CN109913462B

CN109913462B - Application research of aptamer recognition and combination with CD171 and related functions thereof

Info

Publication number: CN109913462B
Application number: CN201711315775.8A
Authority: CN
Inventors: 上官棣华; 王林林; 邴涛; 刘祥军; 张楠; 沈璐瑶
Original assignee: Institute of Chemistry CAS
Current assignee: Institute of Chemistry CAS
Priority date: 2017-12-12
Filing date: 2017-12-12
Publication date: 2022-09-13
Anticipated expiration: 2037-12-12
Also published as: WO2019114675A1; CN109913462A

Abstract

The invention discloses an application study of aptamer recognition and combination with CD171 and related functions thereof. The aptamer and the derivative thereof can be applied to identifying high-expression tumors of L1 cell adhesion molecule protein or preparing kits, molecular probes and targeting media for detecting high-expression tumors of L1 cell adhesion molecule protein, as well as to designing and preparing nucleic acid probes for detecting L1 cell adhesion molecule protein and applying the nucleic acid probes to neurite growth inhibition. Compared with the prior art, the aptamer of the invention has the advantages of higher affinity and specificity, no immunogenicity, capability of chemical synthesis, small molecular weight, stability, easy storage and marking and the like.

Description

Application research of aptamer recognition and combination with CD171 and related functions thereof

Technical Field

The invention belongs to the technical field of biochemical analysis, and particularly relates to an application study of a nucleic acid aptamer for recognizing and combining L1 cell adhesion molecules (L1CAM, CD171) and related functions thereof.

Background

L1 cell adhesion molecule (neuronal cell adhesion molecule L1(L1CAM), also called CD171) is a member of the L1 protein family, identified for the first time in 1984 by Melita Schachner in studying the post-mitotic phase of mouse neurons, the cells expressing the L1 cell adhesion molecule are numerous, not only neuronal cells but also some non-neuronal cells, the cells which have been reported to express the L1 cell adhesion molecule are immature oligodendrocytes and Schwann cells, T-cells, B-cells and monocytes, cerebellar granulocytes and Purkinje cells, which also express it in a variety of tumor cells such as melanoma and lung cancer cells, the L1 cell adhesion molecule is a transmembrane protein consisting of three transmembrane, transmembrane and intracellular domains (200-220kDa), the extracellular domain consists of five leukotypic domains linked to six immunoglobulin III domains, linked to the intracellular domain by a transmembrane helix. Its main functions are cell migration, adhesion, neurite outgrowth, myelination and neuronal differentiation. In terms of cell adhesion, the L1 cell adhesion molecule has a homeostatic function as a cell adhesion molecule linking different cells, and the fasciculate spontaneous contractions of the neurites control the adhesion between neurons and thus the growth of the neurites. In terms of cell migration, which promotes growth regulation of nerve cells during neuronal development, L1 cell adhesion molecules are present in developing neuronal cells and play an important role in guiding new neurons into the correct position, assisting axonal growth and connecting to other neurons, and in addition, L1 cell adhesion molecules are involved in synaptic plasticity, i.e., enhancement or reduction of synaptic plasticity ability, and play a role in synaptic regeneration after trauma. Several studies have demonstrated that L1 cell adhesion molecules play an important role in tumor growth, tumor invasion, melanoma, metastasis of ovarian and colon cancers due to the overexpression of L1 cell adhesion molecules, thereby increasing the cell migration capacity of malignant cells. L1 cell adhesion molecules have the effect of promoting homophilic interactions, i.e., adhesion molecules on one cell interact with the same molecule on another cell; l1 cell adhesion molecules also have heterophilic interactions, i.e., adhesion molecules on one cell act as receptors linked to different molecules on another cell, and these interactions promote the regulation of cell adhesion and signal transduction. In addition, the L1 cell adhesion molecule is involved in myelination of neurons, and in the proliferation of myelin sheaths in the nervous system (especially the progressive myelination of nerve axonal fibers) by mediating schwann cell growth along axons. It also has a key role in tumor resistance. Mutations in the adhesion molecule of L1 cells often cause neurological syndromes, referred to as MASA syndrome, also called CRASH syndrome, mainly dysgenosis of the corpus callosum, developmental block, aphasia, spastic paraplegia and hydrocephalus.

Aptamer (aptamer) is a single-chain oligonucleotide composed of 10-150 bases, can recognize a certain ligand molecule or a certain class of ligand molecules with high binding force and high selectivity, and is called as a chemically synthesized antibody. The aptamer can be DNA, RNA, peptide nucleic acid or other chemically modified nucleic acid; since the nucleic acid can form different three-dimensional structures such as hairpin, pseudoknot, G-quadruplex and the like by virtue of intermolecular interactions such as van der Waals force, hydrogen bond, electrostatic interaction, hydrophobic interaction and the like, the aptamer can realize the binding effect with high affinity and high specificity with a target substance. The technology of screening the aptamer which is efficiently and specifically combined with the target substance is called the EXponential enrichment ligand Systematic Evolution (SELEX) technology. The target substances of the aptamers screened by the SELEX technology are wide in range, including inorganic metal ions, organic small molecules, biological macromolecules, viruses, bacteria, cells, tissue sections and the like, and hundreds of aptamers have been reported so far.

The aptamer aiming at complex targets such as living cells, tissues and the like, which can be screened by utilizing the SELEX technology, has a similar recognition function with the monoclonal antibody, but has the following advantages compared with the monoclonal antibody:

(1) in vitro screening, animals or cells need not be immunized;

(2) low toxicity or immunogenicity, good water solubility, and capability of subcutaneous intravenous injection or focal injection with large dose;

(3) can be prepared by a large amount of chemical synthesis, and has small batch difference;

(4) the structure is easy to modify, mark or immobilize, and the stability in serum is increased;

(5) the stability is good, and the storage and the transportation are easy;

(6) the molecular weight is small, and is generally 4-50 KD.

Based on the advantages, the aptamer serving as a novel bionic recognition element is widely applied to a plurality of fields such as disease diagnosis and treatment, drug screening, molecular recognition, analysis and detection and the like.

Disclosure of Invention

An object of the present invention is to provide a nucleic acid aptamer.

The aptamer provided by the invention is A) or B):

A) a single-stranded DNA molecule shown in sequence 1;

B) removing 1-20 nucleotides of the nucleotide sequence shown in A) including the first nucleotide residue at the 5 'end from the 1 st nucleotide at the 5' end, and removing 10-20 nucleotides of the nucleotide sequence shown in A) including the first nucleotide residue at the 3 'end from the 1 st nucleotide at the 3' end, wherein the remained nucleotide residues form the nucleic acid aptamer.

In the above aptamer, the aptamer represented by B is any one of the following 1) to 7):

1) a single-stranded DNA molecule shown in sequence 2;

2) a single-stranded DNA molecule shown in sequence 3;

3) a single-stranded DNA molecule shown in sequence 4;

4) a single-stranded DNA molecule shown in sequence 5;

5) a single-stranded DNA molecule shown in sequence 6;

6) a single-stranded DNA molecule shown in sequence 7;

7) a single-stranded DNA molecule shown in sequence 8.

It is another object of the present invention to provide derivatives of the above-mentioned nucleic acid aptamers.

