A dna-programmed virus neutralizing multivalent aptamer agent and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a DNA programmed virus neutralizing multivalent aptamer medicament and a preparation method thereof.The medicament comprises a circular DNA as a template, and a DNA multivalent neutralizing aptamer particle is synthesized through a rolling circle amplification reaction, wherein the DNA template can be designed to be directed against a plurality of virus antigen epitope neutralizing aptamers.The DNA programmed virus neutralizing multivalent aptamer medicament provided by the application is simple to operate, raw materials are easy to obtain, has good biocompatibility, and has a wide application range, can effectively inhibit the invasion of virus particles into the cell interior, can capture and close the virus particles, thereby inhibiting the infection of the virus particles on the host cells, and can play a safe and effective virus neutralizing role and block the transmission and infection of the virus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a DNA-programmed virus-neutralizing multivalent aptamer agent and its preparation method. Background Technology

[0002] For millennia, viral pandemics have posed a significant threat to human health. This century has witnessed a series of severe pandemic health crises, such as the 2003 Severe Acute Respiratory Syndrome Coronavirus Disease (SARS-CoV-2), the 2013-2016 West African Ebola outbreak, the 2015 Zika virus outbreak, and the COVID-19 pandemic. Developing neutralizing agents to block the specific interaction between viral antigens and host cell surface receptors has been considered an effective method for inhibiting viral infection. Although monoclonal antibodies exhibit strong neutralizing activity against viral infections, their fatal drawback is limited efficacy against viral variants. Furthermore, cumbersome manufacturing processes, complex storage conditions, and high dosages required for clinical treatment further hinder the widespread application of monoclonal antibodies. Simultaneously, they may induce antibody-dependent enhancement (ADE), leading to increased viral infection in monocytes or macrophages, resulting in severe clinical symptoms. Therefore, researchers have been dedicated to exploring broad-spectrum antiviral platforms, but to date, safe and highly effective antiviral agents remain lacking to address the serious threat posed to global public health by the spread of multiple viruses.

[0003] Nucleic acid aptamers are single-stranded DNA or RNA molecules that can bind to target substances by folding into a three-dimensional structure. Compared with antibodies, neutralizing aptamers exhibit comparable or even higher affinity for target molecules. Furthermore, neutralizing aptamers can be easily synthesized and modified chemically with minimal batch-to-batch variation. For example, DNA aptamers targeting the SARS-CoV-2 S protein RBD have been reported to effectively block SARS-CoV-2 virus infection of host cells by inhibiting the binding of the S protein to the ACE2 receptor on host cells. Notably, universal aptamers targeting multiple mutant SARS-CoV-2 S proteins, such as the MSA52 aptamer, have been screened and can effectively prevent immune escape from mutant strains. Recently, literature has shown that multivalent aptamers can bind to two or more identical targets simultaneously compared to monovalent aptamers, exhibiting higher binding affinity; therefore, multivalent aptamers show stronger neutralizing ability against variants. However, previously reported materials were either too small to match the shape of the virus or too soft to effectively capture and enclose the entire viral particle, which was not conducive to achieving effective antiviral effects.

[0004] Rolling circle amplification (RCA) is a reaction that uses a circular DNA molecule as a template. Under the action of DNA polymerase, four deoxyribonucleotides are used as starting materials to synthesize DNA fragments from the 3' hydroxyl end of the primer, following a base-complementary pairing pattern. After the synthesis of one DNA template fragment is complete, the template is released under the action of DNA polymerase, allowing for the amplification of the next fragment, ultimately producing a long single-stranded DNA molecule with tandem repeat sequences. RCA has several advantages: First, it is an isothermal amplification reaction, suitable for DNA fragment amplification; second, nucleic acid nanostructures prepared by RCA can effectively improve the stability of nucleic acid molecules, such as DNA nanogels, DNA nanococoons, and DNA nanoflowers, exhibiting good resistance to nuclease degradation; finally, RCA synthesis is simple and readily yields the target product. Therefore, using RCA to synthesize multivalent aptamers, and further mineralizing and assembling them during the reaction process, can produce more stable structures. By controlling the reaction time, the particle size and surface morphology of the product can be regulated, thereby developing formulations with structure and function compatible.

