Alpha-thrombin cyclic NU nucleic acid aptamer, preparation method, antidote and application

Abstract

The invention relates to an alpha-thrombin cyclic NU nucleic acid aptamer, a preparation method, an antidote and application. The preparation method of the alpha-thrombin cyclic NU nucleic acid aptamer comprises the following steps: mixing and annealing an aptamer to be cyclized and a double-hairpin auxiliary chain to obtain a first mixed solution, adding T4 ligase for temperature control reaction to obtain a second mixed solution, concentrating, adding glycerol and formamide for mixing and dissolving to obtain a third mixed solution; and purifying the third mixed solution to obtain the alpha-thrombin cyclic NU nucleic acid aptamer. The invention also provides the alpha-thrombin cyclic NU nucleic acid aptamer prepared by the preparation method. The invention also provides an antidote of the alpha-thrombin cyclic NU nucleic acid aptamer. The problems that the blood half-life period is short and the thermal stability is poor due to the fact that a linear alpha-thrombin NU nucleic acid aptamer is easily hydrolyzed by nuclease are solved. According to the invention, efficient reversion of the anticoagulation effect of the alpha-thrombin cyclic NU nucleic acid aptamer is realized.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to an alpha-thrombin cyclic NU nucleic acid aptamer, a preparation method, an antidote and applications. Background Art

[0002] Aptamers, as a new type of biorecognition molecule, are composed of short-chain DNA or RNA, have a unique three-dimensional folded structure, and can bind to various target molecules with high affinity and specificity. Their application range covers a wide range of fields from simple metal ions, organic dyes, amino acids, peptides, enzymes, proteins, whole cells to complex organisms (such as bacteria and viruses). Compared with traditional antibodies, aptamers show significant advantages, including smaller size, lower immunogenicity, stability over a wide range of pH and temperature, and the ability to be easily synthesized and chemically modified. These advantages together lay the foundation for the widespread application of aptamers in many fields such as biomedicine, diagnostics and biotechnology.

[0003] Especially in the field of biomedicine, targeting the key target of α-thrombin, thrombin aptamers obtained by in vitro SELEX technology screening, such as NU172, have become a hot topic of research. These aptamers not only show the ability to bind thrombin with high affinity, but also stabilize the G-quadruplex region to a certain extent and improve the resistance to enzymatic hydrolysis through their unique double-stranded structure. However, the exposed terminal bases in the double-stranded region of the thrombin aptamer will still be quickly recognized by nucleases, which limits its half-life in the physiological environment and cannot meet the long-term anticoagulation requirements in vivo.

[0004] In order to overcome this challenge, the scientific research community has explored and reported a variety of strategies to extend the in vivo half-life of aptamers. For example, chemical modification of the aptamer skeleton, design of multivalent aptamer structure, and covalent terminal connection of aptamers by introducing exogenous molecules, etc. Although these strategies have improved the in vivo stability of aptamers to a certain extent, they are also accompanied by problems such as high cost, complex preparation process, unsatisfactory yield, and easy introduction of exogenous compound residues, which limit their widespread promotion in practical applications. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide an α-thrombin cyclic NU nucleic acid aptamer, a preparation method and an application thereof, so as to solve the problem that the linear α-thrombin nucleic acid aptamer NU172 is easily hydrolyzed by nucleases, resulting in a short blood half-life and poor thermal stability. Another purpose of the present invention is to provide an antidote and a preparation method thereof, so as to achieve efficient reversal of the anticoagulant effect of the α-thrombin cyclic NU nucleic acid aptamer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] α-thrombin cyclic NU nucleic acid aptamer, wherein the α-thrombin cyclic NU nucleic acid aptamer is cNUDx+y+1;

[0008] The nucleotide sequence of cNUDx+y+1 is shown in SEQ ID NO: 15, and the values ​​of x and y are positive integers.

[0009] Among them, SEQ ID NO: 15 single-stranded nucleic acid:

[0010] (N) x TTTT(N') x (N) y cTAGGTTGGGTAGGGTGGTg(N') y ; x and y are positive integers, N and N' correspond to any base among A, T, C, G; (N) x Sum (N) y Respectively with (N') x and (N') y Complementary pairing.

[0011] The α-thrombin circular NU nucleic acid aptamer is cNUDx+y+1, which is obtained by improving the structure and sequence of the existing linear aptamer NU172. The sequence of NU172 is shown in SEQ ID NO:1.

[0012] Among them, SEQ ID NO: 1 single-stranded nucleic acid: CGCCtaGGttGGgtaGGgtGGtGGCG.

[0013] Among them, the α-thrombin circular NU nucleic acid aptamer cNUD2 corresponds to the α-thrombin circular NU nucleic acid aptamer prepared by the aptamer to be circularized NUD2 and the double hairpin auxiliary chain NUC2.

[0014] The α-thrombin circular NU nucleic acid aptamer cNUD4 corresponds to the α-thrombin circular NU nucleic acid aptamer prepared by the aptamer to be circularized NUD4 and the double hairpin auxiliary chain NUC4.

[0015] The α-thrombin circular NU nucleic acid aptamer cNUD6 corresponds to the α-thrombin circular NU nucleic acid aptamer prepared by the aptamer to be circularized NUD6 and the double hairpin auxiliary chain NUC6.

[0016] The α-thrombin circular NU nucleic acid aptamer cNUD8 corresponds to the α-thrombin circular NU nucleic acid aptamer prepared by the aptamer to be circularized NUD8 and the double hairpin auxiliary chain NUC8.

[0017] The α-thrombin cyclic NU nucleic acid aptamer cNUD10 corresponds to the aptamer to be cyclized NUD10 and the double hairpin auxiliary chain NUC10 to prepare the α-thrombin cyclic NU nucleic acid aptamer. The preparation method of the α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0018] S1, mixing and annealing the aptamer to be circularized and the double-hairpin auxiliary chain to obtain a first mixed solution;

[0019] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition, to obtain a second mixed solution;

[0020] S3, concentrating the second mixed solution under the second temperature condition, and then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution;

[0021] S4. Purify the third mixed solution by gel electrophoresis to obtain α-thrombin cyclic NU nucleic acid aptamer.

[0022] According to the above technical means, by using T4 ligase to carry out a mixed reaction with a rationally designed aptamer to be circularized and a double-hairpin DNA auxiliary chain, the terminal bases of the two linear aptamer nucleic acid sequences are connected in a manner to generate endogenous phosphodiester bonds, thereby constructing a closed circular nucleic acid backbone, i.e., an α-thrombin circular NU nucleic acid aptamer, which has higher thermal stability and blood half-life than linear aptamers, as well as higher affinity for α-thrombin under normal physiological conditions.