The derivative of the aptamer provided by the invention is any one of the following (1) to (6):

(1) deleting or adding one or more nucleotides to the aptamer to obtain a derivative of the aptamer with the same function as the aptamer;

(2) carrying out nucleotide substitution or modification on the aptamer to obtain a derivative of the aptamer with the same function as the aptamer;

(3) transforming the skeleton of the aptamer into a phosphorothioate skeleton to obtain a derivative of the aptamer with the same function as the aptamer;

(4) obtaining a derivative of the aptamer with the same function as the aptamer from the RNA molecule coded by the aptamer;

(5) obtaining derivatives of the aptamer with the same function as the aptamer from the peptide nucleic acid coded by the aptamer;

(6) and (2) adding a signal molecule and/or an active molecule and/or a functional group to one end or middle of the aptamer to obtain the derivative of the aptamer with the same function as the aptamer.

In the above derivatives, the modification is phosphorylation, methylation, amination, sulfhydrylation or isotopic amination; the functional group is a fluorescent group, a biotin group, a radioactive substance, a therapeutic substance, digoxin, a nano luminescent material or an enzyme label.

In the above derivatives, the derivative of the aptamer is a derivative of the aptamer obtained by labeling a fluorescein group, a cyanine dye group, or a biotin group to the 5' -end of the aptamer shown in sequence 8.

The use of the above-mentioned aptamer or the above-mentioned derivative in at least one of the following (B1) to (B19) is also within the scope of the present invention:

(B1) recognizing or aiding in recognizing the L1 cell adhesion molecule protein;

(B2) binds to or aids in binding to L1 cell adhesion molecule protein;

(B3) preparing a protein product for identifying or assisting in identifying the L1 cell adhesion molecule;

(B4) preparing a protein product which binds or assists in binding L1 cell adhesion molecules;

(B5) detecting or assisting to detect whether the sample to be detected contains L1 cell adhesion molecule protein;

(B6) preparing a product for detecting or assisting in detecting whether the sample to be detected contains the L1 cell adhesion molecule protein;

(B7) detecting or assisting to detect the content of the L1 cell adhesion molecule protein in the sample to be detected;

(B8) preparing a product for detecting or assisting in detecting the content of the L1 cell adhesion molecule protein in a sample to be detected;

(B9) detecting or assisting in detecting the tumor or tumor cell expressing the L1 cell adhesion molecule protein;

(B10) preparing a product for detecting or assisting in detecting the tumor or the tumor cell expressing the L1 cell adhesion molecule protein;

(B11) identifying or aiding in the identification of a tumor or tumor cell;

(B12) binding or aiding binding to a tumor or tumor cell;

(B13) preparing a product for identifying or assisting in identifying tumors or tumor cells;

(B14) preparing a product that binds or assists in binding to a tumor or tumor cell;

(B15) preparing a product of the targeted L1 cell adhesion molecule protein;

(B16) inhibiting neurite outgrowth of neuroblastoma cells;

(B17) preparing a product for inhibiting neurite outgrowth of neuroblastoma cells;

(B18) identifying neurite outgrowth of neuroblastoma cells;

(B19) a product was prepared that recognized the growth of neurites of neuroblastoma cells.

In the above application, the sample to be tested is a cell; the cells are specifically neuroblastoma cells, human colon cancer cells, human cervical cancer cells, human breast cancer cells, adriamycin-resistant human breast cancer cells or liver cancer cells.

The tumor is neuroblastoma, human colon cancer, human cervical cancer, human breast cancer, adriamycin-resistant human breast cancer or liver cancer.

In the above application, the tumor or tumor cell expressing the L1 cell adhesion molecule protein is a tumor or tumor cell (e.g., a human colon cancer cell or a human neuroblastoma cell) with high expression of the L1 cell adhesion molecule protein.

In the application, the product is a kit or a probe or a targeting substance or a medicament.

The 3 rd object of the present invention is to provide a product having the following functions.

The invention provides a product with the following functions, wherein the active ingredients of the product are the aptamer or the derivative;

the function is at least one of the following 1) to 9):

1) recognizing or aiding in recognizing the L1 cell adhesion molecule protein;

2) binds to or assists in binding to L1 cell adhesion molecule protein;

3) detecting or assisting to detect whether the sample to be detected contains L1 cell adhesion molecule protein;

4) detecting or assisting to detect the content of the L1 cell adhesion molecule protein in the sample to be detected;

5) detecting or assisting in detecting a tumor or tumor cell expressing the L1 cell adhesion molecule protein;

6) identifying or aiding in identifying a tumor or tumor cell;

7) binding or aiding binding to a tumor or tumor cell;

8) the preparation method is used for preparing the medicine targeting the L1 cell adhesion molecule protein;

9) inhibiting neurite outgrowth of neuroblastoma cells;

10) identifying neurite outgrowth of neuroblastoma cells.

In the above, the product is a kit or a probe or a targeting substance or a drug.

Compared with the prior art, the invention has the advantages that: the aptamer obtained by screening has high affinity; no immunogenicity; can be chemically synthesized in vitro, has small molecular weight, can modify and replace different parts, has stable sequence and is easy to store; convenient labeling (no labeled secondary antibody required), etc. When the aptamer is used for detecting the L1 cell adhesion molecule protein, the operation is simpler and quicker, and the synthesis cost of the aptamer is lower than that of antibody preparation, the period is short and the reproducibility is good.

Other features and embodiments of the present invention will become more fully apparent from the following detailed description and appended claims.

Drawings

FIG. 1 shows the enrichment process of aptamers with increasing number of rounds of selection in example 1. The peak-shaped curve shows the cell fluorescence distribution of the differentiated neuroblastoma cell SH-SY 5Y; blank cells were used as a control, and differentiated neuroblastoma cells SH-SY5Y were combined with a Fluorescein (FAM) -labeled nucleic acid library, the initial library, round 1, round 3, round 5, and round 6 binding events.

FIG. 2 shows the confocal binding fluorescence of the target cells (differentiated neuroblastoma cells D-SH-SY5Y) in rounds 1, 4, 6 and 8 of the enriched library selected in example 2.

FIG. 3 is a measurement of the binding dissociation constants of nucleic acid aptamer ylQ3, nucleic acid aptamer yly1, nucleic acid aptamer yly2, nucleic acid aptamer yly3, nucleic acid aptamer yly4, nucleic acid aptamer yly10, nucleic acid aptamer yly11, and nucleic acid aptamer yly12 to colon cancer cells LoVo in example 3.

FIG. 4 shows the results of flow cytometry experiments in example 5 in which multiple tumor cells were co-infected with L1 cell adhesion molecule protein antibody (anti-CD171) and aptamer yly 12. A is LoVo cell, B is SH-SY5Y cell, C is PC-3 cell.

FIG. 5 shows the results of flow cytometry experiments on LoVo cells stained with L1 cell adhesion molecule protein antibody (anti-CD171) and aptamer yly12 after siRNA in example 6 interfered with the expression of L1 cell adhesion molecule protein by LoVo cells of human colon cancer.

FIG. 6 shows the confocal binding fluorescence of neuroblastoma D-SH-SY5Y differentiated from target cells co-infected with L1 cell adhesion molecule protein antibody (anti-CD171) and aptamer yly12 in example 7.

FIG. 7 shows the confocal binding fluorescence of LoVo cells co-infected with L1 cell adhesion molecule protein antibody (anti-CD171) and aptamer yly12 in example 8.

FIG. 8 shows fluorescent staining of human brain tissue sections with aptamer yly12 and a control sequence as in example 9.

FIG. 9 shows fluorescent staining of olfactory neuroblastoma tissue sections with nucleic acid aptamer yly12 and a control sequence as in example 9.

FIG. 10 shows the inhibition of neurite outgrowth of SH-SY5Y in neuroblastoma cells by nucleic acid aptamer yly12 in example 10.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The nucleic acid sequences in the following examples were synthesized by Shanghai Biotechnology, Inc.

