CN115074367A

CN115074367A - Group of nucleic acid aptamers combined with brain-derived neurotrophic factor with high affinity and application thereof

Info

Publication number: CN115074367A
Application number: CN202210708211.5A
Authority: CN
Inventors: 高顺祥; 吴继红; 孙兴怀; 张圣海; 李倩
Original assignee: Eye and ENT Hospital of Fudan University
Current assignee: Eye and ENT Hospital of Fudan University
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2022-09-20

Abstract

The invention relates to the field of ophthalmology, in particular to a group of aptamers capable of being combined with brain-derived neurotrophic factor (BDNF) with high affinity. The invention obtains the high-affinity aptamer capable of specifically recognizing BDNF by adopting SELEX technology screening, and further improves the performance of the aptamer by combined application of optimization strategies such as truncation, dimerization and the like. The aptamer has wide application prospect, and can be used for capturing BDNF in a system, detecting in vivo and in vitro BDNF, developing BDNF treatment drugs in related diseases, constructing a targeted drug delivery system and the like. The nucleic acid aptamer of the present invention has a great potential for practical use.

Description

Group of nucleic acid aptamers combined with brain-derived neurotrophic factor with high affinity and application thereof

Technical Field

The invention relates to the field of ophthalmology, in particular to a group of nucleic acid aptamers combined with brain-derived neurotrophic factor (BDNF) with high affinity and application thereof.

Background

Glaucoma is the first irreversible blindness-causing eye disease in the world, and is a group of neurodegenerative diseases which are characterized by atrophy and depression of optic papilla, visual field loss and visual deterioration. Statistically, about 8000 ten thousand glaucoma patients are present in 2020 globally. Because glaucoma has the characteristics of occult morbidity, difficult early diagnosis, irreversible visual function damage and the like, the blindness rate of glaucoma patients over 40 years old in China is more than 30 percent. Therefore, early screening, early diagnosis and early intervention have become important means to reduce the risk of blindness in glaucoma.

At present, glaucoma is diagnosed mainly by tonometry, morphology and functional examinations. However, the intraocular pressure varies greatly from patient to patient, and the visual function of the patient is often damaged by more than 50% when glaucoma is diagnosed by means of morphology, functional science and the like, which makes it difficult to meet the needs of clinical early diagnosis.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family and is also the currently accepted glaucoma early diagnosis marker with the most application prospect. Numerous studies have demonstrated that BDNF has a significant dose-dependent relationship with the onset and progression of glaucoma. The BDNF derived from tears or serum is used as a target spot for early diagnosis of glaucoma, has the advantages of no wound, convenience, easy obtainment, high specificity and the like, and can provide help for screening, diagnosing and intervening of glaucoma.

The problem to be solved is to obtain a biological recognition molecule that specifically binds to BDNF. The aptamer is used as a novel molecular recognition tool, and can be specifically recognized and bind to a target molecule with high affinity. It is usually ssDNA or RNA obtained from a library of nucleic acid molecules by using the in vitro screening technique, the exponential enrichment ligand phylogenetic technique (SELEX). The aptamer has the advantages of chemical synthesis, easy labeling and modification, wide target identification range, low immunogenicity and toxicity, high affinity, strong specificity and the like, can be used for capturing, detecting, in-vivo delivery, nucleic acid drug development and the like of specific targets, and has wide application prospects in the fields of analysis, diagnosis, medicine and the like.

Disclosure of Invention

The invention aims to provide a group of high-affinity aptamers capable of specifically binding to brain-derived neurotrophic factor (BDNF). It is another object of the invention to provide the set of nucleic acid aptamers in the preparation of a BDNF detection reagent, kit or sensor; in preparing BDNF capture, isolation and purification formulations; the application in the preparation of BDNF diagnostic tools, targeted drug delivery systems, neutralizing or blocking drugs and the like.

In order to achieve the purpose, the main technical scheme of the invention is as follows: (1) obtaining the aptamer specifically combined with BDNF by screening through a magnetic bead SELEX technology; (2) obtaining a core sequence of the aptamer through truncation optimization; (3) by constructing dimers of aptamers, dimeric aptamers with very high targeting affinity are obtained.

