A group of nucleic acid aptamers binding to brain-derived neurotrophic factor with high affinity and their application

Abstract

The present invention relates to the field of ophthalmology, and specifically to a group of nucleic acid aptamers that bind to brain-derived neurotrophic factor (BDNF) with high affinity. The present invention uses SELEX technology to screen and obtain high-affinity nucleic acid aptamers that specifically recognize BDNF, and further improves the performance of nucleic acid aptamers by combining and applying optimization strategies such as truncation and dimerization. The nucleic acid aptamers of the present invention have broad application prospects and can be used for capturing BDNF in a system, detecting BDNF in vivo and in vitro, and developing BDNF therapeutic drugs for related diseases and constructing a targeted drug delivery system. The nucleic acid aptamers of the present invention have great potential for practical applications.

Description

Technical Field

[0001] The invention relates to the field of ophthalmology, in particular to a group of nucleic acid aptamers that bind to brain-derived neurotrophic factor (BDNF) with high affinity and applications thereof. Background Art

[0002] Glaucoma is the world's leading irreversible blinding eye disease. It is a group of neurodegenerative diseases characterized by optic disc atrophy and depression, visual field defects and decreased vision. Therefore, early screening, early diagnosis and early intervention have become important means to reduce the risk of blindness caused by glaucoma.

[0003] At present, the diagnosis of glaucoma mainly relies on intraocular pressure measurement, morphological and functional examinations. However, intraocular pressure varies greatly from patient to patient, and when glaucoma is diagnosed by morphological or functional means, the patient's visual function damage is often more than 50%, which is difficult to meet the needs of early clinical diagnosis.

[0004] Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family and is currently recognized as the most promising marker for early diagnosis of glaucoma. A large number of studies have confirmed that BDNF has a significant dose-dependent relationship with the occurrence and development of glaucoma. By using tear or serum-derived BDNF as a target for early diagnosis of glaucoma, it has the advantages of being non-invasive, convenient, easy to obtain, and highly specific, which can help with the screening, diagnosis, and intervention of glaucoma.

[0005] The problem that needs to be solved urgently is to obtain a biorecognition molecule that specifically binds to BDNF. As a new type of molecular recognition tool, nucleic acid aptamers can specifically recognize and bind to target molecules with high affinity. It is usually obtained from ssDNA or RNA from a nucleic acid molecule library using an in vitro screening technology - Systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers can be used for the capture, detection, in vivo delivery, nucleic acid drug development, etc. of specific targets due to their advantages such as chemical synthesis, easy labeling and modification, wide recognition of targets, low immunogenicity and toxicity, high affinity, and strong specificity. They have broad application prospects in many fields such as analysis, diagnosis, and medicine. Summary of the invention

[0006] The present invention aims to provide a group of high-affinity nucleic acid aptamers that specifically bind to brain-derived neurotrophic factor (BDNF). Another object of the present invention is to provide the group of nucleic acid aptamers for use in preparing BDNF detection reagents, kits or sensors; in preparing preparations for capturing, separating and purifying BDNF; in preparing BDNF diagnostic tools, targeted drug delivery systems, neutralizing or blocking drugs, etc.

[0007] In order to achieve the above objectives, the main technical scheme of the present invention is: (1) a nucleic acid aptamer that specifically binds to BDNF is screened by magnetic bead SELEX technology; (2) the core sequence of the nucleic acid aptamer is obtained by truncation optimization; (3) a dimeric nucleic acid aptamer with extremely high targeting affinity is obtained by constructing a dimer of the nucleic acid aptamer.

[0008] In a first aspect of the present invention, a group of high-affinity nucleic acid aptamers that specifically bind to brain-derived neurotrophic factor (BDNF) are provided, and the nucleotide sequences thereof are shown in SEQ ID No. 1 to SEQ ID No. 7 (Table 1).

[0009] Table 1: Aptamer sequences and their binding affinities

[0010]

[0011] The second aspect of the present invention provides the use of the high-affinity nucleic acid aptamer specifically binding to brain-derived neurotrophic factor (BDNF) as described above in the preparation of a brain-derived neurotrophic factor (BDNF) detection reagent, kit or sensor.

[0012] The third aspect of the present invention provides the use of the high-affinity nucleic acid aptamer that specifically binds to brain-derived neurotrophic factor (BDNF) as described above in the preparation of brain-derived neurotrophic factor (BDNF) capture, separation and purification preparations.