[0023] Preferably, in S1, the secondary structure of the aptamer to be cyclized contains a hairpin structure of four T bases, and the hairpin structure contains 1 to 10 base pairs.

[0024] Preferably, in S1, the secondary structure of the double-hairpin auxiliary chain contains two hairpin structures, and the sequence length of the complementary region between the hairpin structure and the aptamer to be circularized is twelve bases.

[0025] Preferably, in S1, the aptamer to be cyclized is NUDx+y+1; x+y+1 represents the number of base pairs of the stem in the hairpin, and the value range of x+y+1 is 1 to 10;

[0026] The nucleotide sequence of NUDx+y+1 is shown in SEQ ID NO:16.

[0027] Among them, SEQ ID NO: 16 single-stranded nucleic acid:

[0028] Phosphate(N) x TTTT(N') x (N) ycTAGGTTGGGTAGGGTGGTg(N') y ; The value range of x is an integer between 0 and 5, and the value range of y is an integer between 0 and 4; N and N' correspond to any base among A, T, C, and G; (N) x Sum (N) y Respectively with (N') x and (N') y Complementary pairing, phosphate means that the 5' end of the sequence is modified with a phosphate group.

[0029] The aptamer to be cyclized NUDx+y+1 indicates that the secondary structure of the aptamer to be cyclized contains a hairpin structure, including a loop of TTTT and a stem of 1 to 10 base pairs.

[0030] Preferably, in S1, when the value of x+y+1 is 2, 4, 6, 8 and 10, the aptamer NUD2 to be cyclized represents a hairpin structure containing four T bases in the secondary structure of the aptamer to be cyclized, and the stem in the hairpin has two base pairs. The nucleotide sequence of the aptamer NUD2 to be cyclized is shown in SEQ ID NO:2.

[0031] The aptamer NUD4 to be cyclized indicates that the secondary structure of the aptamer to be cyclized contains a hairpin structure with four T bases, and the stem of the hairpin has four base pairs. The nucleotide sequence of the aptamer NUD4 to be cyclized is shown in SEQ ID NO:3.

[0032] The aptamer NUD6 to be cyclized indicates that the secondary structure of the aptamer to be cyclized contains a hairpin structure with four T bases, and the stem of the hairpin has six base pairs. The nucleotide sequence of the aptamer NUD6 to be cyclized is shown in SEQ ID NO:4.

[0033] The aptamer NUD8 to be cyclized indicates that the secondary structure of the aptamer to be cyclized contains a hairpin structure with four T bases, and the stem of the hairpin has eight base pairs. The nucleotide sequence of the aptamer NUD8 to be cyclized is shown in SEQ ID NO:5.

[0034] The aptamer NUD10 to be cyclized indicates a hairpin structure containing four T bases in the secondary structure of the aptamer to be cyclized, and the stem of the hairpin has ten base pairs. The nucleotide sequence of the aptamer NUD10 to be cyclized is shown in SEQ ID NO:6.

[0035] Wherein, SEQ ID NO:2 single-stranded nucleic acid: phosphateGttttCCtaGGttGGgtaGGgtGGtG;

[0036] SEQ ID NO:3 Single-stranded nucleic acid: phosphate AGttttCTTCtaGGttGGgtaGGgtGGtGA;

[0037] SEQ ID NO:4 Single-stranded nucleic acid: phosphate CAGttttCTGATCtaGGttGGgtaGGgtGGTgat;

[0038] SEQ ID NO:5 single-stranded nucleic acid: phosphate GTAGttttCTACAGTCtaGGttGGgtaGGgtGGtGACT;

[0039] SEQ ID NO:6 Single-stranded nucleic acid: phosphate GTAGttttCTACAGTCtaGGttGGgtaGGgtGGtGACT.

[0040] Preferably, in S1, the double hairpin auxiliary chain is NUCx+y+1; the nucleotide sequence of NUCx+y+1 is shown in SEQ ID NO: 17;

[0041] Among them, SEQ ID NO: 17 single-stranded nucleic acid: AGTCttatGACTAAA(N') x (N) y CACCAGGACttttGTCC; x is an integer between 0 and 5, and y is an integer between 0 and 4; N and N' correspond to any base among A, T, C, and G; (N) x Sum (N) y Respectively with (N') x and (N') y Complementary pairing.

[0042] The double-hairpin auxiliary chain NUCx+y+1 represents the double-hairpin DNA auxiliary chain corresponding to the aptamer NUDx+y+1 to be circularized.

[0043] Preferably, in S1, when the value of x+y+1 is 2, 4, 6, 8 and 10, the double hairpin auxiliary chain NUC2 represents the double hairpin DNA auxiliary chain corresponding to the aptamer NUD2 to be cyclized. The nucleotide sequence of the double hairpin auxiliary chain NUC2 is shown in SEQ ID NO:7.

[0044] The double hairpin auxiliary chain NUC4 represents the double hairpin DNA auxiliary chain corresponding to the aptamer NUD4 to be circularized. The nucleotide sequence of the double hairpin auxiliary chain NUC4 is shown in SEQ ID NO:8.

[0045] The double hairpin auxiliary chain NUC6 represents the double hairpin DNA auxiliary chain corresponding to the aptamer NUD6 to be circularized. The nucleotide sequence of the double hairpin auxiliary chain NUC6 is shown in SEQ ID NO:9.

[0046] The double hairpin auxiliary chain NUC8 represents the double hairpin DNA auxiliary chain corresponding to the aptamer NUD8 to be circularized. The nucleotide sequence of the double hairpin auxiliary chain NUC8 is shown in SEQ ID NO:10.

[0047] The double hairpin auxiliary chain NUC10 represents the double hairpin DNA auxiliary chain corresponding to the aptamer NUD10 to be circularized. The nucleotide sequence of the double hairpin auxiliary chain NUC10 is shown in SEQ ID NO:11.

[0048] Wherein, SEQ ID NO:7 single-stranded nucleic acid: AGTCttatGACTGaaaaCCACCACCGGACttttGTCC;

[0049] SEQ ID NO: 8 Single-stranded nucleic acid: AGTCttatGACTaaaaCTTCACCACGGACttttGTCC;

[0050] SEQ ID NO:9 Single-stranded nucleic acid: AGTCttatGACTaaaCTGATCACCAGGACttttGTCC;

[0051] SEQ ID NO: 10 Single-stranded nucleic acid: AGTCttatGACTaaCTACAGTCACCGGACttttGTCC;

[0052] SEQ ID NO: 11 Single-stranded nucleic acid: AGTCttatGACTaCTCAGAGATCACGGACttttGTCC.

