CN110684774A - Aptamer specifically binding to DEK protein and application thereof - Google Patents

Abstract

The invention relates to the research field of rheumatoid arthritis drugs, in particular to a nucleic acid aptamer specifically binding DEK protein and application thereof. The aptamer provided by the invention has obviously improved stability on the basis of retaining high-affinity binding with DEK protein. The aptamer can inhibit the ability of neutrophils to form NETs by binding with DEK protein, thereby inhibiting inflammation. The nucleic acid aptamer has wide application prospect, is expected to become a novel nucleic acid molecule for treating rheumatoid arthritis, and has great potential in clinical application.

Description

Aptamer specifically binding to DEK protein and application thereof

Technical Field

The invention relates to the research field of rheumatoid arthritis drugs, in particular to a nucleic acid aptamer specifically binding DEK protein and application thereof.

Background

Rheumatoid Arthritis (RA) is a chronic immunological disorder of the whole body characterized by joint pain, swelling, stiffness and destruction of bone and cartilage tissues, even loss of mobility in the critically ill, resulting in severe deterioration of the quality of life and shortened life span of patients. The pathological changes of the rheumatic arthritis are mainly the hyperplasia of synovial cells in joint cavities, inflammatory cell erosion of inflamed joints, angiogenesis in joint cavities, network disorder of inflammatory factors in joint cavities, generation of a series of enzymes, erosion of cartilage and cartilage tissues and joint damage and deformity. At present, the rheumatoid arthritis is controlled by the clinically commonly used medicines, and the rheumatoid arthritis cannot be completely cured. Therefore, the research and development of an efficient, specific and stable treatment method has important significance for preventing and treating the rheumatoid arthritis.

With the continuous and deep research on the pathogenesis of RA, neutrophils play an important role in the local inflammation development of RA joints and the systemic immune response process. Neutrophil Extracellular Traps (NETs) are a network structure containing DNA, histones, myeloperoxidase, Neutrophil elastase, cathepsin G, which are released extracellularly by neutrophils. NETs can externalize inflammatory factors and various autoantigens, are sources of citrullinated autoantigens, can induce the production of ACPA, and activate fibroblast-like synoviocytes and B cells. ACPA and inflammatory factors (TNF-alpha, IL-8 and the like) can stimulate the enhancement of NETosis, so that the vicious circle is generated, and the autoimmune response is gradually increased. Therefore, the inhibition or elimination of abnormal NETs in time can be expected to reduce the inflammatory response of rheumatoid arthritis. The DEK protein is a phosphorylatable nucleoprotein rich in multicellular organisms, is involved in chromatin structure and gene regulation, and can be translocated to cytoplasm and then secreted out of cells. Both phosphorylation and ADP-ribosylpolymerization allow DEK proteins to enter the cell from apoptotic nuclei and become autoantigens. Activated neutrophils at the site of inflammation secrete DEK proteins out of the cell, which is important for the production of NETs. Therefore, the DEK protein is expected to be a novel target for treating rheumatoid arthritis.

Aptamers (aptamers) are single-stranded DNA or RNA molecules capable of tightly binding to a specific target. The aptamer has the advantages of high specificity, low immunogenicity, easiness in large-scale synthesis, reasonable price, almost no or no batch change, good physical stability, easiness in chemical modification and the like, and has wide application prospects in the fields of analysis and diagnosis, medical research and the like.

However, since the unmodified aptamer is easily degraded by nuclease in vivo, the molecular weight is small and the aptamer is easily filtered by the kidney, and the like, the application of the aptamer as a therapeutic agent is limited. Methods for solving this problem include chemical modification of the ribose 2' position of aptamers, nucleic acid backbone modification, nucleic acid end group modification (e.g., PEG, cholesterol), and the like. The treatment of rheumatoid arthritis is a long-term process and unmodified DEK aptamers do not exert good efficacy in vivo. At present, aptamers targeting the DEK protein have not been reported for the treatment of rheumatoid arthritis.

