CN111979248B

CN111979248B - Protective fragment of aptamer and aptamer containing protective fragment

Info

Publication number: CN111979248B
Application number: CN202010704574.2A
Authority: CN
Inventors: 谭蔚泓; 邱丽萍; 谢斯滔
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2022-04-08
Anticipated expiration: 2040-07-21
Also published as: CN111979248A

Abstract

The invention relates to the technical field of molecular biology, in particular to a protective fragment of a nucleic acid aptamer and the nucleic acid aptamer containing the protective fragment. The invention provides a protective fragment of a nucleic acid aptamer, and provides a nucleic acid aptamer containing the protective fragment and application thereof. In the present invention, the end of the aptamer (aptamers) can form end-protected aptamer (MT-aptamers) after ATP aptamer ligation protection, and MT-aptamers can specifically improve the biostability in target tissues or organs and can be metabolized in normal tissues or organs. Experiments prove that the protective fragment provided by the invention can play a role in improving the stability of various aptamers.

Description

Protective fragment of aptamer and aptamer containing protective fragment

Technical Field

The invention relates to the technical field of molecular biology, in particular to a protective fragment of a nucleic acid aptamer and the nucleic acid aptamer containing the protective fragment.

Background

Aptamer (aptamer) refers to a single-stranded DNA or RNA oligonucleotide molecule that is obtained by in vitro screening and can bind various biological molecules (such as ions, small molecules, proteins, etc.) and other targets with certain affinity and specificity.

Since aptamers composed of natural base elements (a/T/C/G/U) are easily degraded by nucleases, scientists have developed for many years by designing and synthesizing various unnatural chemical modifications (different in chemical structure from natural base elements and therefore difficult to recognize and degrade by natural enzymes, hereinafter referred to as unnatural base elements) and introducing unnatural base elements into aptamers to improve the nuclease degradation resistance (mainly referred to as biostability) of aptamers (hereinafter collectively referred to as unnatural aptamers).

In the application of the aptamer for the purpose of disease diagnosis or treatment, it is most desirable that the aptamer exerts its diagnostic or therapeutic effect only in an abnormal tissue or organ, whereas the biostability of the unnatural aptamer is improved in all tissues or organs, with a potential adverse effect on the normal tissues or organs. The initiation of adverse effects (e.g., inflammatory responses, immune stimulatory responses) is primarily due to unnatural chemical modifications that are difficult to recognize and degrade by natural enzymes. Moreover, the non-natural base elements are not efficient in synthesis compared with natural bases and are not easy to produce; the introduction of the non-natural base elements can greatly change the structure of the target aptamer so as to influence the function of the aptamer, and the method is not strong in universality.

In order to understand the potential adverse effect of the aptamer modified by the unnatural base element on normal tissues or organs, some reports in the prior art include that a linker sequence and a regulatory sequence are connected to one end of the aptamer, so that the aptamer forms a stable structure under an acidic condition, and does not form a stable structure under a neutral condition, so that the function of the aptamer in binding with cancer cells is weakened. However, the function of the living organism is not clearly defined.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a protective fragment of a nucleic acid aptamer and a nucleic acid aptamer containing the same, wherein the stability of the nucleic acid aptamer, and the specificity and stability in detection of living organisms are improved by using the protective fragment.

First, the present invention provides: I) III) as a protective fragment of the aptamer:

I) a fragment having a nucleotide sequence shown as SEQ ID NO. 1;

II. A fragment in which one or more nucleotides are substituted, deleted or added in the fragment of I);

III, a fragment which is partially or fully complementary to I) or II).

In the invention, the protection fragment of the aptamer is a nucleic acid fragment connected to the tail end of the aptamer and is composed of natural base elements, and the two protection fragments form a special structure, so that when a target molecule is embedded or interacts with the target molecule, the degradation of the aptamer can be avoided.

The invention also provides a protective fragment of the aptamer, which comprises an X chain and a Y chain;

wherein the X chain has a nucleotide sequence shown as SEQ ID NO. 1, or a sequence obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown as SEQ ID NO. 1;

at least 4bp of the Y chain is reversely complementary with the X chain.

The X chain and the Y chain can be complemented to form a double chain and exist, and can also exist on the same DNA molecular chain and exist in a single chain form.

In the invention, the nucleotide sequence of SEQ ID NO. 1 is ACCTGGGGGAG TAT; the nucleotide sequence of the Y chain is shown as SEQ ID NO. 2, which is TGCGGAGGAAGGT.

In some embodiments, both ends of the X chain further comprise fragment X3 and/or fragment X5;

both ends of the Y chain further comprise fragment Y3 and/or fragment Y5;

the nucleotide sequences of the fragment X3, the fragment X5, the fragment Y3 and the fragment Y5 are the same or different, and the length is 0-50 bp;

the fragment X3 and fragment Y5 are complementary or not complementary or partially complementary;

the fragment X5 and the fragment Y3 are complementary or not complementary or partially complementary.