[0005] Based on the above characteristics and advantages of RCA, a new multivalent aptamer formulation for virus neutralization was developed through the design and screening of neutralizing aptamer fragments, thereby achieving the blocking of the transmission of multiple viruses.

[0006] To address the problems raised in the background art, those skilled in the art have proposed a DNA-programmed virus-neutralizing multivalent aptamer agent and its preparation method. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a DNA-programmed virus-neutralizing multivalent aptamer agent and its preparation method, thereby resolving the issues in the prior art.

[0008] A DNA-programmed virus-neutralizing multivalent aptamer agent includes synthesizing DNA multivalent neutralizing aptamer particles via rolling circle amplification reaction using circular DNA as a template, wherein the circular DNA is obtained by circularizing a mixture of template sequence SEQ ID NO.1 and primer sequence SEQ ID NO.2, and the virus is SARS-CoV-2 virus.

[0009] Preferably, the particle size and surface pore size of the multivalent neutralizing aptamer particles can be controlled by adjusting the rolling ring amplification reaction time and substrate concentration.

[0010] A method for preparing a DNA-programmed virus-neutralizing multivalent aptamer agent includes the following steps:

[0011] S1. The designed linear DNA template containing the neutralizing aptamer sequence is annealed and circularized, and the circularized gap is ligated with T4 DNA ligase.

[0012] S2. Using the circular DNA prepared in step S1 as a template and four deoxyribonucleotides as substrates, the mixture is incubated with phi29 DNA polymerase under shaking to obtain DNA-programmed multivalent neutralizing aptamer drug particles.

[0013] Preferably, in step S2, the rotational speed of the oscillation is 200-800 rpm, wherein the rotational speed of the oscillation is preferably 500 rpm.

[0014] Preferably, in step S2, the reaction temperature for obtaining the DNA-programmed multivalent neutralizing aptamer drug particles is 25-40°C, wherein the reaction temperature is preferably 30°C.

[0015] Preferably, in step S2, the reaction time for obtaining the DNA-programmed multivalent neutralizing aptamer drug particles is 6-24 hours, wherein the reaction time is preferably 24 hours.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. This invention provides a DNA-programmed virus-neutralizing multivalent aptamer agent, which is simple to operate, uses readily available raw materials, has good biocompatibility, and has a wide range of applications. On the one hand, the size of the synthesized multivalent aptamer agent particles and the size of the surface pores can be controlled according to the RCA reaction time to make them compatible with the size of the virus particles to be captured. While capturing virus particles, they are not easily taken up by cells, thereby effectively inhibiting the invasion of virus particles into the cell. On the other hand, due to the programmability of the DNA sequence, the DNA aptamer encoded in this agent can be designed for different antigenic phenotypes. For example, aptamer sequences encoding SARS-CoV-2 NTD domains or surface high-mannose glycans, as well as aptamer sequences targeting antigenic sites of various prevalent viruses, can all be synthesized in this way to capture and block virus particles, thereby inhibiting their infection of host cells, exerting a safe and effective virus neutralization effect, and blocking the spread and infection of viruses. This multivalent aptamer agent can be applied in the biomedical field of neutralizing various viruses. Attached Figure Description

[0018] Figure 1 This is an electrophoresis diagram of the long DNA chain in Example 1, where 1: ladder; 2: template DNA in Example 1; 3: circular DNA in Example 1; 4: DNA multivalent aptamer product in Example 1.

[0019] Figure 2 SEM image of the DNA-programmed multivalent neutralizing aptamer prepared in Example 1.

[0020] Figure 3The graph shows the results of in vivo virus neutralization by the DNA-programmed multivalent neutralizing aptamer prepared in Example 1. Figure 3 (A) Images of bioluminescence in mice injected with different drugs, captured by a 3D imager. Figure 3 (B) is Figure 3 (A) Statistical analysis results of bioluminescence in mice injected with different drugs. Detailed Implementation

[0021] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0022] like Figures 1 to 3 As shown:

[0023] Example 1:

[0024] Preparation of a DNA-programmed virus-neutralizing multivalent aptamer agent

[0025] (1) Preparation of long DNA chains containing DNA neutralizing aptamers

[0026] A long DNA chain containing viral neutralizing aptamers was designed and obtained commercially. The DNA chain was dissolved in water and shaken to obtain a final concentration of 100 μM DNA aqueous solution.