The L1 cell adhesion molecule protein antibody (anti-CD171) in the following examples is a product of BD Biosciences, Catalogue No. 554273.

Antibody isotype control (normal mouse IgG) in the examples described below ₁ ) Is a product of ANTA CRUZ company, and has catalog number sc-3877.

The phycoerythrin-modified secondary antibody (rabbitant anti-mouse IgG-PE) in the examples described below is a product of ANTA CRUZ company under the catalog number sc-358926.

The siRNA in the examples described below is a product of suma germa gene inc.

Binding buffer in the following examples (pH 7.4): containing 137mM NaCl, 5mM MgCl ₂ 、2.7mM KCl、2mM KH ₂ PO ₄ 、10mM Na ₂ HPO ₄ 25mM glucose, 1. mu.g/ml BSA, 0.1. mu.g/ml herring sperm DNA and 0.01% (v/v) Tween-80.

Wash buffer in the following examples (pH 8.0): containing 137mM NaCl, 5mM MgCl ₂ 、2.7mM KCl、2mM KH ₂ PO ₄ 、10mM Na ₂ HPO ₄ And 25mM glucose.

siRNA solubilizers in the following examples: DEPC water.

Dicyclohexylcarbodiimide (DCC) in the following examples is a product from the company carbofuran under the catalog No.: 36650, respectively; 4-Dimethylaminopyridine (DMAP) is a product from carbofuran, catalog number: 117147.

example 1 screening and preparation of aptamers

Culture of neuroblastoma cells and differentiated neuroblastoma cells

1. Culture of neuroblastoma cells

Neuroblastoma cells (SH-SY5Y) (purchased from the institute of basic medicine of Chinese academy of medical sciences (Beijing), catalog number: 3111C0001CCC000026)) were cultured with RPMI1640 (containing 15% inactivated fetal bovine serum, 1% penicillin/streptomycin).

2. Culture of differentiated neuroblastoma cells

The differentiated neuroblastoma cells (D-SH-SY5Y) were cultured for three days in RPMI1640 (containing 10. mu.M tretinoin A, RA), and then cultured for four days in RPMI1640 (containing 10. mu.M tretinoin A and 10ng/ml brain-derived nerve growth factor BDNF).

All the cells were routinely cultured in an incubator (37 ℃ C., 5% CO) ₂ ) Passage every two days.

Design of random nucleic acid library

A random library was designed that included 20 fixed nucleotides at both ends and 45 nucleotides in the middle as follows: 5' -AAGGAGCAGCGTGGAGGATA-N ₄₅ -TTAGGGTGTGTCGTCGTGGT-3'; wherein, N ₄₅ Representing 45 random nucleotide sequences of A, T, C or G.

Screening of aptamer

1. Library pretreatment

Dissolving 18nmol random nucleic acid library (synthesized in the second step) in binding buffer, denaturing at 95 ℃ for 5min, cooling on ice for 10min, and renaturing at room temperature for 30min to obtain the pretreated ssDNA library.

2. Reverse sieve

A dish of neuroblastoma cells SH-SY5Y was taken and a pool of pretreated ssDNA (decreasing DNA content with increasing screening pressure) was added to the cells and incubated at 4 ℃ with shaking for 1h (increasing incubation time with increasing number of screening rounds) before 2-8 rounds of incubation with the target cells (differentiated neuroblastoma cells D-SH-SY 5Y).

3. Positive sieve

The supernatant from the reverse screening process was incubated with one dish of differentiated neuroblastoma cells D-SH-SY5Y at 4 ℃ for a period of time with shaking (the incubation time was gradually decreased as the number of screening rounds was increased). And then washing the cells for several times by using the binding buffer solution, blowing the cells down by using a pipette, centrifuging at 4000RPM to remove cell bodies, adding the supernatant solution into a new centrifuge tube, centrifuging at 12000RPM to collect precipitates and obtain neurites, adding 200 mu L of water, heating at 95 ℃ for 5min, and eluting ssDNA bound on the differentiated neuroblastoma cell D-SH-SY5Y neurites. The eluted ssDNA is then PCR amplified. The primers for PCR amplification were:

5’-FAM-AAGGAGCAGCGTGGAGGATA-3’；

5’-Biotin-ACCACGACGACACACCCTAA-3’。

PCR amplification procedure: 3min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃, 30s at 72 ℃ and 10 cycles; 72 ℃ for 5 min.

The FAM-labeled single-stranded dna (ssdna) sequence was isolated from the PCR product using streptavidin agarose beads. The ssDNA obtained was desalted using NAP-5 column (general electric medical group, Sweden) and dried under vacuum for the next round of screening.

In order to improve the affinity and specificity of the aptamer, the washing times are gradually increased, the number of positive sieve cells D-SH-SY5Y is reduced, and the number of negative sieve cells SH-SY5Y is increased in the screening process, so that the screening pressure is increased. After eight rounds of screening, PCR amplification was performed with primers (5'-ACGCTCGGATGCCACTACAG-3' and 5'-GTCACCAGCACGTCCATGAG-3') using the screened products as templates and the sixth round PCR products were sequenced. The final selected aptamer 1 (also known as aptamer ylQ3) was as follows:

5'-AAGGAGCAGCGTGGAGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTGTTAGGGTGTGTCGTCGTGGT-3' (SEQ ID NO: 1).

The enrichment process of the aptamer along with the number of screening rounds is shown in figure 1, the nucleotide sequence bound with the positive-screened cell D-SH-SY5Y is obviously enriched in the 1 st round, and after gradual pressurization, the binding sequence is further enriched in the 3 rd round, the 5 th round and the 6 th round.

4. Optimization of aptamers

The aptamer obtained in the step 3 is long, a series of truncated nucleic acid sequences are designed and synthesized after structural analysis, the binding capacity of the nucleic acid sequences and the positive-sieve cell D-SH-SY5Y is investigated through fluorescent dye modification, the sequences with the strongest binding capacity are selected for further application, and the optimized sequences are only 51-85 nucleotides in length.

The resulting truncated aptamer sequence was as follows:

aptamer 2 (also known as aptamer yly 1):

5'-AAGGAGCAGCGTGGAGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTGTTAGGG-3' (SEQ ID NO: 2); aptamer 2 is a nucleotide sequence obtained by cutting aptamer 1 by 14 nucleotides from the 3' end, while keeping the other sequences of aptamer 1 unchanged.

Aptamer 3 (also known as aptamer yly 2):

5'-TGGAGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTGTTAGGGTGTGTCGTCGTGGT-3' (SEQ ID NO: 3); the aptamer 3 is a nucleotide sequence obtained by cutting 11 nucleotides from the 5' end of the aptamer 1 while keeping the other sequences of the aptamer 1 unchanged.

Aptamer 4 (also known as aptamer yly 3):

5'-TGGAGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTGTTAGGG-3' (SEQ ID NO: 4); aptamer 4 is a nucleotide sequence obtained by cleaving aptamer 1 by 11 nucleotides from the 5 'end and by 14 nucleotides from the 3' end, while keeping the other sequences of aptamer 1 unchanged.

Aptamer 5 (also known as aptamer yly 4):

5'-TGGAGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTGTTA-3' (SEQ ID NO: 5); the aptamer 5 is a nucleotide sequence obtained by cutting 11 nucleotides from the 5 'end of the aptamer 1 and 17 nucleotides from the 3' end of the aptamer 1, and keeping the other sequences of the aptamer 1 unchanged.