The fragment X3 is located at the 3 'end of the X chain, and the fragment X5 is located at the 5' end of the X chain. That is, the structure of the X chain is: X5-X-X3.

The fragment Y3 is located at the 3 'end of the Y chain, and the fragment Y5 is located at the 5' end of the Y chain. That is, the structure of the Y chain is: Y5-Y-Y3.

In the present invention, the sequences of fragment X3, fragment X5, fragment Y3 and fragment Y5 are random.

In some embodiments, fragment X5 is 6bp in length, which is reverse complementary to fragment Y3; specifically, the sequence of X5 is CATCGC, and the sequence of Y3 is GCGATG.

In some embodiments, fragment X3 is 1bp in length, which is a (guanine); fragment B5 was 0bp in length.

In some embodiments, the X chain sequence of the aptamer protective fragment is shown as SEQ ID NO. 3, and the Y chain sequence is shown as SEQ ID NO. 4. The invention also provides a nucleic acid aptamer, which comprises an aptamer fragment and the protective fragment.

In the present invention, the number of aptamer fragments is 1 or 2; and the two ends of the aptamer fragment are respectively connected with the X chain and the Y chain of the protection fragment.

The targets of the aptamer include: at least one of metal ions, adenosine, proteins, drugs, pathogenic microorganisms, cells or tissues.

The nucleic acid aptamer further comprises a drug, a chemical marker and/or a biomarker;

the drug is a chemical drug or an RNAi molecule;

the chemical label is a fluorophore, isotope and/or immunotoxin;

the biomarker is a biotin, avidin, or enzyme label.

The target of the aptamer is a tumor cell; also included are fluorophores.

The nucleic acid sequence is shown in any one of SEQ ID NO 5-8; the 5' end of the fluorescent probe is modified with FAM fluorescent group or Cy5 fluorescent group.

The nucleic acid aptamer is applied to the preparation of a target detection reagent.

The invention also provides a target detection reagent, which comprises the aptamer.

The detection reagent also comprises a solid phase carrier or a non-solid phase carrier.

The nucleic acid aptamer is applied to the preparation of medicines for treating diseases.

The invention also provides a medicament for treating diseases, which comprises the aptamer.

In the medicament, the aptamer serves as a targeting molecule or serves as a therapeutic agent.

The aptamer is applied to preparation of a target area imaging agent.

The invention also provides an imaging agent which comprises the nucleic acid aptamer.

The imaging agent also comprises an acceptable auxiliary material in the imaging agent.

The invention provides a protective fragment of a nucleic acid aptamer, and provides a nucleic acid aptamer containing the protective fragment and application thereof. In the present invention, the end of the aptamer (aptamers) can form end-protected aptamer (MT-aptamers) after ATP aptamer ligation protection, and MT-aptamers can specifically improve the biostability in target tissues and can be metabolized in normal tissues or organs. Experiments prove that the protective fragment provided by the invention can well improve the stability of various aptamers.

Drawings

FIG. 1 shows (a) stability assay of aptamer at an ATP incubation concentration of 7.5mM, (b) binding assay of the aptamer sample in (a) to CCRF-CEM cells at a DNA concentration of 20 nM;

FIG. 2 (a) stability assay of DNA at an ATP incubation concentration of 7.5mM, (b) binding assay of DNA sample in (a) to CCRF-CEM cells at DNA concentrations of 20 nM;

FIG. 3 (a) stability assay of MT-sgc8c at different ATP concentrations, (b) binding assay of the sample in (a) to CCRF-CEM cells, both at 20nM DNA concentration;

FIG. 4 (a) binding analysis of DNA samples to CCRF-CEM, K562 and Ramos cells. DNA concentrations were all 20nM, (b) confocal imaging analysis of Sgc8c and MT-sgc8c with CCRF-CEM, K562 and Ramos cells, with a scale bar of 20 μm in length;

FIG. 5 thermal stability analysis of DNA in different concentrations of ATP;

FIG. 6 (a) stability assay of aptamers incubated at 10% fetal calf serum for various periods of time at an ATP incubation concentration of 7.5mM, (b) binding assay of the sample in (a) to CCRF-CEM cells at DNA concentrations of 20 nM;

FIG. 7 shows (a) stability analysis of aptamer (MT-XQ-2d) at an ATP incubation concentration of 7.5mM, (b) binding analysis of aptamer in (a) to CCRF-CEM cells at a DNA concentration of 20nM, and (c) confocal imaging analysis of DNA binding to CCRF-CEM cells at a scale bar of 20 μm in length;

