CN115976016A

CN115976016A - Multi-aptamer-based target leukemia cell in-situ microRNA DNA nanocage probe and kit and method thereof

Info

Publication number: CN115976016A
Application number: CN202310058427.6A
Authority: CN
Inventors: 高铭萱; 刘钱; 陈鸣; 包静; 刘璐; 申曼; 徐含青; 冯柳; 吴显兰; 夏涵; 王靖雪; 赵雅霞; 王小; 肖婷; 张利改
Original assignee: First Affiliated Hospital of Army Medical University
Current assignee: First Affiliated Hospital of Army Medical University
Priority date: 2023-01-18
Filing date: 2023-01-18
Publication date: 2023-04-18

Abstract

The invention discloses an in-situ microRNA DNA nanocage probe based on multi-aptamer targeted leukemia cells, a kit and a method thereof, wherein the probe comprises a cross-section octahedral DNA nanocage and a self-assembly reaction hairpin structure; the cross-section octahedron DNA nano cage probe is formed by self-assembling 8 strands serving as main body frameworks to form an octahedron, wherein a leukemia tumor cell DNA aptamer sequence extends from the 3' end of at least one framework strand, the self-assembly reaction hairpin structure comprises a hairpin I and a hairpin II, and the hairpin I specifically identifies target miRNAs. The invention designs the probe by combining the DNA aptamer and the CHA sensing method, realizes the target of leukemia cells and the in-situ detection of miRNA in the target cells, effectively improves the stability of the aptamer and the detection hairpin by the probe with the octahedral DNA nanocage main body framework, ensures that the detection probe is assembled in one step, has the assembly efficiency of more than 90 percent, and has important significance for the detection of leukemia.

Description

Multi-aptamer-based targeted leukemia cell in-situ microRNA DNA nanocage probe and kit and method thereof

Technical Field

The invention relates to the field of detection, in particular to an in-situ microRNA DNA nanocage probe based on multi-aptamer targeted leukemia cells, and also relates to a kit and a method containing the probe.

Background

While alterations in leukemia cell membrane receptor expression levels and/or function can lead to systemic dysfunction, the same molecular markers are often shared by different subpopulations of cells, and multiple markers are required to accurately identify cancer cell subpopulations that do not have a specific marker. By analyzing high or low expression levels of various membrane receptors, the properties of individual cells can be more precisely identified, thereby enabling more accurate diagnosis and treatment of diseases. Therefore, it is of great interest to develop methods that can analyze multiple tumor molecular markers to identify cancer cell subsets.

Aptamers are single-stranded nucleic acid strands that are a class of DNA or RNA with unique three-dimensional conformations, also known as "chemical antibodies," that bind to a variety of targets with high specificity and affinity. Can be used as a commonly used ligand, can specifically recognize and be tightly combined with a membrane protein receptor, and a molecular probe with an aptamer can be used for cancer diagnosis and targeted therapy through the overexpression of the membrane protein of a targeted cancer cell. Aptamer-based targeted detection suffers from a number of limitations and challenges, and further improvements are needed. Key issues that hinder better clinical applications include: 1. the aptamer is a single-stranded nucleic acid chain, has short half-life and is easy to degrade; 2. the flexibility or structural instability of single-stranded nucleic acids leads to poor experimental reproducibility associated with aptamers in complex environments.

Abnormal expression of intracellular microRNAs (miRNAs) is closely associated with many healthy diseases, making it a potential marker for early clinical diagnosis. Therefore, it is very necessary to develop a highly sensitive, specific and reliable miRNA analysis method.

Over the past several decades, many strategies have been proposed to measure mirnas with high sensitivity, specificity and rapidity. Compared with the traditional miRNA detection method: compared with Northern blotting analysis, microarray technology, qRT-PCR technology, SPR sensing strategy and colorimetric sensing method, the signal amplification sensing strategy based on Catalytic Hairpin Assembly (CHA) has the advantages of no enzyme, high sensitivity and selectivity, rapidness, no need of expensive and precise instruments and the like. CHA-based fluorescence methods are not limited to quantification, but intracellular imaging and dynamic monitoring have also been reported.

The CHA-based intracellular miRNA sensing strategies suffer from a number of limitations and challenges, and further improvements are needed. First, current CHA-based sensing strategies focus primarily on single miRNA monitoring, which leads to high throughput and difficulty in multiplex detection of mirnas. Secondly, most sensing strategies involve complex instrumentation and multiple reaction steps, which add complexity and thus limit their practical application. Third, single-chain CHA assay hairpins are susceptible to degradation.

