DNA nanocage probes for leukemia cell multispecific membrane protein logic circuit analysis, kits and methods thereof

Abstract

The application discloses a DNA nanocage probe for leukemia cell multi-specific membrane protein logic circuit analysis, a kit and a method thereof, and realizes in-situ imaging analysis on three target membrane proteins by using a probe of a cross-section octahedral DNA nanocage main frame, stable structure, and designing a three-specific recognition biological computing DNA molecule logic gate based on a nucleic acid aptamer (Sgc4f, TC01, Sgc8c), which provides a new tool and idea for early diagnosis of tumors and biomedical application of the DNA molecule logic gate in a complex cell system.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, in particular to a DNA nanocage probe for analyzing leukemia cell multi-specific membrane protein logic circuit, and also relates to a leukemia cell multi-specific membrane protein detection kit and method. BACKGROUND

[0002] DNA molecule logic gate can be widely used in the detection of various biological substances in the field of biochemical analysis, such as DNA, RNA, protein, small molecule and ion, etc. The molecular logic gate can realize two or more complex operations at the molecular level, similar to the logic gate in digital circuit, and the output signal is the corresponding logic signal obtained by operation, which is usually called Boolean logic.

[0003] There are many limitations and challenges in designing detection probes based on DNA molecule logic gate, including: 1. For complex cell samples, due to the influence of cell metabolism and other factors, the physiological environment inside and outside the cell is complex and changeable. At the same time, due to the existence of various organelle structures in the cell, the cell has multiple solid-liquid interfaces, which puts forward higher requirements for the application of DNA molecule logic gate in cell analysis. 2. The use of DNA molecule logic gate for membrane protein interaction analysis still lacks a scheme that matches the important application in the field of biomedicine. Therefore, the development of DNA molecule logic gate suitable for the needs of biomedicine and its application in clinical diagnosis is a key problem in current biomedical research.

[0004] The change of leukemia cell membrane receptor expression level and / or function can cause systemic dysfunction, and the same molecular marker is often shared by different cell subgroups. To accurately identify cancer cell subgroups without specific markers, multiple markers are needed. By analyzing the high or low expression level of multiple membrane receptors, the properties of individual cells can be more accurately identified, so as to realize more accurate disease diagnosis and treatment. Therefore, it is of great significance to develop a method for analyzing multiple tumor molecular markers to identify cancer cell subgroups.

[0005] Aptamer is a single-stranded nucleic acid chain, which is a kind of DNA or RNA with unique three-dimensional conformation, also known as "chemical antibody", which can bind with various targets with high specificity and affinity. It can be used as a common ligand and can specifically recognize and tightly bind with membrane protein receptors. The molecular probe with aptamer can target the overexpressed membrane protein of cancer cells and be used for cancer diagnosis and targeted therapy.

[0006] Designing DNA molecule logic gate based on nucleic acid aptamer and constructing detection probe for targeted analysis of leukemia cell membrane protein is a hot and difficult point in current research.

[0007] DNA geometric polyhedron (DNA-GPs): including tetrahedron, prism, octahedron, dodecahedron, icosahedron, buckyball, etc., high programmability, good biocompatibility and diversified functionalization method, plus stable framework and hollow interior, providing broad opportunities for multidisciplinary research. However, the assembly detection strategy based on DNA-GPs is subject to many limitations, the key problems are: 1. DNA tetrahedron and other simpler geometric polyhedron modification space is insufficient; 2. The modification space of more complex geometric polyhedron such as dodecahedron is larger, the assembly steps are complex, and the assembly rate is low. Therefore, a probe that is simple to assemble, has sufficient modification space and can detect leukemia with high sensitivity is urgently needed. SUMMARY

[0008] Therefore, one of the purposes of the present application is to provide a DNA nanocage probe for leukemia cell multispecific membrane protein logic circuit analysis; the second purpose of the present application is to provide the use of the DNA nanocage probe for leukemia cell multispecific membrane protein logic circuit analysis in preparing a leukemia membrane protein in situ imaging analysis kit; the third purpose of the present application is to provide a kit containing the probe; and the fourth purpose of the present application is to provide a method for using the probe for leukemia membrane protein in situ imaging analysis.

[0009] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0010] 1. A DNA nanocage probe for leukemia cell multispecific membrane protein logic circuit analysis, the probe comprising a cross-octahedral DNA nanocage and a DNA molecule logic calculation sequence designed based on a nucleic acid aptamer.