FIG. 8 shows (a) stability analysis of aptamer (MT-KK1B10) with ATP at an incubation concentration of 7.5mM, (B) binding analysis of aptamer in (a) to K562 cells at a DNA concentration of 20nM, and (c) confocal imaging analysis of DNA binding to K562 cells at a scale of 20 μm in length;

FIG. 9 (a) stability analysis of aptamer (MT-TDO5) at an ATP incubation concentration of 7.5mM, (b) binding analysis of aptamer to Ramos cells at a DNA concentration of 20nM in (a), (c) confocal imaging analysis of DNA binding to Ramos cells at a scale of 20 μm in length;

FIG. 10: (a) binding analysis of DNA samples to HCT116 and HEK293 cells at DNA concentrations of 20nM, (b) confocal imaging analysis of Sgc8c and MT-sgc8c with HCT116 and HEK293 cells, with a scale bar of 20 μm in length;

FIG. 11 shows an analysis of ATP content in different tissues in a mouse model of tumors seeded with HCT116 cells;

FIG. 12 shows binding assays of aptamers to CCRF-CEM cells at 4 ℃;

FIGS. 13, 14 show imaging analysis of different tissues in a seeded tumor mouse model, the mouse having an organ removed at 5 hours;

FIG. 15 (a) imaging analysis of DNA in HCT116 cell seeded tumor mouse model, (b) imaging analysis of different tissues in tumor mouse model seeded in (a) mice organs were harvested at 5 hours;

FIG. 16 shows imaging analysis of DNA in a normal mouse model;

FIG. 17 shows an imaging analysis of different tissues in a normal mouse model, with the mouse having an organ taken at 5 hours;

FIG. 18 shows the principle of aptamer design;

FIG. 19 shows the structure of the aptamer of the invention.

Detailed Description

The invention provides a protective fragment of a nucleic acid aptamer and a nucleic acid aptamer containing the protective fragment, and a person skilled in the art can use the content to appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the present invention, the aptamer (aptamer), also called aptamer, is a single-stranded oligonucleotide (DNA, RNA, and nucleic acid derivatives) capable of specifically binding to various target molecules with a certain high affinity. The combination of aptamers with various target molecules is based on the diversity of nucleotide structures and spatial conformations, and the aptamers can be adaptively folded through pairing among certain complementary bases in a chain, electrostatic interaction, hydrogen bond interaction and the like to form stem-loop structures, hairpin structures, pseudoknot structures or tetramer structures and the like. Based on these stable three-dimensional structures, aptamers are able to recognize a target or target molecule by structural complementarity, base stacking forces, van der waals forces, hydrogen bonding, or electrostatic interactions. In the art, aptamer screening is usually performed by SELEX technology, which includes five major steps: combining, separating, eluting, amplifying and identifying. In the present invention, the aptamer is a single-stranded oligo-DNA. In the invention, the aptamer fragment is single-stranded oligo DNA, and 5-6 bases at the 3 'end and the 5' end of the aptamer fragment are complementary.

First, the present invention provides:

I) III) as a protective fragment of the aptamer:

I) a fragment having a nucleotide sequence shown as SEQ ID NO. 1;

III, a fragment which is partially or fully complementary to I) or II).

1 is an aptamer fragment of ATP which is capable of specifically binding to ATP, thereby increasing the stability of the aptamer in the presence of ATP.

at least 4bp of the Y chain is reversely complementary with the X chain.

both ends of the Y chain further comprise fragment Y3 and/or fragment Y5;

the nucleotide sequences of the fragment X3, the fragment X5, the fragment Y3 and the fragment Y5 are the same or different, and the length is 0-50 bp; in some embodiments, the fragments are 0-10 bp in length. Namely, the length of the fragment X3 is 0-50 bp, or 0-10 bp; the length of the fragment X5 is 0-50 bp, or 0-10 bp; the length of the fragment Y3 is 0-50 bp, or 0-10 bp; the length of the fragment Y5 is 0-50 bp or 0-10 bp; .

In the present invention, the sequences of fragment X3, fragment X5, fragment Y3 and fragment Y5 are random. The lengths of the two can be 0 at the same time, and can also be independent any length of 0-50 bp. The fragment X3 and fragment Y5 are complementary or not complementary or partially complementary; the fragment X5 and the fragment Y3 are complementary or not complementary or partially complementary.

In some embodiments, the X chain sequence of the aptamer protective fragment is shown as SEQ ID NO. 3, and the Y chain sequence is shown as SEQ ID NO. 4. Namely:

the sequence of the X chain is:

the sequence of the Y chain is: 5' -TGCGGAGGAAGGTGCGATG-3’，

The aptamer formed by connecting the protective fragment of the invention with the tail end of the aptamer fragment has good stability.

