CN114317544B

CN114317544B - Aptamer specifically binding to CD133, screening method and application thereof

Info

Publication number: CN114317544B
Application number: CN202210019655.8A
Authority: CN
Inventors: 裴仁军; 李文静
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2022-01-10
Filing date: 2022-01-10
Publication date: 2023-08-25
Anticipated expiration: 2042-01-10
Also published as: CN114317544A

Abstract

The invention discloses a nucleic acid aptamer specifically binding to CD133, a screening method and application thereof. The aptamer is a plurality of CD133 aptamer candidate sequences obtained by multiple rounds of screening by an engineering cell SELEX screening method. The nucleic acid aptamer has a nucleotide sequence shown in any one of SEQ ID No.1-SEQ ID No. 6. The secondary structure prediction shows that the nucleic acid aptamer has stable stem-loop or hairpin-shaped structures, has good affinity and specificity, can be synthesized artificially, and has the advantages of low cost, short production period, good repeatability among different batches, high stability, long-term storage and convenience in chemical modification. The screened aptamer can be specifically and stably combined with CD133, and has wide application prospects in aspects of CD133 related tumor diagnosis and treatment and the like.

Description

Aptamer specifically binding to CD133, screening method and application thereof

Technical Field

The invention relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer specifically combined with CD133, a screening method and application thereof, and belongs to the technical field of biology.

Background

CD133, also known as Promin-1, is a five transmembrane, highly glycosylated glycoprotein originally found in hematopoietic stem and progenitor cells, and is associated with cholesterol in the plasma membrane. In recent years, it has been identified as a tumor surface marker for a variety of Cancer Stem Cells (CSCs), including brain cancer, liver cancer, ovarian cancer, prostate cancer, colorectal cancer, and the like. CD133 expression, when bound to other potential CSC markers, is often associated with clinical outcome in a variety of tumor patients. The development of an effective CD133 affinity molecule, such as a nucleic acid aptamer, can be used as a probe for recognizing CD133 protein on the surface of tumor cells, which would greatly help solve the problems of disease diagnosis and targeted therapy. In particular to the diagnosis and treatment of tumors with drug resistance, differentiation potential and high tumorigenicity of radiotherapy and chemotherapy.

Nucleic acid aptamers, also known as aptamers, etc., are short single-stranded DNA or RNA screened in vitro from synthetic nucleic acid libraries, capable of binding to specific targets, such as ions, small molecules, proteins and cells, with high affinity and selectivity. The aptamer mainly forms various secondary structures (such as hairpins, stem loops, bulges and the like) through matching of spatial structures, ionic interactions, hydrogen bonds, van der Waals forces and hydrophobicity, and further forms a unique three-dimensional structure to specifically recognize target molecules so as to realize functions similar to antibodies. However, aptamers have unique chemical and biological properties, unlike traditional antibodies, such as ease of chemical synthesis, high chemical stability, small molecular weight, low immunogenicity, low cost, ease of modification, and the like. Therefore, aptamers have shown great potential in the fields of cancer diagnosis, biomarker discovery, drug delivery therapy and treatment, and the like.

SELEX is the process of exponential enrichment of ligand system evolution technology to obtain specific aptamers from a random oligonucleotide library. Many aptamers developed using traditional SELEX lack the binding affinity and specificity required for therapeutic applications. Most aptamers are screened by using purified proteins, which may not recognize the native conformation of the cell surface protein, as the molecular structure and morphology of the purified form of the cell surface protein may differ from the native molecular structure and morphology of the cell membrane. In addition, there are technical challenges in separating and purifying low abundance membrane proteins.

Disclosure of Invention

The present invention is directed to a nucleic acid aptamer specifically binding to CD133, a screening method thereof and an application thereof, so as to solve the problems set forth in the background art.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a nucleic acid aptamer specifically binding to CD133, which has a nucleotide sequence shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

The embodiment of the invention also provides application of the nucleic acid aptamer specifically binding to CD133 in preparing a product capable of specifically recognizing and binding to target protein CD133.

The embodiment of the invention also provides a kit which comprises the nucleic acid aptamer specifically binding to CD133.

The embodiment of the invention also provides application of the aptamer specifically combined with CD133 or the kit in preparing a tumor detection reagent or a tumor treatment drug.

The embodiment of the invention also provides a screening method of the nucleic acid aptamer, which comprises the following steps:

1) Cell transfection is carried out on CHO-K1 cells by using a lentiviral system to obtain a CD133 cell line with stable expression of CD133, which is used for positive screening of target cells;

2) Selecting a CHO-K1 cell line control cell which is not transfected for negative screening of target cells;

3) Screening the CD133 nucleic acid aptamer by using a cell-SELEX technology to obtain the nucleic acid aptamer specifically binding to CD133.