[0011] The cross-octahedral DNA nanocage probe is self-assembled from 8 strands as the main skeleton to form a cross-octahedron, wherein 3' end of 3 skeleton chains is extended with a leukemia tumor cell DNA aptamer sequence, and the DNA aptamer sequence has 3 short chain DNAs that are complementary to each other, and 3' end of 3 skeleton chains is extended with a connection sequence of a DNA molecule logic calculation sequence;

[0012] The DNA molecule logic calculation sequence is assembled from 9 sequences to form 3 groups of logic calculations with different fluorescent light emitting groups and quenching groups, respectively, and is named as R1, R2 and R3, and is connected with the connection sequence of the DNA molecule logic calculation sequence at the 3' end of the cross-octahedral DNA nanocage.

[0013] When the 3 aptamers of the probe are combined with leukemia cell specific membrane proteins, the short chain DNAs complementary to the aptamer part will fall off, and any two or more short chain DNAs will further trigger the R1, R2 and R3 logic calculations, resulting in the generation of corresponding fluorescent signals.

[0014] Preferably, the DNA molecule logic calculation sequence is as shown in SEQ ID NO. 9 to SEQ ID NO. 17.

[0015] Preferably, the main skeleton sequence of the cross-section octahedral DNA nanocage probe is composed of bases 1 to 75 of SEQ ID NO. 1 to SEQ ID NO. 8.

[0016] Preferably, the leukemia tumor cell DNA aptamer is at least one of Sgc4f, TC01 and Sgc8c.

[0017] Preferably, the sequences of the three short-chain DNAs are as shown in SEQ ID NO. 18 to SEQ ID NO. 20.

[0018] 2. Application of the DNA nanocage probe for leukemia cell multi-specific membrane protein logic circuit analysis in the preparation of a leukemia membrane protein in-situ imaging analysis kit.

[0019] 3. A kit containing the probe.

[0020] Preferably, the kit further comprises an assembly solution, and the assembly solution is a TAM buffer solution, and the concentrations of the components are as follows: pH 7.0, 40 mM Tris acetate, 12.6 mM magnesium acetate.

[0021] 4. A method for using the probe for leukemia membrane protein in-situ imaging analysis, comprising the following steps:

[0022] 1) 8 main skeleton chains with leukemia tumor cell DNA aptamer sequences and DNA molecule logic calculation sequence connection sequences are self-assembled to form an octahedral DNA nanocage probe;

[0023] 2) 9 DNA molecule logic calculation sequences are annealed at 95 DEG C for 5 minutes according to logic calculation R1, R2 and R3, and are assembled by gradient cooling;

[0024] 3) The self-assembled cross-section octahedral DNA nanocage probe and the assembled probe main body and logic calculation parts R1, R2 and R3 are mixed at a ratio of 1:1, and are reacted at room temperature for 1 hour to obtain a leukemia cell multi-specific membrane protein logic circuit analysis DNA nanocage probe, which is then added to the white blood cells to be tested, and after sufficient reaction, the leukemia cell membrane protein is analyzed according to fluorescence.

[0025] Preferably, the self-assembly conditions are that equal molar amounts of main skeleton chains are mixed at a concentration of 1 μM in a TAM buffer solution, heated at 95 DEG C for 10 minutes, then heated at 80 DEG C for 5 minutes, cooled to 60 DEG C, and finally slowly cooled to 4 DEG C.

[0026] The application has the advantages that the application provides a DNA nanocage probe for leukemia cell multi-specific membrane protein logic circuit analysis DNA, a probe for forming a cross-section octahedral DNA nanocage main frame is designed by reasonably designing a cross-section octahedral DNA nanocage skeleton sequence, the structure is stable, and the assembly efficiency is greater than 90%; then three-specific recognition biological computing DNA molecules are designed based on nucleic acid aptamers (Sgc4f, TC01, Sgc8c), and a logic gate is realized, in-situ imaging analysis of three target membrane proteins of leukemia cells is realized, and new tools and ideas are provided for early diagnosis of tumors and biomedical application of DNA molecule logic gates in complex cell systems. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to make the purpose, technical scheme and beneficial effects of the application clearer, the application provides the following drawings for description:

[0028] Figure 1 The main structure of the probe, logic computing components and assembly are shown.

[0029] Figure 2 The logic computing truth table of the probe, logic circuit design is shown.

[0030] Figure 3 The logic computing detection principle of the probe is shown.

[0031] Figure 4 The three-dimensional diagram of the DNA nanocage probe for leukemia cell multi-specific membrane protein logic circuit analysis is shown.

[0032] Figure 5 The DNA nanocage probe for leukemia cell multi-specific membrane protein logic circuit analysis is shown.