[0013] The fourth aspect of the present invention provides the use of the high-affinity nucleic acid aptamer that specifically binds to brain-derived neurotrophic factor (BDNF) as described above in developing a new method for early rapid diagnosis of glaucoma.

[0014] Furthermore, the novel method for diagnosing eye diseases is based on the high-affinity binding of the above-mentioned nucleic acid aptamer to BDNF, and can be used for early and rapid diagnosis of glaucoma.

[0015] The fifth aspect of the present invention provides the use of the high-affinity nucleic acid aptamer that specifically binds to brain-derived neurotrophic factor (BDNF) as described above in the preparation of an early diagnosis reagent or kit for glaucoma.

[0016] Furthermore, the early diagnosis reagent or kit detects the concentration of BDNF in body fluids through the high-affinity binding of the above-mentioned nucleic acid aptamer to BDNF, and is used for early and rapid diagnosis of glaucoma.

[0017] In a sixth aspect, the present invention provides the use of the high-affinity nucleic acid aptamer specifically binding to brain-derived neurotrophic factor (BDNF) as described above in constructing an eye disease targeted drug delivery system.

[0018] Furthermore, the targeted drug delivery system is based on the strong specific recognition of BDNF by the above-mentioned nucleic acid aptamer and is used for targeted transportation and fixed-point release of drugs.

[0019] In a seventh aspect, the present invention provides use of the high-affinity nucleic acid aptamer specifically binding to brain-derived neurotrophic factor (BDNF) as described above in the preparation of a drug for treating glaucoma.

[0020] Furthermore, the drug can alleviate or treat related clinical diseases mediated by BDNF by neutralizing or blocking BDNF.

[0021] The advantages of the present invention are:

[0022] 1. The present invention fixes BDNF protein on the surface of magnetic beads, screens nucleic acid aptamers through magnetic bead SELEX technology, obtains a group of high-affinity nucleic acid aptamers that specifically bind to BDNF, and further improves the performance of nucleic acid aptamers through the combined application of optimization strategies such as truncation and dimerization.

[0023] 2. As a molecular tool for targeting BDNF, the nucleic acid aptamer of the present invention has the advantages of high affinity, strong specificity, good stability, low immunogenicity, low preparation cost, easy modification and labeling, etc. It can be used for the capture of BDNF in the system, the detection of BDNF in vivo and in vitro, the development of BDNF therapeutic drugs in related diseases, and the construction of targeted drug delivery systems, etc., and has broad application prospects. Therefore, the nucleic acid aptamer of the present invention has great potential for practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Schematic diagram of magnetic bead SELEX technology screening.

[0025] Figure 2 . Recovery of ssDNA bound to BDNF.

[0026] Figure 3 .Binding and dissociation curves of nucleic acid aptamer F1 and BDNF.

[0027] Figure 4 .Binding and dissociation curves of nucleic acid aptamer F2 and BDNF.

[0028] Figure 5 .Prediction diagram of the secondary structure of nucleic acid aptamer.

[0029] Figure 6 .Binding and dissociation curves of dimeric nucleic acid aptamer F1T-1T and BDNF. DETAILED DESCRIPTION

[0030] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0031] The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only.

[0032] Example 1: Construction of nucleic acid aptamer screening library and primers thereof

[0033] 1. Construction of 80-nucleotide ssDNA library

[0034] 5′-AGCAGCACAGAGGTCAGATG-N 40 -CCTATGCGTGCTACCGTGAA-3′ (SEQ ID No. 8); wherein N represents any one of the bases A, T, C, and G. 40 Represents a random sequence of 40 nucleotides in length.

[0035] 2. Primer Construction

[0036] Upstream primer: 5′-AGCAGCACAGAGGTCAGATG-3′ (SEQ ID No. 9);

[0037] Downstream primer 1: 5′-TTCACGGTAGCACGCATAGG-3′ (SEQ ID No. 10);

[0038] Downstream primer 2: 5′-poly(dA20)-Spacer18-TTCACGGTAGCACGCATAGG-3′ (SEQ ID No. 11).

[0039] Example 2: Screening of BDNF nucleic acid aptamers

[0040] like Figure 1 As shown, in order to obtain a high-affinity nucleic acid aptamer that specifically binds to BDNF, the present invention fixed BDNF on the surface of magnetic beads by EDC / NHS chemical coupling, and then performed 12 rounds of screening. In order to improve the efficiency of screening, reverse magnetic beads were introduced from the 6th round for negative screening, and competitive reverse targets (ATP, GTX, HSA, BSA, PDGF and ANGPTL4) were gradually added to the forward system for co-incubation to further improve the specificity of screening.

