DNA aptamer inhibitors

Abstract

To provide a means to rapidly and efficiently inhibit the activity of DNA aptamers and avoid side effects caused by aptamer drugs. [Solution] An inhibitor of a DNA aptamer or a DNA aptamer in which one or more thymines in the base sequence of the DNA aptamer are replaced with DNA uracil, comprising uracil DNA glycosylase (UdgX) as the active ingredient.

Description

Technical Field

[0001] The present invention relates to a DNA aptamer inhibitor.

Background Art

[0002] A DNA aptamer is a nucleotide sequence having high binding ability and specificity, and is expected to be used as a molecular recognition element in biosensors and as a pharmaceutical. Especially in the application to pharmaceuticals, it binds to cancer cells, pathogenic viruses, etc., and since it can be easily prepared by chemical synthesis, it is expected to be used as a molecular target drug like an antibody. In addition to antisense pharmaceuticals, the development of aptamer pharmaceuticals is also progressing. However, when applying a DNA aptamer as a therapeutic drug, side effects during use may be a problem. When severe side effects occur, it is important to immediately inhibit the drug efficacy to suppress the side effects and promote detoxification. For example, for thrombosis, etc., an anticoagulant aptamer is expected as a pharmaceutical, but when internal bleeding occurs, it is necessary to quickly weaken its effect. With the rise of nucleic acid pharmaceuticals, it has been proposed that its complementary strand can be used as an inhibitor (Non-Patent Document 1, etc.), and further development of DNA pharmaceuticals is expected, but the efficiency of the complementary strand as an inhibitor is not necessarily high.

[0003] On the other hand, DNA covalent binding proteins are broadly classified into HUH endonucleases (histidine-hydrophobic residue-histidine-endonuclease: HUH-endonuclease) and uracil DNA glycosylase X (UdgX) derived from Mycobacterium smegmatis. However, the former undergoes a reverse reaction during covalent binding to DNA, resulting in less efficient complexation. In contrast, UdgX derived from Mycobacterium smegmatis, reported in recent years, is an enzyme that recognizes and removes uracil on DNA and then forms a glycosidic bond at the abasic site via a histidine residue, without the reverse reaction. In fact, it has been confirmed that the covalent binding of UdgX to DNA is highly efficient and irreversible, and it has been reported that it can be used as a versatile tool to connect nucleic acid molecules and proteins (Patent Document 1, Non-Patent Document 2). However, there are no examples of UdgX being used as an inhibitor of DNA aptamers. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2024 / 048599 [Non-patent literature]

[0005] [Non-Patent Document 1] Nature 2002, 419, 90-94 [Non-Patent Document 2] J. Am. Chem. Soc., 146(6), 4087-4097 (2024) [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a means to rapidly and efficiently inhibit the activity of DNA aptamers and to avoid side effects caused by aptamer drugs. [Means for solving the problem]

[0007] The inventors of this invention have conducted extensive research to solve the above problems and have found that uracil DNA glycosylase X (UdgX) efficiently inhibits the aptamer activity of RE31 mutants, in which thymine in the base sequence of RE31, a thrombin-binding DNA aptamer, is replaced with DNA uracil. Furthermore, they have found that the substitution with DNA uracil does not affect the topology of RE31, that RE31 retains its original function of binding to thrombin and suppressing thrombin activity, and that the substitution with DNA uracil has the secondary effect of improving the function of RE31 itself. This invention was completed based on these findings.

[0008] In other words, the present invention encompasses the following inventions. (1) A DNA aptamer inhibitor containing uracil DNA glycosylase (UdgX) as the active ingredient. (2) The DNA aptamer inhibitor according to (1), characterized in that one or more thymines in the base sequence of the DNA aptamer are replaced with DNA uracil. (3) The DNA aptamer inhibitor according to (1) or (2), wherein the DNA aptamer is a thrombin-binding DNA aptamer. (4) A DNA aptamer inhibitor as described in (3), used to reduce the anticoagulant activity of the DNA aptamer. (5) The DNA aptamer inhibitor according to (1) or (2), wherein the DNA aptamer is a bevacizumab-binding DNA aptamer. (6) A DNA aptamer inhibitor as described in (5), used to reduce the binding of the DNA aptamer to the antibody drug bevacizumab. (7) The DNA aptamer inhibitor according to (1) or (2), wherein the UdgX is a fused UdgX in which intermolecular binding modules are fused. (8) The DNA aptamer inhibitor according to (7), wherein the intermolecular binding module is a spycatcher (SC) and / or a spytag (ST). [Effects of the Invention]