[0033] Figure 6 The probe aptamer targets leukemia cell membrane protein CF, CS short chain free promotion R1 logic computing (green fluorescence) is shown.

[0034] Figure 7 The probe aptamer targets leukemia cell membrane protein CQ, CS short chain free promotion R2 logic computing (orange fluorescence) is shown.

[0035] Figure 8 The probe aptamer targets leukemia cell membrane protein CF, CQ short chain free promotion R3 logic computing (red fluorescence) is shown.

[0036] Figure 9 The probe aptamer targets leukemia cell membrane protein CF, CQ, CS short chain free promotion R1, R2, R3 logic computing (three-color fluorescence) is shown.

[0037] Figure 10 The probe assembly verification is shown.

[0038] Figure 11 For three groups of logic components, respectively, in different test tubes, single group single operation function verification is carried out.

[0039] Figure 12 For three groups of logic components, three groups of three operation function verification is carried out in the same test tube.

[0040] Figure 13 For three groups of logic components, three groups of three operation function verification is carried out in the same test tube.

[0041] Figure 14 For the probe, the leukemia cell lines CEM and JURKAT are targeted and logical operation is carried out.

[0042] Figure 15 For the probe, the leukemia cell lines CEM and JURKAT are targeted and logical operation is carried out.

[0043] Figure 16 For the probe, the leukemia cell lines CEM and JURKAT are targeted and logical operation is carried out. DETAILED DESCRIPTION

[0044] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and can implement it, but the examples are not as a limitation on the present application.

[0045] The DNA nanocage probe for leukemia cell multispecific membrane protein logic circuit analysis in the embodiment of the present application is a probe based on a cross octahedral DNA nanocage main frame, which is stable in structure, and three specificity recognition biological computing DNA molecules are designed based on nucleic acid aptamer (Sgc4f, TC01, Sgc8c) to realize in-situ imaging analysis of three target membrane proteins, which provides a new tool and idea for early diagnosis of tumors and biomedical application of DNA molecular logic gate in complex cell systems.

[0046] Example 1, design of DNA nanocage probe for leukemia cell multispecific membrane protein logic circuit analysis

[0047] The detection probe of the embodiment can be divided into two parts: a middle main body part and a logic calculation part. The middle main body part is assembled by 11 primers, and is taken the cross octahedral DNA nanocage as a skeleton, and includes 1-①-TC01, 1-②-R2, 1-③-Sgc4f, 1-④-R1, 1-⑤, 1-⑥-R3, 1-⑦-Sgc8c, 1-⑧. The bold sequence is the main skeleton sequence of the cross octahedral DNA nanocage, and the italicized sequence represents three short-chain DNAs which are complementary to the three adaptors, and the corresponding relationship is TC01-CQ, Sgc4f-CF, Sgc8c-CS. The logic calculation part is assembled by 9 primers, and includes R1V-S1-F1, R2V-Q1-F2, R3V-F2-Q2. The related primer sequences are shown in Table 1, and the assembly effect is shown in Figure 1 .

[0048] Table 1, related primer sequences of the DNA nanocage probe for analyzing the multi-specific membrane protein logic circuit of leukemia cells

[0049]

[0050]

[0051] The detection principle of the logic calculation part (see Figures 2 to 8 ): when the three adaptors (Sgc4f, TC01 and Sgc8c) of the probe are combined with the leukemia cell specific membrane protein, the short-chain DNAs (CF, CS and CQ) which are complementary to the adaptor part will fall off, and any two or more short-chain DNAs will further trigger the R1, R2 and R3 logic calculation, resulting in the generation of corresponding fluorescence signals.

[0052] The logic gate design scheme of the multi-specific leukemia cell membrane protein logic circuit analysis DNA nanocage probe.

[0053] As shown in A of Figure 2 , the logic calculation simple circuit diagram, any two free short-chain DNAs (CF, CS and CQ) will trigger the R1, R2 and R3 logic calculation, resulting in the generation of corresponding fluorescence signals; as shown in B of Figure 2 , the reaction relationship between the short-chain DNAs (CF, CS and CQ) and the R1, R2 and R3 logic calculation; as shown in Figure 2As shown in Figure C, the truth table for logic calculations describes in tabular form the relationship between the binding of any two aptamers of probes Sgc4f, TC01, and Sgc8c to leukemia cell membrane proteins and the fluorescence signal. Specifically, when the three aptamers (Sgc4f, TC01, and Sgc8c) bind to specific leukemia cell membrane proteins, it leads to the shedding of short DNA strands (CF, CS, CQ) partially complementary to the aptamers. The release of any two or more of these short strands further triggers logic calculations R1, R2, and R3, subsequently generating the corresponding fluorescence signals (F1, F2, F3). Figure 2 As shown in Figure D, the logic circuit is consistent with the truth table of the logic calculation in Figure C, describing the relationship between the binding of any two aptamers of the probe to leukemia cell membrane proteins and the fluorescence signal.

