CN107815453B - Nucleic acid aptamer and application thereof - Google Patents

Abstract

The invention provides a nucleic acid aptamer, wherein the nucleotide sequence of the nucleic acid aptamer is shown as one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. The nucleic acid aptamer can be specifically and efficiently combined with PDGF-CC, so that the angiogenesis promotion effect of the PDGF-CC under pathological conditions is blocked, and the nucleic acid aptamer plays a role in treating diseases caused by hyperproliferation of new blood vessels.

Description

Nucleic acid aptamer and application thereof

Technical Field

The invention relates to a nucleic acid aptamer and application thereof.

Background

1.PDGF-CC

The platelet-derived growth factor (PDGF) family is one of the earliest discovered growth factor families and plays a crucial role in the physiological and pathological processes of vascular systems and other organ tissues. In 1974, Harker et al discovered PDGF-A. In 1977, PDGF-B was discovered by Westermark et al. Scientists have since for more than twenty years considered only two members of the PDGFs family, and until 2000 and 2001, applicants' li xu-day professor and colleagues discovered two new family members in succession: PDGF-C and PDGF-D. Experiments show that PDGF-C is a strong angiogenesis promoting factor except VEGF, and the expression of PDGF-C is increased in a laser-induced Choroidal Neovascularization (CNV) animal model, which suggests that PDGF-C angiogenesis promoting may be a new mechanism for ocular neovascularization and a new target for preventing and treating ocular neovascular diseases. Ocular neovascularization is a major cause and common pathological process of blindness in many intractable blinding eye diseases. These diseases include wet age-related macular degeneration (AMD), Diabetic Retinopathy (DR), retinopathy of prematurity (ROP), Retinal Vein Occlusion (RVO), and the like.

2. Nucleic acid aptamer

In 1990, Tuerk et al and Ellington et al reported that RNA fragments capable of specifically binding to bacteriophage T4DNA polymerase and organic dye were selected using exponential enrichment of ligand system evolution (systematic evolution of ligands by exogenic enzyme, SE L EX) and named "aptamers" or "aptamers", respectively, nucleic acid aptamers were single-stranded oligonucleotides (DNA or RNA) capable of binding to targets with high specificity and high affinity, aptamers could form a variety of thermodynamically stable three-dimensional structures due to their base sequence diversity and structural diversity, such as pockets, hairpins, collars, pseudoknots and G-tetramers, which could form stable complexes with the target molecules through van der Waals, hydrogen bonding, etc., gametes were considered as an in vitro synthesized "antibody" because they could bind tightly and specifically to the target molecules, affinity and specificity of aptamers were somewhat superior to antibodies, showing a large scale binding to antibodies, which showed a high specificity of specificity, even a low binding affinity to target molecules, high specificity, low binding to target molecules, high specificity of aptamers, small molecular specificity, small molecular binding to target molecules, small molecular aptamers, high specificity, targeting molecules binding to target molecules, small targeting molecules.

3. Aptamer screening technique-SE L EX

SE L EX is a screening technology capable of obtaining oligonucleotides which are highly selective and combined with target molecules with high affinity, most aptamers are screened from an oligonucleotide library which is synthesized by chemical random and has a library capacity of about 1014 to 1016 by a SE L EX technology, the SE L EX technology is repeatedly screened by a plurality of rounds or tens of rounds, and finally oligonucleotide sequences which are specifically combined with the target molecules are obtained, each round of screening comprises 3 main steps of (1) combining the target molecules with an oligonucleotide library of random, and (2) separating, namely forming oligonucleotide-target complexes and separating unbound oligonucleotides, (3) amplifying, namely amplifying the bound oligonucleotide sequences by PCR and preparing a new screened oligonucleotide sub-library, wherein ideal aptamers with high specificity and affinity can be obtained by more than ten rounds of screening, along with the increase of the number of screening rounds, the oligonucleotides which are gradually enriched in the binding affinity to the target molecules can be cloned, and finally the nucleic acid aptamer L EX technology has very wide sequencing target molecules, and the target molecules comprise proteins, small proteins, polysaccharides, and the important nucleic acid molecules which are obtained by a plurality of capillary electrophoresis and the biological target molecule screening methods such as the biological target molecules, and the biological target molecules.