In the first aspect of the invention, a group of high affinity aptamers specifically binding to Brain Derived Neurotrophic Factor (BDNF) is provided, and the nucleotide sequences of the aptamers are respectively shown as SEQ ID No. 1-SEQ ID No.7 (Table 1).

Table 1: aptamer sequences and binding affinities thereof

In a second aspect of the present invention, there is provided an application of the high affinity aptamer specifically binding to brain-derived neurotrophic factor (BDNF) in preparing a brain-derived neurotrophic factor (BDNF) detection reagent, kit or sensor.

In a third aspect of the present invention, there is provided the use of a high affinity aptamer that specifically binds to Brain Derived Neurotrophic Factor (BDNF) as described above in the preparation of a Brain Derived Neurotrophic Factor (BDNF) capture, isolation and purification formulation.

In a fourth aspect of the present invention, there is provided the use of a high affinity aptamer that specifically binds to brain-derived neurotrophic factor (BDNF) as described above in the development of a novel method for the early and rapid diagnosis of glaucoma.

Further, the novel method for diagnosing eye diseases is based on the binding effect of the aptamer on BDNF high affinity, and can be used for the early and rapid diagnosis of glaucoma.

In a fifth aspect of the invention, there is provided a use of the high affinity aptamer specifically binding to Brain Derived Neurotrophic Factor (BDNF) as described above in the preparation of a glaucoma early diagnosis reagent or kit.

Furthermore, the early diagnosis reagent or the kit detects the concentration of BDNF in body fluid through the binding effect of the aptamer on the high affinity of BDNF, and is used for the early rapid diagnosis of glaucoma.

In a sixth aspect, the invention provides an application of the high-affinity aptamer specifically binding to brain-derived neurotrophic factor (BDNF) in constructing an ocular disease targeted drug delivery system.

Furthermore, the targeted drug delivery system is based on the recognition effect of the aptamer on the strong specificity of BDNF, and is used for targeted transportation, site-specific release and the like of drugs.

In a seventh aspect of the invention, there is provided a use of the high affinity aptamer specifically binding to Brain Derived Neurotrophic Factor (BDNF) as described above in the preparation of a medicament for the treatment of glaucoma.

Further, the drug can relieve or treat BDNF-mediated relevant clinical diseases by neutralizing or blocking BDNF.

The invention has the advantages that:

1. according to the invention, BDNF protein is fixed on the surface of a magnetic bead, aptamer screening is carried out by a magnetic bead SELEX technology, a group of high-affinity aptamers capable of specifically binding BDNF is obtained, and the performance of the aptamer is further improved by combined application of optimization strategies such as truncation and dimerization.

2. The aptamer serving as a BDNF-targeting molecular tool has the advantages of high affinity, strong specificity, good stability, low immunogenicity, low preparation cost, easiness in modification and marking and the like. The method can be used for capturing BDNF in the system, detecting BDNF in vivo and in vitro, developing BDNF treatment drugs in related diseases, constructing a targeted drug delivery system and the like, and has wide application prospect. Therefore, the nucleic acid aptamer of the present invention has a great potential for practical use.

Drawings

FIG. 1 shows the screening of magnetic bead SELEX technology.

FIG. 2. recovery of BDNF-bound ssDNA.

FIG. 3 shows the binding dissociation curve of aptamer F1 to BDNF.

FIG. 4 shows the binding dissociation curve of aptamer F2 to BDNF.

FIG. 5 is a diagram showing the prediction of the secondary structure of an aptamer.

FIG. 6 shows the binding dissociation curve of the dimeric aptamer F1T-1T with BDNF.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1: aptamer screening library and construction of primers thereof

1. Construction of ssDNA library of 80 nucleotides in length

5′-AGCAGCACAGAGGTCAGATG-N ₄₀ -CCTATGCGTGCTACCGTGAA-3' (SEQ ID No. 8); wherein N represents any one of bases A, T, C and G, and N ₄₀ Representing a random sequence of 40 nucleotides in length.

2. Construction of primers

An upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3' (SEQ ID No. 9);

a downstream primer 1: 5'-TTCACGGTAGCACGCATAGG-3' (SEQ ID No. 10);

a downstream primer 2: 5 '-poly (dA20) -Spacer 18-TTCACGGTAGCACGCATAGG-3' (SEQ ID No. 11).

