2 Preparation of the network CMs system

[0099] Encoder fabrication based on cryo-labeling method (mini) -A package is built into modules and encoders. (mini) The -B package is used to build modules. This includes modules containing a 100μL encoder. (mini) Package A is a building module, a 100μL encoder. (mini) External DNA stimulant (10 μM, 1 μL) was added to a mixture of the B-coated building block and 100 μL TNak buffer to achieve a polyA-induced network structure. The mixture was incubated at 37°C for 3 h to form the network structure.

[0100] Example 2: Validation of DNA circuit-encoded assembly of CMs

[0101] 1. Validation of the dimer CMS system

[0102] The dimer CMs system obtained in Example 1 was verified using transmission electron microscopy (TEM), as follows: Figure 2 The results show that stimulus-response building blocks of different sizes can form dimer structures; such as Figure 3 The measured center-to-center distances of the two 13nm AuNPs and the two 5nm AuNPs were 13.3nm and 5.4nm, respectively.

[0103] like Figure 2 Representative TEM image of dimer CMs structure, scale bar, 50 nm.

[0104] Figure 2 A is a dimerized CMs system formed by 13nm AuNPs;

[0105] Figure 2B is the CMs system with dimeric structure formed by 5 nm AuNPs.

[0106] As shown in Figure 3 , the statistical distribution of the center-to-center distance of the dimeric CMs system particles assembled by AuNPs with different particle sizes (measured by TEM images). N ~ 50. Among them

[0107] Figure 3 A is the statistical distribution of the center-to-center distance of the dimeric structure assembled by 13 nm AuNPs;

[0108] Figure 3 B is the statistical distribution of the center-to-center distance of the dimeric structure assembled by 5 nm AuNPs;

[0109] 2. Verification of linear CMs system

[0110] The linear CMs system obtained in Example 1 was verified by transmission electron microscopy (TEM), as shown in Figure 4 , the formed CMs have a linear structure, and it is captured that the CMs assembled into a linear structure contain different numbers (n) of AuNPs (n = 3, 4, 5, 6, 7 and 11; from left to right).

[0111] As shown in Figure 4 : linear structure CMs encoded by CHA.

[0112] Figure 4 A-E are schematic diagrams and TEM images of linear structures with different numbers (n) of AuNPs (n = 3, 4, 5, 6, 7 and 11); from left to right). Scale bar, 20 nm. The size of AuNPs is 13 nm.

[0113] 3. Verification of network CMs system

[0114] The linear CMs system obtained in Example 1 was verified by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurement, as shown in Figure 5 , the assembled network structure can be seen in different fields of view by TEM imaging, and the DLS results show that the diameter of the network structure is about 157 nm, which is consistent with the calculated size of the typical structure imaged by TEM.

[0115] Figure 5 A is a representative TEM image of the network structure. Scale bar, 100 nm.

[0116] Figure 5 B is the DLS measurement of the network structure. The size of AuNPs is 13 nm.

[0117] Example 3 DNA circuit encoded assembly of CMs and optimization of its freezing conditions

[0118] The inventors further optimized the freezing reaction time, which is an important condition for the freezing labeling method, by selecting at least five points from low to high for experiments.

[0119] To investigate the effect of reaction time in the amplification system on the freezing labeling method, the labeling reaction containing different freezing labeling times (10, 20, 30, 45, 50, and 60 min) was used, and then the color change was observed. Figure 6 It is shown that the optimal reaction time for freezing labeling is 45 min.

[0120] As shown in Figure 6 : Evaluation of the time required to complete the freezing-based labeling process at -20°C.

[0121] Example 4 Analysis of catalytic activity of DNA circuit encoded CMs

[0122] To evaluate the catalytic activity of DNA circuit encoded CMs, the peroxidase-like activity of the prepared samples was analyzed by catalyzing the oxidation of TMB in the presence of H2O2. Specifically, 30 μL of the prepared sample to be tested, TMB (1 mM, 100 μL), and H2O2 (10 M, 2.5 μL) were mixed at 37°C. After 10 min, the absorbance of the mixture was measured at a wavelength range of 400-750 nm using a UV-visible absorption spectrophotometer. To determine the absorbance of oxTMB in real time, 30 μL of the prepared sample was added to 102.5 μL of the mixture (containing 0.98 mM TMB, 0.24 M H2O2), and the absorbance at 652 nm was recorded to monitor the absorbance of the mixture. All experiments were repeated three times.