4. Current research and development status of nucleic acid aptamer drugs

Since the first publication of this study by Tuerk and Ellington et al in 1990, aptamers have become one of the hot spots for the development of targeted therapeutic drugs. The aptamer-based targeted therapeutic drug serving as an 'antibody analogue' with low immunogenicity is applied to treatment of various diseases such as cancers, macular degeneration, von willebrand disease and diabetes, and some drugs are approved to be on the market through FDA certification and show great application prospects in the field of biological medicines.

The first aptamer drug, Macugen, is an RNA aptamer specifically targeting 27 ribonucleotides of vascular endothelial growth factor 165 (VEGF 165) and is administered by ocular injection to treat age-related macular degeneration (AMD), approved by the FDA in the united states in 2004. The aptamer is transcribed by modifying 2 '-fluoropyrimidine and 2' -oxy-methylpurine, a 3 '-connected deoxythymidine terminal cap is added to increase the stability of the aptamer, and a 40kDa polyethylene glycol (PEG) molecule is connected to the 5' end of the aptamer to prolong the half-life of the aptamer.

AS1411 (also known AS AGRO100) is a guanine-rich aptamer that was the earliest entry into clinical trials for cancer, and is also the earliest drug specific for nucleolus, and can form a G-quadruplex structure by itself and thus has many specific biological activities. The aptamer is administrated by intravenous injection, can be combined with the nucleolar membrane protein external domain excessively expressed on the surface of a tumor cell, inhibits the activity of nucleolus, and plays an important role in cell survival, growth, proliferation, nuclear transportation and transcription.

REG-1, the first nucleic acid aptamer called regulatable, consists of an RNA aptamer against factor IX a (RB006) and a single-stranded RNA oligonucleotide (RB007) complementary to RB006, and competes with each other to alter the structure of REG-1, thereby inhibiting factor IX a and achieving an anticoagulant effect. The medicine is administrated by intravenous injection, and shows good medical value in patients with acute coronary syndrome.

Roth et al reported an RNA aptamer that blocked interleukin 4 receptor alpha (I L-4 alpha) in mice or humans, targeted to myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), and effectively inhibited tumor-model mouse tumor cell growth.

Disclosure of Invention

The invention aims to provide a nucleic acid aptamer and also provides application of the nucleic acid aptamer.

In order to realize the purpose, the technical scheme is as follows: a nucleic acid aptamer has a nucleotide sequence shown in one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.

Preferably, the nucleotide sequence of the nucleic acid aptamer is shown as one of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.

Preferably, the nucleotide sequence of the nucleic acid aptamer is shown in one of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 6.

Preferably, the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO. 4 or SEQ ID NO. 6.

The invention provides application of the nucleic acid aptamer in preparing a reagent for inhibiting or blocking the combination of PDGF-CC and PDGFR α.

The invention provides application of the nucleic acid aptamer in preparation of a kit for inhibiting or blocking the combination of PDGF-CC and PDGFR α.

The invention provides application of the nucleic acid aptamer in preparation of a reagent for inhibiting phosphorylation of PDGFR α Tyr988 locus.

The invention provides application of the nucleic acid aptamer in preparation of a kit for inhibiting phosphorylation of PDGFR α Tyr988 locus.

The invention provides application of the nucleic acid aptamer in preparing a medicament for treating diseases caused by hyperproliferation of new blood vessels.

The invention has the beneficial effects that: the nucleic acid aptamer can be specifically and efficiently combined with PDGF-CC, so that the angiogenesis promotion effect of the PDGF-CC under pathological conditions is blocked, and the nucleic acid aptamer plays a role in treating diseases caused by hyperproliferation of new blood vessels.

Drawings

FIG. 1 shows the results of competitive binding assays based on Surface Plasmon Resonance (SPR) in example 1 of the present invention;

FIG. 2 shows the results of blocking the binding of PDGF-CC to PDGFR α with different concentrations of nucleic acid aptamers (PF-1, -2, -4, and-6) in example 1 of the present invention;

FIG. 3 shows the results of a single-cycle analysis of binding of an aptamer to PDGF-CC using surface plasmon resonance in example 1 of the present invention; wherein (A): dynamic data (aptamer antagonism of PDGF-CC) (B): dynamic data (control Adaplets) — consistent with expectations, no binding;

FIG. 4 shows the results of a full cycle analysis of the binding of aptamers to PDGF-CC using surface plasmon resonance in example 1 of the present invention;

FIG. 5 shows the result of Western blot detection in example 1 of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

