DNA microsphere for regulating and controlling peroxidase activity of hemin and application of DNA microsphere

Abstract

The invention provides a DNA (deoxyribonucleic acid) microsphere for regulating and controlling peroxidase activity of chlorhematin and application of the DNA microsphere. Dynamic programming of a physical microenvironment and a biological function of the DNA microsphere is realized by adding a self-crosslinking nucleic acid sequence into the DNA microsphere coded by DNA; therefore, reversible regulation of the activity of the chlorhematin peroxidase is realized through the new modified DNA microspheres, a chlorhematin catalytic system with adjustable activity is developed, and the application of chlorhematin in scientific research and clinical detection is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a DNA microsphere for regulating the peroxidase activity of hemin and a use thereof. Background Art

[0002] Peroxidases are a class of biological enzymes that catalyze the oxidation of substrates using hydrogen peroxide (H2O2) as an electron acceptor. They can directly catalyze the oxidation of phenolic or amine compounds. These natural enzymes, primarily derived from plants or microorganisms, play a vital role in fields such as analytical chemistry and immunoassays. However, the extraction and purification of natural peroxidases is often complex and expensive, and they require stringent reaction conditions and are prone to denaturation and loss of activity. These shortcomings significantly limit their practical applications.

[0003] Hemin is a coenzyme molecule derived from hemoglobin. It is inexpensive and has stable chemical properties. It is often used as a peroxidase mimetic and can catalyze the oxidation reactions of a variety of substrates. For example, Hemin can catalyze the conversion of 3,3',5,5'-tetramethylbenzidine (TMB) from colorless to blue. This reaction is widely used in analytical chemistry and enzyme-linked immunosorbent assay systems. However, when a single Hemin molecule is used as a mimetic enzyme, its peroxidase activity is extremely limited, making it difficult to meet the sensitivity requirements of actual detection. In addition, different application scenarios (such as detection of biomarkers of different abundances) often have different requirements for catalytic activity, which places higher demands on the regulation of mimetic enzyme activity. Therefore, the development of a Hemin catalytic system with controllable activity is of great significance for promoting its expanded application in scientific research and clinical testing.

[0004] Currently, there are relatively few technical approaches to regulating hemin activity. Current research focuses on two approaches: one is to indirectly replace hemin by optimizing the structure and function of native peroxidase proteins; the other is to use in vitro screening techniques to obtain DNA G-tetramer structures that specifically bind to hemin, thereby regulating its activity. However, existing methods still have certain limitations, and there is an urgent need to develop new and more efficient technologies for regulating hemin activity to meet diverse practical needs. Summary of the Invention

[0005] In view of the defects of the prior art, the object of the present invention is to provide a DNA microsphere for regulating the peroxidase activity of hemin and its use. The DNA-encoded DNA microsphere provided by the present invention can be used to effectively regulate the peroxidase activity of hemin, which is of great significance for production, scientific research, clinical and other applications based on the peroxidase activity characteristics of hemin.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides a DNA microsphere for regulating the peroxidase activity of hemin, wherein the DNA microsphere comprises a regulatory chain for downregulating or upregulating the peroxidase activity of hemin, and the regulatory chain is complementary to a portion of the template chain of the DNA microsphere.

[0008] The inventor applied for domestic patent CN116251621A in 2022, disclosing that DNA-encoded DNA microspheres can enhance the peroxidase activity of hemin. In previous studies, this research team found that two DNA microspheres, LAC and GAC, which only differed in partial base sequences, had differences in enhancing Hemin catalytic activity, but the sequence of synthetic GAC contained an additional palindromic sequence, resulting in a higher degree of cross-linking inside GAC than LAC, thereby forming a different physical microenvironment inside the DNA microspheres. Therefore, the present invention considers precisely regulating the degree of cross-linking of DNA chains inside DNA microspheres to achieve regulation of Hemin peroxidase activity, and experiments have shown that introducing a palindromic sequence inside LAC will change the biological function of LAC, but its overall regulatory effect is limited. Therefore, the present invention further considers introducing a self-crosslinking nucleic acid sequence with a more rigid structural domain to enhance the regulatory effect of DNA microspheres on Hemin catalytic activity.