Nucleic acid aptamer 6 (also known as nucleic acid aptamer yly 10):

5'-TGGAGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTG-3' (SEQ ID NO: 6); aptamer 6 is a nucleotide sequence obtained by cleaving aptamer 1 by 11 nucleotides from the 5 'end and by 20 nucleotides from the 3' end, while keeping the other sequences of aptamer 1 unchanged.

Aptamer 7 (also known as aptamer yly 11):

5'-AGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTGTTA-3' (SEQ ID NO: 7); aptamer 7 is a nucleotide sequence obtained by cleaving aptamer 1 by 14 nucleotides from the 5 'end and 17 nucleotides from the 3' end, while keeping the other sequences of aptamer 1 unchanged.

Nucleic acid aptamer 8 (also known as nucleic acid aptamer yly 12):

5'-AGGATAGGGGGTAGCTCGGTCGTGTTTTTGGGTTGTTTGGTGGGTCTTCTG-3' (SEQ ID NO: 8); aptamer 8 is a nucleotide sequence obtained by cleaving aptamer 1 from the 5 'end by 14 nucleotides and from the 3' end by 20 nucleotides, while leaving the other sequences of aptamer 1 unchanged.

The optimization result shows that the aptamer 6 has the strongest binding affinity with cells and the highest fluorescence intensity; the nucleic acid aptamer 5 is arranged next, and then the nucleic acid aptamer 7, the nucleic acid aptamer 8, the nucleic acid aptamer 1, the nucleic acid aptamer 4, the nucleic acid aptamer 3 and the nucleic acid aptamer 2 are arranged in sequence from high to low in binding effect.

Example 2 enrichment of libraries and Co-focal imaging of differentiated neuroblastoma cells

The enriched library obtained in example 1 was subjected to PCR amplification in round 1, round 4, round 6 and round 8, respectively, to prepare single-stranded DNAs, each single-stranded DNA (FAM-labeled DNA sequence) was dissolved in a binding buffer, the concentration was calibrated by UV absorption, and then heated at 95 ℃ for 5min, placed on ice for 10min, and placed at room temperature for 30 min. The denatured-renatured DNA was diluted with binding buffer to 200nmol/L DNA solution (containing 1. mu.g/ml BSA, 0.1. mu.g/ml herring sperm DNA), added to the cells of D-SH-SY5Y that had been separately cultured in a confocal dish, incubated on ice for 30min, washed once with PBS and observed under a confocal microscope 100-fold microscope.

It is evident from FIG. 2 that the aptamers in rounds 4, 6 and 8 bind well to the neurites of differentiated SH-SY5Y cells.

Example 3 binding ability of nucleic acid aptamer to human colon cancer cell LoVo

Nucleic acid aptamer 1, nucleic acid aptamer 2, nucleic acid aptamer 3, nucleic acid aptamer 4, nucleic acid aptamer 5, nucleic acid aptamer 6, nucleic acid aptamer 7, and nucleic acid aptamer 8 obtained in example 1 were labeled with a Fluorescein (FAM) molecule at the 5' end, respectively, to obtain FAM-labeled nucleic acid aptamer 2, FAM-labeled nucleic acid aptamer 3, FAM-labeled nucleic acid aptamer 4, FAM-labeled nucleic acid aptamer 5, FAM-labeled nucleic acid aptamer 6, FAM-labeled nucleic acid aptamer 7, and FAM-labeled nucleic acid aptamer 8 (synthesized by shanghai bio corporation), respectively. Dissolving each single-stranded DNA (FAM labeled aptamer) by using a binding buffer solution, calibrating the concentration according to ultraviolet absorption, heating at 95 ℃ for 5min, standing on ice for 10min, and standing at room temperature for 30min to obtain denatured-renatured DNA; then, the DNA solution (molecular probe solution) was diluted with the binding buffer to have concentration gradients of 0.1nmol/L, 0.25nmol/L, 0.5nmol/L, 1nmol/L, 2.5nmol/L, 5nmol/L, 10nmol/L, 50nmol/L, 100nmol/L and 200 nmol/L.

Taking colon cancer cells LoVo in a logarithmic growth phase, digesting the colon cancer cells LoVo into monodisperse cell suspension by using 0.2% EDTA, evenly dividing the suspension into a plurality of parts, respectively incubating the parts with the molecular probe solution on ice for 30min, washing the parts twice by using a washing buffer solution, and measuring the fluorescence intensity on the cell surface by using a FACSCalibur flow cytometer of BD company. The average fluorescence intensity on the cell surface and the aptamer concentration were plotted, and the equilibrium dissociation constant of the aptamer was calculated using the formula Y ═ BmaxX (Kd + X).

The binding curves of aptamer 1, aptamer 2, aptamer 3, aptamer 4, aptamer 5, aptamer 6, aptamer 7 and aptamer 8 to colon cancer cells LoVo are shown in fig. 3. In FIG. 3, the abscissa represents the single-stranded DNA concentration (nmol/L), and the ordinate represents the mean fluorescence intensity after deduction of the autofluorescence of cells. It is shown that the nucleic acid aptamer 1, the nucleic acid aptamer 2, the nucleic acid aptamer 3, the nucleic acid aptamer 4, the nucleic acid aptamer 5, the nucleic acid aptamer 6, the nucleic acid aptamer 7 and the nucleic acid aptamer 8 can be combined with the colon cancer cell LoVo.

The equilibrium dissociation constants of the above aptamers 1 to 8 are shown in Table 1. The result shows that the equilibrium dissociation constants of the aptamer 1, the aptamer 4, the aptamer 5, the aptamer 6, the aptamer 7 and the aptamer 8 are all in nanomolar level, and the affinity is high.

TABLE 1 equilibrium dissociation constants of aptamers 1-8

Example 4 Mass Spectrometry identification of aptamer 8 (also known as aptamer yly12) specifically recognizing and binding to L1 cell adhesion molecule protein

Preparation of isotope-labeled LoVo cell and aptamer 8 derivative

1. Preparation of isotopically labeled LoVo cells

The heavy isotope-labeled LoVo cell (institute of basic medicine of medical academy of sciences (Beijing)) used lysine (, [ 2 ]) labeled with a heavy isotope ¹³ C ₆ , ¹⁵ N ₂ ]-L-lysine and heavy isotope labeled arginine ([ alpha ], [ beta ] -lysine) ¹³ C ₆ ]-L-arginine) in RPMI1640 medium; the light isotope-labeled Jurkat E6-1 cell uses a protein comprising light isotope-labeled lysine (, [ 2 ] ¹² C ₆ , ¹⁴ N ₂ ]L-lysine and light isotope labeled arginine (, "" L "") ¹² C ₆ ]L-arginine) (Thermo company, medium cat No.: 89982). Culturing the cells for 6-7 generations for later use.

2. Preparation of aptamer 8 derivatives

(1) Biotin-labeled aptamer 8

The biotin-labeled aptamer 8 is obtained by coupling a biotin group to the 5' -end of the aptamer 8, dissolving the biotin-labeled aptamer 8 in a binding buffer, calibrating the concentration (100nM) according to ultraviolet absorption, heating at 95 ℃ for 5min, standing on ice for 5min, and standing at room temperature for 15 min.

(2) Biotin-labeled control nucleic acid sequence L45(L45-Bio)

The biotin-labeled control nucleic acid sequence L45(L45-Bio) was obtained by coupling a biotin group to the 5' end of the control nucleic acid sequence L45, dissolving L45-Bio in a binding buffer, calibrating the concentration (100nM) based on the ultraviolet absorption, heating at 95 ℃ for 5min, standing on ice for 5min, and standing at room temperature for 15 min. Nucleotide sequence of control nucleic acid sequence L45: TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN are provided.