The DNA nucleic acid aptamers which are specifically combined with PDGF-CC are screened by using an exponential enrichment ligand phylogeny (SE L EX) technology, wherein the sequence of PF-1 is shown as SEQ ID NO. 1, the sequence of PF-2 is shown as SEQ ID NO. 2, the sequence of PF-3 is shown as SEQ ID NO. 3, the sequence of PF-4 is shown as SEQ ID NO. 4, the sequence of PF-5 is shown as SEQ ID NO. 5, the sequence of PF-6 is shown as SEQ ID NO. 6, the sequence of PF-7 is shown as SEQ ID NO. 7, the sequence of PF-8 is shown as SEQ ID NO. 8, and the results are shown in Table 1.

TABLE 1 leader sequence + bioinformatics analysis of SE L EX-NGS screening

A competitive binding detection based on Surface Plasmon Resonance (SPR) is that PDGF receptor α (PDGFR α) is fixed on a sensing sheet and is incubated with 200nM nucleic acid aptamers (PF 1-PF 8) first, and then PDGF-CC is added, and the results are shown in figure 1, and it is found that PF-4 and PF-6 can obviously and effectively block the binding of PDGF-CC and PDGFR α, and then PF-1, PF-8 and PF-7.

Competitive binding experiments based on Surface Plasmon Resonance (SPR), namely, binding of PDGF-CC and PDGFR α is blocked by using nucleic acid aptamers (PF-1, -2, -4 and-6) at different concentrations, and as a result, as shown in FIG. 2, PF-4 and PF-6 have the strongest blocking effect on PDGF-CC, the action intensity is dose-dependent, and the blocking effect of PF-1 is weaker.

As can be seen from the results of FIG. 3, the surface plasmon resonance assay showed that the nucleic acid aptamers PF-1, PF-4 and PF-6 can bind with high affinity to PDGF-CC. PDGF-CC is fixed on the surface of a CM5 sensing piece and is incubated with a nucleic acid aptamer. Binding of the aptamer to PDGF-CC was detected by surface plasmon resonance. The long arrows represent experimental values and the short arrows represent fitted values.

As can be seen from the results of FIG. 4, the nucleic acid aptamers PF-1, PF-2, PF-4 and PF-6 can bind with high affinity to PDGF-CC. PDGF-CC is fixed on the surface of a CM5 sensing piece and is incubated with a nucleic acid aptamer. Binding of the aptamer to PDGF-CC was detected by surface plasmon resonance. Fig. 4 shows the results of the full cycle analysis.

NIH3T3 cells are starved and cultured for 4 hours in a serum-free medium, 10nM nucleic acid aptamers are added for 1 hour, 50ng/ml PDGF-CC is added for 10 minutes for incubation, cell protein is lysed, and 30 mu g of protein is subjected to PAGE electrophoresis, wherein the primary antibody is PDGFR α antibody (phospho-Tyr 988) and PDGFR alpha antibody respectively, and Western blot detection results show that the nucleic acid aptamers PF-4 and PF-6 can inhibit phosphorylation of PDGFR α Tyr988 site (as shown in figure 5).

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

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Claims (9)

1. A nucleic acid aptamer is characterized in that the nucleotide sequence of the nucleic acid aptamer is shown as one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.

2. The nucleic acid aptamer according to claim 1, wherein the nucleotide sequence of the nucleic acid aptamer is shown as one of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.

3. The nucleic acid aptamer according to claim 1, wherein the nucleotide sequence of the nucleic acid aptamer is shown as one of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 6.

4. The nucleic acid aptamer according to claim 1, wherein the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO. 4 or SEQ ID NO. 6.

5. Use of the nucleic acid aptamer according to any one of claims 1 to 4 for the preparation of a reagent for inhibiting or blocking the binding of PDGF-CC to PDGFR α.

6. Use of the nucleic acid aptamer according to any one of claims 1 to 4 for the preparation of a kit for inhibiting or blocking the binding of PDGF-CC to PDGFR α.

7. Use of the nucleic acid aptamer according to any one of claims 1 to 4 for the preparation of a reagent for inhibiting phosphorylation of the PDGFR α Tyr988 site.

8. Use of the nucleic acid aptamer according to any one of claims 1 to 4 for the preparation of a kit for inhibiting phosphorylation of the PDGFR α Tyr988 site.

9. Use of the nucleic acid aptamer according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of diseases caused by hyperproliferation of new blood vessels.

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