[0009] Furthermore, when the DNA microspheres are used to downregulate the peroxidase activity of hemin, the regulatory chain includes any one or more DNA sequences shown in SEQ ID NO.1 to SEQ ID NO.13.

[0010] In some embodiments, the present invention targets the internal structure of LAC and designs 13 DNA sequences 1, represented by SEQ ID NOs. 1 to 13, using GTAA as the base sequence unit. The effect of extending the sequence by 0-9 bases on peroxidase activity in the LAC-Hemin system was explored. The results showed that the addition of sequences 1 with different base compositions resulted in varying degrees of decrease in enzyme activity, with three sequences significantly reducing peroxidase activity, confirming that the addition of sequence 1 to the LAC microspheres can downregulate Hemin peroxidase activity.

[0011] Furthermore, when the DNA microspheres are used to upregulate the peroxidase activity of hemin, the regulatory chain includes the DNA sequences shown in SEQ ID NO.6 and SEQ ID NO.19.

[0012] In theory, adding a DNA sequence that is partially complementary to sequence 1 can achieve the de-crosslinking of the DNA chains inside the DNA microspheres, thereby potentially restoring the physical properties and biological functions of the DNA microspheres. Therefore, this study further speculated that adding a DNA sequence complementary to sequence 1 could achieve reversible regulation of Hemin peroxidase activity. Using the base mismatch mechanism, complementary sequences 2 corresponding to 13 sequences 1 shown in SEQ ID NO.14 to SEQ ID NO.26 were designed. Through TMB oxidation reaction, it was found that sequence 1 shown in SEQ ID NO.6 and sequence 2 shown in SEQ ID NO.19 could significantly enhance peroxidase activity when added to LAC. Sequence 2 shown in SEQ ID NO.19 could effectively reverse the downregulation of Hemin peroxidase activity by sequence 1 shown in SEQ ID NO.6.

[0013] Furthermore, the DNA microspheres include the template chain sequences shown in SEQ ID NO. 27 and SEQ ID NO. 28.

[0014] In some embodiments, the 5' end of the template strand of the DNA microsphere is modified by phosphorylation.

[0015] On the other hand, the present invention provides a preparation system for the DNA microspheres as described above, comprising a ligase, an exonuclease, a polymerase, a buffer, a dNTP mixture, a cyclization primer and an amplification primer, wherein the cyclization primer has the sequence shown as SEQ ID NO.29 and SEQ ID NO.30, and the amplification primer has the sequence shown as SEQ ID NO.31 and SEQ ID NO.32.

[0016] Preferably, the ligase includes T4 ligase, the exonuclease includes exonuclease I and exonuclease III. The polymerase includes Φ 29 Polymerase, buffer including T4 ligase buffer, Φ 29 At least one of the polymerase buffers.

[0017] In another aspect, the present invention provides a method for regulating the peroxidase activity of hemin, wherein the peroxidase activity of hemin is regulated by using the DNA microspheres as described in any one of claims 1 to 4.

[0018] In some embodiments, the experimental research results of the present invention demonstrate that, after adding two template chain-encoded DNA microspheres (named LAC) to the hemin-catalyzed TMB reaction system, the reaction rate of the entire system is significantly improved. After further adding DNA sequence 1 shown in SEQ ID NO. 6 to the DNA microsphere LAC, a new DNA microsphere (named SAC) is obtained, and the reaction rate of the system decreases significantly. Further adding DNA sequence 2 shown in SEQ ID NO. 19 to SAC further produces a new DNA microsphere (named DAC), which significantly improves the reaction rate of the system. This indicates that different DNA-encoded microspheres (LAC, SAC, DAC) can be used as a good biomaterial for regulating the peroxidase activity of hemin.

[0019] In another aspect, the present invention provides a method for down-regulating the peroxidase activity of hemin, using DNA microspheres containing a regulatory chain represented by any one of SEQ ID NO.1 to SEQ ID NO.13 to reduce the peroxidase activity of hemin.

[0020] In another aspect, the present invention provides a method for upregulating the peroxidase activity of hemin, using DNA microspheres containing the regulatory chains shown in SEQ ID NO. 6 and SEQ ID NO. 19 to increase the peroxidase activity of hemin.

[0021] On the other hand, the present invention provides a use of a regulatory chain for preparing a reagent for downregulating the peroxidase activity of hemin, wherein the regulatory chain is shown in any one of SEQ ID NO.1 to SEQ ID NO.13, and the peroxidase activity of hemin is reduced by DNA microspheres containing the regulatory chain.