The invention also provides a nucleic acid aptamer, which comprises an aptamer fragment and the protective fragment.

The aptamer is an oligo-DNA single strand, and comprises the following components:

chain X-aptamer fragment-chain Y;

or aptamer fragment-X strand-aptamer fragment-Y strand;

or chain X-aptamer fragment-chain Y-aptamer fragment;

in the invention, the preparation method of the aptamer adopts chemical synthesis, and can also be synthesized through biosynthesis or enzyme transcription.

In the invention, the target of the aptamer is also called a target molecule of the aptamer, and the aptamer shows high specificity and affinity to the target molecule, so that the target range is wide. In the present invention, the target of the aptamer includes: at least one of metal ions, adenosine, proteins, drugs, pathogenic microorganisms, cells or tissues.

The adenosine is ATP. The proteins include disease diagnostic markers such as tumor markers, hormones, markers of pathogen infection. Wherein, the tumor marker comprises carcinoembryonic antigen, alpha-fetoprotein, carbohydrate antigen, CYFRA21-1, beta-microglobulin, ferritin and the like. The hormones include four major classes: the first are steroids such as adrenocorticoids (cortisol, aldosterone, etc.), sex hormones (estrogens, progestins, androgens, etc.). The second group is amino acid derivatives, such as thyroxine, adrenomedullary hormone, pineal hormone, etc. The third class of hormones are peptides and proteins, such as hypothalamic hormones, pituitary hormones, gastrointestinal hormones, insulin, calcitonin, and the like. The fourth class is fatty acid derivatives, such as prostaglandins. The pathogen infection marker includes a viral infection marker, a phage infection, a bacterial infection marker, or a fungal infection marker. For example, hepatitis B surface antigen or antibody, hepatitis B core antigen or antibody, etc. formed after hepatitis B virus infection.

The drug includes antibiotics, including, for example: quinolone antibiotics, beta-lactam antibiotics, macrolides, aminoglycoside antibiotics.

The pathogenic microorganism includes pathogenic bacteria, pathogenic fungi or viruses. The cell includes insect cell, animal cell or plant cell, wherein the animal cell includes human cell, etc., wherein the human cell may include cancer cell, white blood cell, red blood cell, lymphocyte. The cancer cells are leukemia lymphocyte T cells, K562 cells or Ramos cells, HCT116 cells or HEK293 cells. The pathogenic microorganisms are human pathogenic microorganisms, animal pathogenic microorganisms or plant pathogenic microorganisms.

In the present invention, the nucleic acid aptamer further comprises a drug, a chemical marker and/or a biomarker;

when the aptamer is not modified by other groups, it can be used to capture the target. Aptamers can be used for target detection when they are modified with other groups so that they can be detected. When the aptamer is combined with a drug, the aptamer can be used for targeted delivery of the drug.

In the present invention, the chemical label or biomarker is a label that enables detection of the aptamer. Means of binding to the nucleic acid aptamer include covalent binding or non-covalent binding. The position at which the chemical label or the biological label is present may be at one end of the nucleic acid aptamer, or embedded in the structure of the nucleic acid aptamer.

The biomarkers are biotin, avidin and enzyme markers. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The avidin is streptavidin or deglycosylated avidin.

The chemical label is a fluorophore, isotope and/or immunotoxin;

in the present invention, the fluorescent group includes acridinium ester compounds or other fluorescent dyes, such as FITC, FAM, Cy3, Cy5 or BODIPY. The isotope is¹³¹I、¹⁴Ba、³²P、⁴⁵Ca、⁵⁶Fe, and the like. The immunotoxin is preferably selected from aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, calycosin, PAP, nystatin, and geloninVegetable or luffa toxin, etc.

The nucleic acid aptamer is applied to the preparation of a target detection reagent. Aptamers for target detection may or may not be chemically or enzymatically labeled. The detection of the invention comprises the enrichment of the target and also comprises the qualitative detection or the quantitative detection of the target.

The invention also provides a target detection reagent, which comprises the aptamer. The detection reagent may contain no carrier or a carrier. The detection reagent without the carrier is mixed with the object to be detected, and the detection of the object to be detected can be realized by detecting the aptamer. The carrier loaded with the nucleic acid aptamer can be used for enriching and detecting the target. Therefore, in the present invention, the detection reagent further comprises a solid phase carrier or a non-solid phase carrier. Also included are reagents for modifying the aptamers of the invention on a solid or non-solid support. In the present invention, the solid medium includes a centrifuge tube, a well plate or a film, or a slide, a microplate or an elisa plate, etc., made of a polymeric material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose). The non-solid medium is selected from colloidal gold, magnetic beads, latex particles and the like.