DNA geometry polyhedra (DNA-GPs): including tetrahedrons, prisms, octahedrons, dodecahedrons, icosahedrons, buckyballs, etc., high programmability, good biocompatibility, diversified functionalization methods, and stable frames and hollow interiors, thereby providing broad opportunities for multidisciplinary research. The assembly detection strategy based on DNA-GPs is limited by a plurality of problems, and the key problems are that: DNA tetrahedron and other simpler geometric polyhedrons have insufficient modification space; 2. dodecahedron and other polyhedrons with complex geometries have larger modification space, complex assembly steps and low assembly rate. Therefore, a probe which is simple to assemble, has enough modification space and can detect microRNAs in cells with high sensitivity is needed.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an in situ microRNA DNA nanocage probe for leukemia cells based on multi-aptamer targeting; the second purpose of the invention is to provide the application of the kit for detecting the in-situ microRNAs of the leukemia cells based on the multi-aptamer targeted leukemia cell in-situ microRNA DNA nanocage probe; it is a further object of the present invention to provide a kit comprising the probe; the fourth purpose of the invention is to provide a method for detecting microRNA in situ by using the probe to target leukemia cells.

In order to achieve the purpose, the invention provides the following technical scheme:

1. based on a multi-aptamer targeted leukemia cell in-situ microRNA DNA nanocage probe, the probe comprises an octahedral DNA nanocage and a self-assembly reaction hairpin structure;

the octahedral DNA nanocage probe is formed by self-assembling 8 strands serving as a main body framework to form an octahedron, wherein the 3 'end of at least one framework strand extends leukemia tumor cell DNA aptamer sequences, the self-assembly reaction hairpin structure comprises a hairpin I and a hairpin II, and the hairpin I specifically identifies target miRNAs and consists of main body framework 3' end extension sequences of unextended aptamer sequences of the octahedral DNA nanocage; the hairpin II is complementary to the hairpin I.

Preferably, the hairpin I comprises a folding region forward sequence, a circular region, a folding region reverse sequence and a free region, wherein the free region and the folding region connected with the free region specifically recognize target miRNAs, and the hairpin II is modified with a fluorescent group and a quenching group and is complementary with the folding region forward sequence, the circular region, the folding region reverse sequence and the free region of the hairpin I.

Preferably, the main framework sequence of the octahedral DNA nanocage probe consists of 1 st to 75 th bases of SEQ ID NO.1 to SEQ ID NO. 8.

Preferably, n is more than or equal to 1 and less than or equal to 3 in the sequence of the leukemia tumor cell DNA aptamer extended by 8 strands of the octahedral DNA nanocage, the 8-n strands of the octahedral DNA nanocage are extended to specifically recognize the hairpin structures of different target miRNAs, and the hairpin structures recognizing the different target miRNAs are marked by different fluorescence.

Preferably, the number of hairpin I for specifically recognizing microRNAs of the same target is at least 2; the leukemia tumor cell DNA aptamer is at least one of Sgc f, TC01 and Sgc c.

2. The application of the in-situ microRNA DNA nanocage probe based on the multi-aptamer targeted leukemia cells in preparing a multi-target microRNAs detection kit is provided.

3. A kit containing the probe.

Preferably, the kit further comprises an assembly solution, wherein the assembly solution is a TAM buffer solution, and the concentration of each component is as follows: pH 7.0, 40mM Tris acetate, 12.6mM magnesium acetate.

4. The method for detecting microRNA in situ by using the probe to target leukemia cells is characterized by comprising the following steps: 8 main skeleton chains with hairpin I and leukemia tumor cell DNA aptamer sequences are self-assembled to form an octahedral DNA nano cage probe, then the octahedral DNA nano cage probe and the hairpin II are added into the separated leukemia cells, and the leukemia cell membrane protein and intracellular miRNAs are detected through fluorescent signals.

Preferably, the self-assembly conditions are to mix equimolar amounts of the main backbone chain in TAM buffer at a concentration of 1 μ M, heat at 95 ℃ for 10 minutes, then heat at 80 ℃ for 5 minutes, cool to 60 ℃, and finally slowly cool to 4 ℃.