[0022] In another aspect, the present invention provides a use of a regulatory chain for preparing a reagent for upregulating the peroxidase activity of hemin, wherein the regulatory chain is shown in SEQ ID NO.6 and SEQ ID NO.19, and the peroxidase activity of hemin is increased by DNA microspheres containing the regulatory chain.

[0023] Furthermore, the DNA microspheres further include template chain sequences shown in SEQ ID NO. 27 and SEQ ID NO. 28.

[0024] The present invention has the following beneficial effects:

[0025] 1. The DNA sequence provided by the present invention can precisely regulate the degree of cross-linking of DNA chains, achieving dynamic programming of the physical microenvironment of DNA microspheres and their biological functions. After being added to DNA microspheres, it can achieve reversible regulation of hemin catalase activity, with significant regulatory effects on enzyme activity.

[0026] 2. The present invention proposes a method for effectively regulating the activity of hemin catalase based on the DNA microspheres provided by the present invention, which is of great significance for production, scientific research, clinical and other applications based on the activity characteristics of hemin peroxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the base sequence composition of LAC and GAC.

[0028] Figure 2a -b shows the effect of introducing LAC into different hybridization sequences on the activity of hemin peroxidase.

[0029] Figure 3 The effects of different sequences 1 on the peroxidase activity of LAC-Hemin, where (a) is 100nM Hemin; (b) is 100nM Hemin added to LAC; (c) is 100nM Hemin added to LAC-S1; (d) is 100nM Hemin added to LAC-S2; (e) is 100nM Hemin added to LAC-S3; (f) is 100nM Hemin added to LAC-S4; (g) is 100nM Hemin added to LAC-S5; (h) is 100nM Hemin added to LAC-S6; (i) is 100nM Hemin added to LAC-S7; (j) is 100nM Hemin added to LAC-S8; (k) is 100nM Hemin added to LAC-S9; (l) is 100nM Hemin was added to LAC-S10; (m) 100 nM Hemin was added to LAC-S11; (n) 100 nM Hemin was added to LAC-S12; (o) 100 nM Hemin was added to LAC-S13; the final concentration of LAC was 13.125 μg / mL.

[0030] Figure 4 The effect of sequence 1 (C6) on the peroxidase activity of LAC-Hemin.

[0031] Figure 5The effects of different combinations of sequence 1 and sequence 2 on the peroxidase activity of LAC-Hemin, wherein (a) is 100nM Hemin; (b) is 100nM Hemin added to LAC; (c) is 100nM Hemin added to LAC-S1-C1; (d) is 100nM Hemin added to LAC-S2-C2; (e) is 100nM Hemin added to LAC-S3-C3; (f) is 100nM Hemin added to LAC-S4-C4; (g) is 100nM Hemin added to LAC-S5-C5; (h) is 100nM Hemin added to LAC-S6-C6; (i) is 100nM Hemin added to LAC-S7-C7; (j) is 100nM Hemin added to LAC-S8-C8; (k) is 100nM Hemin was added to LAC-S9-C9; (l) 100nM Hemin was added to LAC-S10-C10; (m) 100nM Hemin was added to LAC-S11-C11; (n) 100nM Hemin was added to LAC-S12-C12; (o) 100nM Hemin was added to LAC-S13-C13; the final concentration of LAC was 13.125μg / mL.

[0032] Figure 6 Microscopic observation photos of DNA microspheres.

[0033] Figure 7 Schematic diagram of the principle of SAC and DAC regulating Hemin peroxidase activity.

[0034] Figure 8 The results show the catalytic ability of DNA microspheres with different structures.