[0053] Preferably, in S1, the buffer for annealing treatment is 40 mM tris(hydroxymethyl)aminomethane, 1.5 mM magnesium chloride, 5 mM potassium chloride and 140 mM sodium chloride, and the pH value of the buffer is 7.4.

[0054] Preferably, in S1, the annealing treatment procedure is: 95°C for 5 min, 65°C for 30 min, 50°C for 30 min, 37°C for 30 min, 25°C for 30 min, and 4°C for 2 h.

[0055] Preferably, in S1, the molar ratio of the aptamer to be cyclized to the double-hairpin auxiliary chain is 1:1 to 1:2.

[0056] Preferably, in S2, the dosage of T4 ligase is 3-6 KU.

[0057] Preferably, in S2, the first temperature is 16 to 25° C., and the reaction time under the first temperature condition is 10 to 24 hours.

[0058] Preferably, in S3, the second mixed solution is concentrated to 10-30 μL, and the pressure is reduced to -0.05 to -0.1 MP under the second temperature condition, the concentration time is 1 to 2 hours, and the concentration temperature is 30 to 40°C.

[0059] Preferably, in S3, the volume ratio of the concentrated solution, glycerol and formamide is 1:1:4 to 1:1:10.

[0060] The present invention also provides an antidote for the α-thrombin cyclic NU nucleic acid aptamer according to the present invention, wherein the antidote is antiNUDx+y+1;

[0061] The nucleotide sequence of antiNUDx+y+1 is shown in SEQ ID No.18;

[0062] Wherein, SEQ ID NO: 18 single-stranded nucleic acid: aaaa (N') x (N) y CACCACCCTACCCAACC; the value range of x is an integer between 0 and 5, and the value range of y is an integer between 0 and 4; N and N' correspond to any base among A, T, C, and G; (N) x Sum (N) y Respectively with (N') x and (N') y Complementary pairing.

[0063] antiNUDx+y+1 is an antidote for the α-thrombin cyclic NU nucleic acid aptamer cNUDx+y+1.

[0064] The antidote of the present invention can achieve efficient reversal of the anticoagulant effect of the corresponding α-thrombin cyclic NU nucleic acid aptamer.

[0065] The antidote is antiNUDx+y+1, which is completely complementary to the G-quadruplex region sequence of the corresponding α-thrombin cyclic NU nucleic acid aptamer cNUDx+y+1.

[0066] Preferably, when the value of x+y+1 is 2, 4 and 6, antiNUD2 is completely complementary to the G-quadruplex region sequence of the corresponding α-thrombin cyclic NU nucleic acid aptamer cNUD2, and the nucleotide sequence of antiNUD2 is shown in SEQ ID NO: 12. antiNUD4 is completely complementary to the G-quadruplex region sequence of the corresponding α-thrombin cyclic NU nucleic acid aptamer cNUD4, and the nucleotide sequence of antiNUD4 is shown in SEQ ID NO: 13. antiNUD6 is completely complementary to the G-quadruplex region sequence of the corresponding α-thrombin cyclic NU nucleic acid aptamer cNUD6, and the nucleotide sequence of antiNUD6 is shown in SEQ ID NO: 14.

[0067] Wherein, SEQ ID NO: 12 single-stranded nucleic acid: GaaaaCCaCCacCCtacCCaaCCta;

[0068] SEQ ID NO: 13 Single-stranded nucleic acid: GaaaaCttCaCCacCCtacCCaaCCtaga;

[0069] SEQ ID NO: 14 Single-stranded nucleic acid: GaaaaCtgatCaCCacCCtacCCaaCCtagat.

[0070] The present invention also provides an application of the α-thrombin cyclic NU nucleic acid aptamer prepared by the preparation method of the present invention, and an application of the α-thrombin cyclic NU nucleic acid aptamer in the preparation of a coagulation inhibition preparation.

[0071] The present invention also provides an application of an antidote for the α-thrombin cyclic NU nucleic acid aptamer of the present invention, and application of the antidote in restoring blood coagulation.

[0072] Beneficial effects of the present invention:

[0073] 1) The method for preparing the α-thrombin circular NU nucleic acid aptamer of the present invention is to use the designed aptamer to be circularized, the designed double hairpin DNA auxiliary chain and T4 ligase as main raw materials, carry out a mixing reaction, and connect the terminal bases of the two linear aptamer nucleic acid sequences in a manner of generating endogenous phosphodiester bonds to prepare the α-thrombin circular NU nucleic acid aptamer with a purity of more than 99%. It has the advantages of simple operation, low cost and high synthesis efficiency.

[0074] 2) The α-thrombin cyclic NU nucleic acid aptamer of the present invention has the advantages of high thermal stability and long blood half-life, as well as high affinity for cyclic α-thrombin under normal physiological conditions, and is superior to the linear α-thrombin nucleic acid aptamer NU172. It effectively solves the problem that the linear α-thrombin nucleic acid aptamer NU172 is easily hydrolyzed by nucleases, resulting in a short blood half-life and poor thermal stability. Compared with linear aptamers, the α-thrombin cyclic NU nucleic acid aptamer has greater clinical application potential and is suitable for anticoagulant treatment of various diseases and surgeries, thereby providing a new idea for clinical anticoagulation.

[0075] 3) The antidote for the α-thrombin cyclic NU nucleic acid aptamer designed by the present invention can achieve efficient reversal of the anticoagulant effect of the α-thrombin cyclic NU nucleic acid aptamer in vitro, and has promotion and application value in the field of biomaterial technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The figure is a flow chart for the preparation of α-thrombin cyclic NU aptamers;

[0077] Figure 2 This is a 20% dPAGE purity verification result diagram of α-thrombin circular NU nucleic acid aptamer;

[0078] Figure 3 Circular dichroism spectra of α-thrombin cyclic NU aptamer at different temperatures;

[0079] Figure 4 It is the melting curve of α-thrombin circular NU nucleic acid aptamer at wavelengths of 260nm and 295nm.

[0080] Figure 5 The graph is a real-time light scattering detection of the activity of α-thrombin cyclic NU nucleic acid aptamer in inhibiting thrombin (T) conversion of fibrinogen;

[0081] Figure 6 This is the degradation curve of α-thrombin circular NU aptamer in 50% serum. DETAILED DESCRIPTION

[0082] The following will describe the embodiments of the present invention with reference to the preferred embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0083] The present invention aims to provide an α-thrombin cyclic NU nucleic acid aptamer, a preparation method and an application thereof, so as to solve the problem that the linear α-thrombin nucleic acid aptamer NU172 is easily hydrolyzed by nucleases, resulting in a short blood half-life and poor thermal stability.

