CN120350014B

CN120350014B - Nucleic acid aptamers, nucleic acid aptamer derivatives and their applications

Info

Publication number: CN120350014B
Application number: CN202510864740.8A
Authority: CN
Inventors: 谭蔚泓; 丁玎
Original assignee: Renji Hospital
Current assignee: Renji Hospital
Priority date: 2025-06-26
Filing date: 2025-06-26
Publication date: 2025-08-22
Anticipated expiration: 2045-06-26
Also published as: CN120350014A

Abstract

This invention belongs to the field of biomedical testing technology and discloses a nucleic acid aptamer, a nucleic acid aptamer derivative, and its application. Using the Systematic Evolution by Exponential Enrichment (SELEX) technique, the present invention screened for a long single-stranded nucleic acid aptamer DNA targeting DLL3 and then optimized its sequence to obtain two additional short strands. The nucleic acid aptamer specifically targets DLL3 on the surface of tumor cells and exhibits specific targeting and recognition of small cell lung cancer cells, such as SHP77 and H69, which highly express DLL3. The present invention also discloses the screening and optimization of the nucleic acid aptamer. The nucleic acid aptamer exhibits advantages such as high affinity and specificity, lacks immunogenicity, is simple to chemically synthesize, is stable, and is easily stored and labeled.

Description

Aptamer, aptamer derivative and application thereof

Technical Field

The invention relates to the technical field of biomedical detection, in particular to a specific targeted Delta-like ligand 3 aptamer, a specific targeted Delta-like ligand derivative and application of the specific targeted Delta-like ligand 3 aptamer and the specific targeted Delta-like ligand derivative.

Background

Delta-like ligand 3 (DLL 3) is a cell surface protein, which is considered a selective marker for high-grade neuroendocrine tumors and is characterized by low or no expression on the surface of normal adult human tissue cells. DLL3 expression was observed in more than 75% of Small Cell Lung Cancer (SCLC) patients, where DLL3 expression is present in various disease stages and treatments. Preclinical studies in the mouse model indicate that DLL3 regulates SCLC tumor growth via the Snail pathway, knocking out DLL3 results in reduced tumor proliferation, while overexpression of DLL3 generally promotes tumor proliferation. Second, tumors that highly express DLL3, whose immune-related signaling pathways are inhibited, DLL3 is used as a target for radioimmunotherapy in a mouse model. Tumor selectivity for DLL3 expression has prompted the development of several therapies targeted for DLL3, which are currently being clinically investigated in various patients with high-grade neuroendocrine cancer.

SC16 is a humanized monoclonal antibody directed against DLL3, selectively binding to human and murine DLL3, and has been used for targeted therapy and molecular imaging in small cell lung cancer and neuroendocrine prostate cancer. Compared with the traditional monoclonal antibody, the aptamer has advantages in tumor diagnosis and accurate treatment. A nucleic acid aptamer is a highly structured DNA or RNA fragment capable of specifically binding to a cellular target molecule, and has a high affinity, specifically recognizing a ligand molecule or class of ligand molecules, known as "chemically synthesizable antibodies" (Chemical antibody). The nucleic acid aptamer can form a specific three-dimensional structure by virtue of intermolecular interactions such as Van der Waals force, hydrogen bond, electrostatic interaction, hydrophobic interaction and the like, such as structures of hairpin, G-quadruplex and the like, so that the nucleic acid aptamer can realize high-affinity and high-specificity binding with a target substance. The exponential enrichment ligand systematic evolution (SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT, SELEX) technique is a technique for screening nucleic acid aptamers that bind efficiently and specifically to target molecules, and the nucleic acid aptamers screened by the SELEX technique can bind to a wide range of target substances such as biological macromolecules, cells, viruses, bacteria, tissues, etc., and various nucleic acid aptamer sequences have been reported so far. The nucleic acid aptamer screened by the SELEX technology has similar specific recognition capability to a target as a monoclonal antibody, but has the advantages that ① belongs to an in-vitro screening system, immune cells or animals are not needed, ② can be prepared by large-scale synthesis, the batch-to-batch difference is small, ③ stability is good, storage and transportation are easy, ④ molecular weight is small (4-50 kDa), ⑤ is easy to mark, modify, structure reform and the like, serum stability is improved, ⑥ toxicity is low, immunogenicity is avoided, and large-dose local or intravenous administration is realized. Based on the advantages, the aptamer has been widely used in various fields such as disease diagnosis and treatment, drug screening, molecular imaging and the like.