[0054] Example 2: Probe Assembly

[0055] Probe body assembly conditions: The cages were assembled using a PCR instrument by mixing equimolar amounts of each strand in 1 μM of TAM buffer (40 mM Tris acetic acid pH 7.0, 12.6 mM magnesium acetate). The samples were heated at 95 °C for 10 min, then at 80 °C for 5 min, cooled to 60 °C (4 min / 1 °C), and finally slowly cooled to 4 °C (6 min / 1 °C).

[0056] Assembly conditions for logic computation components R1, R2, and R3: R1V-S1-F1, R2V-Q1-F2, and R3V-F2-Q2 were paired, annealed at 95 degrees Celsius for 5 minutes using a PCR instrument, and then assembled using a gradient cooling method. The assembled probe body and logic computation components R1, R2, and R3 were mixed in a 1:1 ratio and reacted at room temperature for 1 hour.

[0057] The assembled 3D model is as follows Figure 9 As shown in the image, the orange, green, and blue strands in the probe correspond to the DNA aptamers Sgc4f, TC01, and Sgc8c, respectively, which can recognize leukemia cell-specific membrane proteins. Each aptamer is complementary to one of three short strands (CF, CQ, and CS). When the probe binds to the leukemia cell-specific membrane protein, the three short strands (CF, CQ, and CS) are released, triggering logical computation. At the top of the image, the three sets of multicolored DNA strands in the probe correspond to three sets of DNA logical computation functional units (R1, R2, and R3). The innermost light blue, light pink, and light green DNA strands are modified with fluorescent groups. When no logical computation is performed, the fluorescent groups are quenched by quenching groups, resulting in no fluorescent signal. When the logical computation is run, the released three short strands (CF, CQ, and CS) are recognized, and the probe's fluorescent groups emit a signal.

[0058] Gel electrophoresis was used to detect probe assembly efficiency, and the results were as follows: Figure 10 As shown in the figure. The results show that the probe assembly efficiency is >90%.

[0059] Example 3, application of the probe

[0060] In the test tube or in the patient isolated leukocytes, the detection probe is added, and the white blood cell membrane protein is analyzed comprehensively in one step at room temperature (the detection results are shown in Figures 11 to 16 ).

[0061] Figure 11 The three groups of logic components are respectively verified in different test tubes for single group single operation function.

[0062] First, three groups of logic calculation components (R1-V(AF488)-S1-F1, R2-V(CY3)-V-Q1-S2, R3-V(Texas red)-F2-Q2) are respectively assembled, and the final concentration is 1 μm. Then, four control groups are set, and the three logic calculation components are equally divided into four parts (①, ②, ③, ④) with a volume of 50 μl: logic calculation R1 (① blank control group added with equal volume of ultrapure water, ② added with short chain CF, ③ added with short chain CS, ④ added with short chain CF, CS), logic calculation R2 (① blank control group added with equal volume of ultrapure water, ② added with short chain CS, ③ added with short chain CQ, ④ added with short chain CS, CQ), logic calculation R3 (① blank control group added with equal volume of ultrapure water, ② added with short chain CQ, ③ added with short chain CF, ④ added with short chain CQ, CF), each group is simultaneously reacted at 25℃ for 1 h, and finally, the reacted liquid is detected by a fluorescence detector for corresponding fluorescence signal, as shown in Figure 11 The results show that, consistent with the logic calculation design of the present scheme ( Figure 2 ), when short chain CF and CS exist simultaneously, logic calculation R1 (AF488) outputs strong fluorescence signal; when short chain CQ and CF exist simultaneously, logic calculation R2 (CY3) outputs strong fluorescence signal; when short chain CS and CQ exist simultaneously, logic calculation R3 (Texas red) outputs strong fluorescence signal. The experimental results confirm that the three groups of logic calculation components designed in the present scheme can effectively play the operation function, and any two short chains of CF, CS and CQ can trigger the operation of the corresponding logic component and output fluorescence signal.