[0035] Figure 9 The results of the LAC catalytic ability test with different concentrations of sequence 1 and sequence 2 are shown. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0037] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0038] Example 1. Screening of regulatory chains using DNA microspheres

[0039] This research team applied for patent CN116251621A in 2022, disclosing that DNA-encoded DNA microspheres can enhance the peroxidase activity of hemin. In addition, in the previous study of this team (Phase Separation of DNA-Encoded Artificial Cells Boosts Signal Amplification for Biosensing. Angew. Chem, 2023, 135 (39): e202306691), it was found that the two DNA-encoded microspheres LAC (DNA microspheres in patent CN116251621A) and GAC had differences in enhancing the catalytic activity of Hemin. Analysis of the structures of LAC and GAC revealed that the two microspheres only differed in some base sequences. Compared with the sequence that constitutes LAC, the sequence of synthetic GAC contains an additional palindrome sequence (ps) in p(A20-m-ps) and p(T20-x-ps) ( Figure 1 ), forming a self-hybridized double-stranded structure. Therefore, the internal cross-linking degree of the synthesized GAC is higher than that of LAC, thereby forming a different physical microenvironment within the DNA microsphere, achieving regulation of Hemin peroxidase activity. Therefore, the present invention hypothesizes that by precisely regulating the degree of cross-linking of the DNA chains within the DNA microsphere, it is possible to dynamically program the physical microenvironment of the DNA microsphere and its biological function, thereby achieving regulation of the enzyme activity therein.

[0040] Therefore, the present invention first locates the sequence inside the DNA microsphere ( Figure 1 The m) in P(A20-m) shown above anchors a palindromic sequence inside LAC and explores its effect on Hemin peroxidase activity. The results show that ( Figure 2a and Figure 2b ), by adjusting the degree of chain cross-linking inside the DNA microsphere LAC, its biological function can be affected, but the palindromic sequence ( Figure 2b , the downregulation effect of LAC-ps on the biological function of LAC is still limited. Considering that rigid nucleic acid chains tend to form more regular arrangements, which increase interactions within the microspheres, thereby affecting the microphysical structure of the microspheres and, in turn, their biological function, the present invention further considers introducing self-crosslinking nucleic acid sequences with more rigid structural domains to further enhance the regulatory effect of DNA microspheres on Hemin catalytic activity.

[0041] DNA oligonucleotides can form multi-stranded helical structures, such as DNA triple helix and DNA quadruple helix, through folding and connection between molecules or within molecules, among which the structural stability (rigidity) of the quadruple helix is higher, such as the imotif DNA quadruple helix and the DNA-G4 quadruple helix structure. Since the folded conformation of DNA-G4 can form a complex with strong peroxidase activity with Hemin, affecting the observation of this process, this study chose to introduce the imotif structure (nucleic acid sequence is CCCCTAACCCC, SEQ ID NO.33) into LAC to observe its effect on the LAC-Hemin catalytic system. Therefore, the present invention targets the single-stranded DNA inside LAC, uses "AAT" as a repeating unit to extend the sequence at both ends of the imotif structure, and designs multiple imotif sequences (sequence 1). The specific sequences are shown in the table below. The catalytic effects of the 13 sequences were then compared through TMB oxidation reaction, and the results are as shown below. Figure 3 shown.

[0042] Table 1 Base sequence of SEQ ID NO: 1

[0043]

[0044] like Figure 3 Compared with the TMB oxidation reaction catalyzed by Hemin alone (group a), the peroxidase activity was significantly improved after the addition of the DNA-encoded microsphere LAC (group b) to the Hemin-catalyzed TMB oxidation system. After the addition of different imotif sequences to the LAC-Hemin system, the enzyme activity decreased to varying degrees. Among them, the decrease in peroxidase activity after the addition of sequences LAC-S1, LAC-S2, and LAC-S3 was the most significant, followed by the sequence LAC-S4. The above results show that the introduction of imotif sequences as regulatory chains into LAC can achieve the downregulation of Hemin peroxidase activity, and the downregulation effects of imotif sequences with different base sequences vary.

[0045] Based on the above results, this study further speculates whether this process is reversible. In theory, the addition of a DNA sequence that is partially complementary to the imotif sequence can achieve the de-crosslinking of the imotif structure inside the DNA microspheres, and thus it is expected to achieve the restoration of the physical properties and biological functions of the DNA microspheres. However, considering that imotif itself is a sequence rich in C bases, its complementary sequence must be a sequence rich in G bases, and it is easy for a sequence rich in G bases to form a DNA G4 structure. It is well known that the DNA G4 structure can form a complex with Hemin, thereby enhancing the peroxidase activity of Hemin, but this affects the observation of this process. Therefore, the present invention uses the base mismatch mechanism to design a sequence 2 that is complementary to the 13 sequences in Table 1, avoiding the formation of DNA-G4 structure by sequence 2 and interfering with the measurement results. Sequence 2 is shown in Table 2. The effects of the 13 sequences 2 on the peroxidase activity of Hemin were then compared through the TMB oxidation reaction. The results showed that the addition of different sequences 2 to the Hemin system and the LAC-Hemin system did not change the enzyme activity of the corresponding system, among which the results of C6 are shown in Table 2. Figure 4 Furthermore, this example measured the catalytic effect of adding sequence 1 and the corresponding sequence 2 to the LAC-Hemin system, and compared the catalytic effect of sequence 2 combined with sequence 1 on the LAC-Hemin system. The results are shown in Figure 5 shown.