[0009] The present invention provides a DNA aptamer inhibitor that inhibits DNA aptamers used in anticoagulants and antitumor drugs. UdgX, the active ingredient of the DNA aptamer inhibitor of the present invention, binds to DNA aptamers into which DNA uracil has been introduced and forms a complex, thereby reducing the ability of DNA aptamers to bind to target substances even at low temperatures of 25°C or below, and can rapidly and efficiently inhibit DNA aptamers. Therefore, the DNA aptamer inhibitor of the present invention is useful in reducing the efficacy of anticoagulants and antitumor drugs using DNA aptamers and avoiding side effects. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1A shows a schematic diagram of the enzymatic reaction of uracil DNA glycosylase (UdgX). Figure 1B shows a schematic diagram of the inhibition of thrombin-binding aptamer (RE31) by uracil DNA glycosylase (UdgX). [Figure 2] Figure 2 shows a schematic diagram of coagulation inhibition (prolonged coagulation time) by the thrombin-binding DNA aptamer (RE31) in blood coagulation (fibrin thrombus formation). [Figure 3] Figure 3 shows the results of fibrin coagulation tests using RE31 and DNA uracil-substituted RE31 (RE31_T11U, RE31_T12U, RE31_T15U, RE31_T17U, RE31_T20U, RE31_T21U). [Figure 4] Figure 4 shows the CD spectra of RE31 and DNA uracil-substituted RE31 (RE31_T11U, RE31_T12U, RE31_T15U, RE31_T17U, RE31_T20U, RE31_T21U). [Figure 5]Figure 5 shows the results of fibrin clotting tests using RE31 and DNA uracil-substituted RE31 (RE31_T11U, RE31_T12U, RE31_T15U, RE31_T17U, RE31_T20U, RE31_T21U) together with UdgX (-: without UdgX, +: with UdgX). [Figure 6] Figure 6 shows the results of comparing the DNA aptamer inhibition of UdgX or BSA against RE31 and DNA uracil-substituted RE31 (RE31_T21U) by measuring fibrinogen clotting time. [Figure 7] Figure 7 shows the results of fibrin clotting tests using DNA uracil-substituted RE31 (RE31_T21U) and UdgX at each mixing ratio. [Figure 8] Figure 8 shows the results of comparing the aptamer inhibition rates of UdgX or complementary strands against DNA uracil-substituted RE31 (RE31_T21U). [Figure 9] Figure 9 shows the results of comparing the DNA aptamer inhibition of UdgX with a fusion molecule added to RE31 and DNA uracil-substituted RE31 (RE31_T21U) by measuring fibrinogen clotting time (UdgX-TrSC: Truncated SpyCatcher (TrSC)-fused UdgX, UdgX: without fusion molecule, UdgX-ST: SpyTag (ST)-fused UdgX). [Figure 10] Figure 10 shows the results of analyzing the change in the binding affinity of RE31 and RE31 mutant (RE31_T21U) to thrombin before and after binding to UdgX by Alpha Assay. [Figure 11] Figure 11 shows the results of dot blot analysis of aptamer inhibition of UdgX against bevacizumab (Bevacizumab) aptamer (A14#_Original) and DNA uracil-substituted A14#1 (A14#1_T12U). MODE FOR CARRYING OUT THE INVENTION

[0011] Hereinafter, the present invention will be described in detail. 1. DNA Aptamer Inhibitor The DNA aptamer inhibitor of the present invention contains uracil DNA glycosylase (UdgX) as an active ingredient.

[0012] "DNA aptamer inhibition" in the present invention refers to inhibiting the binding of a DNA aptamer to a target substance and suppressing the function of the target substance.