The invention has the beneficial effects that: the invention designs the probe by combining the multi-aptamer targeted leukemia cell in-situ microRNA DNA nano cage probe with the DNA aptamer and CHA sensing method, realizes leukemia cell targeting and target cell miRNA in-situ detection, has a cross section of the probe of the octahedron DNA nano cage main body frame, effectively improves the aptamer and detection hairpin stability, realizes one-step assembly of the detection probe, has assembly efficiency of more than 90 percent, and has important significance for leukemia detection.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a schematic diagram of CHA sensing H1, H2 hairpin probes.

FIG. 2 is a wireframe diagram of a microRNA DNA nanocage probe for aptamer-targeted leukemia cell in-situ detection and an aptamer-based leukemia cell-targeted CHA-based sensing detection scheme.

FIG. 3 is a diagram showing the assembly effect of a microRNA DNA nanocage probe for in situ detection of multi-aptamer targeted leukemia cells.

FIG. 4 shows the result of electrophoresis of the assembled product.

FIG. 5 shows the result of AFM detection.

FIG. 6 shows the detection efficiency of miRNA with different concentrations (A: CHA hairpin probe detection result; B: octahedral DNA nanocage probe with hairpin H1 cross section).

FIG. 7 shows the detection efficiency of different H1 hairpin probes for miRNA with the same concentration (A: 2H 1 hairpins; B: 3H 1 hairpins; C: 4H 1 hairpins).

FIG. 8 shows CEM cell lines of leukemia cells with a targeting/non-targeting probe.

FIG. 9 shows miRNA detection in situ by CEM, jurkat and K562 cell probes.

FIG. 10 shows the detection of leukemia and normal human leukocyte surface specific membrane proteins by targeting probe aptamers (Sgc f-Texas Red, TC01-Alexa Fluor 488 and Sgc c-HEX).

FIG. 11 shows the probe aptamer target detection of leukemia human and normal human leukocyte miRNA-146-AF488 and miRNA-17-Cy5.5.

Detailed Description

The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can implement the present invention, but the embodiments are not to be construed as limiting the present invention.

Example 1 construction of MicroRNA DNA nanocage probes for in situ detection of Multi-aptamer-targeted leukemia cells

The detection probe structure of the embodiment comprises: a section octahedral DNA nanocage main body framework comprises 1- (1) -TC01, 1- (2) -H1/H2, 1- (3) -Sgc f, 1- (4) -H1/H2, 1- (5), 1- (6) -H1/H2, 1- (7) -Sgc c and 1- (8) -H1/H2, wherein a black body sequence is a section octahedral DNA nanocage main body framework sequence, and is shown in Table 1. H1 and H2 are CHA detection probes for detecting microRNA.

TABLE 1 sequence of related primers of microRNA DNA nanocage probe for in-situ detection of multi-aptamer targeted leukemia cells

The detection probe of the embodiment firstly enters leukemia tumor cells through the aptamer-bound cell specific membrane protein, the probe after entering the cells detects hairpin through H1 and H2, and the miRNAs in the cells are detected in situ.

The detection probe is formed by assembling 8 primers, a cross-section octahedral DNA nano cage is used as a framework, an H1 hairpin probe extends from the 3' end of a cross-section octahedral DNA nano cage framework chain, the cross-section octahedral DNA nano cage is provided with 8 framework chains, functional components extending from a main framework structure comprise a leukemia tumor cell DNA aptamer (Sgc f, TC01 and Sgc c) targeting functional component and at most 5 miRNAs detection hairpin H1/H2 functional components in a cell, based on a CHA sensing method, H1 and H2 detection probes matched with a target are designed according to selected target miRNAs, and the CHA sensing principle is shown in figure 1. As shown, the two hairpin probes H1 and H2 are in a closed state and thus can stably coexist in a solution. When a catalytic strand of C1 (target miRNAs) is added, C1 binds to a portion of H1, opening the H1 hairpin, exposing a single-stranded sequence in H1. This single stranded sequence then serves as a "foothold" for H2 to H1 hybridization, allowing H1 and H2 to hybridize. Since H2/H1 hybridization is more stable than C1/H1 hybridization, C1 strands are competed for the next catalytic assembly cycle while H2 hybridizes to H1. The net result of this amplification reaction is the production of a large number of H1/H2 hybrid duplexes. By introducing specific sequences or groups into H1 or H2, CHA can be used for analytical signal generation and amplification.