[0084] Among them, Figure 1 As shown, the preparation method of α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0085] S1, mixing the aptamer to be cyclized with the double-hairpin auxiliary chain, and annealing to obtain a first mixed solution, specifically comprising:

[0086] On the basis of the aptamer NU172, the sequences of its G-quadruplex domain and hinge domain were kept unchanged, the sequence of its double-stranded domain was shortened or extended, and it was redesigned and replaced with a closed hairpin structure, and the double-stranded length of the hairpin structure was designed to be x+y+1bp, and the enzymatic ligation site of T4 ligase was set in the middle of the double-stranded structure, thereby obtaining a rationally designed aptamer to be circularized NUDx+y+1, which is a linear structure at this time. At the same time, based on the secondary structure of NUDx+y+1, a matching double-hairpin auxiliary chain NUCx+y+1 was designed, and its single-stranded region of the non-hairpin structure can complementarily pair with the bases at both ends of the NUDx+y+1 enzymatic site. The designed aptamer to be cyclized NUDx+y+1 and the designed double hairpin auxiliary chain NUCx+y+1 are mixed at a molar ratio of 1:1.1, and then a buffer is added for annealing to obtain a first mixed solution; the buffer is 40mM tris(hydroxymethyl)aminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value of the buffer is 7.4; the annealing procedure is: 95°C for 5min, 65°C for 30min, 50°C for 30min, 37°C for 30min, 25°C for 30min, and 4°C for 2h;

[0087] The value range of x is an integer between 0 and 5, and the value range of y is an integer between 0 and 4;

[0088] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition to obtain a second mixed solution, specifically comprising:

[0089] Add T4 ligase to the first mixed solution obtained in S1, vortex and shake to completely dissolve it, and then place it in a metal bath at 22° C. to control the temperature for 16 hours to obtain a second mixed solution;

[0090] S3, concentrating the second mixed solution, then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution, specifically comprising:

[0091] The second mixed solution obtained in S2 was concentrated under reduced pressure to 10 μL, the pressure in the instrument was reduced to -0.1 MPa, the concentration time was 1 h, and the concentration temperature was 37° C., and then the concentrated solution, glycerol and formamide were mixed in a volume ratio of 1:1:5, and vortexed to mix evenly to obtain a third mixed solution;

[0092] S4, purifying the third mixed solution by gel electrophoresis to obtain α-thrombin cyclic NU nucleic acid aptamer, specifically comprising:

[0093] The α-thrombin cyclic NU nucleic acid aptamer cNUDx+y+1 was obtained after purification by 20% denaturing polyacrylamide gel electrophoresis (dPAGE) and gel recovery.

[0094] Figure 1 In this table, NU / Dx+y+1 and NUDx+y+1 have the same meaning; NU / Cx+y+1 and NUCx+y+1 have the same meaning; cNU / Dx+y+1 and cNUDx+y+1 have the same meaning.

[0095] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer, the preparation method of the α-thrombin cyclic NU nucleic acid aptamer of the present invention will be further described in detail below in combination with specific embodiments and drawings. Obviously, the specific embodiments described are only part of the embodiments of the present application, not all of them. The following description of at least one exemplary embodiment is actually only illustrative and is by no means a limitation to the present application and its applications. Based on the specific embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present application.

[0096] If no specific techniques or conditions are specified in the specific embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0097] Example 1

[0098] like Figure 1 As shown, a method for preparing an α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0099] S1, mixing the aptamer to be cyclized with the double-hairpin auxiliary chain, and annealing to obtain a first mixed solution, specifically comprising:

[0100] On the basis of the aptamer NU172, the sequences of its G-quadruplex domain and hinge domain were kept unchanged, the sequence of its double-stranded domain was shortened or extended, and it was redesigned and replaced with a closed hairpin structure. The double-stranded length of the hairpin structure was designed to be 2bps, and the enzymatic ligation site of T4 ligase was set in the middle of the double-stranded structure, thereby obtaining a rationally designed aptamer NUD2 to be circularized, which is now a linear structure. At the same time, based on the secondary structure of NUD2, a matching double-hairpin auxiliary chain NUC2 was designed, and its single-stranded region of the non-hairpin structure can complementarily pair with the bases at both ends of the NUD2 enzymatic site;

[0101] The designed aptamer NUD2 to be cyclized and the designed double-hairpin auxiliary chain NUC2 are mixed at a molar ratio of 1:1.1, and then a buffer is added for annealing to obtain a first mixed solution; wherein the nucleotide sequence of the designed aptamer NUD2 to be cyclized is shown in SEQ ID NO:2; the nucleotide sequence of the designed double-hairpin auxiliary chain NUC2 is shown in SEQ ID NO:7; the buffer is 40mM tris(hydroxymethyl)aminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value of the buffer is 7.4; the annealing treatment procedure is: 95°C for 5min, 65°C for 30min, 50°C for 30min, 37°C for 30min, 25°C for 30min, and 4°C for 2h;

[0102] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition to obtain a second mixed solution, specifically comprising:

[0103] Add T4 ligase to the first mixed solution obtained in S1, vortex and shake to completely dissolve it, and then place it in a metal bath at 22° C. to control the temperature for 16 hours to obtain a second mixed solution;

[0104] S3, concentrating the second mixed solution, then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution, specifically comprising:

[0105] The second mixed solution obtained in S2 was concentrated under reduced pressure to 10 μL, the pressure in the instrument was reduced to -0.1 MPa, the concentration time was 1 h, and the concentration temperature was 37° C., and then the concentrated solution, glycerol and formamide were mixed in a volume ratio of 1:1:5, and vortexed to mix evenly to obtain a third mixed solution;

[0106] S4, purifying the third mixed solution by gel electrophoresis to obtain α-thrombin cyclic NU nucleic acid aptamer, specifically comprising:

[0107] Purification was performed by 20% denaturing polyacrylamide gel electrophoresis (dPAGE), and the α-thrombin circular NU nucleic acid aptamer cNUD2 was obtained after gel recovery;

[0108] Wherein, SEQ ID NO:2 single-stranded nucleic acid: phosphateCGCCtaGGttGGgtaGGgtGGtGGCG;

[0109] SEQ ID NO: 7 Single-stranded nucleic acid: AGTCttatGACTGaaaaCCACCACCGGACttttGTCC.