[0041] The specific screening process is as follows: (1) Take 50 μL of BDNF magnetic beads, rinse several times with screening buffer (PBS containing 2 mM MgCl2, pH 7.2), and then add blocking buffer (screening buffer containing 0.1 mg / ml yeast tRNA and 1 mg / mL BSA) for blocking. (2) Dissolve the ssDNA library in screening buffer, incubate at 95°C for 10 minutes, cool in ice bath for 5 minutes, place at room temperature for 10 minutes, add to the blocked magnetic beads, and incubate at room temperature with low speed rotation. (3) After the incubation, rinse several times with screening buffer to remove unbound ssDNA, then add 100 μL of enzyme-free water, incubate at 95°C for 10 minutes, and recover the ssDNA that specifically binds to BDNF. (4) The eluted ssDNA was used as a template for PCR amplification. The reaction system included: 10 μL of Hotstart premix (5x); 2.5 μL of upstream and downstream primers (10 μM); 5 μL of template; finally, sterile water was added to replenish the system to 50 μL, for a total of 40 tubes. The amplification conditions were: 94°C, pre-denaturation for 1 min; 95°C, denaturation for 30 s; 60°C, annealing for 30 s; 72°C, extension for 30 s; and finally 72°C, extension for another 2 min; for a total of 20 cycles. (5) Urea-denatured loading buffer was added to the amplified PCR library, incubated at 95°C for 10 min, cooled in an ice bath for 5 min, and placed at room temperature for 5 min before loading onto a 12% urea-denatured polyacrylamide gel well and electrophoresing was performed at a constant voltage of 300 V. (6) After electrophoresis, add 20 ml of ddH2O and 5 μL of nucleic acid fluorescent dye to a clean dish, mix thoroughly, place the gel in it, shake gently on a horizontal shaker, and stain for 10-20 minutes. (7) Place the polyacrylamide gel on a gel imaging system for imaging, cut the gel to recover the ssDNA library at the bottom into a 2 mL test tube, add 1.5 mL of ddH2O, boil the gel in boiling water for 30 minutes, and centrifuge to recover the supernatant. (8) The kit recovers ssDNA in the purified supernatant and re-dissolves it in the screening buffer for the next round of screening.

[0042] The above screening process was repeated. By the 12th round, the recovery rate of ssDNA increased significantly and entered a plateau phase ( Figure 2 ). Therefore, the screening was stopped, and the enriched library was cloned, sequenced, and analyzed by multiple sequence alignment to obtain nucleic acid aptamers F1 and F2.

[0043] Example 3: Determination of molecular interactions using biomembrane interferometry

[0044] Biomembrane interferometry is a real-time analysis method for molecular interactions. The principle is that the instrument emits white light to the sensor surface, and the light will be reflected after passing through the biomembrane layer of the sensor, and the frequency of the reflected light will be affected by the thickness of the biomembrane layer. Some frequencies of reflected light will produce constructive interference with the incident light, while others will be destructively interfered. These interfering light waves are detected by the spectrometer to form an interference spectrum, which is displayed as the relative displacement intensity of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the change in the thickness of the biomembrane on the sensor surface. That is, when the nucleic acid aptamer fixed on the biomembrane of the sensor interacts with the BDNF in the solution, the thickness of the biomembrane layer changes, resulting in a relative displacement. This relative displacement increases or decreases with the increase or decrease in the amount of BDNF binding, and finally reaches an equilibrium state and gives the corresponding binding curve, dissociation curve and affinity constant in real time.

[0045] The specific implementation process is as follows: (a) After the biotin-labeled aptamer is dissolved in the screening buffer, it is placed in a 95°C water bath for 10 minutes, ice-cooled for 5 minutes, and left at room temperature for 10 minutes to form a stable spatial structure; (b) 200 μL of screening buffer, aptamer, BDNF protein and screening buffer are added to a 96-well plate in sequence; (c) The streptavidin-coated sensor is immersed in each reaction well in sequence according to the program set by the instrument, and goes through five steps: sensor balancing, aptamer solidification, rinsing, and BDNF binding and dissociation. The results are shown in Figure 2. Figure 3 and Figure 4 As shown, the binding affinity constants of nucleic acid aptamers F1 and F2 to BDNF are 1.32 and 7.31 nM, respectively.