[0013] (Uracil DNA glycosylase: UdgX) Uracil DNA glycosylase (hereinafter referred to as "UdgX") is an enzyme that forms a glycosidic bond at an abasic site via a histidine residue after recognizing and removing uracil from ssDNA (single-stranded DNA: ssDNA) or double-stranded DNA (dsDNA) (Figure 1A; Sang, P. B.; Srinath, T.; Patil, A. G.; Woo, E. J.; Varshney, U., A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily. Nucleic acids research, 2015, 43(17), 8452-8463).

[0014] The UdgX used in this invention can be of any origin as long as it has the above-mentioned DNA aptamer inhibitory activity, but UdgX derived from Mycobacterium (Mycolicibacterium) smegmatis is preferred. The amino acid sequence of UdgX derived from Mycobacterium (Mycolicibacterium) smegmatis is publicly known (NCBI Reference Sequence: WP_011726794.1), and the amino acid sequence is shown in Sequence ID No. 10 of the sequence listing. The UdgX used in this invention may be an amino acid sequence having 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity with the amino acid sequence of Sequence ID No. 10, and such homologous proteins are also included in the UdgX referred to in this invention and can be used in the same manner. For example, UdgX molecules with more than 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 include, but are not limited to, those derived from Mycolicibacterium goodii (NCBI Reference Sequence: WP_214394510.1, 98% sequence identity), Mycolicibacterium mageritense (NCBI Reference Sequence: WP_286215220.1, 98% sequence identity), Mycobacteriaceae bacterium (NCBI Reference Sequence: MBV9639477.1, 96% sequence identity), Rhodococcus ruber (NCBI Reference Sequence: WP_372446897, 97% sequence identity), Mycolicibacterium houstonense (NCBI Reference Sequence: WP_066900809, 97% sequence identity), etc.

[0015] The method for producing UdgX is not particularly limited and may include extraction from living organisms, production by genetic engineering, or production by organic synthesis. In particular, production by genetic engineering is advantageous for industrial production, as the gene encoding UdgX can be isolated from an organism that produces UdgX (for example, Mycobacterium smegmatis mentioned above), and the desired UdgX can be easily produced by genetically modifying a host such as E. coli.

[0016] Furthermore, since UdgX can be fused with an intermolecular binding module to achieve the same DNA aptamer inhibitory effect, it can be used in the form of fused UdgX. Examples of intermolecular binding modules, though not limited to those mentioned above, include spycatchers (SC) and spytags (ST).

[0017] (DNA aptamer) In this invention, "DNA aptamer" refers to single-stranded DNA that specifically binds to a target substance. DNA aptamers are ligand molecules that bind specifically and strongly to a target substance through their three-dimensional structure, as complementary sequences within the DNA oligonucleotide molecule form complementary strands, causing the single-stranded DNA oligonucleotide to form secondary and tertiary structures. By binding with a DNA aptamer, it is possible to suppress the function of the target substance.

[0018] The length of the nucleotides constituting the DNA aptamer targeted by the DNA aptamer inhibitor of the present invention is not limited, but is preferably 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, 40 bases or less, and 20 bases or more, 25 bases or more, or 30 bases or more.

[0019] The DNA aptamers targeted by the DNA aptamer inhibitor of the present invention are preferably DNA aptamer mutants in which one or more thymines in the base sequence are replaced with DNA uracil. The number and position of the bases replaced with DNA uracil are not limited and can be appropriately selected depending on the structure and length of the base sequence of the DNA aptamer.

[0020] For example, in the case of RE31, a thrombin-binding DNA aptamer which is a preferred embodiment of the DNA aptamer in the present invention, it is preferable to replace one thymine at any of the positions 11, 12, 15, 17, 20, and 21 in the base sequence of RE31 (SEQ ID NO: 1) with DNA uracil, more preferably to replace one thymine at any of the positions 11, 12, 20, and 21 with DNA uracil, and even more preferably to replace one thymine at any of the positions 11, 20, and 21 with DNA uracil. Alternatively, two or more thymines at positions 11, 12, 15, 17, 20, and 21 may be replaced with DNA uracil.