[0027] Table 1. Sequences for preparing DNA multivalent aptamer drugs

[0028]

[0029] The DNA template sequence SEQ ID NO.1 is divided into segments 1-1, 1-2, 1-3, 1-4, and 1-5 from the 5' end to the 3' end. Segments 1-1 and 1-5 are primer complementary sequences. Segment 1-1 is anticomplementary to the 5' end of the primer sequence SEQ ID NO.2 of SEQ ID NO.1, with 14 nucleotides. Segment 1-5 is anticomplementary to the 3' end of the sequence SEQ ID NO.2, with 10 nucleotides. The DNA primer sequence SEQ ID NO.2 has a total of 24 nucleotides. Segments 1-2 and 1-4 are spacer sequences with a total of 25 nucleotides. Segment 1-3 is a neutralizing aptamer sequence targeting the RBD domain of the S protein on the surface of the SARS-CoV-2 virus, with 46 nucleotides, for a total of 95 nucleotides.

[0030] (2) Preparation of DNA agents containing multivalent neutralizing aptamer sequences

[0031] Using the sequence SEQ ID NO.1 designed in (1) as a template and four deoxyribonucleotides (dNTPs) as raw materials, the template sequence SEQ ID NO.1 and primer sequence SEQ ID NO.2 were first mixed at a molar ratio of 1:1.5, with final concentrations of 10 μM and 15 μM, respectively, and the template sequence was circularized by annealing. Next, the gap was ligated using T4 ligase. The circularized template sequence from the previous step was mixed with T4 DNA ligase and T4 DNA ligase buffer (1×), and reacted at 16°C for 12 h to obtain circular DNA with a final concentration of 1 μM. Finally, the DNA multivalent aptamer structure was prepared using rolling circle amplification technology. The circular DNA synthesized in the previous step (final concentration 0.3 μM) was mixed with phi 29 DNA polymerase (final concentration 1 U / μL), phi 29 DNA polymerase buffer (1×), dNTPs (final concentration 1 mM), KCl (final concentration 50 mM), and recombinant protein (final concentration 0.2 mg / mL), and reacted at 30 °C and 500 rpm for 24 h to prepare a long DNA chain containing a large number of viral neutralizing aptamer repeat sequences. The DNA multivalent aptamer drug particles were obtained by mineralization self-assembly with magnesium pyrophosphate generated during the reaction.

[0032] Example 2:

[0033] Preparation of a DNA-programmed virus-neutralizing multivalent aptamer agent

[0034] (1) Design and synthesize long DNA chains;

[0035] Long DNA chains were synthesized using rolling circle amplification (RCA). First, the template sequence (SEQ ID NO.3) and primer sequence (SEQ ID NO.2) required for preparing the long DNA chains were designed and synthesized, dissolved in water, and shaken to obtain a single-stranded DNA aqueous solution with a final concentration of 100 μM.

[0036] Table 2. Sequences for preparing DNA multivalent aptamer drugs

[0037]

[0038] The DNA template sequence SEQ ID NO.3 is divided into segments 1-1, 1-2, 1-3, 1-4, and 1-5 from the 5' end to the 3' end. Segments 1-1 and 1-5 are primer complementary sequences. Segment 1-1 is anticomplementary to the 5' end of the primer sequence SEQ ID NO.2 of SEQ ID NO.3, and has 14 nucleotides. Segment 1-5 is anticomplementary to the 3' end of the sequence SEQ ID NO.2, and has 10 nucleotides. The DNA primer sequence SEQ ID NO.2 has a total of 24 nucleotides. Segments 1-2 and 1-4 are spacer sequences, with a total of 21 nucleotides. Segment 1-3 is a neutralizing aptamer sequence targeting the NTD domain of the SARS-CoV-2 virus, with 50 nucleotides, for a total of 95 nucleotides.