[0110] Example 2

[0111] like Figure 1 As shown, a method for preparing an α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0112] S1, mixing the aptamer to be cyclized with the double-hairpin auxiliary chain, and annealing to obtain a first mixed solution, specifically comprising:

[0113] On the basis of the aptamer NU172, the sequences of its G-quadruplex domain and hinge domain were kept unchanged, the sequence of its double-stranded domain was shortened or extended, and it was redesigned and replaced with a closed hairpin structure. The double-stranded length of the hairpin structure was designed to be 4bps, and the enzymatic ligation site of T4 ligase was set in the middle of the double-stranded structure, thereby obtaining a rationally designed aptamer NUD4 to be circularized, which is now a linear structure. At the same time, based on the secondary structure of NUD4, a matching double-hairpin auxiliary chain NUC4 was designed, and its single-stranded region of the non-hairpin structure can complementarily pair with the bases at both ends of the NUD4 enzymatic site;

[0114] The designed aptamer NUD4 to be cyclized and the designed double-hairpin auxiliary chain NUC4 are mixed at a molar ratio of 1:1.1, and then a buffer is added for annealing to obtain a first mixed solution; wherein the nucleotide sequence of the designed aptamer NUD4 to be cyclized is shown in SEQ ID NO:3; the nucleotide sequence of the designed double-hairpin auxiliary chain NUC4 is shown in SEQ ID NO:8; the buffer is 40mM tris(hydroxymethyl)aminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value of the buffer is 7.4; the annealing treatment procedure is: 95°C for 5min, 65°C for 30min, 50°C for 30min, 37°C for 30min, 25°C for 30min, and 4°C for 2h;

[0115] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition to obtain a second mixed solution, specifically comprising:

[0116] Add T4 ligase to the first mixed solution obtained in S1, vortex and shake to completely dissolve it, and then place it in a metal bath at 22° C. to control the temperature for 16 hours to obtain a second mixed solution;

[0117] S3, concentrating the second mixed solution, then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution, specifically comprising:

[0118] The second mixed solution obtained in S2 is concentrated under reduced pressure to 10 μL, the pressure in the instrument is reduced to -0.1 MPa, the concentration time is 1 h, and the concentration temperature is 37° C., and then the concentrated solution, glycerol and formamide are mixed in a volume ratio of 1:1:5, and vortexed to mix evenly to obtain a third mixed solution; S4, the third mixed solution is purified by gel electrophoresis to obtain an α-thrombin cyclic NU nucleic acid aptamer, specifically comprising:

[0119] Purification was performed by 20% denaturing polyacrylamide gel electrophoresis (dPAGE), and the α-thrombin circular NU nucleic acid aptamer cNUD4 was obtained after gel recovery;

[0120] Wherein, SEQ ID NO:3 single-stranded nucleic acid: phosphateAGttttCTTCtaGGttGGgtaGGgtGGtGA;

[0121] SEQ ID NO: 8 Single-stranded nucleic acid: AGTCttatGACTaaaaCTTCACCACGGACttttGTCC.

[0122] Example 3

[0123] like Figure 1 As shown, a method for preparing an α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0124] S1, mixing the aptamer to be cyclized with the double-hairpin auxiliary chain, and annealing to obtain a first mixed solution, specifically comprising:

[0125] On the basis of the aptamer NU172, the sequences of its G-quadruplex domain and hinge domain were kept unchanged, the sequence of its double-stranded domain was shortened or extended, and it was redesigned and replaced with a closed hairpin structure. The double-stranded length of the hairpin structure was designed to be 6bps, and the enzymatic ligation site of T4 ligase was set in the middle of the double-stranded structure, thereby obtaining a rationally designed aptamer NUD6 to be circularized, which is now a linear structure. At the same time, based on the secondary structure of NUD6, a matching double-hairpin auxiliary chain NUC6 was designed, and its single-stranded region of the non-hairpin structure can complementarily pair with the bases at both ends of the NUD6 enzymatic site;

[0126] The designed aptamer NUD6 to be cyclized and the designed double-hairpin auxiliary chain NUC6 are mixed at a molar ratio of 1:1.1, and then a buffer is added for annealing to obtain a first mixed solution; wherein the nucleotide sequence of the designed aptamer NUD6 to be cyclized is as shown in SEQ ID NO:4; the nucleotide sequence of the designed double-hairpin auxiliary chain NUC6 is as shown in SEQ ID NO:9; the buffer is 40mM tris(hydroxymethyl)aminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value of the buffer is 7.4; the annealing treatment procedure is: 95°C for 5min, 65°C for 30min, 50°C for 30min, 37°C for 30min, 25°C for 30min, and 4°C for 2h;

[0127] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition to obtain a second mixed solution, specifically comprising:

[0128] Add T4 ligase to the first mixed solution obtained in S1, vortex and shake to completely dissolve it, and then place it in a metal bath at 22° C. to control the temperature for 16 hours to obtain a second mixed solution;

[0129] S3, concentrating the second mixed solution, then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution, specifically comprising:

[0130] The second mixed solution obtained in S2 is concentrated under reduced pressure to 10 μL, the pressure in the instrument is reduced to -0.1 MPa, the concentration time is 1 h, and the concentration temperature is 37° C., and then the concentrated solution, glycerol and formamide are mixed in a volume ratio of 1:1:5, and vortexed to mix evenly to obtain a third mixed solution; S4, the third mixed solution is purified by gel electrophoresis to obtain an α-thrombin cyclic NU nucleic acid aptamer, specifically comprising:

[0131] Purification was performed by 20% denaturing polyacrylamide gel electrophoresis (dPAGE), and the α-thrombin circular NU nucleic acid aptamer cNUD6 was obtained after gel recovery;

[0132] Wherein, SEQ ID NO:4 single-stranded nucleic acid: phosphateCAGttttCTGATCtaGGttGGgtaGGgtGGTgat;

[0133] SEQ ID NO: 9 Single-stranded nucleic acid: AGTCttatGACTaaaCTGATCACCAGGACttttGTCC.

