Mechanical stimulation pressing targeting aptamer and application of derivative thereof

Abstract

The invention relates to application of a mechanical stimulation pressing targeting aptamer and a derivative thereof. The core sequence of the mechanical stimulation pressing targeting aptamer is a nucleic acid fragment shown in the specification (the specific sequence is optimized according to research), and can also be a similar sequence with higher homology or a derivative obtained by chemical modification or molecular engineering means on the basis of the sequence. The aptamer and the derivative thereof have high specificity and high affinity binding capacity for a specific target (such as a force sensitive protein, a specific molecular marker or a tissue surface structure) triggered by mechanical stimulation pressing. The aptamer and the derivative thereof have the following advantages: 1, high specificity and high affinity: a specific molecular target triggered by mechanical stimulation can be accurately recognized and combined; 2, the property is stable: the stability is kept in a complex environment; 3, the molecular weight is small, and tissue penetration and signal transmission are facilitated; 4, the biocompatibility is good, and no toxic effect is generated on cells and tissues; 5, chemical synthesis and preservation are easy, and large-scale preparation and long-term preservation have economic feasibility.

Description

Technical Field

[0001] The present invention relates to an aptamer and its derivatives, and in particular to an application of a mechanical stimulation pressing targeting aptamer and its derivatives Background Art

[0002] Mechanical stimulation refers to the effect of external physical forces such as pressure, tension, and shear force on biological molecules or cells. This force can cause conformational changes in molecules and thus regulate their biological functions. In the biomedical field, mechanical stimulation has been widely used in research on cell biology, drug delivery, disease diagnosis, etc., especially in molecular recognition and sensor design. Mechanical stimulation provides a new idea for improving molecular recognition capabilities. By designing targeted aptamers that can respond to external pressure, the binding ability to target molecules can be enhanced under specific stimulation conditions, thereby improving the accuracy and efficiency of diagnosis and treatment.

[0003] As a chemically synthesized highly specific recognition molecule, aptamers have been widely used in molecular detection, disease diagnosis, and drug delivery. The preparation of aptamers usually relies on the systematic evolution of nucleic acids (SELEX), which enables the screening of aptamers with specific binding ability to target molecules from a large number of oligonucleotide libraries. However, traditional aptamers only rely on chemical interaction forces to identify targets, and fail to fully utilize the regulatory effect of external mechanical stimulation on molecular structure.

[0004] In recent years, with the development of biomechanics, the effect of mechanical stimulation on aptamers has gradually become a research hotspot. Mechanical pressure can significantly enhance the affinity and selectivity of aptamers by changing their three-dimensional structure. For example, by applying mechanical stimulation, aptamers can regulate their binding ability with targets, thereby improving the efficiency of molecular recognition. Designing targeted aptamers that can respond to mechanical stimulation and enhance binding ability has broad application potential.

[0005] Nevertheless, in the prior art, there are still few targeting aptamers that can be adjusted under mechanical stimulation, and their mechanisms are relatively complex, and efficient and adjustable molecular recognition has not yet been achieved. Therefore, developing a mechanical stimulation pressing targeting aptamer that can respond to mechanical stimulation and has high specificity, is easy to synthesize, and has good biocompatibility is still a technical problem that needs to be solved urgently. Summary of the invention

[0006] The purpose of the present invention is to provide a mechanical stimulation compression targeting aptamer and its derivatives which can respond to mechanical stimulation, have high specificity and high affinity to bind to target molecules, are easy to chemically synthesize, have good biocompatibility, have a small molecular weight, are stable and easy to store.

[0007] The present invention provides a mechanical stimulation and pressing targeting aptamer, characterized in that the nucleotide sequence of the aptamer is the sequence shown in SEQ ID No: 1 in the sequence table.

[0008] Furthermore, a certain position on the nucleotide sequence of the aptamer is phosphorylated, oxygen-methylated, methylated, aminated, sulfhydrylated, fluorinated or isotopized.