The invention also provides a target detection method, which is to contact and incubate the reagent and the target. The incubation condition is 0-37 ℃, and the incubation time is 0-4 hours.

The aptamer is applied to preparation of a target area imaging agent. As mentioned above, chemical markers or biological markers can be modified in the aptamer, and the aptamer after the modification of the marker can be detected by immunodetection or other means, so that the aptamer can be used for imaging the target position recognized by the aptamer.

The invention also provides an imaging agent which comprises the nucleic acid aptamer. The imaging agent also comprises an acceptable auxiliary material in the imaging agent. The target region of the present invention includes pathological regions such as tumor tissue and the like.

The invention also provides a method of imaging a target area comprising administering the imaging agent of the invention. The mode of administration includes injection. In some embodiments, the aptamer is present in the injected imaging agent at a concentration of 5nmol/100 μ L.

The invention also provides application of the aptamer in preparation of a medicament for treating diseases.

Aptamers bind to targets with very high specificity and affinity, similar to antigen-antibody binding. And the aptamer has small molecular size, and the modification of the medicine is easier. Aptamer drugs are currently used in a variety of applications, including anticancer, anti-infection, anticoagulant, anti-inflammatory, and immunotherapy. However, the aptamer drugs used in the past are not widely applicable because they are difficult to stably exist in the digestive system. In the present invention, the drug is a chemical drug or an RNAi molecule.

In the medicament, the aptamer serves as a targeting molecule or serves as a therapeutic agent. The aptamer provided by the invention has higher stability, so that the application range of aptamer medicaments is expanded. Some aptamers are capable of promoting the activation of immune cells by themselves, thereby exerting a therapeutic effect, based on the fact that the aptamers themselves can specifically bind to the immune cells or the markers. In addition, based on the specific binding of the aptamer to the target, it can be targeted as a targeting molecule for drug delivery to the target site. The drug for treating diseases is a nucleic acid aptamer (marked as aptamer drug) loaded with the drug, wherein the drug is connected with the aptamer through a covalent bond or a non-covalent bond or is embedded into the structure of the aptamer. After the aptamer drug enters the body, the aptamer plays a role of a target molecule, and the drug plays a therapeutic role, so that the specificity of the drug on the target is improved, and the toxicity to non-target substances is reduced. In the present invention, the disease is a disease caused by a pathogen that can be recognized by the aptamer. The method comprises the following steps: viral infection, bacterial infection, fungal infection, phage infection, autoimmune disease, tumor, cardiovascular disease and cerebrovascular disease, etc. The medicine for treating diseases also comprises pharmaceutically acceptable auxiliary materials. The medicine also comprises other therapeutic agents, and the treatment mechanism of the other therapeutic agents can be the same as or different from that of the aptamer medicine. The other therapeutic agent can be a drug for treating the same disease as the aptamer drug or a drug for treating different diseases from the aptamer drug. The dosage form of the medicine comprises injection, granules, pills, powder, tablets, capsules, oral liquid or syrup.

The invention also provides a method for treating diseases, which is to administer the medicine for treating diseases. The route of administration includes oral administration or injection.

In some embodiments, the aptamer is targeted to a tumor cell; in some embodiments, the aptamer fragment is selected from Sgc8c, XQ-2d, KK1B10, or TDO 5. The aptamer further comprises a fluorescent group.

The nucleic acid sequence of the polypeptide is shown in any one of SEQ ID NO 5-8; the 5' end of the fluorescent probe is modified with FAM fluorescent group or Cy5 fluorescent group.

The invention provides a protective fragment of a nucleic acid aptamer, and provides a nucleic acid aptamer containing the protective fragment and application thereof. In the present invention, the end of the aptamer (aptamers) can form end-protected aptamer (MT-aptamers) after ATP aptamer ligation protection, and MT-aptamers can specifically improve the biological stability in tumor tissues and can be metabolized in normal tissues or organs. Experiments prove that the protective fragment provided by the invention can well improve the stability of various aptamers.

The reagents and consumables adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

examples

1. Description of materials:

the DNA sequences used in the invention are all purchased from Biotechnology engineering (Shanghai) GmbH, and other related materials are all purchased from the same company; in principle, agents or materials which have the same chemical composition or structure obtained by other means can also be used.