[0134] Example 4

[0135] like Figure 1As shown, a method for preparing an α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0136] S1, mixing the aptamer to be cyclized with the double-hairpin auxiliary chain, and annealing to obtain a first mixed solution, specifically comprising:

[0137] On the basis of the aptamer NU172, the sequences of its G-quadruplex domain and hinge domain were kept unchanged, the sequence of its double-stranded domain was shortened or extended, and it was redesigned and replaced with a closed hairpin structure. The double-stranded length of the hairpin structure was designed to be 8bps, and the enzymatic ligation site of the T4 ligase was set in the middle of the double-stranded structure, thereby obtaining a rationally designed aptamer to be circularized NUD8, which is a linear structure at this time. At the same time, based on the secondary structure of NUD8, a matching double-hairpin auxiliary chain NUC8 was designed, and its single-stranded region of the non-hairpin structure can complementarily pair with the bases at both ends of the NUD8 enzymatic site;

[0138] The designed aptamer NUD8 to be cyclized and the designed double-hairpin auxiliary chain NUC8 are mixed at a molar ratio of 1:1.1, and then a buffer is added for annealing to obtain a first mixed solution; wherein the nucleotide sequence of the designed aptamer NUD8 to be cyclized is as shown in SEQ ID NO:5; the nucleotide sequence of the designed double-hairpin auxiliary chain NUC8 is as shown in SEQ ID NO:10; the buffer is 40mM tris(hydroxymethyl)aminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value of the buffer is 7.4; the annealing treatment procedure is: 95°C for 5min, 65°C for 30min, 50°C for 30min, 37°C for 30min, 25°C for 30min, and 4°C for 2h;

[0139] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition to obtain a second mixed solution, specifically comprising:

[0140] Add T4 ligase to the first mixed solution obtained in S1, vortex and shake to completely dissolve it, and then place it in a metal bath at 22° C. to control the temperature for 16 hours to obtain a second mixed solution;

[0141] S3, concentrating the second mixed solution, then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution, specifically comprising:

[0142] The second mixed solution obtained in S2 is concentrated under reduced pressure to 10 μL, the pressure in the instrument is reduced to -0.1 MPa, the concentration time is 1 h, and the concentration temperature is 37° C., and then the concentrated solution, glycerol and formamide are mixed in a volume ratio of 1:1:5, and vortexed to mix evenly to obtain a third mixed solution; S4, the third mixed solution is purified by gel electrophoresis to obtain an α-thrombin cyclic NU nucleic acid aptamer, specifically comprising:

[0143] Purification was performed by 20% denaturing polyacrylamide gel electrophoresis (dPAGE), and the α-thrombin circular NU nucleic acid aptamer cNUD8 was obtained after gel recovery;

[0144] Wherein, SEQ ID NO:5 single-stranded nucleic acid: phosphateGTAGttttCTACAGTCtaGGttGGgtaGGgtGGtGACT;

[0145] SEQ ID NO: 10 Single-stranded nucleic acid: AGTCttatGACTaaCTACAGTCACCGGACttttGTCC.

[0146] Example 5

[0147] like Figure 1 As shown, a method for preparing an α-thrombin cyclic NU nucleic acid aptamer comprises the following steps:

[0148] S1, mixing the aptamer to be cyclized with the double-hairpin auxiliary chain, and annealing to obtain a first mixed solution, specifically comprising:

[0149] On the basis of the aptamer NU172, the sequences of its G-quadruplex domain and hinge domain were kept unchanged, the sequence of its double-stranded domain was shortened or extended, and it was redesigned and replaced with a closed hairpin structure. The double-stranded length of the hairpin structure was designed to be 10bps, and the enzymatic ligation site of T4 ligase was set in the middle of the double-stranded structure, thereby obtaining a rationally designed aptamer NUD10 to be circularized, which is now a linear structure. At the same time, based on the secondary structure of NUD10, a matching double-hairpin auxiliary chain NUC10 was designed, and its single-stranded region of the non-hairpin structure can complementarily pair with the bases at both ends of the NUD10 enzymatic site;

[0150] The designed aptamer NUD10 to be cyclized and the designed double-hairpin auxiliary chain NUC10 are mixed at a molar ratio of 1:1.1, and then a buffer is added for annealing to obtain a first mixed solution; wherein the nucleotide sequence of the designed aptamer NUD6 to be cyclized is shown in SEQ ID NO:6; the nucleotide sequence of the designed double-hairpin auxiliary chain NUC10 is shown in SEQ ID NO:11; the buffer is 40mM tris(hydroxymethyl)aminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value of the buffer is 7.4; the annealing treatment procedure is: 95°C for 5min, 65°C for 30min, 50°C for 30min, 37°C for 30min, 25°C for 30min, and 4°C for 2h;

[0151] S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition to obtain a second mixed solution, specifically comprising:

[0152] Add T4 ligase to the first mixed solution obtained in S1, vortex and shake to completely dissolve it, and then place it in a metal bath at 22° C. to control the temperature for 16 hours to obtain a second mixed solution;

[0153] S3, concentrating the second mixed solution, then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution, specifically comprising:

[0154] The second mixed solution obtained in S2 is concentrated under reduced pressure to 10 μL, the pressure in the instrument is reduced to -0.1 MPa, the concentration time is 1 h, and the concentration temperature is 37° C., and then the concentrated solution, glycerol and formamide are mixed in a volume ratio of 1:1:5, and vortexed to mix evenly to obtain a third mixed solution; S4, the third mixed solution is purified by gel electrophoresis to obtain an α-thrombin cyclic NU nucleic acid aptamer, specifically comprising:

[0155] Purification was performed by 20% denaturing polyacrylamide gel electrophoresis (dPAGE), and the α-thrombin circular NU nucleic acid aptamer cNUD10 was obtained after gel recovery;

[0156] Wherein, SEQ ID NO:6 single-stranded nucleic acid: phosphateGTAGttttCTACAGTCtaGGttGGgtaGGgtGGtGACT;

[0157] SEQ ID NO: 11 Single-stranded nucleic acid: AGTCttatGACTaCTCAGAGATCACGGACttttGTCC.

[0158] Detection and analysis

[0159] 1) Purity verification

[0160] The purity of the α-thrombin circular NU nucleic acid aptamers prepared in Examples 1 to 5 and the pre-ordered control linear aptamer NU172 was verified by 20% denaturing polyacrylamide gel electrophoresis (dPAGE). The verification results are as follows: Figure 2 shown.

[0161] The nucleotide sequence of the control linear aptamer NU172 is shown in SEQ ID NO:1.

[0162] SEQ ID NO: 1 Single-stranded nucleic acid: CGCCtaGGttGGgtaGGgtGGtGGCG.

[0163] from Figure 2 It can be seen from the analysis that the purity of the α-thrombin cyclic NU nucleic acid aptamers prepared in Examples 1 to 5 is above 99%.

[0164] 2) Circular dichroism analysis of α-thrombin cyclic NU aptamer

[0165] The α-thrombin cyclic NU nucleic acid aptamer prepared in Examples 1 to 5, as well as the pre-ordered control linear aptamer NU172, were recorded with a micro-quartz cuvette on a Jasco J-1500 spectropolarimeter equipped with a temperature control system for circular dichroism (CD) spectra. The scanning parameters are as follows: the scanning range is set to 220–320 nm, the data spacing is set to 1 nm, the bandwidth is set to 2 nm, the response time is set to 4 s, the temperature is set to 25°C, 37°C and 50°C, respectively, the scanning speed is set to 100 nm / min, and a baseline correction is performed. The concentration of the samples to be tested is 10 μM, and they are temporarily stored at 4°C after programmed annealing. The results of the CD scanning results after data processing are as follows. Figure 3 shown.