Disclosure of Invention

The invention provides an aptamer specifically binding DEK protein and application thereof, and solves the technical problems that the aptamer in the prior art is low in stability and bioavailability and cannot be used for preparing a medicine for preventing and treating rheumatic arthritis.

The technical scheme of the invention is realized as follows:

an aptamer specifically binding to DEK protein, wherein the aptamer is an optimized aptamer obtained by chemical modification, group modification or/and sequence extension on the basis of the sequence of an original aptamer, and the sequence of the original aptamer is shown as SEQ ID No. 1.

The chemical modification refers to 2' -OMe modification on a nucleic acid aptamer.

The sequence extension refers to the addition of trans-deoxythymidine at the 3' end of the aptamer.

The group modification refers to the addition of cholesterol at the 5' end of the nucleic acid aptamer.

The optimized aptamer sequence is obtained by modifying 2 ' -OMe + 3' end idT at positions 8-13 of SEQ ID No.1 or modifying 2 ' -OMe + 3' end idT at positions 22-27 of SEQ ID No.1 or modifying 2 ' -OMe + 3' end idT at positions 8-13 and 22-27 of SEQ ID No.1 or modifying 2 ' -OMe + 3' end idT at positions 1-41 of SEQ ID No.1 or modifying 5' end cholesterol + 2 ' -OMe + 3' end idT at positions 1-41 of SEQ ID No. 1.

The nucleic acid aptamer specifically binding the DEK protein is applied to preparation of drugs for inhibiting formation of NETs by neutrophils.

The nucleic acid aptamer specifically binding the DEK protein is applied to preparation of drugs for preventing and treating rheumatoid arthritis.

The invention has the beneficial effects that:

(1) the invention carries out optimized modification on the aptamer (the sequence is shown as SEQ ID No: 1) specifically binding DEK, and the modification method is shown as the following table 1:

TABLE 1 methods for modification of DEK aptamers

SEQ ID No.2-SEQ ID No.6 are single-stranded RNA analogues containing 42 basic groups, can spontaneously form a spatial structure to be combined with DEK protein secreted by activated neutrophils in a high specificity manner, and can remarkably inhibit the neutrophils from generating NETs. The aptamer is subjected to chemical modification and group modification, the stability of the aptamer in vivo is improved, the stability is important for the aptamer to be capable of preparing intravenous injection medicines, and the modified aptamer solves the technical problem that the unmodified aptamer medicines cannot treat rheumatoid arthritis in an intravenous injection administration mode.

(2) The invention improves the stability of the aptamer by carrying out 2 ' -OMe modification on the aptamer DTA1 which can be specifically combined with DEK protein and increasing the optimization strategy of 3' -end trans-deoxythymidine and 5' -end cholesterol. Provides a nucleic acid molecule with strong specificity, high stability and high affinity, which is easy to prepare, for inhibiting NETs at inflammatory parts. The nucleic acid aptamer has wide application prospect, and can be used for diagnosis of related diseases such as rheumatoid arthritis and construction of a targeted drug delivery system.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a secondary structure prediction diagram of aptamers targeting DEK proteins.

FIG. 2 is a line graph of the binding kD of aptamer DTA1-DTA1.4 to DEK protein.

FIG. 3 shows the electrophoresis chart of the stability test of aptamer DTA1-DTA1.4 under the in vitro cell culture condition within 6 h.

FIG. 4 is a line graph showing stability test of aptamer DTA1-DTA1.4 under in vitro cell culture conditions for 6 h.

FIG. 5 shows the electrophoresis chart of the stability test of aptamer DTA1-DTA1.4 in 90% mouse serum within 24 h.

FIG. 6 shows the electrophoresis chart of the stability test of aptamer DTA1.4 in 90% mouse serum within 72 h.

FIG. 7 histogram of NET-DNA growth rate of aptamer DTA1-DTA1.4 under PMA stimulation in vitro.

Figure 8 is a line graph of clinical scores of mouse joints after dosing.

FIG. 9 is a micro CT image of mouse joints after administration.