[0046] Table 2 Base sequence of sequence 2

[0047] serial number Serial number 5’-3’ C1 SEQ ID NO.14 ATT ATT GGTG TTA TGG T ATT ATT C2 SEQ ID NO.15 ATT ATT GTGT TTA TGG T ATT ATT C3 SEQ ID NO.16 ATT ATT GGTG TTA GGG T ATT ATT C4 SEQ ID NO.17 GGTG TTA TGG T C5 SEQ ID NO.18 C GGTG TTA TGG TC C6 SEQ ID NO.19 AC GGTG TTA TGG T AC C7 SEQ ID NO.20 TAC GGTG TTA TGG T TAC C8 SEQ ID NO.21 TTA C GGTG TTA TGG T TTA C C9 SEQ ID NO.22 CTT AC GGTG TTA TGG T CTT AC C10 SEQ ID NO.23 ACT TAC GGTG TTA TGG T ACT TAC C11 SEQ ID NO.24 TAC TTA C GGTG TTA TGG T TAC TTA C C12 SEQ ID NO.25 TTA CTT AC GGTG TTA TGG T TTA CTT AC C13 SEQ ID NO.26 CTT ACT TAC GGTG TTA TGG T CTT ACT TAC

[0048] Depend on Figure 5It can be seen that compared with the Hemin catalytic system with the addition of LAC, the enzyme activity in the LAC-Hemin reaction system after adding different sequences 2 and combining with the corresponding sequence 1 changed to varying degrees. Among them, the peroxidase activity in the system was significantly improved after the addition of LAC-S6-C6, and there was no obvious change in the enzyme activity after the addition of LAC-S4-C4. The peroxidase activity of other groups decreased, indicating that only the sequence combination of S6-C6 can achieve the increase in enzyme activity, while sequence 2 of other sequences cannot reverse the downregulation of enzyme activity by sequence 1 and significantly increase enzyme activity. In summary, sequence 1 shown in SEQ ID NO.1 to SEQ ID NO.13 can downregulate the peroxidase activity in the LAC-Hemin system, and sequence 2 (C6) shown in SEQ ID NO.19 can upregulate the peroxidase activity after combining with the corresponding sequence 1 (S6) shown in SEQ ID NO.6. The sequential addition of S6 sequence and C6 sequence to the LAC-Hemin system can achieve reversible regulation of peroxidase activity. Therefore, sequence 1 (S6) and its complementary sequence 2 (C6) are preferred as regulatory chain sequences for modifying the internal structure of DNA microspheres and preparing new DNA-encoded micromaterials.

[0049] Example 2: Synthesis of DNA microspheres

[0050] The synthesis of DNA microspheres was modified based on the reports of Andreas Walther et al. (Nat. Nanotech., 2018, 13, 730-738; Nat. Nanotech., 2020, 15, 914-921; Adv. Sci., 2021, 8, 2003740; Nat. Commun., 2022, 13, 3968). The specific experimental steps are as follows:

[0051] (1) Preparation of template chain cyclization products

[0052] The stock solutions of the two template chains (Tp(A20-m) and Tp(A20-n)) that make up the microspheres and their corresponding circularization primers (50 mM, dissolved in TE buffer containing 100 mM NaCl) (see Table 3 below) were mixed in equimolar amounts, then heated to 85°C at 18°C / min and held for 5 minutes, and then cooled to room temperature using the same temperature gradient. 20 μL of 10XT4 ligase buffer, 1 μL of T4 ligase (NEB, 400 U / μL), and 79 μL of DEPC water were added to the system in sequence, mixed, and shaken at room temperature for 4 hours, then kept at 70°C for 20 minutes to inactivate the T4 ligase. After cooling, 20 μL of nuclease I (NEB, 10 U / μL) and 4 μL of nuclease III (NEB, 200 U / μL) were added to the mixture and shaken at 37°C overnight. The reaction mixture was then heated at 80°C for 40 minutes to inactivate the nuclease. The product was purified using a 10 kD ultrafiltration tube and washed three times with TE buffer to obtain a concentrated template chain cyclization product, which was diluted to a mother solution with a concentration of 1 μM and stored at -20°C.