Currently, no research report of specific targeting DLL3 aptamer exists, and development of a novel nucleic acid aptamer with high specificity and high stability aiming at DLL3 is needed to be applied to tumor diagnosis and treatment.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a specific targeted Delta-like ligand 3 aptamer, a specific targeted Delta-like ligand derivative and application thereof. The aptamer can specifically target the tumor cell surface DLL3, and has specific targeting recognition effect with small cell lung cancer cells SHP77, H69 and the like with high expression of the DLL 3. The invention also discloses screening and optimizing of the aptamer. The aptamer has the advantages of high affinity and specificity, no immunogenicity, simple and stable chemical synthesis, easy preservation and marking, and the like.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a nucleic acid aptamer specifically targeting Delta-like ligand 3, the nucleotide sequence of which is selected from any one of the following:

i. The nucleotide sequence of the aptamer D3A1 is shown as SEQ ID NO. 1;

The nucleotide sequence of the aptamer D3A2 is shown as SEQ ID NO. 2;

The nucleotide sequence of the aptamer D3A1T is shown as SEQ ID NO. 3;

The nucleotide sequence of the aptamer D3A2T is shown as SEQ ID NO. 4;

v. an oligonucleotide sequence having a homology of 60% or more to any of the nucleic acid aptamers of i-iv;

vi, i or ii a truncated sequence of said aptamer;

DNA sequences hybridising to any of the nucleic acid aptamers of i-iv;

viii, i-iv a reverse transcribed RNA sequence of the nucleic acid aptamer of any one of claims.

The invention screens the long single chain of the nucleic acid aptamer DNA of the targeting DLL3 through an exponential enrichment system evolution (SELEX) technology, and optimizes the sequence to obtain two other short chains. Through in vitro affinity detection and small cell lung cancer tumor cell level targeting detection, and a series of experiments prove that the nucleic acid aptamer provided by the invention can be used for carrying out specific targeting recognition on DLL3 high-expression tumor cells such as SHP77, H69 and the like, and can be used for carrying out molecular diagnosis on the DLL3 high-expression tumor in a cell level and tumor-bearing mouse model.

As a preferred embodiment of the aptamer specifically targeting the Delta-like ligand 3 according to the present invention, the nucleotide sequence is an oligonucleotide sequence having a homology of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% or more with any of the aptamers of i-iv.

Preferably, the aptamer is lyophilized powder, and the aptamer is synthesized by a solid phase synthesis method, preferably in the form of lyophilized powder.

In a second aspect, the invention provides a nucleic acid aptamer derivative specifically targeting Delta-like ligand 3, which is modified on the basis of any one of the nucleic acid aptamers in the above-mentioned i-iv.

As a preferred embodiment of the aptamer derivative of the specific targeting Delta-like ligand 3 according to the present invention, the modification comprises a marker label or a chemical modification.

As a further preferred embodiment of the aptamer derivative of the specific targeting Delta-like ligand 3 according to the present invention, the label comprises at least one of an isotopic label, a fluorescent label, a biotin label, an enzymatic label and a chemiluminescent label.

As a further preferred embodiment of the aptamer derivative of the specific targeting Delta-like ligand 3 according to the invention, the chemical modification comprises at least one of methylation modification, amination modification, sulfhydrylation modification, phosphorylation modification, thio modification, carboxylation modification and isotopic modification.

In one embodiment, the aptamer derivative is a derivative of a aptamer obtained by labeling a cyanine dye group at the 5' end of any of the aptamers i to iv.

Preferably, the aptamer derivative is freeze-dried powder, and the aptamer derivative is synthesized by a solid phase synthesis method, preferably in the form of freeze-dried powder.

In a third aspect, the invention provides a reagent for detecting Delta-like ligand 3, comprising said aptamer and/or said aptamer derivative.