FIG. 10 is a photograph of pathological sections stained for mouse joints H & E after administration.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Measuring the intermolecular interaction by a biomembrane interference technology:

the kinetics of aptamer binding to DEK proteins was determined by forteBIO Octet Red96 biomolecular interaction detection platform. By means of light interference, the inside of the instrument has biosensor with the bottom covered with biological film. When visible light with a certain width is vertically emitted to the biomembrane layer, the light forms interference waves after being reflected by two interfaces of the biomembrane layer. When the immobilized aptamer molecules bind to DEK protein in kinetic buffer, the biofilm thickness increases and binding kinetic data is calculated from the phase shift of the light wave.

All aptamers used in the experiment were biotin-modified, and the obtained biotinylated aptamer powder was diluted to 100nM with kinetic buffer, and the DEK recombinant protein was assigned to Wuhan Huamei bioengineering Co., Ltd. The purchased DEK protein powder was dissolved in an appropriate amount of kinetic buffer and diluted to a range of concentrations of 400, 200, 100, 50, 25, 12.5 nM. Prior to the experiment, 200. mu.l of kinetic buffer, Streptavidin (SA) Dip and Read Biosensors was hydrated in wells corresponding to the sensor positions in the pre-wetted plates for ten minutes. Setting the temperature at 30 ℃, shaking at 1000rpm, detecting a baseline for 5min, fixing the hydrated sensor with the biotinylated aptamer for 5min, and setting the binding time and the dissociation time to be 5 min. Data was converted to 1 using forteBIO Octet data analysis software: 1 in the binding model. Kinetic constants were determined by integrating the experimental data using the equation dR/dt = ka · C · (Rmax-R) -kd · R to obtain ka and kd values simultaneously (R = observed response, Rmax = maximum response at saturation, C = analyte concentration, ka = association rate constant, kd = dissociation rate constant). Then, the dissociation constant KD = KD/ka was derived from the ratio between KD and ka, and as a result, as shown in fig. 2, KD =68.87 ± 4.72 for DTA1, KD =69.33 ± 0.95 for DTA1.1, KD =79.60 ± 7.67 for DTA1.2, KD =73.2 ± 6.77 for DTA1.3, and KD =91.57 ± 7.31 for DTA 1.4. The data show that the KD of the 2 '-OMe modified aptamer (SEQ ID No.2-SEQ ID No. 5) is not greatly improved compared with that of the unmodified aptamer (SEQ ID No. 1), and the data show that the DTA1.1-DTA1.4 aptamer maintains the characteristic of high-affinity binding with DEK protein after 2' -OMe modification.

Example 2

Denaturing urea polyacrylamide gel electrophoresis determination of the stability of aptamer DTA1-DTA1.4 under in vitro cell culture conditions over 6 hours:

the aptamers were diluted to 2.5. mu.M in RPMI1640 medium containing 2% BSA and incubated at 37 ℃ for 0, 1, 2, 4, 4.5, 6 hours, respectively, and after the time of arrival, 3 Xformamide gel loading buffer was added immediately and mixed well with the samples at a ratio of 1:2, and the mixture was stored at-20 ℃.

Assembling a gel glass plate, detecting leakage for 10 min, preparing 12% of denatured urea polyacrylamide gel: 4 g of urea, 4mL of 30% acrylamide and 2mL of 5 XTBE were added to a beaker and dissolved in a 50 ℃ water bath. After dissolving, cooling to room temperature, adding 0.1 mL of 10% ammonium persulfate and 4 mu L of TEMED, mixing uniformly, injecting the glue between glass plates, inserting a comb, and polymerizing for 30 min. Installing an electrophoresis device, filling the electrophoresis tank with 1 xTBE electrophoresis solution, pulling out a comb, sucking the electrophoresis solution by using a liquid transfer device to wash the sample adding hole, setting the voltage to be 110V, and pre-running the gel at 55 ℃ for 30 minutes. After the pre-operation is finished, the sample mixed with the 3 Xformamide gel loading buffer solution is denatured at 95 ℃ for 5min, then placed on ice, immediately loaded, and the loading hole is washed by the electrophoresis solution again before loading. Electrophoresis is carried out for 70min under the condition of 110V, the gel is placed in gelred staining solution for staining for 70min, water washing is carried out for 5min, the gel is photographed by a Bio-Rad gel imager and the strip brightness is analyzed. Percent intact aptamer = (strip intensity at time t/strip intensity at 0 h) × 100%. The electropherograms and the percentage of intact aptamer versus time line plots are shown in fig. 3 and 4, respectively. The results show that the percentage of the whole aptamer accounting for 0h in the 2% BSA RPMI1640 culture medium at the time t is more than 80%, and DTA1.4 has no obvious degradation under the condition.