[0123] 1. Modulating AuNPs peroxidase-like activity by blocking surface catalytic sites.

[0124] As shown in Figure 7 A, oxTMB is formed after TMB oxidation, which is blue and has increased optical absorption at around 652 nm. As shown in Figure 7 B-F, the absorbance intensity gradually decreases from 0 to 555 nM as the concentrations of the encoder and effector increase, which corresponds to the change in the color of oxTMB from dark to light, indicating that the designed encoder blocks the surface catalytic sites of AuNPs through polyA-Au coordination, resulting in a gradual decrease in the peroxidase-like activity of AuNPs. When the encoder is replaced with ssDNA containing no polyA domain, the color and the corresponding absorbance value change little (as shown in Figure 8 ). The above results show that the ssDNA containing polyA used in the present application can accurately regulate the peroxidase-like catalytic activity of AuNPs.

[0125] As shown inFigure 7 : Modulating peroxidase-like activity of AuNPs by blocking surface catalytic sites with dual-domain encoders containing polyA domain (15A) and different concentrations of A effectors.

[0126] Figure 7 A is the AuNPs-mediated TMB oxidation related chemical reaction.

[0127] Figure 7 B-F are the concentrations of encoders and effectors used to anchor AuNPs, respectively 0 nM, 4.4 nM, 22 nM, 111 nM, 555 nM.

[0128] As shown in Figure 8 : ssDNA without polyA domain and A effectors at different concentrations to treat AuNPs.

[0129] Curves a to d (from bottom to top), ssDNA without polyA domain and A effectors at concentrations of 4.4 nM, 22 nM, 111 nM, 555 nM, respectively.

[0130] 2. Assembling modulating peroxidase-like activity of AuNPs by CHA coding

[0131] The present application explores whether inter-particle interaction can affect the catalytic function of AuNPs. As shown in Figure 9 , in the CHA coding dimeric CMs system, the colorimetric reaction of the stimulus-responsive building block with TMB produced a significant blue color (curve c and inset c). When the non-specific DNA stimulus was mixed with the stimulus-responsive building block, the mixture also produced a significant blue color (curve b and inset b), with an absorption intensity at 652 nm similar to that of the stimulus-responsive building block (curve b and c). This indicates that the DNA chain without a polyA domain has little effect on the catalytic activity of the structural module. However, the CHA reaction assembly with a dimeric structure triggered by external DNA stimulus produced little color change (see curve a and inset a), indicating that the dimeric CMs structure inhibits the peroxidase-like catalytic activity of AuNPs, which is due to the fact that part of the catalytic surface sites are blocked by the encoder. In addition, this phenomenon was also found in the CHA coding linear and network structure systems (as shown in Figure 10 and 10 ). These results show that inter-particle interaction can inhibit the peroxidase-like catalytic activity of AuNPs, providing a new way to change the catalytic function of nanomaterials.

[0132] As shown in Figure 9 : UV-Vis absorption spectra and color change of TMB in the CHA coding dimeric structure system.

[0133] Curve a is the dimeric structure;

[0134] Curve b is building block + non-specific DNA stimulus;

[0135] Curve c is building block.

[0136] As shown in Figure 10 : UV-Vis absorption spectra and color change of TMB in CHA-encoded linear structure system.

[0137] Curve a is linear structure;

[0138] Curve b is building block + non-specific DNA stimulus;

[0139] Curve c is building block.

[0140] As shown in Figure 11 : UV-Vis absorption spectra and color change of TMB in CHA-encoded mesh structure system.

[0141] Curve a is mesh structure;

[0142] Curve b is building block + non-specific DNA stimulus;

[0143] Curve c is building block.

[0144] The present application also explores whether the peroxidase-like catalytic activity of CHA-encoded CMs system can be adjusted by changing the concentration of external DNA stimulus. As shown in Figure 10 : In CHA-encoded dimer structure system, the absorption intensity at 652 nm gradually decreases with the increase of DNA stimulus concentration from 0 to 1 μM, indicating that the catalytic activity of the system gradually weakens with the increase of dimer structure number. Therefore, by simply changing the concentration of external DNA stimulus, the peroxidase-like catalytic activity of CHA-encoded CMs system can be accurately adjusted.