In a fourth aspect, the invention provides a method for detecting a Delta-like ligand 3, wherein a sample to be detected is mixed with the aptamer and/or the aptamer derivative, and the Delta-like ligand 3 in the sample is detected.

In a fifth aspect, the invention applies said aptamer, said aptamer derivative, said reagent in the preparation of a product for detecting or purifying Delta-like ligand 3.

As a preferred embodiment of the application according to the invention, the product comprises at least one of a kit, a biosensor, a detection chip.

In a sixth aspect, the invention applies said aptamer, said aptamer derivative, said agent in the identification of binding and/or tumor cells for non-therapeutic or diagnostic purposes, in the preparation of a medicament for diagnosis and/or treatment of tumors.

As a preferred embodiment of the application of the invention, the tumor is small cell lung cancer and the tumor cell is small cell lung cancer cell.

Compared with the prior art, the invention has the beneficial effects that:

The aptamer obtained by screening has high affinity, no immunogenicity, capability of in vitro chemical synthesis, small molecular weight, capability of marking or modifying different parts, stable sequence and easy preservation. The nucleic acid aptamer of the invention is used for detecting the expression of the Delta-like ligand 3 on the surface of the tumor cells, and has high efficiency and simple and rapid operation. Compared with the antibody preparation cost, the synthesis cost of the aptamer is low, the period is short, and the repeatability is good.

Drawings

FIG. 1 is a schematic diagram of the SELEX screening principle in example 1;

FIG. 2 shows the enrichment of aptamer with increasing number of rounds of screening in example 1;

FIG. 3 is a schematic diagram showing the preparation process of the aptamer in example 3;

FIG. 4 is a high resolution mass spectrum of the aptamer of example 3;

FIG. 5 is an in vitro serum stability assay for the aptamer of example 4;

FIG. 6 shows the structure of the aptamer of example 5;

FIG. 7 shows the affinity detection (surface plasmon resonance SPR) of two long-chain nucleic acid aptamers in example 6;

FIG. 8 shows the affinity detection (surface plasmon resonance SPR) of two truncated nucleic acid aptamers of example 6;

FIG. 9 is a western blot experiment of DLL3 protein expression in small cell lung cancer tumor cells of example 7;

FIG. 10 is a high resolution mass spectrum of a cyanine dye group labeled aptamer of example 8;

FIG. 11 is a flow cytometry experimental result of aptamer recognition of small cell lung cancer cells SHP77 and H69 in example 9;

FIG. 12 shows the results of confocal experiments in which the aptamer identified small cell lung cancer cells SHP77 and H69 in example 9;

FIG. 13 shows the results of flow cytometry detection of the affinity of aptamer for SHP77, a small cell lung cancer cell, in example 10;

FIG. 14 shows the results of in vivo fluorescence imaging of aptamer D3A1T of example 11 on SHP77 tumor bearing mice of small cell lung carcinoma.

FIG. 15 shows the results of in vivo nuclide imaging of aptamer D3A1T of example 11 on SHP77 tumor-bearing mice.

Detailed Description

In the present invention, the nucleic acid aptamer refers to an aptamer comprising a nucleic acid, that is, a ligand molecule having the ability to specifically block or inhibit a function such as a physiological activity of a target substance by firmly and specifically binding to the target substance using a steric structure formed by a secondary structure, and further a tertiary structure of a single-stranded nucleic acid via a hydrogen bond or the like. As the nucleic acid aptamer, an RNA aptamer composed of only RNA and a DNA aptamer composed of only DNA are generally known, but the nucleic acid constituting the nucleic acid aptamer in the present specification is not particularly limited. Examples thereof include a DNA aptamer, an RNA aptamer, an aptamer comprising a combination of DNA and RNA, an aptamer comprising a modified nucleic acid in a part of them, an aptamer comprising only a modified nucleic acid, and the like. Preferably DNA aptamers. The nucleic acid is a biopolymer in which nucleotides are principally used as constituent units and they are linked by phosphodiester bonds. The DNA may be DNA obtained by ligating deoxyribonucleotides having only any one of adenine, guanine, cytosine and thymine, RNA obtained by ligating ribonucleotides having only any one of adenine, guanine, cytosine and uracil, or a combination thereof.