Example 3

Denaturing urea polyacrylamide gel electrophoresis determination of the stability of aptamer DTA1-DTA1.4 in 90% mouse serum over 24 hours:

aptamers were mixed with mouse serum to give a final aptamer concentration of 2.5 μ M and a final serum concentration of 90%. Incubate at 37 ℃ for 0, 2, 4, 6, 8, 12, 24 hours, respectively, and immediately after the time of arrival, add an equal volume of phenol: chloroform: isoamyl alcohol P: C: I (25: 24: 1), fully shaking, centrifuging at 4 ℃ and 10000RPM for 10 minutes, taking out supernatant, fully mixing the supernatant with 3 times formamide gel loading buffer solution according to the proportion of 2:1, and storing at-20 ℃. Denaturing urea polyacrylamide gel electrophoresis was performed as described in example 2. The electrophorograms are shown in FIG. 5, DTA1, DTA1.1, DTA1.2, DTA1.3 degraded almost completely at 24 hours, whereas DTA1.4 did not degrade significantly under these conditions. The result shows that the aptamer modified by the 2' -methoxyl group at the whole site has better stability in serum.

Example 4

Denaturing urea polyacrylamide gel electrophoresis determination of the stability of aptamer DTA1.4 in 90% mouse serum over 72 hours:

aptamer DTA1.4 was mixed with mouse serum to give a final aptamer concentration of 2.5. mu.M and a final serum concentration of 90%. Incubate at 37 ℃ for 0, 12, 24, 36, 48, 72 hours, respectively. Sample treatment was as described in example 3. The electrophoretogram is shown in FIG. 6. The result shows that the aptamer DTA1.4 with 41 nucleotides modified by 2' -OMe has no obvious degradation in 90% of mouse serum within 72 hours and has excellent stability.

Example 5

Quantitative determination of DNA in cell supernatant NETs DTA1-DTA1.4 Effect in vitro inhibition of NETs:

1. extraction of mouse neutrophils

(1) The femur and tibia were isolated from the mice, soaked in a petri dish with medical alcohol for 30s, and then transferred to a petri dish with RPMI1640 for soaking.

(2) The bone at two sides of the femur of the mouse is cut off by the scissors to be leaked out of the hollow part in the middle, the needle head of the disposable sterile syringe is inserted into the bone, PBS is used for repeatedly and skillfully washing the inside of the bone, and the bone marrow cells in the bone are washed out of the bone until the bone is white and transparent. The 10ml centrifuge tube containing the bone marrow cell suspension was placed in a centrifuge and centrifuged at 200g for 3 minutes at 4 ℃.

(3) Discarding the supernatant, adding 3-5 times of erythrocyte lysate, blowing and beating uniformly, cracking for 1-2 minutes, centrifuging for 5 minutes at 4 ℃ and 450 g. If the cleavage is incomplete, the cleavage step is repeated.

(4) The supernatant was discarded and at least 5 volumes of PBS were added to the cell pellet to resuspend the cells. After centrifugation at 450g for 3 minutes at 4 ℃ and washing 1-2 times, 2ml of PBS was added to the total cell pellet for resuspension.

(5) The method of discontinuous density gradient centrifugation is used for separating and purifying the neutrophils in the bone marrow. 78%, 65% and 55% percoll solutions were slowly added to the centrifuge tube using a 1ml disposable sterile syringe, the cell suspension was aspirated with the syringe, slowly added to the 55% percoll solution along the wall of the centrifuge tube, and centrifuged at 1600g for 30 minutes at 25 ℃.