Example 2: screening for BDNF aptamers

As shown in FIG. 1, in order to obtain a high-affinity aptamer specifically binding to BDNF, the present invention performs 12 rounds of screening after fixing BDNF on the surface of magnetic beads by EDC/NHS chemical coupling. To increase the efficiency of the screening, reverse magnetic beads were introduced for negative screening from round 6 and were incubated with competing reverse targets (ATP, GTX, HSA, BSA, PDGF and ANGPTL4) in a forward system in steps to further increase the specificity of the screening.

The specific screening process is as follows: (1) 50 μ L of BDNF magnetic beads were washed with selection buffer (containing 2mM MgCl) ₂ PBS, pH 7.2) was washed several times and blocking was performed by adding blocking buffer (screening buffer containing 0.1mg/mL yeast tRNA and 1mg/mL BSA). (2) And dissolving the ssDNA library in a screening buffer, carrying out water bath at 95 ℃ for 10min, carrying out ice bath quenching for 5min, standing at room temperature for 10min, adding the ssDNA library into the sealed magnetic beads, and carrying out low-speed rotation incubation at room temperature. (3) After the incubation was completed, the cells were rinsed several times with a screening buffer to remove unbound ssDNA, 100. mu.L of enzyme-free water was added, and after a water bath at 95 ℃ for 10min, ssDNA specifically bound to BDNF was recovered. (4) Performing PCR amplification by using the eluted ssDNA as a template, wherein the reaction system comprises: 10 μ L of Hot start premix (5 ×); 2.5. mu.L of upstream and downstream primers (10. mu.M); 5 μ L of template; finally, sterile water was added to replenish the system to 50 μ L for a total of 40 tubes. The amplification conditions were: pre-denaturation at 94 ℃ for 1 min; denaturation at 95 ℃ for 30 s; annealing at 60 ℃ for 30 s; stretching at 72 ℃ for 30 s; finally, the temperature is 72 ℃, and the elongation is carried out for 2min(ii) a For a total of 20 cycles. (5) Adding a urea-denatured loading buffer solution into the amplified PCR library, carrying out water bath at 95 ℃ for 10min, carrying out ice bath quenching for 5min, standing at room temperature for 5min, loading the sample into 12% urea-denatured polyacrylamide gel pores, and carrying out electrophoresis at a constant voltage of 300V. (6) After the electrophoresis was finished, 20ml of ddH was added to a clean dish ₂ And O and 5 mu L of nucleic acid fluorescent dye, fully and uniformly mixing, placing the gel in the gel, slightly shaking on a horizontal shaking table, and dyeing for 10-20 min. (7) The polyacrylamide gel was imaged on a gel imaging system, the lower ssDNA library was recovered by cutting the gel into 2mL tubes, and 1.5mL ddH was added ₂ And O, boiling the gel in boiling water for 30min, and centrifuging to recover supernatant. (8) By passing

The kit recovers ssDNA from the purified supernatant and redissolves it in the screening buffer for the next round of screening.

The screening process was repeated as above, and by round 12 recovery of ssDNA increased significantly and entered a plateau (fig. 2). Thus, the screening was stopped and the enriched library was subjected to cloning, sequencing and multiple sequence alignment analysis to obtain aptamers F1 and F2.

Example 3: measurement of intermolecular interactions by biofilm interference technique

The biofilm interference technique is a real-time analysis method of intermolecular interaction. The principle is that white light is emitted to the surface of the sensor by the instrument, the light is reflected after passing through a biological film layer of the sensor, and the frequency of the reflected light is influenced by the thickness of the biological film layer. Some frequencies of reflected light interfere constructively with incident light, while others undergo destructive interference. The interference light waves are detected by the spectrometer to form an interference spectrum, and the interference spectrum is displayed by the relative displacement intensity of the interference spectrum. Thus, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer detects a shift in the interference spectrum in real time, which directly reflects the change in the biofilm thickness on the sensor surface. When the nucleic acid aptamer fixed on the sensor biomembrane interacts with BDNF in a solution, the thickness of the biomembrane layer is changed, so that relative displacement is generated, the relative displacement is increased and decreased along with the increase and decrease of the binding capacity of the BDNF, and finally, an equilibrium state is reached, and a corresponding binding curve, a dissociation curve and an affinity constant are given in real time.