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

The test methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available. In the examples, the aptamer sequence and cyanine dye group-labeled aptamer were synthesized by Shanghai Biotechnology, inc., DLL3 protein was purchased from Abcam, inc., catalog number RP02041, anti-Delta-like ligand 3 antibody was purchased from Abcam, inc., catalog number ab229902, washing buffer was DPBS solution containing 5mM Mgcl ₂, 4.5 mg/mL glucose, and binding buffer was prepared by adding 0.1 mg/mL tRNA and 1 mg/mL bovine serum albumin to washing buffer.

Example 1 screening of nucleic acid aptamers

DLL3 protein and HSA protein were coupled. Taking 300 mu L of carboxyl magnetic beads, washing with ultrapure water for 4 times, removing supernatant, adding prepared EDC and NHS (mixing uniformly in advance), and incubating at room temperature for 15-20 min. Separating magnetic beads by a magnetic separator, removing supernatant, washing twice by DPBS, adding 100ug protein (prepared in advance by NaAC), incubating at room temperature for 20-60: 60 min, separating magnetic beads by a magnetic separator, and removing supernatant. Adding ethanolamine for blocking, and incubating at room temperature for 5-10 min. The beads were separated in a magnetic separator, the supernatant was removed, and the mixture was washed with DPBS for 4 times. The repeated screening process (see fig. 1) is then further carried out, taking the first round of screening as an example, the following steps are performed:

(1) Taking a Lib library dry powder (random DNA sequence library with the base number of 76 bp), centrifuging at 14000 rpm to 10min, adding DPBS, performing vortex oscillation dissolution, centrifuging at 14000 rpm to 5 to 10min, and performing renaturation (95 ℃ for 5 to 10min, 4 ℃ for 10 to 20min, and balancing to room temperature);

(2) Adding the library into a positive sieve magnetic bead, slowly and uniformly blowing by using a gun, and incubating at room temperature for 30-45 min;

(3) Separating magnetic beads by a magnetic separator, adding the supernatant into positive sieve magnetic beads, incubating for 30-45 min at room temperature, and cleaning with DPBS for 3 times;

(4) Adding 5-10 min parts of ultra-pure water boiling water bath into the magnetic beads, separating the magnetic beads by a magnetic separator, and reserving the supernatant;

(5) Performing PCR amplification on the supernatant, wherein the cycle number of each amplification cycle is determined by denaturing PAGE;

(6) Separating single chain by streptavidin magnetic bead method after amplification;

(7) The subsequent screening can be adjusted according to the screening result of each round, and the operation steps are the same as the above.

Finally, as shown in FIG. 2, the library obtained from each round of screening was subjected to flow cytometry for affinity determination.

Finally, the nucleic acid aptamers D3A1 and D3A2 with strong affinity are screened, and the nucleotide sequences are as follows:

the nucleotide sequence of D3A1 is:

5’-TCCAGCACTCCACGCATAACCCTGGGGGAGGGAGTTACGTTTGGGTGGGTCGAGGGGTTATGCGTGCTACCGTGAA-3’;

the nucleotide sequence of D3A2 is:

5’-TCCAGCACTCCACGCATAACGGGGGTGGGGCTTTTAAATATGGTTGGGGGGGTCGAGTTATGCGTGCTACCGTGAA-3’。

example 2 optimization of nucleic acid aptamers

The primer-end sequence is truncated, and the primer binding regions at both ends of the original sequence of the aptamer obtained by screening in example 1 are removed.