(6) Cells were aspirated at the interface between 78% and 65%, washed with an equal volume of cold PBS, centrifuged at 1000RPM for 5 minutes at 4 ℃ and repeated three times. Finally, the appropriate amount of RPMI1640(2% BSA) was added for resuspension and counting.

2. Inhibiting formation of NETs

Inoculating fresh neutrophils in a 96-well cell culture plate, incubating for 1h in an incubator, adding each aptamer to make the final concentration of the aptamer respectively 4, 20 and 100nM, incubating for 30min, adding PMA with the final concentration of 100nM, incubating for 4h, carefully discarding cell supernatant, adding 100 μ l of pre-heated medium, carefully washing once, and discarding. Mu.l of 5U/ml Micrococcus nuclease was added, the mixture was incubated in an incubator for 30min, 50. mu.l of 15mM EDTA was added to stop the digestion, and the supernatant was pipetted into a 1.5ml EP tube and centrifuged at 4 ℃ for 5min at 300 g. Sucking the supernatant and storing in a refrigerator at-20 deg.C.

Quantification of NET-DNA

(1) Quantitative determination of content of free DNA in supernatant by using Picogreen ds DNA detection kit

The stock solutions and samples of the standards were diluted with reference to kit instructions and added to a 96-well plate at 100. mu.l/well, 100. mu.l of the diluted dye working solution was added to each well, and the final concentrations of the standards were 1000 ng/ml, 500 ng/ml, 200 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml and 0ng/ml in that order. And (3) keeping out of the sun for 2-5 min at room temperature, detecting the signal intensity by using a fluorescence reading instrument, exciting light to 485nm, and emitting light to 525 nm. In order to prevent fluorescence quenching from being operated as soon as possible, a standard curve is obtained. Mixing the solution to be detected and the dye working solution according to the proportion of 1: 1, mixing, standing in the dark at room temperature for 5min, measuring the fluorescence value, substituting the measured fluorescence value into a regression equation to calculate the dsDNA/NETs content (ng/m 1), and comparing the groups with the condition that the neutrophils generate the NETs as shown in FIG. 7. NET-DNA growth rate (%) after PMA stimulation (%) = (NET-DNA content in each group-NET-DNA content in control group)/NET-DNA content in control group 100%. The results show that DTA1.4 can inhibit the formation of NETs under in vitro cell culture conditions, and the unmodified aptamer DTA1 has no obvious effect.

Example 6

The CIA mouse model tested the anti-inflammatory effects of DTA1 and DTA1.4 in vivo.

Establishment of CIA mouse model

(1) Preparation of emulsions

Fully cleaning the stirring head of the emulsifying machine and disinfecting with 100 ℃ boiled water. 3 mL of complete Freund's adjuvant was added to the beaker and the beaker was placed in an ice bath, at which time the emulsifier was stirred at low speed and 3 mL of type II chicken collagen was added dropwise with stirring. After the dropwise addition, the stirring speed is increased to 18000 rpm, stirring and emulsification are carried out for 5min, and cooling is carried out for 5 min. This procedure was repeated three times to fully emulsify it. The obtained emulsion is stored for later use at 4 degrees.

(2) First immunization of mice

Mice were placed in a holder and the prepared emulsion was injected subcutaneously into the tail root at 100 μ L. Mice were scored clinically every 7 days after the first immunization and the status of the mice was closely observed. The scoring standard of the clinical index of the joint of the mouse is 0 point: no red swelling; 1 minute: slight red swelling of the joints; and 2, dividing: mild redness and swelling of all paws and joints; and 3, dividing: moderate redness and swelling of all paws and joints; and 4, dividing: severe inflammation of all the paw and joints with dysfunction.

(3) Immune enhancement of mice

21 days after the first immunization, an emulsion consisting of incomplete Freund's adjuvant and collagen type II of chicken was prepared, and the mice were injected subcutaneously into the tail roots again. Mice were then clinically scored every 2 days for the next week, with close attention to the mice's morbidity.