[0021] In the present invention, "target substance" refers to a biomolecule to which a DNA aptamer is bound, and the binding of the DNA aptamer inhibits, suppresses, or enhances a specific biological function. Here, specific biological functions include catalytic function or gene expression regulatory function (including control of transcription, translation, transport, etc.), apoptosis regulatory function, and biomolecular interactions such as protein-protein interactions that carry out cell signaling. Examples of target substances include peptides (not particularly limited in amino acid composition, but including oligopeptides, dipeptides, and tripeptides), polypeptides (proteins), nucleic acids, lipids, sugars (including sugar chains), or small molecule compounds, but peptides are preferred, and polypeptides (proteins) are more preferred. If the target substance is a protein, it may be a fusion protein with a tag sequence fused to it. Examples of tag sequences include hexahistidine (His), FLAG, HA, and GFP.

[0022] Examples of target proteins include enzymes, antibodies, antigen proteins, structural proteins, hormones, cell surface receptors, and tumor-associated proteins. More specifically, but not limited to, examples include thrombin and antibody drugs (such as bevacizumab, an anti-VEGF antibody; infliximab, an anti-TNF-α antibody; tocilizumab, an anti-IL-6 antibody; and trastuzumab, an antibody against the HER2 oncogene).

[0023] UdgX, the active ingredient of the DNA aptamer inhibitor of the present invention, forms a complex with the RE31 mutant, in which thymine of the thrombin-binding DNA aptamer RE31 is replaced with DNA uracil, when mixed with the RE31 mutant, and inhibits the thrombin-binding activity of RE31 (Figure 1B). This inhibitory effect has been confirmed by reducing the fibrinogen clotting time, as shown in the examples below. Therefore, since the DNA aptamer inhibitor of the present invention can reduce the anticoagulant activity of thrombin-binding DNA aptamers, it can be used to reduce the efficacy of anticoagulants using DNA aptamers and avoid side effects such as internal bleeding.

[0024] The mixing ratio of DNA aptamer to UdgX is not limited as long as the inhibitory effect of UdgX on the DNA aptamer is obtained, but 1:0.5 to 1:20 is preferred, 1:1 to 1:10 is more preferred, and 1:2 to 1:5 is even more preferred.

[0025] The DNA aptamer inhibitor of the present invention is administered in a therapeutically effective amount to mammals that require a reduction in the efficacy of anticoagulants or antitumor drugs using DNA aptamers. Examples of mammals include humans, dogs, cats, sheep, goats, cattle, horses, and pigs. The specific dosage should be appropriately increased or decreased depending on the route of administration, the age and weight of the recipient, and the nature and severity of adverse events caused by the aptamer drug.

[0026] Possible forms of administration include intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, local, intratumoral, oral, transdermal, rectal, vaginal, nasal, and sublingual administration.

[0027] When formulating the DNA aptamer inhibitor of the present invention, UdgX can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various formulations such as tablets, powders, granules, fine granules, capsules, oral solutions (suspensions, syrups, emulsions, etc.), topical solutions (injections, sprays / aerosols, inhalants, ointments, etc.), injections, drips, and suppositories using methods known in the art. Pharmacologically and pharmaceutically acceptable additives may include, depending on the dosage form and application, formulation bases or carriers, excipients, diluents, binders, lubricants, coatings, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, colorants, etc., which can be appropriately added and prepared into various formulations that can be administered orally or parenterally systemically or locally using various known methods. The DNA aptamer inhibitors of the present invention, prepared in various formulations, can be administered orally or parenterally, systemically or topically. When the DNA aptamer inhibitors of the present invention are administered orally, they may be formulated as tablets, capsules, granules, powders, pills, oral solutions, suspensions, emulsions, syrups, etc., or as a dried product that is redissolved before use. When the DNA aptamer inhibitors of the present invention are administered parenterally, they may be formulated as intravenous injections (including infusions), intramuscular injections, intraperitoneal injections, intrathecal injections, subcutaneous injections, suppositories, etc., and in the case of injection formulations, they may be provided in unit dose ampoules or multi-dose containers.