1-11 Nucleotides comprising the first nucleotide residue at the 5 'end from the 1 st nucleotide at the 5' end of the nucleotide sequence of D3A1 are removed, and 1-11 nucleotides comprising the first nucleotide residue at the 3 'end from the 1 st nucleotide at the 3' end of the nucleotide sequence of D3A1 are removed, wherein the remaining nucleotide residue constitutes a nucleic acid aptamer, denoted as D3A1T, having the nucleotide sequence:

5’-ACGCATAACCCTGGGGGAGGGAGTTACGTTTGGGTGGGTCGAGGGGTTATGCGT-3’。

1-12 nucleotides comprising the first nucleotide residue at the 5 'end from the 1 st nucleotide at the 5' end of the nucleotide sequence of D3A2 are removed, and 1-12 nucleotides comprising the first nucleotide residue at the 3 'end from the 1 st nucleotide at the 3' end of the nucleotide sequence of D3A2 are removed, wherein the remaining nucleotide residue constitutes a nucleic acid aptamer, denoted as D3A2T, having the nucleotide sequence:

5’-CGCATAACGGGGGTGGGGCTTTTAAATATGGTTGGGGGGGTCGAGTTATGCG-3’。

example 3 preparation of nucleic acid aptamer

The nucleic acid aptamers of example 1 and example 2 were synthesized using a solid phase synthesis method, DNA containing a solid phase carrier and a protecting group was synthesized through a multi-step solid phase synthesis, and finally the final product was obtained by ammonolysis deprotection and purification. A schematic of the preparation process is shown in FIG. 3. Oligonucleotide sequences were synthesized using phosphoramidite technology in solid phase synthesis.

The solid phase synthesis method in this example is specifically as follows:

Firstly, according to the scale, using a nucleic acid synthesizer to synthesize on a solid support (CPG carrier) made of glass with controllable porosity, all phosphoramidite monomers can be removed under alkaline conditions, all phosphoramidite monomers are dissolved in anhydrous acetonitrile (100 mM) and added with molecular sieve to dry, acetonitrile solution of 5-ethylmercapto tetrazole (0.6M) is used as an activator solution, the coupling time is 200 seconds, acetic anhydride, N methylimidazole, pyridine and acetonitrile mixed reagent (N methylimidazole/acetonitrile is 1:4; acetic anhydride/pyridine/acetonitrile is 2:3:5) are used as capping reagent for unreacted active groups, and the oligomer combined by the solid support is cracked and deprotected;

Next, after the completion of the solid phase synthesis, the dried solid support was treated with an aqueous ammonia solution at 55 ℃ for 16 hours, the solution was evaporated and the solid residue was redissolved in water, and the target nucleic acid strand was obtained by HPLC purification. As shown in FIG. 4, the results of the quality inspection using high resolution mass spectrometry showed that the D3A1 molecular weight was 23628.27 Da, the D3A2 molecular weight was 23642.29 Da,D3A1T molecular weight was 16925.95 Da,D3A2T molecular weight was 16322.57 Da, indicating that all DNA sequences were correct.

Example 4 in vitro serum stability detection of nucleic acid aptamers

The stability of the aptamer determines the possibility of subsequent practical application, the prepared aptamers of example 1 and example 2 were respectively dissolved in RPMI1640 medium containing 10% FBS at a concentration of 3 μm, split into 8 tubes, and incubated 0h,1 h,2 h,4 h,6 h,8 h,12 h and 48 h in 37 ℃ incubator, respectively. Immediately after the incubation, the mixture was put into a metal bath with constant temperature of 95 ℃ for denaturation for 5 minutes, cooled on ice and then put into-80 ℃ for preservation. After all samples were collected, agarose gel electrophoresis was run and imaged.

As shown in FIG. 5, the in vitro stability of the short-chain nucleic acid aptamers D3A1T and D3A2T obtained by truncation optimization was relatively high compared to long-chain D3A1 and D3A 2.

Example 5 detection of the Structure of aptamer

The three-dimensional structure of the G-quadruplexes as aptamers may be important because they improve the properties of the nucleic acids, such as higher resistance to nuclease degradation. For the characterization of the tetrad type and its formation, circular Dichroism (CD) spectroscopy is one of the most commonly used identification methods.

The prepared aptamer samples of example 1 and example 2 were placed in a constant temperature metal bath for heating at 95 ℃ for 5 minutes, then left at room temperature for 10 minutes at 4 ℃ and equilibrated to room temperature. Samples were diluted to 1 μm in DPBS (supplementary 5mM MgCl ₂) solution and then scanned from 200 nm to 300 nm (protected under nitrogen throughout the experiment) in 1mm, 400 μl assay dishes.