Compared with the prior art, the technical scheme provided by the invention has the advantages that at least:

1) Compared with the traditional method which takes purified protein or artificially synthesized protein as a target, the aptamer obtained by screening through a cell-SELEX technology has high affinity and strong specificity, and can be better combined with cells expressing CD133 in a natural state;

2) The CD133 aptamer screened by the invention can be efficiently and specifically combined with target protein CD133, has obvious affinity, and provides an effective tool and means for early diagnosis, parting analysis, treatment and other aspects of cancer.

Drawings

In order to more clearly illustrate the technical solutions of the present invention, the following brief description will be given of the drawings used in the embodiments or the description of the prior art, it being obvious that the drawings described below are only examples of the invention herein, and that other drawings can be obtained from these drawings without the inventive effort of a person skilled in the art.

FIG. 1 is a schematic representation of an engineered high expression CD133 stably transformed cell line according to an exemplary embodiment of the invention;

FIG. 2 is a schematic diagram of screening for CD133 nucleic acid aptamers using the cell-SELEX method in an exemplary embodiment of the present invention;

FIG. 3 is a schematic representation of the secondary structure of the aptamers C1, cs1, C5, cs5, C53, and Cs53 simulated using the Mfold software in an exemplary embodiment of the present invention;

FIGS. 4 a-4 f are flow charts of the binding of each aptamer to CD133 cells or CHO-K1 cells in example 1 of the invention;

FIG. 5 is a graph showing the dissociation constant curve-fitting of aptamer Cs1 to CD133 cells in example 2 of the invention;

FIG. 6 is a graph showing the dissociation constant curve-fitting of the nucleic acid aptamer Cs5 to CD133 cells in example 2 of the present invention;

FIG. 7 is a graph showing the dissociation constant curve-fitting of the nucleic acid aptamer Cs53 to CD133 cells in example 2 of the invention;

FIG. 8 is a fluorescence confocal micrograph of aptamer Cs5 and CD133 cells of example 2 of the invention;

FIG. 9 is a fluorescence confocal micrograph of aptamer Cs5 and intestinal cancer cell HCT116 in example 3 of the invention.

Detailed Description

As described above, in view of the many drawbacks of the prior art, the present inventors have long studied and practiced in a large number, and have proposed the technical solution of the present invention, and have adopted the modified cell-SELEX method to obtain CD133 candidate aptamers. The invention constructs the CHO-K1 cell over-expressing CD133 as target cell, and the CHO-K1 cell as cell-SELEX control cell. This cell-SELEX approach based on engineered cell lines has its unique advantages, such as the isolated aptamer can specifically bind to cell surface proteins with native conformations, reflecting their biological information more accurately; moreover, the aptamer may interact directly with the membrane protein to recognize the whole cell.

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

In one aspect, the invention provides a nucleic acid aptamer specifically binding to CD133, which has a nucleotide sequence shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

In some embodiments, the aptamer that specifically binds CD133 has a nucleotide sequence set forth in any one of SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6, more preferably as set forth in SEQ ID No. 5.

In some embodiments, the secondary structure prediction shows that the nucleic acid aptamer has a stable stem loop or hairpin structure.

In some embodiments, the nucleic acid aptamer is capable of specifically recognizing and stabilizing binding to the target protein CD133. Furthermore, the aptamer has good affinity and specificity, can be synthesized artificially, and therefore has the advantages of low cost, short production period, good repeatability among different batches, high stability, long-term storage and convenience for chemical modification.

In another aspect, embodiments of the present invention provide the use of the aptamer specifically binding to CD133 for the preparation of a product capable of specifically recognizing and binding to the target protein CD133.

In another aspect of embodiments of the invention, a kit is provided that includes the aforementioned aptamer that specifically binds to CD133.

In another aspect, the embodiment of the invention further provides an application of the aptamer or the kit specifically binding to CD133 in preparing a tumor detection reagent or a tumor therapeutic drug.

Further, the nucleic acid aptamer can be used for identifying CD133 and applied to tumor diagnosis and treatment.

Wherein the tumor includes, but is not limited to, intestinal cancer, pancreatic cancer, lung cancer, gastric cancer, breast cancer, liver cancer, skin cancer, nasopharyngeal cancer, ovarian cancer, brain cancer, etc.

In another aspect, the embodiment of the invention also provides a screening method of the aptamer, which mainly uses a cell-SELEX technology, uses CHO-K1 stably expressing CD133 by slow virus transfection as a target cell, and uses normal cell CHO-K1 as a control cell to screen the aptamer of the CD133.