[0028] When administering the DNA aptamer inhibitor of the present invention, the dosage will vary depending on the symptoms, purpose, age, and method of administration of the recipient, but for adults, the dosage is usually 10 μg / kg to 1 mg / kg, preferably 50 μg / kg to 0.5 mg / kg, of the active ingredient UdgX per day. There are no particular restrictions on the number of administrations, but typically 1 to 5 times a day, preferably 3 to 5 times, can be exemplified. [Examples]

[0029] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0030] The reagents and equipment used in the following examples are shown below. <Reagents> Dulbecco's Phosphate Buffered Saline (DPBS) (Nacalai Tesque) Ultrapure water (In this example, Milli-Q water (MQ) was used as the ultrapure water) Fibrinogen (derived from bovine serum) (Fujifilm Wako Pure Chemical Industries) Thrombin (derived from bovine serum) (Fujifilm Wako Pure Chemical Industries) <Equipment> Circular dichroism dispersion meter J-725 (JASCO) Spectrophotometer U-2900 (HITACHI) Electrophoresis apparatus WSE-1150 (ATTO) Ultra-trace ultraviolet / visible spectrophotometer nanodrop (Thermo Fisher scientific) Chemiluminescence detection device Image Quant 500 (Cytiva) Thermal Cycler TP-650 (Takara) Centrifuge MX-307 (TOMY) Vortex Mixer VTX-3000L (LMS) Thermo minder 50 mini (TAITEC) Electronic balance (Sartorius)

[0031] (Example 1) Introduction of DNA uracil into thrombin-binding aptamer RE31 and evaluation of its effects In this example, DNA uracil was introduced into RE31 in order to bind UdgX to the DNA aptamer RE31, which binds to thrombin, inhibits its enzymatic activity, and has anticoagulant activity (antithrombin activity).

[0032] The effects of DNA uracil introduction on RE31 were evaluated from both a functional and structural perspective. Functionally, anticoagulant activity was assessed by measuring the fibrinogen clotting time. Structurally, changes in topology were evaluated using circular dichroism (CD) spectroscopy.

[0033] <RE31 mutant used for evaluation> The RE31 mutant (hereinafter referred to as "RE31 mutant"), into which DNA uracil was introduced, was synthesized by contract manufacturing at Eurofins Genomics. For experimental use, the mutant was diluted to 10 μM, then heat-treated at 95°C for 5 minutes, and folded by slowly cooling to 30°C at a rate of 1°C per minute. For introduction into the RE31 sequence, thymines within the three loops of RE31 (positions 11, 12, 15, 17, 20, and 21) were selected and substituted with DNA uracil. The sequences of RE31 and the RE31 mutant are shown below (Table 1, bold U: substituted uracil).

[0034] [Table 1]

[0035] 1. Method (1) Measurement of fibrinogen coagulation time The samples (RE31 and RE31 mutant) were diluted with DPBS to a concentration of 0.5 μM. 40 μL of 10 unit / mL thrombin was added to 40 μL of the sample (RE31 and RE31 mutant), followed by 420 μL of DPBS. The mixture was pipetted and incubated at 25°C for 5 minutes. After incubation, 500 μL of 2 mg / mL fibrinogen solution was added, vortexed for 5 seconds, and then the absorbance at 380 nm was measured every second for 5 minutes using a spectrophotometer. Since the absorbance increases with fibrinogen coagulation, a time-dependent absorbance curve was created from the obtained absorbance values. The time at which the rate of increase in absorbance was maximized when differentiated using the absorbance values ​​for 5 seconds before and after the target time was defined as the fibrinogen coagulation time.

[0036] (2) CD spectrum measurement The samples (RE31 and RE31 mutants) were diluted to 1 μM with DPBS and then added to a quartz cell with a path length of 1 cm. The CD spectra from 220 nm to 320 nm were measured using a circular dichroism dispersometer. The measurement conditions were: sensitivity 100 mdeg, scan rate 100 nm / min, bandwidth 1.0 nm, and number of integrations 5 times. The CD spectra of each obtained sequence were determined by subtracting the spectrum obtained with DPBS alone as a blank. The influence on topology was evaluated by comparing the measured spectra with the spectrum of the original RE31 sequence.

[0037] 2.Results (1) Fibrinogen coagulation time Thrombin cleaves fibrinogen to form fibrin, promoting coagulation. When the thrombin-binding aptamer (RE31) binds to thrombin, its activity is suppressed, thus prolonging the time to coagulation (Figure 2).