As shown in FIG. 6, the nucleic acid aptamers D3A1, D3A2, D3A1T and D3A2T all had G-quadruplex structural characteristic peaks.

Example 6 affinity detection of aptamer

And determining the affinity difference between the target protein and the aptamer by an SPR detection method, and evaluating the binding dissociation kinetic characteristics of the aptamer and the target protein. The specific experimental steps are as follows:

(1) Coupling of target protein DLL3 sensor surface was activated by injection with a mixture of 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) using a CM5 chip for 7 min. The target protein DLL3 was diluted to 30. Mu.g/mL with 10 mM Acetate,pH 4.5, and the target coupled to 1000 RU at a flow rate of 10. Mu.L/min, and immobilized on the CM5 chip surface. Finally the surface was blocked with 1M ethanolamine (pH 8.5).

(2) The test conditions of the aptamer D3A1 and D3A2 are that the binding characteristics of the target protein DLL3 and the target proteins D3A1 and D3A2 are primarily measured and evaluated through a manual mode, 800 nM is determined as the highest analysis concentration of the target protein D3A1 and the target protein D3A2, and 6 analysis concentrations are set in total through 2-time gradient dilution. The concentration gradients were 0nM, 50 nM, 100 nM, 200 nM, 400nM, 800 nM, respectively, the flow rate was set at 30 μl/min, the binding time was 120 s, and the dissociation time was 720 s during sample analysis.

(3) The test condition of the aptamer D3A1T is that the binding characteristics of the target proteins DLL3 and D3A1T are primarily measured and evaluated through a manual mode, 200 nM is determined as the highest analysis concentration of the D3A1T, 2-time gradient dilution is carried out, and 9 analysis concentrations are set. The concentration gradients were 0nM, 1.56 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, respectively, and the flow rates were set at 30 μl/min, the binding time at 120 s, and the dissociation time at 720 s during sample analysis.

(4) Testing conditions for nucleic acid D3A2T the binding properties of the target proteins DLL3 and D3A2T were initially determined and evaluated by manual mode, 200 nM was determined as the highest analytical concentration of D3A2T, 2-fold gradient dilution, 10 total analytical concentrations were set. The concentration gradients were 0nM, 0.78 nM, 1.56 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, respectively, the flow rate was set at 30 μl/min, the binding time was 120 s, and the dissociation time was 720 s at the time of sample analysis.

(4) And the dynamic parameter measurement is that the experiment adopts multi-cycle operation, the response signal of the multi-cycle operation takes analysis time as an abscissa and the response value as an ordinate. The obtained data are fitted through BIAcore T200 analysis software, a fitting model is a 1:1 Langmuir binding model, and the kinetic constants such as a binding rate constant, a dissociation rate constant, a binding dissociation constant and the like are determined.

As shown in FIGS. 7 and 8, the nucleic acid aptamers D3A1, D3A2, D3A1T and D3A2T had binding dissociation constants of 82.5 nM,54.8 nM,2.358 nM and 2.668 nM, respectively, and D3A1T had the highest affinity for DLL3 protein.

Example 7 detection of DLL3 protein expression in tumor cells

The expression of DLL3 in each cell line of small cell lung cancer was assessed for subsequent use of the aptamer. Protein expression of DLL3 was detected using a western blot experiment, and whole cell extracts were obtained from the cell lines. The method comprises the following steps:

20 μg of whole protein of small cell lung cancer cells SHP77 and H69 was mixed with protein loading buffer and boiled at 100℃for 10 min. Proteins were isolated using a 10% SDS-AGE gel (constant pressure 100V, 80 min). Proteins were transferred onto PVDF membranes (Millipore) and further incubated with anti-DLL 3 antibodies. Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used for protein chemiluminescent visualization detection.

As shown in fig. 9, small cell lung cancer cells SHP77 and H69 highly expressed DLL3.