The scheme is shown in a figure 2 in a line frame diagram of a probe and a detection scheme based on CHA sensing, the sequences of related primers are shown in a table 1, and the assembly effect is shown in a figure 3.

Example 2 Assembly and functional verification of the Probe

The construction process of the sensing platform is simplified, and the operation steps are reduced;

the cage was assembled by mixing equimolar amounts of each strand in TAM buffer (40 mM Tris acetate pH 7.0, 12.6mM magnesium acetate) at a concentration of 1. Mu.M using a PCR apparatus one-step procedure, with the sample heated at 95 ℃ for 10 minutes, then at 80 ℃ for 5 minutes, cooled to 60 ℃ (4 min/1 ℃) and finally slowly cooled to 4 ℃ (6 min/° C), to give cross-sectional octahedral DNA nanocage probes with hairpin H1 recognizing different miRNAs. The assembled product was subjected to electrophoretic detection, and the results are shown in fig. 4. The results show that the cross-sectional octahedral DNA nanocage probe with hairpin H1 can be assembled in one step. AFM examination and gel electrophoresis showed >90% higher probe assembly efficiency (fig. 5).

The CHA hairpin probe detection efficiency is verified, 1H 1 hairpin cross-section octahedral DNA nano cage probe is used for respectively detecting BA (target miRNA 146) with the concentration of 100nm, 10nm, 1nm,100pm and 10pm, and the CHA hairpin probe is directly used as a control to detect, and the results are shown as A and B in figure 6. The result shows that the detection sensitivity of the cross-section octahedral DNA nano cage probe using the hairpin H1 is higher and is improved by 10 times compared with that of the single CHA hairpin probe.

The detection efficiencies of 2, 3 and 4 same H1-hairpin probes for miRNA at the same concentration were then determined, respectively, and the results are shown in fig. 7. The result shows that the sensitivity of detecting the hairpin target containing 2 and 3H 1 can reach 10pm target miRNA, and is interfered by the background signal generated by H1 and H2 hairpin nonspecific reaction, 4H 1 detection hairpins are contained, and the sensitivity of detecting the target reaches 100pm target miRNA. Therefore, according to the experimental result, the probe with 2 identical H1 hairpins is finally selected, and two target miRNAs can be simultaneously detected.

Example 3 clinical application of Probe

Adding detection probe (octahedral DNA nanocage-aptamer-H1 probe with 50nm section + octahedral DNA nanocage-aptamer-H1 probe with 50nm probe section) into leukocyte separated from patient, and reacting at room temperature in one step (200 μ l reaction system PBS buffer solution, 3 × 10 ⁵ Leukemia cells) to detect leukemia cell membrane proteins and intracellular miRNAs.

(1) The probe is targeted to leukemia cells CEM (Sgc f +, TC01+, sgc c +) for verification, and the result is shown in FIG. 8. The result shows that compared with the probe without the aptamer, the probe assembled with 3 aptamers (3 APT) has obviously enhanced fluorescence signals in CEM cells, and the fact that the detection probe with the aptamer target can enter leukemia cells expressed by specific membrane proteins more efficiently and detect the intracellular miRNA target molecules more effectively is proved.

(2) Leukemia cells CEM (Sgc f +, TC01+, sgc c +), JURKAT (Sgc f-, TC01-, sgc c-), K562 (Sgc f-, TC01-, sgc c-) are detected in situ by targeting leukemia probes miRNA-146a and miRNA-17, respectively, and the results are shown in FIG. 9. The result shows that the CEM leukemia cells highly express 3 aptamers and specifically bind to the membrane protein, so that the miRNA target molecules are effectively detected by the detection probes entering the cells, and the JURKAT and K562 cells cannot effectively enter the cells due to no or low expression of the related membrane protein related to the related membrane protein. The combination of the DNA aptamer and the probe designed by the CHA sensing method is proved to realize leukemia cell targeting expressed by specific membrane protein and target cell miRNA in-situ detection.