[0053] (2) Preparation of ssDNA polymers

[0054] To synthesize ssDNA polymers, 10 μL of the above template chain cyclization product was mixed with 20 μL of 10XΦ 29 Polymerase buffer, 2 μL 10 μM amplification primer, 4 μL Φ 29 Polymerase (NEB, 10 U / μL), pyrophosphatase (NEB, 0.2 U / μL), 10 μL of 100 mM dNTP mix (Sangon Biotech, the percentage of each base corresponds to the expected sequence composition in the ssDNA polymer), and 144 μL of ultrapure nuclease-free water were added. The reaction mixture was incubated on a shaker at 37°C for 60 h. The ssDNA polymer was purified using a 30 kD ultrafiltration tube and washed three times with TE buffer.

[0055] (3) Self-assembly of DNA microsphere LAC

[0056] The above two ssDNA polymers were mixed at a mass concentration ratio of m:n of 1:4, then heated to 95°C and maintained for 15 minutes. After cooling, 1M magnesium acetate solution was added to make the final concentration 50mM. After mixing evenly, the mixture was heated to 95°C at 0.1°C / min and maintained for 12 minutes. The mixture was cooled to room temperature using the same cooling gradient to obtain DNA microspheres, which were named LAC.

[0057] Table 3 Nucleic acid chain sequences constituting DNA microspheres

[0058]

[0059] The morphology and size of LAC were characterized under a laser confocal microscope using green fluorescent labeling. Figure 6 As shown in the figure, the DNA microspheres are regular spherical and have a particle size of 1-10 μm.

[0060] Example 3: Characterization of catalase activity regulated by DNA microspheres in response to hemin

[0061] In order to verify that sequence 1 and its complementary sequence 2 obtained by screening in Example 1 can be used to prepare new DNA microspheres to effectively regulate the activity of hemin peroxidase, the present invention added the desired DNA microspheres LAC and 875nM sequence 1 (S6, the sequence is shown in SEQ ID NO. 6) to a 250 μL centrifuge tube, and added TE buffer containing 100mM NaCl to a final volume of 20 μL. After mixing evenly, the mixture was incubated at room temperature for 2 hours to complete the assembly process. The final concentration of LAC was 13.125 μg / mL, and a new DNA microsphere was obtained, named SAC.

[0062] Then, 50 μL of reaction buffer (containing 26.6 mM Na3C6H5O7, 51.4 mM Na2HPO4, 20 mM KCl, pH 5.0) was added thereto, shaken thoroughly, and incubated at room temperature for 30 min to allow the imotif structure to form. Then, the corresponding concentration of complementary sequence 2 (C6, sequence as shown in SEQ ID NO.19) was added and incubated for 1 h to obtain new microspheres, named DAC. The process is as follows Figure 7 As shown. Hemin was then added to a final concentration of 100 nM and incubated at room temperature with shaking for 1 hour. 100 μL of reaction buffer, 15 μL of 0.5% TMB, and 15 μL of 3% H2O2 were then added to the prepared solution in sequence, mixed thoroughly, and the resulting solution was incubated at room temperature with shaking for 40 minutes. The UV-visible absorption spectrum at 450-850 nm was measured. The absorbance at a wavelength of 652 nm was measured at 40 minutes. Normalization was performed based on the activity of Hemin, and each data point in the curve represents the average of three parallel experiments.

[0063] Depend on Figure 8It can be seen that compared with the TMB color development reaction catalyzed by Hemin alone, the reaction rate of the entire system was significantly improved after the addition of DNA microspheres LAC to the Hemin-catalyzed TMB color development system, while the reaction rate of the system was significantly decreased after the addition of sequence S6 to LAC (i.e., SAC); after the addition of the corresponding complementary sequence C6 (i.e., DAC), the rate of the entire reaction was significantly improved again; at the same time, the microspheres LAC / SAC / DAC alone could not catalyze the TMB color development reaction, indicating that the DNA-encoded DNA microspheres themselves do not have peroxidase activity, and they mainly control the reaction rate by regulating the peroxidase activity of Hemin.