[0145] As shown in Figure 12 : In CHA-programmed CMs with dimer structure, the typical TMB catalytic curve responds to the external DNA stimulus concentration of 0-1 μM.

[0146] Curve a is external DNA stimulus concentration 0 nM;

[0147] Curve b is external DNA stimulus concentration 10 nM;

[0148] Curve c is external DNA stimulus concentration 100 nM;

[0149] Curve d is external DNA stimulus concentration 1 μM.

Claims

1. A construction method for DNA circuit encoded assembly of CMs, characterized by: The construction of DNA circuit encoded CMs is realized by integrating dynamic DNA circuit-CHA into AuNPs containing ssDNA patterns with alternating polyA and functional domains; based on the freeze-labeling strategy, ssDNA containing polyA domains are adsorbed on the surface of AuNPs to construct a stimulus-responsive module, by changing the number and length of polyA domain and functional domain, the module is automatically assembled and forms dimeric CMs, linear CMs or reticular CMs; the preparation steps of the dimeric CMs system are as follows: the dual-domain encoder is added into effector A or effector B to anneal, forming encoder-A or encoder-B double complex; then the AuNPs solution is mixed with the encoder-A or encoder-B double complex, and the coordination is frozen at-30 to-15 DEG C for 10 to 60 min; after thawing, centrifugation is carried out at 3 to 6 DEG C with a buffer A at 10000 to 15000 rpm / min for 20 to 40 min, and the precipitate is washed; the precipitate is resuspended in buffer A, TNak buffer solution for dissolving external DNA stimulator is added, and incubation is carried out at 35 to 40 DEG C for 2 to 4 h to form a dimeric CMs system; The sequence of the dual-domain encoder of the dimeric CMs system (SEQ ID NO: 1) is: (5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA-3'); The sequence of effector A of the dimeric CMs system (SEQ ID NO: 2) is: (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CT-3'); The sequence of effector B of the dimeric CMs system (SEQ ID NO: 3) is: (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT ATC TAC ACA TGG TAT TGC ACA TTA GAC CAT GTG TAGA-3'); The sequence of the DNA stimulator (SEQ ID NO: 4) is: (5'-TATTGCACATTACTAAGTTGCA). The preparation steps of the linear CMs system are as follows: adding a three-domain encoder into a mixed solution containing effector A and effector A1, annealing to form an A-encoder-A1 triple-chain complex; preparing a B-encoder-B1 complex in the same way; then mixing the A-encoder-A1 and B-encoder-B1 complexes, and then mixing with an AuNPs solution, freezing and coordinating at-30 to-15°C for 10 to 60 min, centrifuging at 3 to 6°C at 10000 to 15000 rpm / min for 20 to 40 min after thawing, and washing the precipitate; resuspending the precipitate in buffer A, adding TNak buffer solution for dissolving an external DNA stimulator, and incubating at 35 to 40°C for 2 to 4 h to form a linear CMs system; Linear CMs system effector A (SEQ ID NO: 5): (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CTA TCT ACC CAT ACA TAA CTC AT-3'); Linear CMs system effector A1 (SEQ ID NO: 6): (5'-TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACA TTA CTA TCT ACC CAT ACA TAA CTC AT-3'); Linear CMs system three-domain encoder (SEQ ID NO: 7): (5'-CCG TAG CGT ACC GTA GCT TAT AAAAAA AAA AAA AAA AAA AAA AAA AAA AAA ATG AGT TAT G TAT GG GT AGAT-3'); Linear CMs system effector B (SEQ ID NO: 8): (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT ATC TAC ACA TGG TAT TGC ACA TTA GAC CAT GTG TAG A-3'); Linear CMs system encoder-B1 (SEQ ID NO: 9): (5'-TGC AAT ATC TAC ACA TGG TAT TGC ACA TTA GAC CAT GTG TAG AAT CTA CCC ATA CAT AAC TCAT-3'); DNA stimulator sequence (SEQ ID NO: 4): (5'-TATTGCACATTACTAAGTTGCA) The preparation steps of the network CMs system are as follows: adding a dual-domain encoder into effector A or effector B, annealing to form an encoder(mini)-A or an encoder(mini)-B; then mixing the encoder(mini)-A and the encoder(mini)-B, and then mixing with an AuNPs solution, freezing coordination at-30 to-15°C for 10 to 60 min, after thawing, centrifuging at 3 to 6°C at 10000 to 15000 rpm / min for 20 to 40 min, and washing the precipitate; collecting the precipitated particles and resuspending them in buffer A at pH 7.4 to obtain a stimulus-responsive encoder(mini)-A coating construction module and an encoder(mini)-B coating construction module, and storing them in a dark environment at 4°C; then adding a TNak buffer solution containing a dissolved external DNA stimulus, and incubating at 35 to 40°C for 2 to 4 h to form the network CMs system. The network CMs system dual-domain encoder (SEQ ID NO: 10); (5'-CCG TAG CGT ACC GTA GCT TAT AAA AAA AAA AAA AAA-3'); The network CMs system effector A (SEQ ID NO: 11); (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAC TTA GTA ATG TGC AAT ACC ATG TGT AGA TAT TGC ACATTACT-3'); The network CMs system effector B (SEQ ID NO: 12); (5'-ATA AGC TAC GGT ACG CTA CGG TGC AAT ATCTACACATGGTATTGCACATTAGACCATGTGTAGA-3'); The DNA stimulus sequence (SEQ ID NO: 4) is (5'-TATTGCACATTACTAAGTTGCA). CHA is catalytic hairpin assembly; polyA is polyadenine; CMs is a colloidal molecule; AuNPs is gold nanoparticles; the TNak buffer solution contains 20 mM Tris, 5 mM KCl, 140 mM NaCl, and pH 7.5; the TA buffer solution contains 40 mM Tris-Ac and 10 mM MgAc2, and pH 8.0; and the buffer A is a buffer solution containing 0.1 M NaCl and 0.01 M phosphate.