The design principle of the aptamer is shown in fig. 18, and the sequence of the aptamer used for verifying the effect is shown in table 1:

TABLE 1 detailed DNA sequence information

2. Sample preparation:

each of the aptamers shown in Table 1 was buffered in the reaction (10mM Tris,10mM MgCl)₂0.1mg/mL BSA, pH7.4) to 90 ℃ for 5 minutes, and then rapidly cooled to 4 ℃; subsequently, the sample was incubated with ATP at 25 ℃ for 60 minutes and then with a nuclease cocktail (0.15U/. mu.LExo I and 0.5U/. mu.LExo III) at 37 ℃ for 15 minutes; finally heated to 95 ℃ for 10 minutes and then stored at 4 ℃ for analysis. For stability assay in serum, ATP-treated samples were treated with 10% fetal bovine serum (FBS, Gibco) at 37 deg.C, heated to 95 deg.C for 10 minutes at various time points, then rapidly cooled to 4 deg.C, and stored at-20 deg.C for analysis.

3. Gel electrophoresis analysis:

to assess the integrity of the aptamers, 5. mu.L of the prepared sample (1.5. mu.M) was mixed with 5. mu.L of urea and analyzed in a 10% polyacrylamide denaturing gel, and the corresponding fluorescent signals were collected by a molecular imaging apparatus (BIO-RAD).

4. Cell culture:

CCRF-CEM, Ramos, K562, HCT116 and HEK293 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum at 37 ℃ with 5% CO₂Culturing in a cell culture box under the conditions of (1).

5. Flow cytometry analysis:

to evaluate the binding ability of the aptamer to the cell, washing buffer (DPBS supplemented with 4.5mg/mLglucose and5 mMMgCl) was used₂) Cells were washed at 4 ℃ (2X 10)⁵) Then, each of the aptamers shown in Table 1 was incubated in a binding buffer (washing buffer supplemented with 0.1mg/ml tRNAand 1mg/ml SA) at 4 ℃ for 1 hour. Washing buffer (DPBS supplemented with 4.5mg/mLglucose and5mM MgCl)₂) Clear at 4 ℃After washing twice, the cells were analyzed using a flow cytometer.

6. Confocal imaging analysis:

to visualize the binding capacity of aptamers to cells, washing buffer (DPBS supplemented with 4.5mg/mL glucose and5mM MgCl) was used₂) Cells were washed at 4 ℃ (2X 10)⁵) Then, they were incubated in a binding buffer (washing buffer supplemented with 0.1mg/mL tRNAand 1mg/mLBSA) with each of the aptamers (300nM) shown in Table 1, respectively, at 4 ℃ for 1 hour. Washed buffer (DPBS supplemented with 4.5mg/mLglucose and5 mMMgCl)₂) After washing twice at 4 ℃, cells were resuspended in confocal dishes for confocal laser imaging analysis.

7. Small animal fluorescence imaging analysis:

to evaluate the biostability of DNA in mice, DNA purchased from a company was lysed using DPBS, and 100 μ L of 5nmol cy 5-labeled DNA was injected into a tumor nude mouse model or a normal healthy nude mouse model (BALB/c) of HCT116 cell transplantation via tail vein of mice. Under anesthesia, imaging analysis was performed on mice using a small animal imager.

8. The experimental results are as follows:

1. the introduction of the ATP aptamer into the end of the target aptamer can greatly improve the biostability of the target aptamer in vitro in the presence of ATP, and has little effect on the ability to bind to the target.

From FIG. 1a it can be seen that MT-sgc8c is stably present in the presence of both nuclease mixture and ATP (lane 5), whereas Sgc8c (lane 2) without protection of the ATP aptamer and MT-sgc8c (lane 4) are completely decomposed by the nuclease mixture in the absence of ATP; the flow results from FIG. 1b also show that MT-sgc8c is resistant to nuclease cleavage in the presence of ATP and retains the ability to bind to CCRF-CEM cells;

furthermore, it can be seen from FIG. 2a that Control-1 (end design with ATP aptamer protection) exhibits some resistance to enzymatic cleavage in the presence of both nuclease mixture and ATP (lane 3); control-2 (end design without ATP aptamer protection) is completely decomposed by the nuclease mixture in the presence of both the nuclease mixture and ATP, proving the necessity of end design with ATP aptamer protection; the importance of the end design protected by the ATP aptamer for improving the biostability of the target aptamer is also laterally demonstrated from the flow results in FIG. 2 b;

furthermore, it can be seen from FIG. 3a that the cleavage resistance of MT-sgc8c increases with increasing ATP concentration (lanes 2-7); this is also demonstrated by the increasing binding shift of MT-sgc8c to CCRF-CEM cells in FIG. 3 b.