[0064] To further investigate the effective regulation of Hemin peroxidase activity in LAC microspheres by different concentrations of sequence 1 (S6) and sequence 2 (C6), sequence S6 was mixed with LAC at concentrations ranging from 6.7 nM to 1005 nM. The final Hemin concentration in each sample was 100 nM, and the final concentration of DNA microsphere LAC was 13.125 μg / mL. Sequence 2 (C6) was added at a concentration twice that of S6. TE buffer containing 100 mM NaCl was added to the samples to a total volume of 20 μL, mixed thoroughly, and incubated at room temperature for 1 hour. Then, 150 μL of reaction buffer (containing 26.6 mM Na3C6H5O7, 51.4 mM Na2HPO4, 20 mM KCl, pH 5.0) and 15 μL of 3% H2O2 were added in sequence and mixed evenly. The absorbance of the reaction system at 652 nm was measured using a UV spectrophotometer after 40 minutes. The absorbance was normalized to the absorbance of the control Hemin and the enzyme activity differences of each sample were compared. The test results are shown in Figure 2. Figure 9 shown.

[0065] As can be seen from the figure, as the added concentration of sequence 1 (S6) gradually increases, the peroxidase activity of the system gradually decreases; and as the added concentration of sequence 2 (C6) gradually increases, the peroxidase activity of the system gradually increases, indicating that the regulation of Hemin peroxidase activity by the regulatory chain and its complementary chain is proportional to its concentration within a certain range. DNA microspheres with added regulatory chains and their complementary chains can be used as a good biomaterial for regulating Hemin peroxidase activity.

[0066] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A DNA microsphere for regulating the peroxidase activity of hemin, characterized in that: The DNA microsphere comprises a regulatory chain, which is used to down-regulate or up-regulate the peroxidase activity of hemin, and the regulatory chain is complementary to a part of the template chain of the DNA microsphere.

2. The DNA microsphere according to claim 1, wherein When the DNA microspheres are used to downregulate the peroxidase activity of hemin, the regulatory chain includes any one or more DNA sequences shown in SEQ ID NO.1 to SEQ ID NO.

13.

3. The DNA microsphere according to claim 1, wherein When the DNA microspheres are used to upregulate the peroxidase activity of hemin, the regulatory chain includes the DNA sequences shown in SEQ ID NO.6 and SEQ ID NO.

19.

4. The DNA microsphere according to claim 1, wherein The DNA microspheres further include template chain sequences shown in SEQ ID NO. 27 and SEQ ID NO.

28.

5. A method for regulating the peroxidase activity of hemin, characterized in that: The peroxidase activity of hemin is regulated by using the DNA microspheres as described in any one of claims 1 to 4.

6. A method for down-regulating the peroxidase activity of hemin, characterized in that: Use the SEQ ID NO. DNA microspheres containing a regulatory chain represented by any one of SEQ ID NO. 1 to SEQ ID NO. 13 reduce the peroxidase activity of hemin.

7. A method for upregulating the peroxidase activity of hemin, characterized in that: DNA microspheres containing regulatory chains shown in SEQ ID NO. 6 and SEQ ID NO. 19 are used to increase the peroxidase activity of hemin.

8. Use of a regulatory chain for preparing a reagent for downregulating the peroxidase activity of hemin, characterized in that: The regulatory chain is represented by any one of SEQ ID NO. 1 to SEQ ID NO. 13, and the peroxidase activity of the hemin is reduced by the DNA microspheres containing the regulatory chain.

9. Use of a regulatory chain for preparing a reagent for upregulating the peroxidase activity of hemin, characterized in that: The regulatory chains are shown in SEQ ID NO. 6 and SEQ ID NO. 19, and the peroxidase activity of the hemin is increased by DNA microspheres containing the regulatory chains.

10. The use according to claim 9, characterized in that The DNA microspheres further include template chain sequences shown in SEQ ID NO. 27 and SEQ ID NO. 28.

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