[0166] from Figure 3 It can be seen from the analysis that the CD curves of the α-thrombin cyclic NU aptamers prepared in Examples 1 to 5 at different temperatures all have strong peak signals. Among them, the CD curve peak signals of the α-thrombin cyclic NU aptamer cNUD6 prepared in Example 3 at different temperatures are stronger than those of other cyclic aptamers and linear aptamers, indicating that the structure of the α-thrombin cyclic NU aptamer cNUD6 prepared in Example 3 is the most stable.

[0167] 3) Melting curve analysis of α-thrombin circular NU aptamer

[0168] The α-thrombin circular NU nucleic acid aptamer prepared in Examples 1 to 5, and the pre-ordered control linear aptamer NU172, were tested for UV absorption using a conventional cuvette on a UV spectrophotometer equipped with a temperature control system. Select the Thermal mode. The scanning parameters are as follows: the scanning rate is set to 1°C / min, the temperature range is set to 25-95°C, and the absorbance changes at wavelengths of 260nm and 295nm are recorded respectively. The concentration of the samples to be tested is 2μM, and they are temporarily stored at 4°C after programmed annealing. The melting curve measured in the Thermal mode is shown in the figure. Figure 4 As shown, the melting temperature (T m The values ​​are shown in Table 1.

[0169] Table 1. Melting temperature T m value

[0170] Aptamers <![CDATA[T m Value(℃)]]> NU172 43.2 cNUD2 37.3 cNUD4 46.5 cNUD6 47.6 cNUD8 45.7 cNUD10 46.1

[0171] from Figure 4 From the comprehensive analysis in Table 1, it can be seen that the α-thrombin cyclic NU aptamers prepared in Examples 1 to 5 all have relatively high melting temperatures. Among them, the α-thrombin cyclic NU aptamer cNUD6 prepared in Example 3 has the highest melting temperature, indicating that the α-thrombin cyclic NU aptamer cNUD6 prepared in Example 3 has the highest thermal stability.

[0172] 4) Affinity analysis of α-thrombin cyclic NU aptamers

[0173] An isothermal titration calorimeter was used to measure the affinity of the α-thrombin cyclic NU nucleic acid aptamer cNUD6 prepared in Example 3 and the pre-ordered control linear aptamer NU172 to α-thrombin. The titration parameters were as follows: the sample pool temperature was set to 37°C, the number of titrations was set to 20 drops, the interval time was set to 120 seconds, the syringe speed was set to 1000r / min, the system compensation power was set to 5μcal / s, and the baseline running time before the formal start of the titration was set to 60 seconds. The titration concentration was 30μM aptamer titrated 2μM α-thrombin. The results of the binding affinity determination of the aptamer and thrombin are shown in Table 2.

[0174] Table 2. Affinity K d value

[0175] Aptamers <![CDATA[K d (nM)]]> NU172 305 cNUD6 130

[0176] From the analysis in Table 2, it can be seen that the affinity of the α-thrombin circular NU nucleic acid aptamer cNUD6 prepared in Example 3 of the present invention to thrombin is 130, which is higher than that of its corresponding linear aptamer NU172.

[0177] 5) Study on the ability of α-thrombin circular NU aptamer to inhibit thrombin activity

[0178] A 0.4 mg / mL fibrinogen solution was prepared, and 3 nM of the α-thrombin circular NU nucleic acid aptamer prepared in Examples 1 to 5 and the pre-ordered control linear aptamer NU172 were programmed for annealing, and then mixed with 1.5 nM thrombin in buffer A respectively; in the buffer system environment, the same volume of primary water was used to replace the aptamer and mixed with 1.5 nM thrombin as a blank control, and finally, these groups of mixed solutions were incubated at 37°C for 1 hour and the fluorescence signal changes were immediately measured. The time scan mode (Time scan) was used, the excitation wavelength (Ex) and the emission wavelength (Em) were both set to 650 nm, the excitation light slit was set to 10 nm, the emission light slit was set to 5 nm, the PMT voltage was set to 700 V, the response time was set to 2.0 s, and the scanning time was set to 1200 s. The ability of different aptamers to inhibit thrombin activity was measured by real-time light scattering as shown in FIG. Figure 5 As shown, buffer A is 40 mM tris, 1.5 mM magnesium chloride, 5 mM potassium chloride and 140 mM sodium chloride, with a pH value of 7.4.

[0179] from Figure 5 It can be seen from the analysis that the α-thrombin cyclic NU aptamers prepared in Examples 1 to 5 all have strong thrombin inhibition activity. Among them, the α-thrombin cyclic NU aptamer cNUD6 prepared in Example 3 has the strongest ability to inhibit thrombin activity.

[0180] 6) Serum stability analysis of α-thrombin cyclic NU aptamer

[0181] 1 μg of the α-thrombin circular NU nucleic acid aptamer prepared in Examples 1 to 5 and the pre-ordered control linear aptamer NU172 were programmed for annealing, and then added with equal volumes of serum and mixed, and incubated at 37°C for 0.5, 1, 2, 4, 8, 16, 32, 64 and 96 hours. After the reaction of the samples at each time point was completed, the incubated samples were immediately placed in a -80°C freezer to terminate the reaction. After the samples at all time points were completely reacted, all samples were thawed and an appropriate amount of glycerol was added. The degradation of the aptamer was verified by 20% dPAGE, and the grayscale value of each band in the gel was counted by Image J software. After the percentage conversion with the control band, the degradation curve and half-life of each aptamer in 50% serum were calculated. The results are shown as follows: Figure 6 As shown in Table 3.

[0182] Table 3. Aptamer half-life

[0183]

[0184]

[0185] from Figure 6 From the comprehensive analysis in Table 3, it can be seen that the serum stability and half-life of the α-thrombin cyclic NU nucleic acid aptamers prepared in Examples 1 to 5 are higher than those of the control linear aptamer NU172, among which the α-thrombin cyclic NU nucleic acid aptamer cNUD6 prepared in Example 3 has the highest serum stability and half-life.

[0186] 7) Design of antidote for α-thrombin cyclic NU aptamer and study of its detoxification ability in vitro

[0187] The antidotes antiNUD2, antiNUD4 and antiNUD6 of the α-thrombin cyclic NU nucleic acid aptamers prepared in Examples 1 to 3 were designed respectively using the principle of complementary base pairing, and the nucleotide sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 respectively.