Further, the nucleic acid aptamer is a nucleic acid aptamer candidate sequence of a plurality of CD133 obtained by multiple rounds of screening by an engineering cell SELEX screening method, an aptamer with high occurrence frequency of a sequencing result is selected, and the combination condition of the full-length sequence and the truncated sequence with the CD133 cells is determined by using a flow cytometry.

In some embodiments, the screening method of the nucleic acid aptamer comprises:

In one embodiment of the invention, the nucleic acid aptamer sequence that specifically binds to CD133 may be isolated by a modified nucleic acid aptamer screening (cell-SELEX) method. Referring to fig. 1-2, the screening method may include the steps of:

(1) Cell transfection is carried out on CHO-K1 cells by using a lentiviral system to obtain a CD133 cell line for stably expressing CD133, and the CD133 antibody is used for carrying out expression condition verification on the CHO-K1 cell line for stably expressing CD133.

(2) Single-stranded DNA library (first round 10nmol m-lib, followed by several rounds of gradual decrease to 0.2 nmol) was denatured at 95℃for 5min, then slowly cooled to Room Temperature (RT).

(3) The single stranded DNA library in the binding buffer was then transferred to a petri dish containing 90% CD133 cells (100X 20mm petri dish for rounds 1-6 and 60X 15mm petri dish for rounds 7-10) and incubated at 4℃for 60min (each round then gradually decreasing to 30 min) to bind the potential sequence to the target cells.

(4) The unbound DNA was removed by washing 3 times with wash buffer and the cells were scraped from the petri dish using 1mL Milli-Q water. After heating at 95℃for 10min, the suspension was centrifuged at 14,000rpm for 10min to separate the sequences from the cells and collected into a centrifuge tube.

(5) The collected library was then PCR amplified using biotin-modified reverse primer biotin-P2 (8-16 cycles, 94℃30s,48℃30s,72℃20s,72℃5 min).

(6) The PCR product was first incubated with streptavidin sepharose beads (150. Mu.L) for 10min in an empty mini-affinity chromatography column (Bio-rad, USA) and repeated 3 times. To eliminate unbound DNA, the column was washed 3 times with 800 μl PBS. After denaturing for 2min with the addition of 0.1M NaOH solution, the desired single-stranded DNA was obtained from the streptavidin sepharose beads. The single-stranded DNA solution was adjusted to pH 6.5-7.5 with 1.5M hydrochloric acid solution, and then purified by ethanol precipitation.

(7) From round 3, a reverse screen was performed to remove non-specific binding sequences. Specific procedures the collected ssDNA library was incubated with negative control CHO-K1 cells (60X 15mm dishes on rounds 3-8, 100X 20mm dishes on rounds 9-10) at 4℃for 30min (time gradually increased to 60min with rounds), sequences that did not bind to CHO-K1 were collected and incubated with CD133 cells.

(8) And (3) carrying out PCR amplification on the finally obtained enriched single-stranded DNA library, then carrying out high-throughput sequencing, analyzing the secondary structure of the sequence, and optimizing the sequence to obtain the optimized nucleic acid aptamer specifically binding to CD133.

Further, the sequence of the aptamer of CD133 obtained by screening in this embodiment is:

5’-ATACCAGCTTATTCAATTGCACCACAGATTGTTATTATTTAGTTTATCTCCTAGTTTAGATAGTAAGTGCAATCT-3’(SEQ ID No.1)；

5’-ATACCAGCTTATTCAATTACATCGAGTGGCTTATAAAGTAGGCGTAGGGCTAGGCGGAGAGATAGTAAGTGCAATCT-3’(SEQ ID No.2)；

5’-ATACCAGCTTATTCAATTGGGACGCTGAACACTATCATGGAGTGATATCTTTCTTGATAGATAGTAAGTGCAATCT-3’(SEQ ID No.3)。

by optimizing the sequences of the nucleic acid aptamers respectively through the software Mfold predicted secondary structure, as shown in fig. 3, the nucleic acid aptamers specifically binding to CD133 with shortened sequences can be obtained respectively, specifically:

5’-ATTGCACCACAGATTGTTATTATTTAGTTTATCTCCTAGTTTAGATAGTAAGTGCAAT-3’(SEQ ID No.4)；

5’-TTACATCGAGTGGCTTATAAAGTAGGCGTAGGGCTAGGCGGAGAGATGTAA-3’(SEQ ID No.5)；

5’-ACACTATCATGGAGTGATATCTTTCTTGATAGATAGTAAGTGC-3’(SEQ ID No.6)。

furthermore, the aptamer screened at the time can be specifically and stably combined with CD133, and has wide application prospects in aspects of CD 133-related tumor diagnosis, treatment and the like.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further described in detail with reference to the accompanying drawings and several preferred embodiments. The above-described aspects are further described below in conjunction with specific embodiments. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The reagents and starting materials used in the following examples were all commercially available, and the test methods in which the specific conditions were not specified were generally conducted under conventional conditions or under the conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in biochemistry, molecular biology, analytical chemistry and related arts.