[0039] (2) Preparation of DNA agents containing multivalent neutralizing aptamer sequences;

[0040] Using the sequence SEQ ID NO.3 designed in (1) as a template and four deoxyribonucleotides (dNTPs) as raw materials, the template sequence SEQ ID NO.3 and primer sequence SEQ ID NO.2 were first mixed at a molar ratio of 1:1.5, with final concentrations of 10 μM and 15 μM, respectively, and the template sequence was circularized by annealing. Next, the gap was ligated using T4 ligase. The circularized template sequence from the previous step was mixed with T4 DNA ligase and T4 DNA ligase buffer (1×), and reacted at 16°C for 12 h to obtain circular DNA with a final concentration of 1 μM. Finally, the DNA multivalent aptamer structure was prepared using rolling circle amplification technology. The circular DNA synthesized in the previous step (final concentration 0.3 μM) was mixed with phi 29 DNA polymerase (final concentration 1 U / μL), phi 29 DNA polymerase buffer (1×), dNTPs (final concentration 1 mM), KCl (final concentration 50 mM), and recombinant protein (final concentration 0.2 mg / mL), and reacted at 30 °C and 500 rpm for 24 h to prepare a long DNA chain containing a large number of viral neutralizing aptamer repeat sequences. The DNA multivalent aptamer drug particles were obtained by mineralization self-assembly with magnesium pyrophosphate generated during the reaction.

[0041] Example 3:

[0042] Preparation of a DNA-programmed virus-neutralizing multivalent aptamer agent

[0043] (1) Design and synthesize long DNA chains;

[0044] Long DNA chains were synthesized using rolling circle amplification (RCA). First, the template sequence (SEQ ID NO.4) and primer sequence (SEQ ID NO.5) required for preparing the long DNA chains were designed and synthesized, dissolved in water, and shaken to obtain a single-stranded DNA aqueous solution with a final concentration of 100 μM.

[0045] Table 3. Sequences for preparing DNA multivalent aptamer drugs

[0046]

[0047] The DNA template sequence SEQ ID NO.4 is divided into segments 1-1, 1-2, 1-3, 1-4, and 1-5 from the 5' end to the 3' end. Segments 1-1 and 1-5 are primer complementary sequences. Segment 1-1 is anticomplementary to the 5' end of the primer sequence SEQ ID NO.4 (SEQ ID NO.5), and contains 14 nucleotides. Segment 1-5 is anticomplementary to the 3' end of the sequence SEQ ID NO.5, and contains 10 nucleotides. The DNA primer sequence SEQ ID NO.5 contains a total of 24 nucleotides. Segments 1-2 and 1-4 are spacer sequences, containing a total of 47 nucleotides. Segment 1-3 is a neutralizing aptamer sequence targeting the high-mannose content on the surface of the SARS-CoV-2 virus, containing 24 nucleotides, for a total of 95 nucleotides.

[0048] (2) Preparation of DNA reagents containing multivalent neutralizing aptamer sequences, including the following steps:

[0049] Using the sequence SEQ ID NO.4 designed in (1) as a template, four deoxyribonucleotides (dNTPs) were used as raw materials. First, the template sequence SEQ ID NO.4 and the primer sequence SEQ ID NO.5 were mixed in a 1:1.5 molar ratio, with final concentrations of 10 μM and 15 μM, respectively, and the template sequence was circularized by annealing. Second, the gap was ligated using T4 ligase, and the circularized template sequence from the previous step was mixed with T4 DNA ligase and T4 DNA ligase buffer (1×), and reacted at a constant temperature of 16 °C for 12 h to obtain circular DNA with a final concentration of 1 μM. Finally, the DNA multivalent aptamer structure was prepared using rolling circle amplification technology. The circular DNA synthesized in the previous step (final concentration 0.3 μM) was mixed with phi 29 DNA polymerase (final concentration 1 U / μL), phi 29 DNA polymerase buffer (1×), dNTPs (final concentration 1 mM), KCl (final concentration 50 mM), and recombinant protein (final concentration 0.2 mg / mL), and reacted at 30 °C and 500 rpm for 24 h to prepare a long DNA chain containing a large number of viral neutralizing aptamer repeat sequences. The DNA multivalent aptamer drug particles were obtained by mineralization self-assembly with magnesium pyrophosphate generated during the reaction.