[0038] Figure 3 shows the results of measuring fibrinogen coagulation time using the above samples. Although there were differences in coagulation time among all the DNA uracil-substituted RE31s, it was shown that the coagulation time increased compared to when no aptamer was added (Figure 3). Therefore, it was shown that RE31 with DNA uracil substituted into the sequence also binds to thrombin and suppresses the coagulation reaction, similar to the original RE31 sequence. Among them, RE31_T21U showed a longer coagulation time than the other DNA uracil-substituted RE31s. This is thought to be because the substitution of T21 with DNA uracil causes the methyl group of T21 to be lost, reducing steric hindrance between T20 and T21, and causing T20 to move towards the thrombin side.

[0039] (2) Topology Figure 4 shows the CD spectrum results for DNA uracil-substituted RE31. The CD spectrum of RE31 with uracil substituted into the sequence almost completely overlaps with that of the original sequence. Therefore, it was confirmed that the substitution of uracil within the sequence did not significantly change the peak wavelength of the spectrum and had no effect on the topology.

[0040] The results above indicate that by selecting thymine, which is structurally similar to uracil, and substituting it with DNA uracil, no effect on the topology of RE31 was observed, and RE31 retained its original function of binding to thrombin and inhibiting its activity. In addition, two RE31 mutants were identified (RE31_T12U and E31_T21U) in which the function of RE31 was enhanced by uracil substitution. This demonstrates that the function of aptamers can be improved by introducing DNA uracil into aptamers or by substituting it with DNA uracil.

[0041] (Example 2) Measurement of fibrinogen coagulation time when UdgX is added In Example 1, the RE31 mutant whose topology and function were confirmed was mixed with UdgX, and the fibrinogen coagulation time was measured to evaluate the aptamer inhibitory effect of UdgX. UdgX-Truncated SpyCatcher (UdgX-TrSC) was used as the UdgX. After recombinant production, UdgX-TrSC was purified by Ni-NTA affinity chromatography and used for testing.

[0042] 1. Method The samples (RE31 and RE31 mutant) were diluted with DPBS to a concentration of 1 μM. 40 μL of 10 unit / mL thrombin was added to 20 μL of the sample (RE31 and RE31 mutant), followed by 420 μL of DPBS. The mixture was pipetted and incubated at 25°C for 5 minutes. After incubation, 20 μL of 5 μM UdgX-TrSC was added, and the mixture was incubated again at 25°C for 5 minutes. Subsequently, 500 μL of 2 mg / mL fibrinogen solution was added, and the mixture was vortexed for 5 seconds. The absorbance at 380 nm was then measured every second for 5 minutes using a spectrophotometer, and an absorbance curve was created from the obtained absorbances. The time at which the rate of increase in absorbance was maximized when the absorbance was differentiated using the absorbances before and after 5 seconds was defined as the fibrinogen coagulation time.

[0043] 2.Results Figure 5 shows the fibrinogen coagulation time with and without UdgX-TrSC addition for DNA uracil-substituted RE31. The data for fibrinogen coagulation time without UdgX-TrSC is the same as that in Figure 3. As shown in Figure 5, a decrease in fibrinogen coagulation time was observed when UdgX-TrSC was added to all DNA uracil-substituted RE31 samples. Therefore, it was shown that UdgX-TrSC inhibited the binding of DNA uracil-substituted RE31 to thrombin, thereby inhibiting the suppression of thrombin activity by RE31.

[0044] On the other hand, RE31_T15U and RE31_T17U showed less reduction in fibrinogen clotting time, indicating that the inhibitory effect on DNA uracil-substituted RE31 varies depending on the binding site of UdgX-TrSC. Since the crystal structure confirms that T15 and T17 in RE31 are located directly opposite the thrombin binding site, it is thought that UdgX binding could not inhibit thrombin binding, resulting in a smaller reduction in fibrinogen clotting time compared to other DNA uracil-substituted RE31.

[0045] Furthermore, RE31_T21U was used as the DNA uracil-substituted RE31, and UdgX or BSA was added as a comparison, and the fibrinogen coagulation test was performed in the same manner as above. With BSA, there was no decrease in coagulation time like with UdgX, and there was no DNA aptamer inhibitory effect (Figure 6).