To further demonstrate the specificity of Sgc8c binding to target cells with MT-sgc8c, it can be seen from FIG. 4a that only Sgc8c bound to MT-sgc8c target cells (CCRF-CEM) and also not to selected Control cells (K562 and Ramos) compared to library sequence design of equivalent length (Lib-41 and Control-1); from the confocal imaging analysis in fig. 4b it was also verified Sgc8c bound to the target cells (CCRF-CEM) with MT-sgc8c and also not to the selected control cells (K562 and Ramos);

as can be seen from the melting temperature analysis of Sgc8c and MT-sgc8c and the corresponding control sequence in FIG. 5, the introduction of ATP concentration did not affect the thermodynamic stability of DNA in the experiment, and the improvement of the biostability of MT-sgc8c was not directly related to the improvement of the melting temperature, which suggests that the improvement of the biostability of MT-sgc8c is due to the binding of ATP to the aptamer-protected end-designed sequence of ATP.

As can be seen from FIG. 6a, MT-sgc8c was stable in vitro for more than 36 hours in the presence of ATP at a certain concentration; as can be seen from FIG. 6b, MT-sgc8c retained the ability to bind to cells for 36 hours only in the presence of a certain concentration of ATP.

As can be seen from FIG. 12, the introduction of the ATP aptamer-protected end design had little effect on the binding ability of MT-sgc8c to target cell CCRF-CEM compared to original Sgc8c, and in addition, the presence of ATP had little effect on the binding ability of MT-sgc8c to target cell CCRF-CEM.

2. The ATP aptamer is introduced into the tail end of the target aptamer, and the tumor microenvironment has higher ATP concentration relative to normal tissues, so that the biological stability of the MT-aptamers can be greatly enhanced, and the action time of the MT-aptamers on a disease part can be prolonged.

First, as can be seen from fig. 10, the aptamers bind well to tumor cells. As can be seen from FIG. 11, the ATP content at the tumor site was much higher than that of other tissues and organs by analyzing the ATP content in different tissues of the mouse model of tumor seeded with HCT116 cells.

As can be seen from fig. 13, MT-sgc8c could be present in the tumor site in the HCT116 cell-seeded tumor mouse model for more than 300 minutes, whereas the group Sgc8c had begun to gradually decrease in fluorescence intensity after 120 minutes; the importance of introducing an ATP aptamer to the end of a target aptamer to improve the biostability of the aptamer was fully demonstrated, since Control-1 did not contain Sgc8c, and thus could not be specifically enriched at the tumor site, and Control-2 did not contain an ATP aptamer-protected end design and thus was not stably present in the tumor region for more than 120 minutes.

As can be seen from FIG. 14, the organofluorescence imaging of the mouse in FIG. 13 after 300 minutes showed the presence of DNA only at the tumor sites of the MT-sgc8c group, illustrating laterally the importance of introducing ATP aptamers to the ends of the target aptamers for improving the biostability of the aptamers. In addition, fluorescence in the mouse kidney and liver is due to normal metabolism in the metabolic organs.

As can be seen from FIG. 15, the fluorescence intensity of the library sequences of the same length (Lib-41 and Lib-79) in the mouse tumor region had substantially disappeared after 120 minutes, and in addition, no fluorescence except for the metabolic organs was shown to indicate that MT-sgc8c could be specifically enriched in the tumor site in the HCT116 cell-seeded tumor mouse model.

As can be seen from FIGS. 16-17, the imaging effect of different aptamers in a mouse body shows the metabolic process of the aptamers in the mouse body.

To demonstrate that fluorescence in the kidney and liver regions of the mouse in FIG. 14 is caused by normal metabolism of the metabolic organs, Sgc8c, MT-sgc8c and library sequences of the same length (Lib-41 and Lib-79) were injected into a healthy mouse model, and from the fluorescence imaging and organ and tissue imaging analysis in FIGS. 15 and 16, it can be seen that the fluorescence change in the kidney and liver regions of the mouse is caused by normal metabolism of the metabolic organs.

3. Introduction of ATP aptamers to the ends of target aptamers improves the versatility of the biostability approach to nucleic acid aptamers, which can in theory be broadened to the adaptation of nucleic acid aptamer sequences not to mention in the present invention.

Due to the design diversity of the DNA sequence, the biostability of any aptamer can be improved by the method mentioned in the present invention by replacing the aptamer sequence in the MT-sgc8c sequence (see Table 1) with any aptamer. In order to demonstrate the versatility of the method, the nucleic acid aptamer sequence in the MT-sgc8c sequence was experimentally replaced with the nucleic acid aptamers XQ-2d, KK1B10 and TDO5 to construct corresponding MT-aptamers (see Table 1), and from the experimental data in FIGS. 7, 8 and 9, it can be seen that MT-XQ-2d, MT-KK1B10 and MT-TDO5 can all exist stably in the presence of both a nuclease mixture and ATP (lane 3 in a), and in the absence of ATP, MT-XQ-2d, MT-KK1B10 and MT-TDO5 are all decomposed completely by the nuclease mixture (lane 2 in a); in addition, the flow experimental data in B also prove that MT-XQ-2d, MT-KK1B10 and MT-TDO5 can stably exist in a nuclease mixture and ATP and can be combined with corresponding target cells; from the c confocal imaging analysis, it can be seen that only MT-XQ-2d, MT-KK1B10 and MT-TDO5 bound the target cells compared to the control sequence Contro-1.