[0050] Figure 1 The image shows a polyacrylamide gel electrophoresis diagram of the DNA multivalent neutralizing aptamer prepared in Example 1. The migration rate of the circular DNA band in lane 3 is lower than that of the DNA template sequence in lane 2, indicating that the DNA template sequence and the DNA primer sequence are circularized. The band in lane 4 shows a pore-blocking phenomenon, indicating that the DNA multivalent neutralizing aptamer structure has been synthesized.

[0051] Figure 2 The image shows a transmission electron microscope (TEM) image of the DNA-programmed multivalent neutralizing aptamer prepared in Example 1. The TEM image shows that the prepared multivalent neutralizing aptamer has a porous granular morphology with a size ranging from 1 to 3 μm and good dispersibility.

[0052] Figure 3 Three-dimensional imaging of mice neutralizing pseudovirus XBB.1.16 in vivo with the DNA-programmed multivalent neutralizing aptamer prepared in Example 1. Pseudovirus XBB.1.16 carries a luciferase gene tag and can express luciferase in host cells, reacting with potassium luciferin to produce bioluminescence. First, 6-8 week old male C57BL / 6J mice were divided into two groups of three. Each mouse in each group was injected intravenously with either XBB.1.16 or XBB.1.16 treated with the DNA multivalent neutralizing aptamer (DNC-24) (37°C, incubation for 2 hours) (DNC-24 / XBB.1.16). Twenty-four hours after infection, each mouse in both groups was injected with an equal volume of D-luciferin potassium solution. Three to five minutes later, mice anesthetized with isoflurane were placed on a three-dimensional imaging system for bioluminescence imaging. The results are shown below. Figure 3 As shown in (A) and (B), treatment with the DNA-programmed multivalent neutralizing aptamer significantly reduced the bioluminescence produced by XBB.1.16 in mice. Statistical analysis of the bioluminescence values ​​in mice treated with the two groups revealed that the bioluminescence value in mice treated with DNC-24 / XBB.1.16 was reduced by approximately 74% compared to mice treated with XBB.1.16. This demonstrates that the DNA-programmed multivalent neutralizing aptamer can significantly inhibit the infection effect of XBB.1.16 in mice, exhibiting a significant virus neutralization effect.

[0053] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A DNA-programmed virus-neutralizing multivalent aptamer agent, characterized in that: The method includes synthesizing DNA multivalent neutralizing aptamer particles using circular DNA as a template via rolling circle amplification reaction, wherein the circular DNA is obtained by mixing and circularizing the template sequence SEQ ID NO.1 and the primer sequence SEQ ID NO.2, and the virus is SARS-CoV-2 virus.

2. The DNA-programmed virus-neutralizing multivalent aptamer agent as described in claim 1, characterized in that: The multivalent neutralizing aptamer particles can have their particle size and surface pore size controlled by adjusting the rolling ring amplification reaction time and substrate concentration.

3. A method for preparing a DNA-programmed virus-neutralizing multivalent aptamer agent according to any one of claims 1-2, characterized in that: Includes the following steps: S1. The designed linear DNA template containing the neutralizing aptamer sequence is annealed and circularized, and the circularized gap is ligated with T4 DNA ligase. S2. Using the circular DNA prepared in step S1 as a template and four deoxyribonucleotides as substrates, the mixture is incubated with phi29 DNA polymerase under shaking to obtain DNA-programmed multivalent neutralizing aptamer drug particles.

4. The method for preparing a DNA-programmed virus-neutralizing multivalent aptamer agent as described in claim 3, characterized in that: In step S2, the rotational speed of the oscillation is 200-800 rpm.

5. The method for preparing a DNA-programmed virus-neutralizing multivalent aptamer agent as described in claim 3, characterized in that: In step S2, the reaction temperature for obtaining the DNA-programmed multivalent neutralizing aptamer drug particles is 25-40°C.

6. The method for preparing a DNA-programmed virus-neutralizing multivalent aptamer agent as described in claim 3, characterized in that: In step S2, the reaction time for obtaining the DNA-programmed multivalent neutralizing aptamer drug particles is 6-24 hours.

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