The specific implementation process is as follows: (a) dissolving the biotin-labeled aptamer by using a screening buffer solution, then carrying out water bath at 95 ℃ for 10min, carrying out ice bath quenching for 5min, and standing at room temperature for 10min to promote the formation of a stable spatial structure; (b) respectively adding 200 mu L of screening buffer solution, aptamer, BDNF protein and screening buffer solution into a 96-well plate in sequence; (c) the streptavidin-coated sensor is sequentially immersed into each reaction well according to the program set by the instrument, and is subjected to sensor equilibrium, aptamer solidification, rinsing, BDNF combination and dissociation. As a result, as shown in FIGS. 3 and 4, the binding affinity constants of the aptamers F1 and F2 to BDNF were 1.32 and 7.31nM, respectively.

Example 4 aptamer optimization and identification

In order to further improve the performance of aptamers, optimization strategies such as sequence truncation and dimer construction were introduced separately. As shown in FIG. 5, the aptamer F1 formed a G-tetramer structure and F2 had a double stem loop structure, based on QGRS and MFold predictions. By truncating the redundant sequences at both ends of the aptamer, core structures F1T and F2T were generated and they maintained a binding strength consistent with BDNF of 4.98 and 4.18nM, respectively. In order to obtain the aptamer with extremely high affinity, the invention connects F1T and F2T through a T30 joint, and obtains F1T-1T, F1T-2T and F2T-2T dimeric aptamer respectively. However, the binding affinity of F1T-2T and F2T-2T to BDNF is similar to that of its monomer (still at nanomolar level), which is probably due to steric hindrance limiting simultaneous folding or binding of the dimeric structure, resulting in a dynamic switching process for monovalent binding. Notably, F1T-1T has 110 times greater binding affinity for BDNF than its monomeric F1T (K) _D 45pM, fig. 6), indicating that dimer F1T-1T can significantly increase the binding strength to BDNF by virtue of its pattern of bivalent binding.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

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Claims

1. The nucleotide sequences of a group of high affinity aptamers specifically binding with the brain-derived neurotrophic factor are respectively shown as SEQ ID No. 1-SEQ ID No. 7.

2. The use of the high affinity aptamer of claim 1 that specifically binds to brain-derived neurotrophic factor for the preparation of a brain-derived neurotrophic factor detection reagent, kit, or sensor.

3. Use of the high affinity aptamer specific for binding to brain-derived neurotrophic factor according to claim 1 in the preparation of a brain-derived neurotrophic factor capture, isolation and purification formulation.

4. Use of the high affinity aptamer according to claim 1 that specifically binds to brain-derived neurotrophic factor for the development of a novel method for the early rapid diagnosis of glaucoma.

5. The use of claim 4, wherein the novel method for the rapid early diagnosis of glaucoma is based on the high affinity binding of the aptamer to the brain-derived neurotrophic factor, and is used for the rapid early diagnosis of glaucoma.

6. Use of the high affinity aptamer according to claim 1, which specifically binds to a brain-derived neurotrophic factor, for the preparation of a glaucoma early diagnosis reagent or kit.

7. The use of claim 6, wherein the early diagnosis reagent or the kit detects the concentration of the brain-derived neurotrophic factor in the body fluid by the high affinity binding of the aptamer to the brain-derived neurotrophic factor, and is used for the early and rapid diagnosis of glaucoma.

8. The use of the high affinity aptamer specific for binding to brain-derived neurotrophic factor according to claim 1 for the construction of a targeted delivery system for ocular diseases.

9. The use according to claim 8, wherein the targeted drug delivery system is based on the recognition of the strong specificity of the aptamer for brain-derived neurotrophic factor for targeted delivery and site-directed release of drugs.

10. Use of the high affinity aptamer according to claim 1, which specifically binds to a brain-derived neurotrophic factor, for the preparation of a medicament for the treatment of glaucoma.