[0046] (Example 3) Investigation of the mixing ratio of DNA aptamer and UdgX RE31_T21U was used as the DNA uracil-substituted RE31, and the fibrinogen coagulation test was performed in the same manner as in Example 2, with the concentration ratio of UdgX to RE31_T21U (1 μM) changed. The results are shown in Figure 7. As shown in Figure 7, an inhibitory effect was obtained by adding 0.5 to 5 times the amount of UdgX-TrSC relative to the DNA aptamer (RE31_T21U), but the effect was more pronounced when 5 times the amount was added.

[0047] (Example 4) Comparison of complementary strand sequences and the RE31 inhibitory effect of UdgX 1. Method RE31_T21U was used as the DNA uracil-substituted RE31. RE31_T21U was diluted to 1 μM with DPBS. 40 μL of 10 unit / mL thrombin was added to 20 μL of RE31_T21U, followed by pipetting of 420 μL of DPBS, and incubation at 37°C for 5 minutes. After incubation, 20 μL of 5 μM UdgX-TrSC was added, and incubation was performed at 25°C for 1, 3, 5, and 10 minutes. Subsequently, 500 μL of 2 mg / mL fibrinogen solution was added, vortexed for 5 seconds, and the absorbance at 380 nm was measured every second for 5 minutes using a spectrophotometer. An absorbance curve was created from the obtained absorbance values. At this time, the time at which the rate of increase in absorbance was maximum when differentiated using the absorbance values ​​before and after 5 seconds was defined as the fibrinogen coagulation time. At this time, if the clotting time was the same as when the RE31 mutant was not added, the aptamer inhibition rate was defined as 100%, and if the clotting time was the same as when only the RE31 mutant was added, the aptamer inhibition rate was defined as 0%, and the aptamer inhibition rate was calculated accordingly. For comparison, the fibrinogen clotting time was measured in the same manner as above using the complementary chain sequence of RE31 instead of UdgX-TrSC, and the aptamer inhibition rate was calculated.

[0048] 2.Results Figure 8 shows the time-dependent aptamer inhibition rates measured using UdgX-TrSC and complementary strand sequences. The horizontal axis represents the incubation time after adding UdgX-TrSC, and the vertical axis represents the DMA aptamer inhibition rate. UdgX-TrSC showed a 100% inhibition rate at 1 minute after addition. It was confirmed that the 100% inhibition rate was maintained even when the incubation time was extended. On the other hand, when using complementary strand sequences, the inhibition rate was about 13% at 1 minute after addition, and 73% after 10 minutes. Therefore, it was confirmed that the inhibitory effect of UdgX on DNA aptamers is higher and faster than the inhibitory effect using complementary strand sequences.

[0049] (Example 5) Effect of changing the molecule fused to UdgX on the RE31 inhibitory effect For RE31 or RE31_T21U, UdgX and UdgX-ST were added instead of UdgX-TrSC, respectively, and a fibrinogen coagulation test was performed in the same manner as in Example 2. In RE31_T21U, a decrease in coagulation time was observed even when UdgX without fused TrSC was added, confirming that it inhibits the suppression of thrombin activity. Furthermore, it was confirmed that the suppression of thrombin activity was inhibited even when UdgX with fused ST was added (Figure 9). Therefore, it was confirmed that the presence or type of intermolecular binding module fused to UdgX does not affect the aptamer inhibitory effect.

[0050] (Example 6) Analysis of binding affinity of RE31 mutant conjugated with UdgX We analyzed the binding affinity of the RE31 mutant to thrombin using alpha assay with RE31_T21U, which was created by substituting uracil for thymine at position 21 of RE31. We also measured the binding affinity of the RE31 mutant conjugated with UdgX.

