An IL-6 / sIL-6R bispecific circular nucleic acid aptamer and kit and application thereof

Abstract

The application belongs to the field of biological medicine, and relates to an IL-6 / sIL-6R bispecific circular nucleic acid aptamer, a kit and application. The nucleotide sequence of the circular nucleic acid aptamer is selected from at least one of a), b) and c): a) the nucleotide sequence shown in SEQ ID NO:1 and connected into a ring at the 5' end and the 3' end; b) a substitution sequence, wherein the substitution sequence is a nucleotide sequence obtained by substitution of the nucleotide sequence shown in SEQ ID NO:1; and c) a deletion sequence, wherein the deletion sequence is a nucleotide sequence obtained by deletion of the nucleotide sequence shown in SEQ ID NO:1. Compared with a monovalent nucleic acid aptamer, the bispecific nucleic acid aptamer has stronger recognition ability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an IL-6 / sIL-6R bispecific cyclic nucleic acid aptamer, a kit containing the nucleic acid aptamer, and the use of the nucleic acid aptamer and the kit in preparing anti-inflammatory agents and / or immune disease treatment agents. Background Art

[0002] Interleukin-6 (IL-6) is a multifunctional cytokine that plays a key role in many inflammatory and immune diseases, such as rheumatoid arthritis, asthma, and Crohn's disease. IL-6 interacts with a soluble form of the IL-6 receptor (sIL-6R), a process known as the trans-signaling pathway, which primarily regulates proinflammatory responses. Blocking the IL-6 / sIL-6R interaction has been shown to be a promising therapeutic strategy for inhibiting various inflammatory and immune diseases. Notably, the IL-6 / sIL-6R interaction has also been reported to be involved in the progression of non-autoimmune diseases, including the cytokine storm associated with coronavirus disease 2019 (COVID-19). Tocilizumab, an immunosuppressive monoclonal antibody targeting sIL-6R, blocks the IL-6 / sIL-6R interaction and alleviates cytokine release syndrome. It has been used to treat severe COVID-19 patients. However, monoclonal antibody-based therapies often have inherent disadvantages, such as high production costs and frequent immune-related adverse events.

[0003] Aptamers are single-stranded nucleic acid probes that can specifically bind to specific molecular targets through their unique secondary or tertiary structures. They are typically isolated from random nucleic acid libraries through an in vitro selection strategy called systematic evolution of ligands by exponential enrichment (SELEX). To improve screening efficiency, several excellent screening methods have been reported, such as microfluidics, droplet amplification, and expanding library diversity through artificial bases and molecular crowding SELEX. Compared with antibodies, aptamers have advantages such as small size, high reproducibility, reversible denaturation, easy controllable modification, high thermal stability, good conformational flexibility, non-immunogenicity, and adaptability to experimental conditions. Therefore, aptamers have great application potential as antibody alternatives in fields such as disease diagnosis and treatment.

[0004] Despite this, when used as therapeutic oligonucleotides, natural linear aptamers are susceptible to nuclease degradation in biological culture media due to their conformational flexibility and are easily affected by a large number of irrelevant substances in the biological culture medium, which seriously hinders the actual therapeutic application of aptamers. To improve the stability of aptamers in biological environments, people have developed many optimization strategies, including chemical modification and nanoparticle modification of aptamers. However, post-selection modifications can affect the specificity and affinity of aptamers, and the preparation process of nanomaterials is complex and has side effects. These problems have caused considerable obstacles to the subsequent application of nucleic acid aptamers in the biomedical field. Summary of the Invention

[0005] The purpose of the present invention is to provide an IL-6 / sIL-6R bispecific cyclic nucleic acid aptamer.

[0006] To achieve the above objectives, the first aspect of the present invention provides an IL-6 / sIL-6R bispecific circular aptamer (BCA), wherein the nucleotide sequence of the circular aptamer is selected from at least one of a) to c):

[0007] a) the nucleotide sequence shown in SEQ ID NO: 1, with the 5' end and the 3' end connected end to form a loop, wherein the nucleotide sequence shown in SEQ ID NO: 1 is 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGGTATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3';

[0008] b) a substituted sequence, wherein the substituted sequence is a nucleotide sequence obtained by replacing the nucleotide sequence shown in SEQ ID NO: 1, and the substituted sequence has more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 1;

[0009] c) a deletion sequence, wherein the deletion sequence is a nucleotide sequence obtained by deleting the nucleotide sequence shown in SEQ ID NO: 1, and the deletion sequence has more than 85% homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0010] According to the present invention, preferably, the substitutions in the substituted sequence relative to the nucleotide sequence shown in SEQ ID NO: 1 are all T base substitutions.