[0046] Example 4. Optimization and identification of nucleic acid aptamers

[0047] In order to further improve the performance of nucleic acid aptamers, optimization strategies such as sequence truncation and dimer construction were introduced. Figure 5As shown, based on the predictions of QGRS and Mfold, the nucleic acid aptamer F1 forms a G-tetramer structure, and F2 has a double stem-loop structure. By truncating the redundant sequences at both ends of the nucleic acid aptamer, the core structures F1T and F2T are generated, and they maintain the same binding strength with BDNF, which are 4.98 and 4.18nM, respectively. In order to obtain a nucleic acid aptamer with extremely high affinity, the present invention connects F1T and F2T through a T30 connector to obtain F1T-1T, F1T-2T and F2T-2T dimeric nucleic acid aptamers, respectively. However, the binding affinity of F1T-2T and F2T-2T to BDNF is similar to that of their monomers (still at the nanomolar level), which may be due to the steric hindrance that limits the simultaneous folding or binding of the dimer structure, resulting in a dynamic conversion process of monovalent binding. It is worth noting that the binding affinity of F1T-1T to BDNF is 110 times that of its monomer F1T (K D 45pM, Figure 6 ), indicating that the dimer F1T-1T can significantly increase the binding strength to BDNF by virtue of its bivalent binding mode.

[0048] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application. Sequence Listing <110> Fudan University Eye, Ear, Nose and Throat Hospital <120> A group of nucleic acid aptamers binding to brain-derived neurotrophic factor with high affinity and their application <130> / <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 40 <212> DNA <213> Artificial <400> 1 tcacgactac aggttggggat aggttggggat aagtagttta 40 <210> 2 <211> 17 <212> DNA <213> Artificial <400> 2 ggttgggata ggttggg 17 <210> 3 <211> 40 <212> DNA <213> Artificial <400> 3 atttagagcg gtggagccgc tttgcgggcg accgctccat 40 <210> 4 <211> 29 <212> DNA <213> Artificial <400> 4 agcggtggag ccgctttgcg ggcgaccgc 29 <210> 5 <211> 64 <212> DNA <213> Artificial <400> 5 ggttgggata ggttgggttt tttttttttt tttttttttt tttttttggt tgggataggt 60 tggg 64 <210> 6 <211> 76 <212> DNA <213> Artificial <400> 6 ggttgggata ggttgggttt tttttttttt tttttttttt tttttttagc ggtggagccg 60 ctttgcgggc gaccgc 76 <210> 7 <211> 88 <212> DNA <213> Artificial <400> 7 agcggtggag ccgctttgcg ggcgaccgct tttttttttt tttttttttt tttttttttt gcggtggagc cgctttgcgg gcgaccgc 88 <210> 8 <211> 80 <212> DNA <213> Manufacturer(Artificial) <220> <221> misc_feature <222> (21)..(60) <223> n is a, c, g, or t <400> 8 agcagcacag aggtcagatg nnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn cctatgcgtg ctaccgtgaa <210> 9 <211> 20 <212> DNA <213> Manufacturer(Artificial) <400> 9 stirring aggtcagatg <210> 10 <211> 20 <212> DNA <213> Manufacturer(Artificial) <400> 10 ttcacggtag cacgcatagg <210> 11 <211> 20 <212> DNA <213> Manufacturer(Artificial) <400> 11 ttcacggtag cacgcatagg

Claims

1. A group of nucleic acid aptamers that specifically bind to brain-derived neurotrophic factor, whose nucleotide sequences are shown in SEQ ID No.1-2 and SEQ ID No.5-6 respectively.

2. Use of the nucleic acid aptamer that specifically binds to brain-derived neurotrophic factor as claimed in claim 1 in the preparation of a brain-derived neurotrophic factor detection reagent, kit or sensor.

3. Use of the nucleic acid aptamer that specifically binds to brain-derived neurotrophic factor as claimed in claim 1 in the preparation of brain-derived neurotrophic factor capture, separation and purification preparations.

4. Use of the nucleic acid aptamer that specifically binds to brain-derived neurotrophic factor as claimed in claim 1 in the preparation of an early diagnostic reagent or kit for glaucoma.

5. The use according to claim 4, characterized in that: The early diagnosis reagent or kit detects the concentration of brain-derived neurotrophic factor in body fluids through the binding effect of the nucleic acid aptamer on brain-derived neurotrophic factor, and is used for early diagnosis of glaucoma.

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