2. The construction method of DNA circuit encoded assembly CMs according to claim 1, wherein: The construction method of the dimer CMs system is that the dual-domain encoder is added into effector A or effector B for annealing to form an encoder-A or an encoder-B double complex; based on the freeze labeling method, the prepared 13 nm AuNPs or 5 nm AuNPs solution is mixed with the encoder-A or the encoder-B, and then frozen at-20°C; after thawing, the particles collected are resuspended in buffer A at pH 7.4, and the obtained stimulus-responsive construction encoder-A coated building block and encoder-B coated building block are stored in a dark environment at 4°C; the external DNA stimulus is added into the mixture containing the encoder-A coated building block, the encoder-B coated building block and the TNak buffer, and then the mixture is incubated at 37°C for 3 h to form a dimer structure.

3. The construction method of claim 2, wherein the DNA circuit encoding assembly CMs are constructed by: The preparation method of the 13 nm AuNPs solution is that all glassware is soaked in chromic acid overnight, and then washed with ddH2O; then sodium citrate is added into a boiling HAuCl4 solution, and the heating is stopped after the color changes from light yellow to wine red, and the solution is cooled to room temperature under stirring; the preparation of the 5 nm AuNPs solution is that ddH2O and trisodium citrate are sequentially added into a round-bottom flask under magnetic stirring, and then HAuCl4 solution is added, and the mixture is stirred in an ice bath for 3-6 min, and then NaBH4 is added, and the stirring is continued until the color changes to orange red.

4. The construction method of DNA circuit encoded assembly CMs as claimed in claim 1, wherein: The construction method of the linear CMs structure system is that the tri-domain encoder is added into effector A and effector A1 for annealing in 1 × TA buffer to form an A-encoder-A1 triple complex; the B-encoder-B1 complex is prepared in the same way; based on the freeze labeling method, the AuNPs solution is mixed with the A-encoder-A1 triple complex or the B-encoder-B1 complex, and then frozen at-20°C; after thawing, the particles collected are resuspended in buffer A at pH 7.4, and the obtained stimulus-responsive construction A-encoder-A1 coated building module and B-encoder-B1 coated building module are stored in a dark environment at 4°C; the external DNA stimulus is added into the mixture containing the A-encoder-A1 coated building module, the B-encoder-B1 coated building module and the TNak buffer, and then the mixture is incubated at 37°C for 3 h to form a linear structure; the buffer A is a buffer containing 0.1 M NaCl and 0.01 M phosphate.