4. The present invention aims to develop a method for specifically improving the biostability of nucleic acid aptamers (consisting of only natural base elements) by utilizing the characteristics of changes in abnormal tissues or organs (relative to normal tissues or organs). Taking the tumor microenvironment with higher ATP concentration relative to normal tissues and taking ATP aptamer as an end protection part as an example, the ATP aptamer end protection part can be replaced by any type of aptamer as the end protection part so as to improve the biological stability of the target aptamer.

As shown in lanes 4 and5 of Control-2 in FIG. 2, the introduction of an arbitrary library sequence at the end of Scg8c did not improve the biostability of Control-2 in the presence of ATP, and it was also demonstrated from the flow-through results that the introduction of an arbitrary library sequence at the end of Scg8c did not affect the function of binding to target cells. The selection of aptamers is generally derived from enrichment screening of any library sequence, and thus, the substitution of any library sequence with any aptamer or functional nucleic acid sequence is not limited to ATP aptamers as the end-protecting moiety.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> university of Hunan

<120> protective fragment of nucleic acid aptamer and nucleic acid aptamer comprising same

<130> MP2016915

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acctggggga gtat 14

<210> 2

<211> 13

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgcggaggaa ggt 13

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

catcgcacct gggggagtat 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgcggaggaa ggtgcgatg 19

<210> 5

<211> 79

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

catcgcacct gggggagtat tctaactgct gcgccgccgg gaaaatactg tacggttaga 60

tgcggaggaa ggtgcgatg 79

<210> 6

<211> 95

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

catcgcacct gggggagtat actcataggg ttaggggctg ctggccagat actcagatgg 60

tagggttact atgagctgcg gaggaaggtg cgatg 95

<210> 7

<211> 93

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

catcgcacct gggggagtat acagcagatc agtctatctt ctcctgatgg gttcctattt 60

ataggtgaag ctgttgcgga ggaaggtgcg atg 93

<210> 8

<211> 84

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

catcgcacct gggggagtat caccgtggag gatagttcgg tggctgttca gggtctcctc 60

cggtgtgcgg aggaaggtgc gatg 84

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41

<210> 10

<211> 79

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

catcgcacct gggggagtat nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

tgcggaggaa ggtgcgatg 79

<210> 11

<211> 79

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

catcgcnnnn nnnnnnnnnn tctaactgct gcgccgccgg gaaaatactg tacggttaga 60

nnnnnnnnnn nnngcgatg 79

Claims

The application of the combination of the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 as the protective fragment of the aptamer.
2. A combination of protective fragments of aptamers comprising an X-chain and a Y-chain;

the X chain sequence is shown as SEQ ID NO. 3, and the Y chain sequence is shown as SEQ ID NO. 4.
3. A nucleic acid aptamer comprising an aptamer fragment in combination with the protective fragment of claim 2.
4. The nucleic acid aptamer according to claim 3, wherein the number of aptamer fragments is 1 or 2; and the two ends of the aptamer fragment are respectively connected with the X chain and the Y chain of the protection fragment.
5. The nucleic acid aptamer according to claim 3 or 4, wherein the target of the aptamer comprises: at least one of metal ions, adenosine, proteins, drugs, pathogenic microorganisms, cells or tissues.
6. The nucleic acid aptamer according to claim 3 or 4, further comprising a drug, a chemical marker and/or a biomarker;

the drug is a chemical drug or an RNAi molecule;

the chemical label is a fluorophore, isotope and/or immunotoxin;

the biomarker is a biotin, avidin, or enzyme label.
7. The aptamer according to claim 6, wherein the nucleic acid sequence is as shown in any one of SEQ ID NO 5-8; the 5' end of the fluorescent probe is modified with FAM fluorescent group or Cy5 fluorescent group.
8. Use of the nucleic acid aptamer of any one of claims 3 to 7 in the preparation of a target detection reagent.
9. A target detection reagent comprising the nucleic acid aptamer according to any one of claims 3 to 7.
10. Use of the nucleic acid aptamer of any one of claims 3 to 7 in the preparation of a medicament for the treatment of a disease.
11. A medicament for treating diseases, comprising the nucleic acid aptamer according to any one of claims 3 to 7.
12. Use of the nucleic acid aptamer of any one of claims 3 to 7 for the preparation of an imaging agent for a target area.
13. An imaging agent comprising the nucleic acid aptamer according to any one of claims 3 to 7.