[0009] Furthermore, the nucleotide sequence of the aptamer is bound with biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, protein or folic acid markers.

[0010] The present invention also provides a mechanical stimulation and pressing targeting aptamer derivative, wherein the aptamer derivative is a phosphorothioate skeleton derived from the skeleton of the nucleotide sequence of the aptamer, or is a corresponding locked nucleic acid or peptide nucleic acid modified from the aptamer.

[0011] The present invention also provides the use of aptamers or aptamer derivatives, which are used to detect specific molecular markers triggered by mechanical stimulation and pressure, for biomechanical signal detection for non-diagnostic or therapeutic purposes.

[0012] The present invention also provides the use of the aptamer or the aptamer derivative, and the aptamer or the aptamer derivative is used to separate and purify mechanical stimulation-related molecular markers for non-diagnostic or therapeutic purposes.

[0013] The present invention also provides the use of the aptamer or the aptamer derivative, and the aptamer or the aptamer derivative is used to prepare a mechanical stimulation detection probe for real-time monitoring of mechanical signal changes of cells or tissues.

[0014] The present invention also provides a mechanical stimulation detection method for detecting mechanical stimulation signals for non-diagnostic or therapeutic purposes, characterized in that the method comprises the following steps:

[0015] Step 1, preparing an aptamer or an aptamer derivative;

[0016] Step 2, preparing a mechanical stimulation pressing targeting aptamer detection probe;

[0017] Step 3, fixing the detection probe on the surface of a surface plasmon resonance chip, and sealing the chip on which the detection probe is fixed with bovine serum albumin;

[0018] Step 4: Utilize the chip on which the detection probe is fixed to detect the molecular markers triggered by the mechanical stimulation pressure signal.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] (1) The mechanical stimulation pressing targeting aptamer of the present invention can adjust its conformation and affinity under external mechanical stimulation, thereby enhancing the recognition ability of the target molecule, and has high specificity, high affinity and good stability.

[0021] (1) The targeting aptamer of the present invention can achieve signal amplification and enhancement through modification, thereby improving detection sensitivity, and is suitable for use in the fields of sensors, food testing, environmental monitoring, etc.

[0022] (1) The targeting aptamer of the present invention is easy to chemically synthesize, modify and label, has a wide range of application potential, and is suitable for large-scale industrial production and wide application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results are the determination results of the protein docking interaction relationship of the mechanical stimulation pressing targeting aptamer and related derivatives in the embodiments of the present invention.

[0024] Figure 2 The results are the determination results of the key proteins that mainly up- and down-regulate the targeting aptamers and their derivatives in the mechanical stimulation pressing and control groups in the embodiments of the present invention.

[0025] Figure 3 This is the enrichment analysis of the actin dynamics regulation gene set corresponding to the mechanical stimulation and compression targeting aptamer and its derivatives in the embodiment of the present invention, and the detection result of analyzing the influence of the coordinated changes of genes in the gene set on the mechanical stimulation phenotypic changes.

[0026] Figure 4 These are the detection results of relevant biological processes, cellular components and molecular functions in the gene ontology corresponding to the mechanical stimulation pressing targeting aptamer and its derivatives in the embodiments of the present invention.

[0027] Figure 5 This is the detection result of the key protein piezo2 playing an important role in the mechanical stimulation pressing targeting aptamer and its derivatives in the embodiments of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific preferred implementations, but the protection scope of the present invention is not limited thereby.

[0029] Example:

[0030] The nucleotide sequence of the mechanical stimulation compression targeting nucleic acid aptamer (hereinafter referred to as "nucleic acid aptamer") of the present invention includes a DNA fragment shown in the following sequence, which can specifically bind to a target molecule (such as a cell surface receptor, a pathogenic microorganism, etc.):

[0031] 5'-ACGTGCTAGGCTCGTGGATCCGTAAGGACCGAGATCGTTAGGAGAGATAGTCAGGATAGGCCGAGTGGTGTTGAG-3'

[0032] The nucleotide sequence of the nucleic acid aptamer is selected from a naturally occurring or artificially synthesized sequence, or any other sequence with high homology, and has high affinity and specificity with the target molecule.