Example 8 cyanine dye group-labeled aptamer

The nucleic acid aptamers D3A1, D3A2, D3A1T and D3A2T were labeled with a cyanine dye group Cy5, respectively. The method comprises the following steps:

Adding dissolved Cy5 activated ester (DMSO is dissolved, equivalent 10) into amino modified aptamer, adding a proper amount of carbonate solution, ultrasonically mixing until the dissolved Cy5 activated ester is completely dissolved, reacting at 25 ℃ for 1h, and purifying by HPLC after the reaction is finished to obtain Cy5 fluorescent-labeled aptamer.

As shown in fig. 10, mass spectrometry quality analysis identified each of the labeled aptamer derivatives.

Example 9 specific Targeted identification and binding experiments of nucleic acid aptamers

(1) The individual nucleic acid aptamers labeled with Cy5 in example 8 were taken and then the human SCLC cell lines SHP77 and H69 were subjected to flow assays, each cell line (5×10 ⁵ cells/tube) was collected separately, the cells were washed twice with wash buffer and then incubated with DNA aptamer (250 nM) in binding buffer for 30min at 4 ℃. After the incubation was completed, the samples were analyzed by BD FACSVerseTM system after three washes with wash buffer.

(2) The nucleic acid aptamer labeled with Cy5 in example 8 was then subjected to confocal imaging assays on the human SCLC cell lines SHP77 and H69, each cell line (5×10 ⁵ cells/tube) was collected separately, the cells were washed twice with wash buffer, and then incubated with DNA aptamer (250 nM) in binding buffer for 30 minutes at 4 ℃. After the incubation was completed, three washes with wash buffer, imaging recordings were performed by Leca Zeiss TCS SP's 8 confocal microscope.

The comparison was also made with a control DNA strand (Con) without binding capacity and a control DNA strand with a random sequence (Lib). The nucleotide sequence adopted by Con is shown as SEQ ID NO.5 (SEQ ID NO.5: CGTACGGTCGACGCTAGCTCTAACTGATTATTATTATTATTATTATT ATTCGGTTAGACACGTGGAGCTCGGATCC), and the nucleotide sequence adopted by Lib is a library with consistent length and random sequence obtained by random combination in the synthesis process after the specific number of bases is 76 in DNA synthesis. As shown in fig. 11 and 12, the nucleic acid aptamers D3A1, D3A2, D3A1T and D3A2T were each able to specifically target the DLL3 highly expressed human SCLC cell lines SHP77 and H69.

Example 10 affinity detection of nucleic acid aptamers to cell surface DLL3

The affinity of the aptamer can be assessed relatively quantitatively by flow cytometry, i.e., by determining its dissociation constant. The Cy 5-labeled aptamer of example 8 was prepared in gradient concentrations, taking care to ensure a final incubation volume of 200. Mu.L. SHP77 cells (5×10 ⁵ cells/tube) were collected, washed twice with wash buffer, then mixed together with pre-prepared gradient concentrations of each aptamer in binding buffer, and incubated at 4 ℃ for 30 min. After the incubation was completed, the samples were analyzed by BD FACSVerseTM system after three washes with wash buffer.

As shown in FIG. 13, the dissociation constants of the nucleic acid aptamers D3A1, D3A2, D3A1T and D3A2T were 114.4 nM,104.6 nM,30.6 nM and 100.5 nM, respectively, consistent with the results in example 6, D3A1T had the highest affinity for the DLL3 protein.

Example 11 aptamer D3A1T specifically recognizes tumor in tumor-bearing mice

And taking the aptamer D3A1T to respectively perform in vivo fluorescence imaging experiments and nuclide imaging in the small animal body, namely, fluorescence and nuclide imaging experiments for specifically recognizing subcutaneous small cell lung cancer by the D3A 1T.

(1) Model construction

The method for constructing the model of the SHP77 tumor-bearing mouse with high DLL3 expression comprises the following steps:

The preparation method comprises the steps of performing subculture on human SCLC tumor cells SHP77 with high expression of DLL3 by using Western blot, digesting and re-suspending the cultured tumor cells in DPBS, performing cell count, mixing with matrigel (Corning) in a ratio of 1:1, placing the mixed cell suspension on ice, performing tumor implantation operation as soon as possible, taking 100 mu L of cell suspension (containing 5X 10 ⁶ cells), injecting the cell suspension into the flank of the right side of a NZG mouse with the age of 5 weeks (Vetolihua), establishing a subcutaneous tumor implantation model, measuring the tumor size by using a vernier caliper, calculating the tumor volume (mm ³) according to the length multiplied by ² multiplied by 0.5, and successfully constructing the model when the tumor size is up to 250-500 mm ³ for in vivo imaging.