[0011] According to a preferred embodiment of the present invention, the substituted sequence comprises 1 to 2 substitutions relative to the nucleotide sequence of SEQ ID NO: 1, each substitution comprising 5 to 21 nucleotides. Each substitution may be continuous or discontinuous, with discontinuity occurring when the corresponding position in SEQ ID NO: 1 is originally T.

[0012] Furthermore, the substitution sequence is selected from at least one of b1) to b2):

[0013] b1) the nucleotide sequence shown in SEQ ID NO: 2, wherein the nucleotide sequence shown in SEQ ID NO: 2 is 5'-ATCTCGA CTATTTT TTTTTTT TTTTTTT TTGGGGA GGCTGTG GTGAGGG TTTTTTT TTTTTTTTTTTTTT GTCTCGGAT-3';

[0014] b2) The nucleotide sequence shown in SEQ ID NO: 3, wherein the nucleotide sequence shown in SEQ ID NO: 3 is 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATTTTTT TTTTTTT TTTTTTT TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3'.

[0015] According to another preferred embodiment of the present invention, the interval between the first substitution site and the last substitution site in the substituted sequence relative to the nucleotide sequence shown in SEQ ID NO: 1 is less than or equal to 3 nucleotides, excluding the first substitution site and the last substitution site.

[0016] Furthermore, the substitution sequence is selected from at least one of b3) to b18):

[0017] b3) the nucleotide sequence shown in SEQ ID NO: 4, wherein the nucleotide sequence shown in SEQ ID NO: 4 is 5'-TTTTTGA CTAGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0018] b4) the nucleotide sequence shown in SEQ ID NO: 5, wherein the nucleotide sequence shown in SEQ ID NO: 5 is 5'-ATCTCTT TTTGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0019] b5) the nucleotide sequence shown in SEQ ID NO: 6, wherein the nucleotide sequence shown in SEQ ID NO: 6 is 5'-ATCTCGA CTATTTT GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0020] b6) the nucleotide sequence shown in SEQ ID NO: 7, wherein the nucleotide sequence shown in SEQ ID NO: 7 is 5'-ATCTCGA CTAGTCA TTTTTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0021] b7) the nucleotide sequence shown in SEQ ID NO: 8, wherein the nucleotide sequence shown in SEQ ID NO: 8 is 5'-ATCTCGA CTAGTCA GTGGTTT TTTCCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0022] b8) the nucleotide sequence shown in SEQ ID NO: 9, wherein the nucleotide sequence shown in SEQ ID NO: 9 is 5'-ATCTCGA CTAGTCA GTGGTGG ATATTTT TTGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0023] b9) the nucleotide sequence shown in SEQ ID NO: 10, wherein the nucleotide sequence shown in SEQ ID NO: 10 is 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATTTTTA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTCGGAT-3';

[0024] b10) the nucleotide sequence represented by SEQ ID NO: 11, which is 5'- ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGTTTTTGTGGTGAGGGTATGGTT GTCTCGGATTGGTTGTCTCGGAT-3';

[0025] b11) the nucleotide sequence represented by SEQ ID NO: 12, which is 5'- ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTTTTTTGAGGGTATGGTT GTCTCGGATTGGTTGTCTCGGAT-3';

[0026] b12) the nucleotide sequence represented by SEQ ID NO: 13, which is 5'- ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTGTGGTTTTTTTATGGTT GTCTCGGATTGGTTGTCTCGGAT-3';

[0027] b13) the nucleotide sequence represented by SEQ ID NO: 14, which is 5'- ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTGTGGTGAGGGTTTTTTT GTCTCGGATTGGTTGTCTCGGAT-3';

[0028] b14) the nucleotide sequence represented by SEQ ID NO: 15, which is 5'- ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTGTGGTGAGGGTATGGTT TTTTCGGATTGGTTGTCTCGGAT-3';

[0029] b15) the nucleotide sequence represented by SEQ ID NO: 16, which is 5'- ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTGTGGTGAGGGTATGGTT GTCTTTTTTTGGTTGTCTCGGAT-3';

[0030] b16) the nucleotide sequence shown in SEQ ID NO: 17, wherein the nucleotide sequence shown in SEQ ID NO: 17 is 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTTTTT GTCTCGGAT-3';

[0031] b17) the nucleotide sequence shown in SEQ ID NO: 18, wherein the nucleotide sequence shown in SEQ ID NO: 18 is 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT TTTTCGGAT-3';

[0032] b18) The nucleotide sequence shown in SEQ ID NO: 19, wherein the nucleotide sequence shown in SEQ ID NO: 19 is 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTG GTGAGGG TATGGTT GTCTCGGATTGGTT GTCTTTTTT-3'.