Mass spectrum identification of aptamer 8 specific recognition and binding to L1 cell adhesion molecule protein

1. Extraction of aptamer 8 target protein

(1) Respectively taking 2 × 10 ⁸ Heavy isotope-labeled LoVo cells and light isotope-labeled LoVo cells in the exponential growth phase were incubated with biotin-labeled aptamer 8(100nM) and biotin-labeled control nucleic acid sequence L45(100nM), respectively, for 30 minutes after PBS washing.

(2) PBS was washed 2 times, 1mL of cell lysate was added, and incubated for 1 hour.

(3) The precipitate was removed by centrifugation at 2000rpm, the supernatant was collected, and streptavidin-modified agarose beads (GE, cat # 17-5113-01) were added thereto, followed by incubation for 1 hour to extract the target protein.

(4) And (3) washing the streptavidin-modified agarose microspheres incubated in the step (3) with PBS for 5 times to obtain heavy isotope-labeled protein extracted from the biotin-labeled aptamer 8, light isotope-labeled protein extracted from a control nucleic acid sequence L45, light isotope-labeled protein extracted from the biotin-labeled aptamer 8 and heavy isotope-labeled protein extracted from a control nucleic acid sequence L45 respectively.

2. Forward and reverse experiments

(1) Forward experiment: and (3) mixing the heavy isotope labeled protein extracted from the biotin-labeled aptamer 8 with the light isotope labeled protein extracted from the control nucleic acid sequence L45 to obtain a mixed system of the heavy isotope labeled protein extracted from the biotin-labeled aptamer 8 and the light isotope labeled protein extracted from the control nucleic acid sequence L45.

(2) Reverse experiment: and (3) mixing the light isotope-labeled protein extracted from the biotin-labeled aptamer 8 with the heavy isotope-labeled protein extracted from the control nucleic acid sequence L45 to obtain a mixed system of the light isotope-labeled protein extracted from the biotin-labeled aptamer 8 and the heavy isotope-labeled protein extracted from the control nucleic acid sequence L45.

3. Enzymolysis and LC-MS identification of protein

(1) And (3) DTT reduction: 200 μ L of 20mM Dithiothreitol (DTT) was added to the mixed system of the heavy isotope-labeled protein extracted from the biotin-labeled aptamer 8 and the light isotope-labeled protein extracted from the control nucleic acid sequence L45, the light isotope-labeled protein extracted from the biotin-labeled aptamer 8 and the heavy isotope-labeled mixed system extracted from the control nucleic acid sequence L45, respectively, and reacted at 56 ℃ for 45 min.

(2) IAA alkylation: the product of step (1) was centrifuged, the supernatant was discarded (DTT was removed), and 200. mu.L of 55mM Iodoacetamide (IAA) was added to each precipitate, followed by reaction at 37 ℃ for 30min with exclusion of light.

(3) The product of step (2) was centrifuged, the supernatant was discarded (IAA removed), and 5. mu.g of mass-produced trypsin (Promega, Cat.: V5111) was added to the precipitate and cleaved with trypsin overnight at 37 ℃ to obtain a cleaved polypeptide.

(4) After the enzyme-cut polypeptide is vacuum-concentrated, 100ul of water is added, and Ziptip C is utilized ₁₈ Desalting with the microcolumn. Before mass spectrometry, the sample was placed in a-20 ℃ freezer.

(5) The product of step (4) was analyzed and identified using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, Calif.) to obtain the original mass spectral data.

(6) Data search analysis

The raw mass spectral data obtained in step (5) was retrieved in the IPI protein database (version number: 3.68) using a MaxQuant search engine (version number: 1.3.0.5). Some parameters of the database search are as follows: the immobilization modification is an alkylation modification on cysteine, and the variable modification is an oxidation modification on methionine and an acetylation modification on the N-terminal of the protein. 2 missed cutting sites are allowed, the fault tolerance of parent ions is 20ppm, and MS/MS fragmentsThe mass error of the ions is 0.5 Da. For the identification of candidate proteins, 2 or more than 2 unique polypeptides are identified with a posterior standard error (PEP) of less than 10 ^-5 . Candidate proteins need to be identified in both forward and reverse experiments.

The results show that: SILAC (stable isotope labeling technique) experiments identified 166 proteins, only L1 cell adhesion molecule protein with the abundance ratio of the extracted protein of aptamer 8 and random control nucleic acid sequence L45 being more than 2, and the abundance ratios of the rest 165 proteins being less than 2. Thus indicating that the aptamer 8 can specifically recognize and bind to the L1 cell adhesion molecule protein.

Example 5 Co-staining of cell lines with aptamer 8 and L1 cell adhesion molecule protein antibody (anti-CD171)

First, pretreatment of cell strain and preparation of aptamer 8 derivative

1. Pretreatment of cell lines

Cell source: human colon cancer cells (LoVo, catalog No. 3111C0001CCC000164), human neuroblastoma cells (SH-SY5Y, catalog No. 3111C0001CCC000026) and human prostate cancer cells (PC-3, catalog No. 3111C0001CCC000115) were purchased from the institute of basic medicine, national academy of medical sciences (Beijing).

One dish of each cell was taken at log phase growth as follows: human colon cancer cells (LoVo), human neuroblastoma cells (SH-SY5Y) and human prostate cancer cells (PC-3) were digested with 0.2% EDTA to form a monodisperse cell suspension, which was washed 2 times with a washing buffer in several portions, each of which was 5X 10 cells ⁴ And (4) respectively.

2. Preparation of aptamer 8 derivatives

(1) Cyanine dye (Cy5) -labeled aptamer 8

The aptamer 8 labeled with cyanine dye (Cy5) is obtained by coupling a cyanine dye (Cy5) group to the 5' end of the aptamer 8, and the aptamer 8 labeled with cyanine dye (Cy5) is dissolved in a binding buffer, and after the concentration is calibrated (20. mu.M) according to ultraviolet absorption, the mixture is denatured by heating at 95 ℃ for 5min, placed on ice for 10min, and placed at room temperature for 30 min.

(2) Cyanine dye (Cy5) -labeled control sequences

The cyanine dye (Cy5) -labeled control sequence was obtained by coupling a cyanine dye (Cy5) group at the 5' end of the control sequence, and the cyanine dye (Cy5) -labeled control sequence was dissolved in a binding buffer, and after calibration of the concentration (20. mu.M) according to ultraviolet absorption, it was denatured by heating at 95 ℃ for 5min, placed on ice for 10min, and placed at room temperature for 30 min. Nucleotide sequence of control sequence: AGAGCAGCGTGGAGGATAGTTGGGGTTTGGCAAGTATTGTGTGCGCTGTTC are provided.

Secondly, specificity detection of aptamer 8

To compare the specific response capabilities of aptamer 8 and the monoclonal antibody to the cell surface L1 cell adhesion molecule protein, 3 cells pretreated in the first step were subjected to the following treatments, each set of three replicates:

treatment 1: taking 5X 10 cells from each of 3 cells ⁴ The individual cells were dispersed in a binding buffer, and then cyanine dye (Cy5) -labeled aptamer 8 (final concentration of 200nmol/L) and L1 cell adhesion molecule protein antibody (anti-CD171, dilution factor 1:50) were added.

And (3) treatment 2: taking 5X 10 cells from each of 3 cells ⁴ The individual cells were dispersed in binding buffer and then cyanine dye (Cy5) labeled control sequences (final concentration 200nmol/L) and isotype controls (IgG) were added ₁ Diluted to a concentration consistent with the concentration of L1 cell adhesion molecule protein antibody).