(4) CIA mouse dosing regimen

On day 29, mice were evenly divided into 6 groups by score (model control group, intravenous unmodified apt high dose group, intravenous modified apt low dose group, intravenous modified apt high dose group, microneedle delivery modified apt low dose group and microneedle delivery modified apt high dose group) with the average score of each group as consistent as possible. The tail back of the mouse is subjected to depilation treatment on the microneedle administration group.

The treatment was administered on day 30, and the administration was completed by tail vein injection at a dose of 0.8 nmol/tube of 200. mu.l each time. The model control group was injected with an equal amount of physiological saline. The treatment period was on alternate days and mice were scored clinically every other two days until treatment was complete. After treatment, joint tissues are taken and placed in the tissue fixing solution for the micro CT and tissue slice examination.

Clinical score as shown in figure 8, the clinical score increased slowly at a dose of 0.8nmol for DTA1.4 during the treatment period, began to decline after the fifth dose, and was significantly lower after the tenth dose than for the other groups. MicroCT images are shown in FIG. 9, with smooth bone surfaces in the 0.8nmol DTA1.4 treated group, and significant bone erosion in the other three groups. H & E stained pathological section as shown in FIG. 10, the cartilage surface of the 0.8nmol DTA1.4 treated group was relatively smooth and free of significant loose cells or pannus, while the ankle cartilage of the other three groups of mice was severely damaged with dense infiltration of inflammatory cells and dense swelling. The results of the three pictures are consistent, which shows that the unmodified aptamer DTA1 is easy to degrade in serum due to poor stability and has no obvious curative effect, while DTA1.4 can overcome the defect of poor stability of single-stranded DNA, has good anti-inflammatory effect under the dosage of 0.8nmol, and has the potential of treating rheumatoid arthritis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

<110> Zhengzhou university

<120> nucleic acid aptamer specifically binding to DEK protein and application thereof

<160>1

<210>1

<211>41

<212>DNA

<213> Artificial sequence

<220>

<221>misc_difference

<222>（1）…（41）

<400>1

ggggttaaat attcccacat tgcctgcgcc agtacaaata g 41

Claims (7)

1. An aptamer that specifically binds to a DEK protein, characterized in that: the aptamer is an optimized aptamer sequence obtained by chemical modification, group modification or/and sequence extension on the basis of the sequence of an original aptamer, wherein the sequence of the original aptamer is shown as SEQ ID No. 1.

2. The aptamer according to claim 1, which specifically binds to a DEK protein, characterized in that: the chemical modification refers to 2' -OMe modification on a nucleic acid aptamer.

3. The aptamer according to claim 2, which specifically binds to a DEK protein, characterized in that: the sequence extension refers to the addition of trans-deoxythymidine at the 3' end of the aptamer.

4. The aptamer according to claim 2, which specifically binds to a DEK protein, characterized in that: the group modification refers to the addition of cholesterol at the 5' end of the nucleic acid aptamer.

5. The aptamer according to any one of claims 2 to 4, which specifically binds to a DEK protein, wherein: the optimized aptamer sequence is obtained by modifying the position 8-13 of 2 ' -OMe + 3' end idT of SEQ ID No.1 or modifying the position 22-27 of 2 ' -OMe + 3' end idT of SEQ ID No.1 or modifying the position 8-13 and the position 22-27 of 2 ' -OMe + 3' end idT of SEQ ID No.1 or modifying the position 1-41 of 2 ' -OMe + 3' end idT of SEQ ID No.1 or modifying the position 5' of cholesterol +1-41 of 2 ' -OMe + 3' end idT of SEQ ID No. 1.

6. Use of the aptamer specifically binding to DEK protein of claim 5 in the preparation of a medicament for inhibiting the formation of NETs by neutrophils.

7. Use of the nucleic acid aptamer specifically binding to DEK protein according to claim 5 for the preparation of a medicament for the prevention and treatment of rheumatoid arthritis.

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