Example 1

The binding of the full length sequence of the aptamer specifically binding to CD133 (sequence shown as SEQ ID No.1-SEQ ID No.3, defined as aptamers C1, C5 and C53) and its truncated aptamer (sequence shown as SEQ ID No.4-SEQ ID No.6, defined as aptamers Cs1, cs5 and Cs 53) to the CD133 cell line was tested using the following procedure:

to determine the binding capacity of the aptamer, 500nM full-length or truncated sequences FAM-C1, FAM-C5, FAM-C53, FAM-Cs1, FAM-Cs5, FAM-Cs53 were prepared and combined with target cells in 200. Mu.L of binding buffer (1X 10 per tube ⁵ Individual cells) were incubated at 4℃for 50min. After incubation, unbound aptamer was washed away with wash buffer and cells were resuspended in 300 μl wash buffer. The fluorescence intensity of each sample was measured using a C6 cytometer (BD, usa). The results are shown in FIGS. 4 a-4 f, and it can be seen that truncated aptamers Cs1, cs5, and Cs53 have no loss of binding capacity to CD133 cells compared to full length aptamers C1, C5, and C53.

Example 2

The affinity of the nucleic acid aptamers of the invention (sequences shown in SEQ ID Nos. 4-6, defined as aptamers Cs1, cs5 and Cs 53) that specifically bind CD133 were tested. The method comprises the following specific steps:

to determine the equilibrium dissociation constant (K) _d ) Value, CD133 cells (1X 10) ⁵ ) The aptamers were incubated with different doses (0, 5, 10, 25, 50, 100, 200, 300, 400, 500 nM) of FAM-labeled aptamer in binding buffer for 50min. The cells were then washed three times and examined using a flow cytometer. The initial single-stranded DNA library (mlib) was used as a negative control. K (K) _d By nonlinear regression determination based on cellular fluorescence intensity, the equation is used: F-F ₀ ＝B _max *X/(K _d +x), wherein F represents the fluorescence intensity of cells bound to FAM-labeled aptamer, F ₀ Represents the fluorescence intensity of cells bound to FAM-labeled m-lib, X represents the aptamer concentration. Dissociation constants (K) for affinity experiments were simulated using GraphPad Prism 5 software _d ) As a result, as shown in FIGS. 5 to 7, the dissociation constants of the obtained aptamers Cs1, cs5, cs53 were 156.3.+ -. 64.8nM, 16.3.+ -. 6.8nM and 134.0.+ -. 24.1nM, respectively. More preferably, the inventors used confocal microscopy to characterize binding of FAM-labeled Cs5, and as shown in fig. 8, cs5 had specific binding to CD133 cells.

Example 3

The nucleic acid aptamers Cs1, cs5 and Cs53 that specifically bound to CD133 were tested for specificity for different cell lines using flow cytometry. The results are shown in Table 1, where all aptamers recognized CD133 cells, but not CHO-K1 cells. The aptamer also has better binding with cancer cells such as HCT116, HT29, HCT8, MCF-7 and the like, which shows that the aptamer has targeted binding affinity and specificity for CD133 positive cells. The inventor uses a confocal microscope to perform binding characterization on FAM marked Cs5 and HCT116 cells, and the result is shown in figure 9, wherein the Cs5 has a specific recognition effect on intestinal cancer cells HCT 116.

Table 1 binding of aptamers to different cell lines

The fluorescence intensity threshold was determined to be below which 95% of the cells had fluorescence intensity after incubation with the FAM-labeled single-stranded DNA library (m-lib). The percentage of cells with fluorescence exceeding a set threshold was used to evaluate the binding capacity of the aptamer to the cells, <10%; 11-35%; ++,36-60%; ++,61-80%; ++ + ++, >81%.

It should be understood that the embodiments described above are some, but not all, embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Sequence listing

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Claims

1. A nucleic acid aptamer that specifically binds CD133, characterized in that: the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID No. 5.

2. The aptamer of claim 1 that specifically binds CD133, wherein: the nucleic acid aptamer has a stable stem-loop or hairpin structure.

3. Use of a nucleic acid aptamer specifically binding to CD133 according to claim 1 or 2 for the preparation of a product capable of specifically recognizing and binding to the target protein CD133.

4. A kit comprising the nucleic acid aptamer of claim 1 or 2 that specifically binds CD133.