[0033] According to a preferred embodiment of the present invention, the deleted sequence is missing 1-10 nucleotides compared to the nucleotide sequence shown in SEQ ID NO: 1, and the missing 1-10 nucleotides are a continuous sequence or two continuous sequences.

[0034] Furthermore, the deleted sequence is selected from at least one of c1) to c3):

[0035] c1) the nucleotide sequence shown in SEQ ID NO: 20, wherein the nucleotide sequence shown in SEQ ID NO: 20 is 5'-ATCTCGA CTAGTCA GGGATAC CGGATGG GGAGGCT GTGGTGA GGGTATG GTTGTCT CGGATTGGTTGTCT CGGAT-3';

[0036] c2) the nucleotide sequence shown in SEQ ID NO: 21, wherein the nucleotide sequence shown in SEQ ID NO: 21 is 5'-ATCTCGA CTAGTCA GTGGTGG ATATGGG GAGGCTG TGGTGAG GGTATGG TTGTCTC GGATTGGTTGTCTC GGAT-3';

[0037] c3) The nucleotide sequence shown in SEQ ID NO: 22, wherein the nucleotide sequence shown in SEQ ID NO: 22 is 5'-ATCTCGA CTAGTCA GGGATAT GGGGAGG CTGTGGT GAGGGTA TGGTTGT CTCGGAT TGGTTGTCTCGGAT-3'.

[0038] The second aspect of the present invention provides a kit for specifically detecting IL-6 / sIL-6R, comprising the aforementioned circular nucleic acid aptamer.

[0039] The third aspect of the present invention provides the use of the circular nucleic acid aptamer or the kit in the preparation of anti-inflammatory agents and / or immune disease treatment agents.

[0040] Most linear nucleic acid molecules are subject to exosome cleavage by endpoints, making them extremely unstable under the action of nucleases in vivo. However, circular nucleic acids, lacking endpoints, cleverly resist the effects of nucleases, allowing them to remain stable in complex biological systems. Furthermore, compared to monovalent aptamers, the bispecific aptamers of the present invention possess stronger recognition capabilities.

[0041] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0043] Figure 1 The stability of circular and linear aptamers in serum is shown.

[0044] Figure 2 The mRNA expression levels of proinflammatory factors (IL-6, IL-1β, COX-2) and sIL-6R in LPS-induced cellular inflammation by the bispecific cyclic aptamers BCA20, BCA21, and BCA22 relative to BCA1 are shown.

[0045] Figure 3 The bispecific cyclic aptamer BCA21 is shown for the mRNA expression levels of proinflammatory factors (IL-6, IL-1β, COX-2) and sIL-6R in LPS-induced cellular inflammation.

[0046] Figure 4 The inhibitory effects of different substances on LPS-induced mRNA are shown. DETAILED DESCRIPTION