Respectively incubating the mixed solution obtained by the treatment 1 and the treatment 2 on ice for 30min, washing twice by using a washing buffer solution, adding phycoerythrin modified secondary antibody, incubating on ice for 30min, washing twice by using the washing buffer solution, and collecting fluorescence intensity data of a second channel and a fourth channel by using a FACSCalibur flow cytometer of BD company as the fluorescence intensity of the cell surface.

The flow cytometry results are shown in fig. 4. In the flow cytometry scatter diagram, the abscissa represents the fluorescence intensity of the fourth channel of the flow cytometer, i.e., the fluorescence intensity of the dye molecule Cy5 modified by the nucleic acid molecule; the ordinate represents the fluorescence intensity of the second channel of the flow cytometer, i.e., the fluorescence intensity of phycoerythrin bound by antibody (anti-CD171) or isotype control. The cross quadrant gate was set based on the fluorescence intensity of the control sequence in the fourth channel and the antibody isotype control in the second channel for each cell treatment 2, such that 95% of the cells in treatment 2 were within the following zone Q4.

According to the results, the cross quadrant gate provided in the scatter plot divides the coordinate area into four areas:

the fluorescence intensity of the upper left corner, namely the second channel, is high, the fluorescence intensity of the fourth channel is low, namely the fluorescence intensity of the antibody is positive, the fluorescence intensity of the aptamer is negative, and the fluorescence intensity is marked as a Q1 area;

the fluorescence intensity of the upper right corner, namely the second channel, is high, and the fluorescence intensity of the fourth channel, namely the antibody positive and the aptamer positive, is marked as a Q2 area;

the lower right corner, namely the fluorescence intensity of the second channel is small, and the fluorescence intensity of the fourth channel is large, namely the fluorescence intensity of the fourth channel is negative for antibodies and positive for aptamers, and is marked as a Q3 area;

the lower left corner, i.e., the second channel, has a low fluorescence intensity, while the fourth channel has a low fluorescence intensity, i.e., antibody-negative and aptamer-negative, and is labeled as the Q4 region.

The percentage of cells with fluorescence intensity exceeding the threshold value of the fluorescence intensity of the treatment 2 after the cells are combined with the aptamer or the antibody is used as the measure of the strength of the combination of the aptamer or the antibody with the cells (-15%; 15-30%; 30-65%; 65-85%; and + ++++ 85%). As shown in Table 2, the binding ability of the aptamer 8 to each cell was evaluated.

TABLE 2 flow cytometry results of aptamer incubation-bound with different cells

(-：<15％；﹢：15-30％；﹢﹢：30-65％；﹢﹢﹢：65-85％；﹢﹢﹢﹢：>85％)

As can be seen from FIG. 4, the number of human colon cancer cells (LoVo, panel A), human neuroblastoma cells (SH-SY5Y, panel B) in the Q2 region, both antibody-positive and aptamer-positive, was greater than 80%; the number of human prostate cancer cells (PC-3, panel C) in the antibody-negative and aptamer-negative region Q4 was greater than 80%. Thus indicating that the expression level of the cell adhesion molecule protein L1 on the surface of the PC-3 cell membrane is lower.

The results show that the aptamer 8 and the monoclonal antibody have the same responsiveness to the L1 cell adhesion molecule protein on the surface of the cell membrane, the two responses are higher to the cells with high expression of the L1 cell adhesion molecule protein, and the two responses do not respond to the cells with low expression of the L1 cell adhesion molecule protein. Compared with an antibody, the aptamer disclosed by the invention is lower in synthesis cost and short in cycle.

Example 6 detection of siRNA interference with expression levels of LoVo cell L1 cell adhesion molecule protein by nucleic acid aptamer and L1 cell adhesion molecule protein antibody

First, preparation of aptamer 8 derivative and LoVo cell after siRNA interference

1. Preparation of aptamer 8 derivatives

(1) Fluorescein (6-FAM) -labeled aptamer 8

The aptamer 8 labeled with fluorescein (6-FAM) is obtained by coupling a fluorescein (6-FAM) group at the 5' end of the aptamer 8, dissolving the aptamer 8 labeled with fluorescein (6-FAM) with a binding buffer solution, calibrating the concentration (20 mu M) according to ultraviolet absorption, heating and denaturing at 95 ℃ for 5min, standing on ice for 10min, and standing at room temperature for 30 min.

(2) Fluorescein (6-FAM) -labeled control sequence

The fluorescein (6-FAM) -labeled control sequence is obtained by coupling fluorescein (6-FAM) group at 5' end of the control sequence, dissolving the fluorescein (6-FAM) -labeled control sequence with binding buffer solution, calibrating concentration (20 μ M) according to ultraviolet absorption, heating and denaturing at 95 deg.C for 5min, standing on ice for 10min, and standing at room temperature for 30 min. Nucleotide sequence of control sequence: AGAGCAGCGTGGAGGATAGTTGGGGTTTGGCAAGTATTGTGTGCGCTGTTC is added.

2. Preparation of LoVo cells after siRNA interference

(1) Preparation of transfection reagents

L1 cellsBoth siRNA (L1CAM, sense strand: 5'-GCUACUCUGGAGAGGACUATT-3'; antisense strand: 5'-UAGUCCUCUCCAGAGUAGCTT-3') and siRNA negative control sequences (NC, sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3'; antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3') of adhesion molecule proteins were synthesized by Suzhou Jima Gene GmbH. Liposome transfection reagents are respectively used as carriers of siRNA sequences and siRNA negative control sequences according to a method provided by a liposome transfection kit manufacturer, siL1CAM sequences and NC sequences are dissolved by DEPC water to be prepared into 20 mu M mother liquor, and transfection reagents containing siRNA sequences (L1CAM) and control RNA sequences (NC) which can be used for transfection are respectively obtained. Liposome transfection kit (

RNAiMAX transfection reagent) is a product of Thermo Fisher Scientific, Inc.

(2) Acquisition of LoVo cells after siRNA interference

LoVo cells were plated evenly in 6-well plates at approximately 1X 10 per 2mL of medium (RPMI1640 medium, Gibco) per well ⁵ Cells, after overnight growth, were treated as follows:

treatment one, 2ml fresh medium was added 250ul of transfection reagent containing control RNA sequences (NC);

treatment two, 2ml fresh medium was added 250ul of transfection reagent containing siRNA sequences (L1 CAM).

And repeating three holes in each treatment mode, and continuously culturing the cells in the six-hole plate in an incubator for 48h to respectively obtain the LoVo cells after siRNA interference and the LoVo cells after control RNA interference. Then, the cells in the six-well plate were digested with 0.2% EDTA into a monodisperse cell suspension, which was washed 2 times with a washing buffer, and the cells were dispersed in a binding buffer, and the cells in each well were divided into four portions.

Secondly, detecting the expression level of the L1 cell adhesion molecule protein of the LoVo cell after siRNA interference

After the treatment in the first step, four cell suspensions in each hole of the six-hole plate are respectively treated as follows:

firstly, adding a fluorescein (6-FAM) labeled control sequence (the final concentration is 200nmol/L) into the cell suspension;

secondly, adding fluorescein (6-FAM) labeled aptamer 8 (the final concentration is 200nmol/L) into the cell suspension;

treatment III, adding isotype control (IgG) into cell suspension ₁ Diluted to a concentration consistent with the concentration of L1 cell adhesion molecule protein antibody);

treatment four, L1 cell adhesion molecule protein antibody in cell suspension (anti-CD171, dilution 1: 50).