[0188] Wherein, SEQ ID NO: 12 single-stranded nucleic acid: GaaaaCCaCCacCCtacCCaaCCta;

[0189] SEQ ID NO: 13 Single-stranded nucleic acid: GaaaaCttCaCCacCCtacCCaaCCtaga;

[0190] SEQ ID NO: 14 Single-stranded nucleic acid: GaaaaCtgatCaCCacCCtacCCaaCCtagat.

[0191] After programmed annealing, 1 μM of the α-thrombin cyclic NU nucleic acid aptamer prepared in Examples 1 to 3 was mixed with 1 μM thrombin in buffer A and incubated at 37°C for 2 hours. Subsequently, the corresponding antidotes were added at a molar ratio of 1:1, 2:1 and 4:1 to the aptamer, and incubated at 37°C for 1 hour. An appropriate amount of glycerol was added to the mixed solution at the end of the reaction, and the in vitro detoxification ability of the antidote was tested by 8% non-denaturing polyacrylamide gel electrophoresis (nPAGE), and the detoxification efficiency was calculated by gray value, and the percentage was converted with the control, and the results are shown in Table 4. Among them, buffer A is 40mM trishydroxymethylaminomethane, 1.5mM magnesium chloride, 5mM potassium chloride and 140mM sodium chloride, and the pH value is 7.4.

[0192] Table 4. Detoxification efficiency of antidotes

[0193] Aptamers Detoxification rate (%) antiNUD2 97.3 antiNUD4 99.2 antiNUD6 98.7

[0194] From the analysis of Table 4, it can be seen that the antidote antiNUDx+y+1 designed by the present invention has a detoxification efficiency of more than 90% in vitro.

[0195] In summary, the method for preparing the α-thrombin circular NU nucleic acid aptamer of the present invention can prepare the α-thrombin circular NU nucleic acid aptamer with a purity of more than 99% by using the designed aptamer to be circularized, the designed DNA double hairpin auxiliary chain and T4 ligase as main raw materials, and has the advantages of simple operation, low cost and high synthesis efficiency.

[0196] The α-thrombin cyclic NU nucleic acid aptamer of the present invention has the advantages of high thermal stability and long blood half-life, as well as high affinity for α-thrombin under normal physiological conditions, and is superior to the linear α-thrombin nucleic acid aptamer NU172. It effectively solves the problem that the linear α-thrombin nucleic acid aptamer NU172 is easily hydrolyzed by nucleases, resulting in a short blood half-life and poor thermal stability, and provides a new idea for clinical anticoagulation.

[0197] The antidote for the α-thrombin cyclic NU nucleic acid aptamer designed by the present invention can achieve efficient reversal of the anticoagulant effect of the α-thrombin cyclic NU nucleic acid aptamer in vitro, and has promotion and application value in the field of biomaterial technology.

[0198] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. An α-thrombin cyclic NU nucleic acid aptamer, characterized in that: The α-thrombin cyclic NU nucleic acid aptamer is cNUDx+y+1; The nucleotide sequence of cNUDx+y+1 is shown in SEQ ID NO: 15, and the values ​​of x and y are positive integers.

2. The method for preparing the α-thrombin cyclic NU nucleic acid aptamer according to claim 1, characterized in that: The following steps are involved: S1, mixing and annealing the aptamer to be circularized and the double-hairpin auxiliary chain to obtain a first mixed solution; S2, adding T4 ligase to the first mixed solution, reacting under the first temperature condition, to obtain a second mixed solution; S3, concentrating the second mixed solution under the second temperature condition, and then adding glycerol and formamide to mix and dissolve, to obtain a third mixed solution; S4. Purify the third mixed solution by gel electrophoresis to obtain α-thrombin cyclic NU nucleic acid aptamer.

3. The method for preparing the α-thrombin cyclic NU nucleic acid aptamer according to claim 2, characterized in that: In S1, the secondary structure of the aptamer to be cyclized contains a hairpin structure with four T bases, and the hairpin structure includes two pairs of base pairs, four pairs of base pairs and six pairs of base pairs; And / or, in S1, the secondary structure of the double-hairpin auxiliary chain contains two hairpin structures, and the sequence length of the complementary region between the hairpin structure and the aptamer to be circularized is twelve bases.

4. The method for preparing the α-thrombin cyclic NU nucleic acid aptamer according to claim 2, characterized in that: In S1, the aptamer to be cyclized is NUDx+y+1; The nucleotide sequence of NUDx+y+1 is shown in SEQ ID NO:

16.

5. The method for preparing the α-thrombin cyclic NU nucleic acid aptamer according to claim 2, characterized in that: In S1, the double hairpin auxiliary chain is NUCx+y+1; The nucleotide sequence of NUCx+y+1 is shown in SEQ ID NO:

17.

6. The method for preparing the α-thrombin cyclic NU nucleic acid aptamer according to claim 2, characterized in that: In the S1, the annealing buffer is 40 mM tris(hydroxymethyl)aminomethane, 1.5 mM magnesium chloride, 5 mM potassium chloride and 140 mM sodium chloride, and the pH value of the buffer is 7.4; And / or, in S1, the annealing treatment procedure is: 95°C for 5 min, 65°C for 30 min, 50°C for 30 min, 37°C for 30 min, 25°C for 30 min, and 4°C for 2 h.

7. The method for preparing the α-thrombin cyclic NU nucleic acid aptamer according to claim 2, characterized in that: In S1, the molar ratio of the aptamer to be cyclized to the double-hairpin auxiliary chain is 1:1-1:2; And / or, in S2, the amount of T4 ligase used is 3-6 KU; And / or, in S2, the first temperature is 16-25° C., and the reaction time under the first temperature condition is 10-24 h; And / or, in S3, the second mixed solution is concentrated under reduced pressure to 10-30 μL, and the pressure is reduced to -0.05-0.1 MP under the second temperature condition, the concentration time is 1-2 h, and the concentration temperature is 30-40 °C; And / or, in S3, the volume ratio of the concentrated solution, glycerol and formamide is 1:1:4~1:1:

10.

8. An antidote for the α-thrombin cyclic NU nucleic acid aptamer according to claim 1, characterized in that: The antidote is antiNUDx+y+1; The nucleotide sequence of antiNUDx+y+1 is shown in SEQ ID No.

18.

9. An application of the α-thrombin cyclic NU nucleic acid aptamer prepared by the preparation method according to any one of claims 2 to 7, characterized in that: Application of the α-thrombin cyclic NU nucleic acid aptamer in the preparation of a blood coagulation inhibition preparation.

10. A use of the antidote of the α-thrombin cyclic NU nucleic acid aptamer as claimed in claim 1, characterized in that: Use of the antidote in restoring blood coagulation.

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