[0047] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0048] Example 1

[0049] SELEX screening steps

[0050] SELEX included six rounds. In the first round, a circular DNA library (5'-ATCTC GACTA N20GGGG AGGCT GTGGTGAGGG N20 TGTCT CGGAT-3', SEQ ID NO: 23) (100 pmol) was denatured at 95°C for 5 minutes, then cooled in ice for 15 minutes and mixed with magnetic beads (1 × 10 8 Beads / mL) were incubated with 100 μL of binding buffer (1×PBSMT, pH 7.2, containing 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, 1 mM MgCl2 and 0.025% Tween-20) at room temperature for 1 hour with rotation. The tube was then placed on a magnetic tube holder, and the supernatant was collected into a new tube and mixed with IL-6-bound magnetic beads (1×10 8The beads (0.5 μg / mL) were incubated with rotation in 100 μL of 1× binding buffer at room temperature for 1 hour. The tube was then placed on a magnetic tube holder, the supernatant removed, and the protein-bound aptamer was washed three times with 100 μL of binding buffer. The aptamer was then incubated with 50 μL of ultrapure water at 95°C for 15 minutes, and magnetic separation and elution of the protein-bound aptamer was performed. After recovery by ethanol precipitation, the circular IL-6 / sIL-6R bispecific aptamer (CIL-6DBA) was enzymatically excised via two rounds of RCA amplification. RCA was typically performed in 50 μL of 1× RCA buffer (prepared from a 10× stock solution, containing 330 mM Tris-acetate, pH 7.9 at 37°C, 100 mM magnesium acetate, 660 mM potassium acetate, 1% (v / v) Tween-20, and 10 mM DTT) containing the CIL-6DBA generated in the previous step, 2 μL of LT1 (100 pmol), and 1 mM dNTPs. After heating at 90°C for 3 minutes and cooling at room temperature for 10 minutes, 0.5 μL of phi29 DNA polymerase (10 U / μL) was added, followed by incubation at 30°C for 30 minutes and heating at 65°C for 10 minutes to inactivate the polymerase. Subsequently, 2 μL of 500 μM LT2 was added to the above RCA reaction mixture. The mixture was heated at 90°C for 3 minutes and cooled at room temperature for 10 minutes. Finally, 10 μL of 10× Fast Digest Buffer (100 mM Tris-HCl, pH 8.0, 50 mM MgCl2, 1 M NaCl, 1 mg / ml BSA) and 5 μL of FastDigest EcoRV (400 U) were added to increase the final reaction volume to 100 μL. The reaction mixture was then incubated at 37°C for 8 hours and heated at 65°C for 10 minutes. The amplified monomeric RCA product was purified by standard ethanol precipitation and 10% dPAGE. The DNA was then eluted and circularized before a second RCA reaction. The reaction conditions were identical to those for the first RCA, except that LT1 was replaced by LT2. For the restriction digest after RCA, LT2 was replaced by LT1. To create a molecularly crowded environment, human serum was added to SELEX rounds 2–6. Simultaneously, the serum amount was increased from 5% in round 2 to 10% in round 3, 20% in round 4, 50% in round 5, and 50% in round 6. The DNA library from round 6 was used for deep sequencing.

[0051] The bispecific circular nucleic acid aptamer BCA1 that specifically targets IL-6 / sIL-6R was screened using SELEX technology. This bispecific circular nucleic acid aptamer has high affinity and specificity for IL-6 / sIL-6R and is highly stable in complex biological systems (such as serum).

[0052] Affinity testing

[0053] Comparison of the affinity of each circular nucleic acid aptamer to IL-6 (left) and to sIL-6 (right). (K d <10nM is marked with ****, 10 <K d <20nM is marked as ***, 20 <K d <50 nM is marked as **, K d >50nM is marked with *)

[0054] BCA1 (SEQ ID NO: 1): 5′-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGA GGCTGTGGTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3′ (****; ****), i.e., affinity K relative to IL-6 d <10 nM, affinity K relative to sIL-6 d <10nM, and the same applies below.

[0055] BCA2 (b1, SEQ ID NO: 2): 5'-ATCTCGA CTATTTT TTTTTTT TTTTTTT TTGGGGAGGCTGTG GTGAGGG TTTTTTT TTTTTTT TTTTTTT GTCTCGGAT-3' (*; *).

[0056] BCA3 (b2, SEQ ID NO: 3): 5'-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATTTTTTTTTTTTT TTTTTTT TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3' (***; *).

[0057] BCA4 (b3, SEQ ID NO: 4): 5'-TTTTTGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3' (*; *).

[0058] BCA5 (b4, SEQ ID NO: 5): 5'-ATCTCTT TTTGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3' (*; *).

[0059] BCA6(b5,SEQ ID NO:6):5’-ATCTCGA CTATTTT GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(**;**)。

[0060] BCA7(b6,SEQ ID NO:7):5’-ATCTCGA CTAGTCA TTTTTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(****;****)。

[0061] BCA8(b7,SEQ ID NO:8):5’-ATCTCGA CTAGTCA GTGGTTT TTTCCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(*;**)。

[0062] BCA9(b8,SEQ ID NO:9):5’-ATCTCGA CTAGTCA GTGGTGG ATATTTT TTGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(****;***)。

[0063] BCA10(b9,SEQ ID NO:10):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATTTTTAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(**;***)。

[0064] BCA11(b10,SEQ ID NO:11):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGTTTTTGTG GTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(***;***)。

[0065] BCA12(b11,SEQ ID NO:12):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTTTT TTGAGGG TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(*;*)。