[0033] It should be noted that the protection scope of the present invention covers all sequences containing the aptamer.

[0034] In this embodiment, the relevant technical solution covers the sequence after a certain position on the nucleotide sequence of the above-mentioned nucleic acid aptamer is phosphorylated, oxygenated, methylated, fluorinated, aminated, thiolated or isotopized.

[0035] In this embodiment, the relevant technical scheme covers the sequence of the nucleotide sequence of the above-mentioned nucleic acid aptamer after binding with biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, peptides, proteins, enzymes or folic acid labels (even connected with radioactive substances, etc.).

[0036] The above-mentioned nucleic acid aptamer can be derived into other nucleic acid aptamers, and the nucleotide sequence of the derived nucleic acid aptamer can be any one of the following three sequences:

[0037] (1) The homology with the nucleotide sequence of the nucleic acid aptamer listed in this embodiment is more than 90% (for example, the above nucleic acid aptamer sequence can be deleted or partially complementary nucleotides can be added);

[0038] (2) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer listed in this example;

[0039] (3) RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer listed in this embodiment.

[0040] The backbone of the nucleotide sequence of the nucleic acid aptamer listed in this embodiment can also be derived into a phosphorothioate backbone, and the above-mentioned nucleic acid aptamer can also be transformed into a corresponding locked nucleic acid or peptide nucleic acid.

[0041] The mechanical stimulation pressing targeting nucleic acid aptamer of this embodiment has the following uses:

[0042] Uses in cell function regulation and targeted drug delivery;

[0043] Use in the design and development of targeted drugs and rehabilitation facilities;

[0044] Use in preparing mechanical stimulation related detection probes.

[0045] The mechanical stimulation target molecules used in this embodiment are receptor proteins, extracellular matrix or pathogenic microorganism surface marker molecules. The specific target molecules bind to their receptors through pressure stimulation, thereby triggering specific recognition and binding reactions.

[0046] Screening process:

[0047] The nucleic acid aptamers in this embodiment are mainly screened by magnetic bead capture method, and the specific screening process includes the following steps:

[0048] (a) Optimizing nucleic acid library:

[0049] The structure of the library is divided into three parts. In the middle is a 40-base random sequence, and on both sides of the random sequence are two DNA arms, which are the necessary sequences for PCR amplification of 20 fixed bases. At the same time, the fixed sequence at the 5' end is complementary to the fixed probe (BDNA). The fixed probe is a DNA chain complementary to the fixed sequence of the library with biotin labeled at one end, which is used to fix the library.

[0050] Random Library Lib:

[0051] 5'-ATTGGCACTCCACGCATAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTATGCGTGCTACCGTGAA-3'

[0052] 5′ primer: 5′-ATTGGCACTCCACGCATAGG-3′;

[0053] 3′ primer: 5′-TTCACGGTAGCACGCATAGG-3′;

[0054] (b) Initial screening:

[0055] Library pretreatment method: 1.3 nmol of library (about 1014 DNA chains) and 2.9 nmol of BDNA were dissolved in 289 μL of DPBS buffer, pre-denatured at 95°C for 10 min, then cooled to 60°C at a rate of 0.1°C per second for 1 min, and then cooled to 25°C at 0.1°C per second for slow denaturation. The hybridization library was added to 1 mL of streptavidin magnetic beads that had been washed 5 times, and incubated at room temperature in a shaker for 30 min. Then, the magnetic beads were adsorbed with a magnet, and the supernatant was removed. The magnetic beads were suspended and washed 6 times with 400 μL of DPBS buffer. Then the target solution was added and incubated at room temperature in a shaker for 40 min. The magnetic beads were adsorbed with a magnet, and the supernatant solution was recovered to obtain a primary screening nucleic acid library.