(2) The established SHP77 tumor-bearing mouse model is adopted to respectively carry out the fluorescent living body imaging and PET imaging experiments of small animals, and the method specifically comprises the following steps:

for in vivo fluorescence imaging, 6 SHP77 tumor-bearing mice were prepared, each mouse was injected with 1 nmol of Cy5-labeled D3AT1 via the tail vein, and after 30min the mice were completely anesthetized with iso fo alkane (3% concentration) and placed in a small animal in vivo fluorescence imager for imaging.

For PET imaging, 3 SHP77 tumor-bearing mice were prepared, each tumor-bearing mouse was injected with a 150 uCi ⁶⁸ Ga-labeled radioactive D3A1T by tail vein injection, tumor-bearing mice were anesthetized with isoflurane mixed with oxygen (3% concentration) at specific time points 30min, 60 min and 150 min after injection, and nude mice put into a deep anesthesia state in a small animal PET/CT imager in a lateral position for imaging recording at different time points.

The fluorescence imaging results are shown in fig. 14, compared with a control chain of a random sequence marked by Cy5 (Cy 5-Lib, the adopted Lib sequence is the random sequence), the Cy5 marked D3A1T is obviously targeted and enriched to the tumor part of the SHP77 tumor-bearing mouse, and in vitro organ distribution shows that the Cy5-D3A1T is mainly metabolized by the liver and the kidney, and the rest normal organs are accumulated.

The PET imaging results of three SHP77 tumor-bearing mice (M1, M2, M3) are shown in fig. 15, with short half-life nuclide ⁶⁸ Ga-tagged D3A1T accumulating significantly in the tumor and most of the nucleic acid aptamer cleared from the kidney or liver, indicating that D3A1T was able to achieve specific targeting recognition of DLL3 positive tumor cells in vivo.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A nucleic acid aptamer characterized in that the nucleotide sequence is selected from any one of the following:

i. The nucleotide sequence of the aptamer D3A1 is shown as SEQ ID NO. 1;

The nucleotide sequence of the aptamer D3A2 is shown as SEQ ID NO. 2;

The nucleotide sequence of the aptamer D3A1T is shown as SEQ ID NO. 3;

the nucleotide sequence of the aptamer D3A2T is shown as SEQ ID NO. 4.

2. A nucleic acid aptamer derivative, characterized by being modified on the basis of the nucleic acid aptamer according to any one of i to iv of claim 1;

the modification includes a marker label or a chemical modification.

3. The aptamer derivative of claim 2, wherein the label comprises at least one of an isotopic label, a fluorescent label, a biotin label, an enzymatic label, and a chemiluminescent label;

the chemical modification includes at least one of methylation, amination, sulfhydrylation, phosphorylation, thio, carboxylation, and isotopic modification.

4. The aptamer derivative according to claim 2, wherein the aptamer derivative is a derivative of a aptamer obtained by labeling a cyanine dye group at the 5' -end of any one of the aptamers according to claim 1.

5. A reagent for detecting Delta-like ligand 3 comprising the aptamer of claim 1 and/or the aptamer derivative of any one of claims 2-4.

6. Use of the aptamer of claim 1, the aptamer derivative of any one of claims 2-4, or the reagent of claim 5 in the preparation of a product for detecting or purifying Delta-like ligand 3.

7. The use of claim 6, wherein the product comprises at least one of a kit, a biosensor, a detection chip.

8. Use of the aptamer of claim 1, the aptamer derivative of any one of claims 2-4, the agent of claim 5 for the recognition of binding to tumor cells for non-therapeutic or diagnostic purposes, wherein the tumor cells are small cell lung cancers.

9. Use of the aptamer of claim 1, the aptamer derivative of any one of claims 2 to 4, the reagent of claim 5 for the preparation of a reagent for diagnosing small cell lung cancer.