Incubating the mixed solution of the first treatment and the second treatment on ice for 30min, and washing twice by using a washing buffer solution; the cells were resuspended in wash buffer and the first channel fluorescence intensity data was collected as fluorescence intensity on the cell surface by a FACSCalibur flow cytometer from BD. Incubating the mixed solution of the third treatment and the fourth treatment on ice for 30min, washing twice by using a washing buffer solution, adding a phycoerythrin modified secondary antibody, incubating on ice for 30min, and washing twice by using the washing buffer solution; the washed cells were resuspended in the washing buffer, and the fluorescence intensity data of the second channel was collected as the fluorescence intensity of the cell surface by the FACSCalibur flow cytometer of BD.

The results of the flow cytometry experiments are shown in FIG. 5. As can be seen from fig. 5, the amount of binding of aptamer 8 (fig. a) to siRNA interfering LoVo cells decreased; the binding capacity of the L1 cell adhesion molecule protein antibody (anti-CD171, panel B) to siRNA interfering LoVo cells is also reduced; the above results indicate that the aptamer 8 and the L1 cell adhesion molecule protein antibody have the same responsiveness to the expression level of the L1 cell adhesion molecule protein on the cell membrane surface, and can be used instead of the L1 cell adhesion molecule protein antibody (FIG. C). Compared with an antibody, the aptamer disclosed by the invention is lower in synthesis cost and short in period; when the detection of the cell surface L1 cell adhesion molecule protein is carried out, the operation is simpler and quicker, and the reproducibility is good.

Example 7 Co-infection of target cells with aptamer yly12 and L1 cell adhesion molecule protein antibody (D-SH-SY5Y)

The target cells (D-SH-SY5Y) were co-stained with the AF 647-labeled aptamer yly12 and the PE-labeled L1 cell adhesion molecule protein antibody, the aptamer yly12(AF 647-labeled DNA sequence) was dissolved in a binding buffer, and after the concentration was calibrated by UV absorption, it was heated at 95 ℃ for 5min, placed on ice for 10min, and placed at room temperature for 30 min. The denatured and renatured DNA was diluted with binding buffer to 200nmol/L DNA solution (containing 1. mu.g/ml BSA, 0.1. mu.g/ml herring sperm DNA, containing PE direct-labeled L1 cell adhesion molecular protein antibody 1:100 dilution), added to the D-SH-SY5Y cells which had been separately cultured in a confocal culture dish, incubated on ice for 30min, washed once with PBS and observed under a confocal microscope 100-fold microscope. As shown in FIG. 6, the additive effect of co-dyeing is very good, and the co-dyeing coefficient is 0.8243.

Example 8 Co-staining of LoVo cells with aptamer yly12 and L1 cell adhesion molecule protein antibody

Cells (LoVo) also expressing the aptamer yly12 labeled with AF647 and co-stained with the PE-labeled L1 cell adhesion molecule protein antibody, aptamer yly12(AF647 labeled DNA sequence) was dissolved in binding buffer, and after calibration of the concentration by UV absorption, it was heated at 95 ℃ for 5min, placed on ice for 10min, and placed at room temperature for 30 min. The denatured-renatured DNA was diluted with binding buffer to 200nmol/L DNA solution (containing 1. mu.g/ml BSA, 0.1. mu.g/ml herring sperm DNA, containing PE direct labeled L1 cell adhesion molecule protein antibody 1:100 dilution), added to LoVo cells that had been cultured in a confocal dish for 24h, incubated on ice for 30min, washed once with PBS and observed under a confocal microscope 100-fold microscope. As shown in FIG. 7, the additive effect of co-dyeing is perfect, and the co-dyeing coefficient is 0.8846.

Example 9 fluorescent staining of aptamer 8 with brain tissue sections and olfactory neuroblastoma sections

The brain tissue section and the olfactory neuroblastoma section were stained with a nucleic acid aptamer 8 labeled with fluorescein (6-FAM) to detect and diagnose the adhesion molecule protein of L1 cells.

Preparation of aptamer 8 derivative and pretreatment of brain tissue section and olfactory neuroblastoma tissue section

1. Preparation of aptamer 8 derivatives

(1) Fluorescein (6-FAM) -labeled aptamer 8

(2) Fluorescein (6-FAM) -labeled control sequences

The fluorescein (6-FAM) -labeled control sequence is obtained by coupling fluorescein (6-FAM) group at 5' end of the control sequence, dissolving the fluorescein (6-FAM) -labeled control sequence with binding buffer solution, calibrating concentration (20 μ M) according to ultraviolet absorption, heating and denaturing at 95 deg.C for 5min, standing on ice for 10min, and standing at room temperature for 30 min. Nucleotide sequence of control sequence: AGAGCAGCGTGGAGGATAGTTGGGGTTTGGCAAGTATTGTGTGCGCTGTTC are provided.

2. Pretreatment of tissue sections

Normal brain tissue (cadaveric brain tissue from 55 year old female); olfactory neuroblastoma tissue (from 17-year-old boy), as a test material, was processed by the following steps:

(1) tissue section dewaxing hydration

1) Baking slices: baking the tissue slices in an oven at 60 deg.C for 60 min;

2) placing in first jar xylene for 15min, and placing in second jar xylene for 15 min;

3) sequentially placing into anhydrous ethanol for 10min, 95% ethanol for 10min, 90% ethanol for 10min, 85% ethanol for 10min, and 70% ethanol for 10 min;

4) the tissue sections were washed with tap water for 5min (in a slow-flowing basin) and rinsed once with distilled water to obtain dewaxed and hydrated tissue sections.

(2) Section staining antigen retrieval

The method for repairing the antigen by using the microwave thermal repairing method comprises the following specific steps: taking a proper amount of TE buffer (0.292 g of EDTA and 6.05g of Tris-base are dissolved in 1000mL of distilled water, the pH value is 8.0), putting the dewaxed and hydrated tissue slice into a container containing a repair liquid (TE buffer), heating the tissue slice in a microwave oven until the tissue slice is boiled, stopping heating, reducing the temperature of the liquid in the container, and keeping the temperature between 95 and 98 ℃ for 15 min. Taking out the container, naturally cooling to room temperature, taking out the section, washing with distilled water, soaking with washing buffer solution for 5min × 3 times (ensuring that the newly-prepared washing buffer solution is soaked for the first time), and obtaining the repaired tissue section.

Secondly, incubating and dyeing step of aptamer

1. Incubating the repaired tissue section of step one 2 with a binding buffer solution containing 20% FBS and 1mg/ml herring sperm DNA for 60min at room temperature;

2. then incubating the sample with 200 mu L of binding buffer solution containing 250nM fluorescein (6-FAM) -labeled aptamer 8 at room temperature for 60min, wherein the staining method of the control sequence is the same, and the blank is not stained;

3. washing three times with washing buffer;

4. drying, sealing with anti-quenching sealing agent, and observing with laser confocal scanning microscope.

The result of microscopic observation of the normal brain tissue section is shown in FIG. 8, and it can be seen from FIG. 8 that the nucleic acid aptamer 8 can bind to the normal brain tissue section, whereas the control sequence hardly binds to the normal brain tissue section. Thus, the aptamer 8 has good identification and detection capability on neurites in normal brain tissues.

As shown in FIG. 9, the result of microscopic observation of the olfactory neuroblastoma tissue section is shown in FIG. 9, and it can be seen from FIG. 9 that the aptamer 8 binds to the olfactory neuroblastoma tissue section, whereas the control sequence hardly binds to the olfactory neuroblastoma tissue section. Thus, the aptamer 8 has good recognition and detection capability on the olfactory neuroblastoma.