5. The construction method of DNA circuit encoded assembly CMs as claimed in claim 1, wherein: The construction of the net CMs structure system: add the dual-domain encoder containing 15 consecutive adenines into the effector A or the effector B, anneal in 1xTA buffer to form the encoder(mini)-A or the encoder(mini)-B double complex; based on the freeze labeling method, mix the AuNPs solution with the encoder(mini)-A or the encoder(mini)-B double complex, and then freeze at-20°C; after thawing, centrifuge at 4°C at a speed of 12,000 rpm / min for 30 min with buffer A, wash three times, resuspend the collected particles in buffer A at pH 7.4, obtain the stimulus-responsive coating encoder(mini)-A coating construction module and the encoder(mini)-B coating construction module, and store in a dark environment at 4°C.

6. The construction method of DNA circuit encoded assembly CMs according to any one of claims 1 to 5, wherein, Prepare according to the following steps: (1) Preparation of 13 nm AuNPs: all glassware is soaked in chromic acid overnight, then rinsed with deionized water; quickly add sodium citrate to the boiling solution of HAuCl4, stop heating the solution when the color changes from light yellow to wine red, continue to stir to cool to room temperature, and store the prepared AuNPs solution in a dark environment at 4°C; (2) Preparation of 5 nm AuNPs: add 18.5 mL deionized water and 0.5 mL trisodium citrate to a clean round-bottom flask in sequence, stir uniformly on a magnetic stirrer, then add 0.5 mL HAuCl4 solution, stir in an ice bath for 5 minutes, then slowly add 0.5 mL of freshly prepared frozen NaBH4, continue to stir until the color turns orange red, and store the prepared AuNPs solution in a dark environment at 4°C; (3) Automatic assembly of the construction module and formation of dimer CMs, linear CMs or net CMs; The construction method of the dimer CMs system is: the dual-domain encoder is added to 50 μL of effector A or effector B to anneal to form the encoder-A or the encoder-B double complex, based on the freeze labeling method, mix the prepared 13 nm or 5 nm AuNPs solution with 1 μL of 10 μM of the encoder-A or the encoder-B, then freeze at-20°C, after thawing, centrifuge at 4°C at a speed of 12,000 rpm / min for 30 min with buffer A, wash three times, resuspend the collected particles in 100 µL of buffer A, and store the obtained stimulus-responsive construction module in a dark environment at 4°C; add 1 μL of external DNA stimulator to the mixture containing 100 μL of the encoder-A coating construction block, 100 μL of the encoder-B coating construction block and 100 uL of TNak buffer, then incubate the mixture at 37°C for 3 h to form a dimer structure; The construction method of the linear CMs structure system is: 20 μL of three-domain encoder is added into the effector A and the effector A1, annealing in 1 × TA buffer to form A-encoder-A1 triple-stranded complex, and B-encoder-B1 complex is prepared in the same way, and the construction module is prepared by using the above freezing marking method; 4 μL of external DNA stimulator is added into a mixture containing 100 μL of A-encoder-A1 coated construction module, 100 μL of B-encoder-B1 coated construction module and 100 uL of TNak buffer, and the mixture is incubated at 37°C for 3 h to form a linear structure; The construction method of the reticular CMs structure system is: 50 μL of double-domain encoder containing 15 consecutive adenines is added into 50 μL of effector A or effector B, annealing in 1 × TA buffer to form encoder(mini)-A or encoder(mini)-B double complex, and the encoder(mini)-A coating construction module and the encoder(mini)-B coating construction module are prepared by using the above freezing marking method; 1 μL of external DNA stimulator is added into a mixture containing 100 μL of encoder(mini)-A coated construction module, 100 μL of encoder(mini)-B coated construction module and 100 μL of TNak buffer, and the mixture is incubated at 37°C for 3 h to form a reticular structure.

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