[0066] BCA13(b12,SEQ ID NO:13):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTTTTTT TATGGTT GTCTCGG ATTGGTT GTCTCGGAT-3’(***;**)。

[0067] BCA14(b13,SEQ ID NO:14):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TTTTTTT GTCTCGG ATTGGTT GTCTCGGAT-3’(***;*)。

[0068] BCA15(b14,SEQ ID NO:15):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT TTTTCGG ATTGGTT GTCTCGGAT-3’(*;***)。

[0069] BCA16(b15,SEQ ID NO:16):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTTTT TTTGGTT GTCTCGGAT-3’(***;***)。

[0070] BCA17(b16,SEQ ID NO:17):5’-ATCTCGA CTAGTCA GTGGTGG ATACCGG ATGGGGAGGCTGTG GTGAGGG TATGGTT GTCTCGG ATTTTTT GTCTCGGAT-3’(*;***)。

[0071] BCA18 (b17, SEQ ID NO: 18): 5'-ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTGTGGTGAGGGTATGGTTGTCTCGGATTGGTTTTTCGGAT-3' (****; ****).

[0072] BCA19 (b18, SEQ ID NO: 19): 5'-ATCTCGACTAGTCAGTGGTGGATACCGGATGGGGAGGCTGTGGTGAGGGTATGGTTGTCTCGGATTGGTTGTCTTTTTT-3' (****; ****).

[0073] BCA20 (cl, SEQ ID NO: 20): 5'-ATCTCGACTAGTCAGGGATACCGGATGGGGAGGCTGTGGTGAGGGTATGGTTGTCTCGGATTGGTTGTCTCGGAT-3' (****; ****).

[0074] BCA21 (c2, SEQ ID NO: 21): 5'-ATCTCGACTAGTCAGTGGTGGATATGGGGAGGCTGTGGTGAGGGTATGGTTGTCTCGGATTGGTTGTCTCGGAT-3' (****; ****).

[0075] BCA22 (c3, SEQ ID NO: 22): 5'-ATCTCGACTAGTCAGGGATATGGGGAGGCTGTGGTGAGGGTATGGTTGTCTCGGATTGGTTGTCTCGGAT-3' (***; ****).

[0076] Specific affinity values are shown in the following table.

[0077]

[0078] Stability testing

[0079] 6 μM cyclic bispecific IL-6 / sIL-6R aptamer BCA21 and the corresponding linear aptamer were incubated in 50% human serum at 37°C for 0, 2, 4, 8, 12, 24, 36, and 48 hours. At the determined time points, 20 μL of sample was heated at 95°C for 10 minutes and then stored at -20°C until all samples were collected. These samples were mixed with 20 μL of 2× loading buffer and analyzed by 10% denaturing polyacrylamide gel electrophoresis (PAGE). Gels were visualized using the TOMOS Molecular Imaging Gel Doc EX system, and bands were quantified using ImageJ software.

[0080] The results are as follows Figure 1 As shown, the circular aptamer BCA21, also targeting IL-6 / sIL-6R, was not degraded in 50% serum until 48 hours later, while the linear aptamer reached its half-life at 4.5 hours and was completely degraded after 12 hours.

[0081] It can be seen that the stability of the circular nucleic acid aptamer BCA21 in complex biological systems is much better than that of existing linear nucleic acid aptamers.

[0082] Inhibition of LPS-induced cellular inflammation

[0083] I. Determine the mRNA expression levels of proinflammatory factors (IL-6, IL-1β, COX-2) and sIL-6R in LPS-induced cellular inflammation by the bispecific cyclic aptamer BCA21.

[0084] RAW264.7 cells were plated at 10 per well 5 Cells were seeded in 24-well plates and cultured overnight at 37°C under 5% CO2. Cells were activated with 1 μg / ml LPS and treated for 12 hours with varying concentrations of BCA1, BCA20, BCA21, and BCA22 circular aptamers, or other positive controls (sIL-6R-specific RNA aptamer AIR-3, IL-6-specific aptamer IL62, CRandom, and Tocilizumab). To extract total RNA, adherent cells were lysed with Trizol reagent. RNA from each sample was converted to cDNA using the Takara PrimeScript RT Kit according to the manufacturer's instructions. qPCR reactions were performed using a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using the following parameters: denaturation at 95°C for 2 minutes, annealing at 55°C for 30 seconds, and extension at 72°C for 45 seconds, for 30 cycles. The expression of target genes, including IL-6, sIL-6, IL-1β, and COX-2, was evaluated.