The above results indicate that the nucleic acid aptamer of the present invention can recognize well tissue sections expressing the L1 cell adhesion molecule protein.

Example 10 inhibition of neurite outgrowth of neuroblastoma cells by aptamer yly12

In 12 well cell culture plates, 5X 10 wells per well ⁴ SH-SY5Y cells. Culturing in normal culture medium (RPMI1640 containing 15% inactivated FBS) for 24 hr, replacing with serum-free RPMI1640 medium (containing differentiation agent RA, BDNF), removing the RPMI1640 medium containing differentiation agent after culturing for 24 hr, washing with PBS, dividing 12-well plate into four groups of A, B, C and D, and dividing each group into two groupsAs shown in FIG. 10, group A was blank (i.e., serum-free RPMI1640 medium), group B was serum-free RPMI1640 medium (containing 1. mu.M yl2 as a control sequence, which did not bind to SH-SY5Y cells and had the series of 5'-ACACACCCAACGCAAAGCCACCTAAGCCAACCTATTAGGGTGTG-3'), group C was serum-free RPMI1640 medium (containing 1. mu.M sg8C as a control sequence, which bound to SH-SY5Y cells and had the series of 5'-TCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'), and group D was serum-free RPMI1640 medium (containing 1. mu.M yl 12). 1.

Rows

2, 3 and 4 represent cultures for 1, 2, 4 and 7 days, respectively, and it is apparent from the figure that neurite outgrowth was significantly inhibited in the group yly12 containing the nucleic acid aptamer.

Further, the mean neurite lengths were calculated and counted using the NeuronJ function in the IamgeJ software, and as shown in a diagram in fig. 10, it could be seen that the mean neurite lengths in the test group containing the nucleic acid aptamer yly12 were much lower than those in the blank control test group, the negative control sequence yl2 test group, and the positive control sequence sg8c test group. The distribution of neurite lengths in different culture systems is shown in the B picture, and it can be seen that the neurite lengths of the experiment groups containing the nucleic acid aptamer yly12 are distributed more uniformly and are all shorter than those of the blank control experiment group, the negative control sequence yl2 experiment group and the positive control sequence sg8c experiment group. In conclusion, the aptamer yly12 has good growth inhibition effect on the growth model of neurites of the neuroblastoma cell SH-SY 5Y.

Sequence listing

<110> chemical research institute of Chinese academy of sciences

<120> application research of aptamer for recognizing and combining CD171 and related functions thereof

<160> 8

<210> 1

<211> 85bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

aaggagcagc gtggaggata gggggtagct cggtcgtgtt tttgggttgt ttggtgggtc 60

ttctgttagg gtgtgtcgtc gtggt 85

<210> 2

<211> 71bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

aaggagcagc gtggaggata gggggtagct cggtcgtgtt tttgggttgt ttggtgggtc 60

ttctgttagg g 71

<210> 3

<211> 74bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 3

tggaggatag ggggtagctc ggtcgtgttt ttgggttgtt tggtgggtct tctgttaggg 60

tgtgtcgtcg tggt 74

<210> 4

<211> 60bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 4

tggaggatag ggggtagctc ggtcgtgttt ttgggttgtt tggtgggtct tctgttaggg 60

<210> 5

<211> 57bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 5

tggaggatag ggggtagctc ggtcgtgttt ttgggttgtt tggtgggtct tctgtta 57

<210> 6

<211> 54bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 6

tggaggatag ggggtagctc ggtcgtgttt ttgggttgtt tggtgggtct tctg 54

<210> 7

<211> 54bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400>7

aggatagggg gtagctcggt cgtgtttttg ggttgtttgg tgggtcttct gtta 54

<210> 8

<211> 51bp

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 8

aggatagggg gtagctcggt cgtgtttttg ggttgtttgg tgggtcttct g 51

Claims

1. A nucleic acid aptamer which is any one of:

1) a single-stranded DNA molecule shown in sequence 1;

2) a single-stranded DNA molecule shown in sequence 2;

3) a single-stranded DNA molecule shown in sequence 3;

4) a single-stranded DNA molecule shown in sequence 4;

5) a single-stranded DNA molecule shown in sequence 5;

6) a single-stranded DNA molecule shown in sequence 6;

7) a single-stranded DNA molecule shown in sequence 7;

8) a single-stranded DNA molecule shown in sequence 8.

2. The derivative of the aptamer of claim 1, wherein: the derivative of the aptamer is obtained by adding a functional group to one end or middle of the aptamer of claim 1 to obtain the aptamer with the same function as the aptamer;

the functional group is a fluorescent group, a biotin group, a radioactive substance, a therapeutic substance, digoxin, a nano luminescent material or an enzyme label.

3. Use of the aptamer of claim 1 or the derivative of claim 2 in at least one of (B1) - (B2) as a non-disease diagnostic treatment:

(B2) binds to or aids in binding to the L1 cell adhesion molecule protein.

4. Use of the aptamer of claim 1 or the derivative of claim 2 in at least one of (B3) - (B9) as follows:

(B5) preparing a product for detecting or assisting in detecting whether the sample to be detected contains the L1 cell adhesion molecule protein or not;

(B6) preparing a product for identifying or assisting in identifying tumors or tumor cells; the tumor cell is specifically a neuroblastoma cell, a human colon cancer cell, a human cervical cancer cell, a human breast cancer cell or a liver cancer cell; the tumor is specifically neuroblastoma, human colon cancer, human cervical cancer, human breast cancer or liver cancer;

(B7) preparing a product that binds or assists in binding to a tumor or tumor cell; the tumor cell is specifically a neuroblastoma cell, a human colon cancer cell, a human cervical cancer cell, a human breast cancer cell or a liver cancer cell; the tumor is specifically neuroblastoma, human colon cancer, human cervical cancer, human breast cancer or liver cancer;

(B8) preparing a product of the targeted L1 cell adhesion molecule protein;

(B9) preparing a product for inhibiting the growth of neurites of neuroblastoma cells.

5. Use according to claim 4, characterized in that:

the sample to be detected is a cell;

or the product is a kit or a probe.

6. Use according to claim 4, characterized in that:

the sample to be detected is a cell;

or the product is a targeting agent.

7. A product having the following functions, wherein the active ingredient is the nucleic acid aptamer of claim 1 or the derivative of claim 2;

the function is at least one of the following 1) to 6):

1) recognizing or aiding in recognizing the L1 cell adhesion molecule protein;

2) binds to or assists in binding to L1 cell adhesion molecule protein;

4) identifying or aiding in the identification of a tumor or tumor cell; the tumor cell is specifically a neuroblastoma cell, a human colon cancer cell, a human cervical cancer cell, a human breast cancer cell or a liver cancer cell; the tumor is specifically neuroblastoma, human colon cancer, human cervical cancer, human breast cancer or liver cancer;

5) binding or aiding binding to a tumor or tumor cell; the tumor cell is specifically a neuroblastoma cell, a human colon cancer cell, a human cervical cancer cell, a human breast cancer cell or a liver cancer cell; the tumor is specifically neuroblastoma, human colon cancer, human cervical cancer, human breast cancer or liver cancer;

6) inhibiting neurite outgrowth of neuroblastoma cells.

8. The product of claim 7, wherein:

the sample to be detected is a cell;

or the product is a kit or a probe.

9. The product of claim 7, wherein:

the sample to be detected is a cell;

or, the product is a targeting substance.