[0051] 1. Method RE31_T21U was used as the DNA uracil-substituted RE31. RE31_T21U was diluted with assay buffer to 2 μM. 10 μL of 10 μM UdgX-TrSC was added to 10 μL of RE31_T21U and incubated at 25°C for 30 minutes. After incubation, 7.5 μL of biotinylated oligonucleotides adjusted to 1 μM were mixed in each well and incubated at 25°C for 10 minutes. Subsequently, serial dilutions were performed to final concentrations of 5, 2.5, 1, 0.5, 0.25, 0.2, 0.15, and 0.1 μM. 10 μL / well of the aptamer_UdgX-TrSc mixture and 10 μL of thrombin-fixed acceptor beads (final concentration: 60 μg / mL) were added to a 96-well plate and mixed with a plate shaker for 1 minute, then incubated in the dark for 60 minutes. Subsequently, 10 μL / well of streptavidin donor beads (final concentration: 60 μg / mL) was added, and the mixture was stirred again in a plate shaker for 1 minute, followed by incubation in the dark for 1 hour. After that, chemiluminescence was measured when irradiated with 680 nm excitation light using a plate reader.

[0052] 2.Results The results of the alpha assay analysis are shown in Figure 10. The RE31 mutant showed decreased binding affinity to thrombin before and after binding to UdgX-TrSC. This indicates that the RE31 mutant loses its ability to bind to thrombin upon binding to UdgX.

[0053] (Example 7) Inhibitory effect of UdgX on bevacizumab DNA aptamer 1. Method We investigated whether UdgX could provide inhibitory effects using the bevacizumab aptamer A14#1 and A14#1_T12U, which is A14#1 with the 12th thymine replaced by uracil, as DNA aptamers. The sequences of A14#1 and A14#1_T12U are shown below (Table 2).

[0054] [Table 2]

[0055] A14#1_Original and A14#1_T12U were biotinylated and dot blotted. First, each DNA was diluted to 2 μM in PBS (pH 7.4) and folded. The folded aptamers were diluted to 1 μM. Bevacizumab was diluted to 100 μM, and 1 μL was dropped onto a nitrocellulose membrane for adsorption and fixation. After air drying, the membrane was blocked by incubation with 2% (w / v) BSA solution at room temperature for 1 hour. The membrane was washed with PBS (PBS-T) with 0.005% (v / v) Tween20, and then incubated with aptamer (A14#1_Original and A14#1_T12U) solution (final concentration: 0.1 μM) at room temperature for 1 hour. After washing the membrane, UdgX solution diluted with PBS-T (final concentrations: 2 μM, 1 μM, 0.5 μM, 0.25 μM) was added and incubated for 1 hour. After incubation, the membrane was washed again, NeutrAvidin-HRP (0.2 μg / mL) was added, incubated at room temperature for 1 hour, and then washed again. The HRP substrate was dropped onto the membrane and allowed to stand in the dark for 5 minutes, and chemiluminescence was detected using Image Quant 500.

[0056] The results of the dot blot analysis are shown in Figure 11. It was confirmed that the adapter activity of A14#1_T12U was inhibited by UdgX (Figure 11). Although the bevacizumab aptamer has a stem-loop structure, unlike the G4 aptamer RE31, it was found to be inhibited by UdgX in the same way as the G4 aptamer. [Industrial applicability]

[0057] This technology can be used in the development and clinical application of molecularly targeted drugs using DNA aptamers.

Claims

1. A DNA aptamer inhibitor containing uracil DNA glycosylase (UdgX) as the active ingredient.

2. The DNA aptamer inhibitor according to claim 1, characterized in that one or more thymines in the base sequence of the DNA aptamer are replaced with DNA uracil.

3. The DNA aptamer inhibitor according to claim 1 or 2, wherein the DNA aptamer is a thrombin-binding DNA aptamer.

4. A DNA aptamer inhibitor according to claim 3, used to reduce the anticoagulant activity of the DNA aptamer.

5. The DNA aptamer inhibitor according to claim 1 or 2, wherein the DNA aptamer is a bevacizumab-binding DNA aptamer.

6. A DNA aptamer inhibitor according to claim 5, used to reduce the binding of the DNA aptamer to the antibody drug bevacizumab.

7. The DNA aptamer inhibitor according to claim 1 or 2, wherein the UdgX is a fused UdgX in which intermolecular binding modules are fused.

8. The DNA aptamer inhibitor according to claim 7, wherein the intermolecular binding module is a spycatcher (SC) and / or a spytag (ST).

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