[0085] The results are as follows Figure 2As shown in the data, at a concentration of 200 ng / mL, BCA1, BCA20, BCA21 and BCA22 all had good inhibitory effects on the expression of genes such as IL-6, sIL-6, IL-1β, and COX-2. Compared with BCA1, BCA20, BCA21 and BCA22 had better inhibitory effects, especially BCA21, which had the best inhibitory effect on genes such as IL-6, sIL-6, IL-1β, and COX-2.

[0086] The results are as follows Figure 3 As shown, at concentrations of 20 ng / mL, 200 ng / mL, and 2 μg / mL, the inhibitory effects of BCA21 on IL-6 were 80%, 60%, and 49%, respectively; the inhibitory effects on sIL-6R were 98%, 77%, and 52%, respectively; the inhibitory effects on IL-1β were 59%, 45%, and 38%, respectively; and the inhibitory effects on COX-2 were 75%, 70%, and 57%, respectively.

[0087] It can be seen that the IL-6 / sIL-6R bispecific circular nucleic acid aptamer BCA21 has the best inhibitory effect on LPS-induced cellular inflammation, and is concentration-dependent.

[0088] II. Taking IL-6 / sIL-6R bispecific cyclic nucleic acid aptamer (BCA21), random cyclic sequence (CRandom), IL-6 specific nucleic acid aptamer (IL-62), sIL-6R specific nucleic acid aptamer (AIR-3) and sIL-6R immunosuppressive monoclonal antibody (Tocilizumab) as examples, the inhibitory effects of these substances on LPS-induced mRNA were determined.

[0089] like Figure 4 As shown in ad, at a concentration of 2 μg / mL, the inhibition rates of BCA21 on IL-6, IL-1β, COX-2 and sIL-6R were 49%, 39%, 57% and 52%, respectively; the inhibition rates of IL-62 on IL-6, IL-1β, COX-2 and sIL-6R were 96%, 80%, 73% and 110%, respectively; the inhibition rates of AIR-3 on IL-6, IL-1β, COX-2 and sIL-6R were The inhibition rates of tocilizumab on IL-6, IL-1β, COX-2 and sIL-6R were 82%, 113%, 89% and 98% respectively; the inhibition rates of CRandom on IL-6, IL-1β, COX-2 and sIL-6R were 126%, 128%, 115% and 130% respectively; BCA21 had the most significant inhibitory effect on the mRNA expression level of pro-inflammatory factors.

[0090] In addition, if Figure 4 As shown in e, after the M1 macrophages were treated with 2 μg / mL of BCA21 and other inhibitors, the M1 macrophages in the control group (CD80 + ) ratio was 71.8%, and the M1 macrophages (CD80 + ) ratio was 55.5%, and the M1 macrophages (CD80 + ) ratio was 67.4%, and the M1 macrophages (CD80 + ) ratio was 65.4%, and the M1 macrophages (CD80 + ) ratio was 70.9%, and the M1 macrophages (CD80 + ) ratio was 71.6%, BCA was effective for M1 macrophages (CD80 + ) has the most significant inhibitory effect. That is, BCA21 has a better inhibitory effect on LPS-induced cellular inflammation than existing nucleic acid aptamers that specifically target IL-6, sIL-6R, and Tocilizumab.

[0091] The above results show that the bispecific cyclic nucleic acid aptamer BCA21 has a much higher inhibitory effect on the mRNA of IL-6, sIL-6R, IL-1β and COX-2 than the random cyclic sequence (CRandom), the existing linear IL-6-specific nucleic acid aptamer (IL-62), the sIL-6R-specific nucleic acid aptamer (AIR-3) and the sIL-6R immunosuppressive monoclonal antibody (Tocilizumab).

[0092] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An IL-6 / sIL-6R bispecific circular nucleic acid aptamer, characterized in that The nucleotide sequence of the circular nucleic acid aptamer is the nucleotide sequence shown in SEQ ID NO: 21, and the nucleotide sequence shown in SEQ ID NO: 21 is 5'-ATCTCGA CTAGTCA GTGGTGG ATATGGG GAGGCTG TGGTGAG GGTATGG TTGTCTC GGATTGGTTGTCTC GGAT-3'.

2. A kit for specifically detecting IL-6 / sIL-6R, comprising the circular nucleic acid aptamer according to claim 1.

3. Use of the circular nucleic acid aptamer according to claim 1 or the kit according to claim 2 in the preparation of an anti-inflammatory agent.

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