[0056] (c) Purification:

[0057] The primary screening nucleic acid library was amplified by PCR, and the amplification primers used were the primers with fluorescent primers at the 5' end and spacer arms at the 3' end listed in step (a). The amplified products were separated by single-stranded preparation using SDS denaturing PAGE, and then the secondary nucleic acid library was formed by boiling the gel, concentrating with n-butanol and dialysis. The secondary nucleic acid library finally obtained was used for the next round of screening.

[0058] (d) Loop:

[0059] Repeat the above-mentioned initial screening and purification steps, use new streptavidin-ylated magnetic beads, and screen out nucleic acid aptamers with high affinity and high specificity binding to the target target in each cycle.

[0060] (e) Screening efficiency evaluation:

[0061] qPCR was used to determine the screening efficiency, i.e. the enrichment of DNA during the screening process. The binding affinity and specificity of the aptamer were evaluated by measuring the concentration changes of nitrobenzene (or other target molecules).

[0062] Affinity and specificity evaluation:

[0063] The affinity of the mechanical stimulation pressing targeting nucleic acid aptamer in this embodiment is measured by the surface plasmon resonance (SPR) method. Different concentrations of target molecules (such as cell surface receptors or microbial surface antigens) are combined with the fixed nucleic acid aptamer, and the binding kinetics are monitored in real time, and the dissociation constant (Kd value) is calculated to evaluate the affinity.

[0064] Specificity evaluation is carried out by fluorescence recovery technology or fluorescence polarization method. The aptamer is labeled with a suitable fluorescent probe and reacts with different concentrations of target molecules to record the change in fluorescence signal. The specific recognition ability of the nucleic acid aptamer is determined by comparing the reaction intensity.

[0065] The mechanical stimulation pressing targeted nucleic acid aptamer of this embodiment can be widely used in the fields of biosensors, targeted drug delivery, environmental monitoring, etc., providing an efficient and sensitive tool for the detection and separation of specific targets.

[0066] The above is only an embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment. All technical solutions under the concept of the present invention belong to the protection scope of the present invention.

Claims

1. A mechanical stimulation pressing targeting aptamer, characterized in that: The nucleotide sequence of the aptamer is the sequence shown in SEQ ID No: 1 in the sequence table.

2. The aptamer according to claim 1, characterized in that A certain position on the nucleotide sequence of the aptamer is phosphorylated, oxygen-methylated, methylated, aminated, sulfhydrylated, fluorinated or isotope-modified.

3. The aptamer according to claim 1, characterized in that The nucleotide sequence of the aptamer is combined with biotin, digoxin, fluorescent substance, nano material, polyethylene glycol, protein or folic acid marker.

4. A mechanical stimulation compression targeting aptamer derivative, characterized in that: The aptamer derivative is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the aptamer in claim 1, 2 or 3, or is a corresponding locked nucleic acid or peptide nucleic acid modified from the aptamer in claim 1, 2 or 3.

5. A use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4, characterized in that: The aptamer or the aptamer derivative is used to detect specific molecular markers triggered after mechanical stimulation and pressing, and is used for biomechanical signal detection for non-diagnostic or therapeutic purposes.

6. A use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4, characterized in that: The aptamer or the aptamer derivative is used to separate and purify mechanical stimulation-related molecular markers for non-diagnostic or therapeutic purposes.

7. A use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4, characterized in that: The aptamer or the aptamer derivative is used to prepare a mechanical stimulation detection probe for real-time monitoring of mechanical signal changes of cells or tissues.

8. A mechanical stimulation detection method for detecting mechanical stimulation signals for non-diagnostic or therapeutic purposes, characterized in that: The method comprises the following steps: Step 1, preparing the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4; Step 2, preparing a mechanical stimulation pressing targeting aptamer detection probe; Step 3, fixing the detection probe on the surface of a surface plasmon resonance chip, and sealing the chip on which the detection probe is fixed with bovine serum albumin; Step 4: Utilize the chip on which the detection probe is fixed to detect the molecular markers triggered by the mechanical stimulation pressure signal.

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