(3) Probe-targeted leukemia cell validation (clinical samples)

The method comprises the steps of firstly separating and extracting lymphocytes of leukemia patients and normal people, and firstly detecting clinical samples by using fluorescence modified aptamer probes (Sgc f-Texas Red, TC01-AF488 and Sgc c-HEX) according to the same method, wherein the result is shown in figure 10, the lymphocyte of the acute gonorrhea leukemia patients is more normal people, and the expression level of cell membrane protein specifically bound by the aptamer is obviously increased. Then collecting and separating and extracting lymphocytes in blood of a normal person, an acute gonorrhea patient in the attack stage, after treatment and after relapse, detecting by using a fluorescence-modified H1 hairpin (H1-miRNA-146-AF 488, H1-miRNA-17-Cy5.5) -aptamer probe according to the method, wherein the result is shown in figure 11, compared with the normal person and the patient after the acute gonorrhea treatment, the related miRNA target molecules in the lymphocytes of the patient in the acute gonorrhea attack stage and the patient after relapse are obviously increased, the probe designed by combining the DNA aptamer and the CHA sensing method is proved, the leukemia cell targeting and the target cell miRNA in-situ detection expressed by specific membrane protein can be realized, and a certain prompting function is provided for the development and regression of leukemia diseases.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. Based on many aptamers target leukemia cell normal position microRNA DNA nanometer cage probe, its characterized in that: the probe comprises an octahedral DNA nanocage and a self-assembly reaction hairpin structure;

the octahedral DNA nano cage probe is formed by self-assembling 8 strands serving as a main body framework to form an octahedron, wherein a leukemia tumor cell DNA aptamer sequence extends from the 3 'end of at least one of the strands, the self-assembly reaction hairpin structure comprises a hairpin I and a hairpin II, the hairpin I specifically identifies target miRNAs and consists of a main body framework 3' end extension sequence of the unextended aptamer sequence of the octahedral DNA nano cage; the hairpin II is complementary with the hairpin I.

2. The in-situ microRNA DNA nanocage probe based on the multi-aptamer targeted leukemia cells according to claim 1, which is characterized in that: the hairpin I comprises a folding region forward sequence, a ring region, a folding region reverse sequence and a free region, the free region and the folding region connected with the free region specifically recognize target miRNAs, and the hairpin II is modified with a fluorescent group and a quenching group and is complementary with the folding region forward sequence, the ring region, the folding region reverse sequence and the free region of the hairpin I.

3. The in-situ microRNA DNA nanocage probe based on the multi-aptamer targeted leukemia cells according to claim 1, which is characterized in that: the main framework sequence of the octahedral DNA nano cage probe is composed of 1-75 th basic groups of SEQ ID NO. 1-SEQ ID NO. 8.

4. The in-situ microRNA DNA nanocage probe based on the multi-aptamer targeted leukemia cells according to claim 1, which is characterized in that: the 8-chain n-chain extension leukemia tumor cell DNA aptamer sequence of the octahedral DNA nano cage is not less than 1 and not more than 3, the 8-n-chain extension of the octahedral DNA nano cage specifically identifies the hairpin structures of different target miRNAs, and the hairpin structures for identifying different target miRNAs are marked by different fluorescence.

5. The in-situ microRNA DNA nanocage probe based on the multi-aptamer targeted leukemia cells according to claim 4, which is characterized in that: the number of hairpin I for specifically recognizing microRNAs of the same target is at least 2; the leukemia tumor cell DNA aptamer is at least one of Sgc f, TC01 and Sgc c.

6. Use of the in-situ microRNA DNA nanocage probe based on the multi-aptamer target leukemia cells according to any one of claims 1 to 5 in preparation of a multi-target microRNAs detection kit.

7. A kit comprising the probe according to any one of claims 1 to 5.

8. The kit of claim 7, wherein: the kit also comprises an assembly liquid, wherein the assembly liquid is a TAM buffer solution, and the concentration of each component is as follows: pH 7.0, 40mM Tris acetate, 12.6mM magnesium acetate.

9. The method for detecting microRNA in situ by targeting leukemia cells by using the probe of any one of claims 1 to 5, is characterized by comprising the following steps: 8 main skeleton chains with the DNA aptamer sequences of the leukemia tumor cells and the hairpin I are self-assembled to form an octahedral DNA nano cage probe, then the octahedral DNA nano cage probe and the hairpin II are added into the separated leukemia cells, and the cell membrane protein and intracellular miRNAs for detecting leukemia are obtained through fluorescent signals.

10. The method of claim 9, wherein: the self-assembly condition is that equimolar main skeleton chains are mixed in TAM buffer solution at the concentration of 1 mu M, heated for 10 minutes at 95 ℃, then heated for 5 minutes at 80 ℃, cooled to 60 ℃, and finally slowly cooled to 4 ℃.