[0063] Figure 12Three groups of logic components were assembled in the same test tube for three-group three-operation function verification. The results showed that three groups of logic computing components (R1-V(AF488)-S1-F1, R2-V(CY3)-V-Q1-S2, R3-V(Texas red)-F2-Q2) were mixed and mixed at a final concentration of 1 um. Eight control groups were set up, and the mixed logic computing components were divided into 8 parts (①, ②, ③, ④, ⑤, ⑥, ⑦, ⑧) with a volume of 50 ul each: ① blank control group added with equal volume of ultrapure water, ② added with short chain CQ, ③ added with short chain CS, ④ added with short chain CF, ⑤ added with short chain CS, CQ, ⑥ added with short chain CF, CS, ⑦ added with short chain CQ, CF, ⑧ CF, CS, CQ. Each group was reacted at 25°C for 1h, and finally the reacted liquid was detected by fluorescence detector for corresponding fluorescence signal. Figure 12 The results showed that the logic computing design of the present scheme was consistent with Figure 2 In ⑥ and ⑧, strong fluorescence signal of logic computing R1(AF488) output was detected, and the output fluorescence signal of the remaining groups was relatively weak; in ⑤ and ⑧, strong fluorescence signal of logic computing R2(CY3) output was detected, and the output fluorescence signal of the remaining groups was relatively weak; in ⑦ and ⑧, strong fluorescence signal of logic computing R3(Texas red) output was detected, and the output fluorescence signal of the remaining groups was relatively weak. The experimental results confirmed that the three groups of logic computing components were mixed, and any two short chains in CF, CS and CQ could trigger the corresponding logic component operation and output fluorescence signal, and the three-group operation did not interfere or interfered very little, and could complete effective detection.

[0064] Figure 13 To verify the three-group three-operation function of the probe three-group logic components in the same test tube. First, the complete detection probe was assembled, i.e. the assembled probe main body and logic computing parts R1-V(AF488)-S1-F1, R2-V(CY3)-V-Q1-S2, R3-V(Texas red)-F2-Q2 were mixed at a ratio of 1:1, reacted at room temperature for 1h, and the final concentration was 0.5 um. Then, eight control groups were set up, and the volume of the assembled complete detection probe was divided into 8 parts (①, ②, ③, ④, ⑤, ⑥, ⑦, ⑧) with a volume of 50 ul each: ① blank control added with equal volume of ultrapure water, ② added with short chain CF, ③ added with short chain CS, ④ added with short chain CQ, ⑤ added with short chain CF, CS, ⑥ added with short chain CS, CQ, ⑦ added with short chain CQ, CF, ⑧ CF, CS, CQ. Each group was reacted at 25°C for 1h, and finally the reacted liquid was detected by fluorescence detector for corresponding fluorescence signal, which was consistent with Figure 12 The results were consistent with Figure 13The results show that strong fluorescent signals are detected in logic calculation R1 (AF488) in 5 and 7, and the rest of the groups output relatively weak fluorescent signals; strong fluorescent signals are detected in logic calculation R2 (CY3) in 6 and 7, and the rest of the groups output relatively weak fluorescent signals; strong fluorescent signals are detected in logic calculation R3 (Texas red) in 6 and 7, and the rest of the groups output relatively weak fluorescent signals. The experimental results confirm that for the assembled complete detection probe, any two short chains in CF, CS and CQ can trigger the corresponding logic component operation and output fluorescent signals, and the three groups of operations do not interfere with or interfere with each other very little, and can complete effective detection.

[0065] Figure 14 The probe targets leukemia cell lines CEM and JURKAT to perform logic operation verification.

[0066] According to the literature report, leukemia cell lines CEM (Sgc4f+, TC01+, Sgc8c+) and JURKAT (Sgc4f-, TC01-, Sgc8c-) are selected, and 5x10 5 cells are taken in 200ul 1xPBS solution, 50ul 0.5um probe is added, incubated at 25°C for 1.5h, washed with PBS for 3 times, then Hoechst cell nucleus dye is added, incubated at room temperature for 10min, washed with PBS for 3 times, finally 200ul PBS is used to resuspend the cells and placed in an imaging dish for fluorescence confocal imaging, as shown in Figure 14 The results show that CEM (Sgc4f+, TC01+, Sgc8c+) logic calculation R1 (AF488), R2 (CY3) and R3 (Texas red) all present strong logic calculation fluorescent signals, and JURKAT (Sgc4f-, TC01-, Sgc8c-) has no logic calculation fluorescent signal output. The research results confirm that the probe can recognize leukemia cell specific membrane proteins and perform logic calculation to output fluorescent signals.

[0067] Figure 15 The probe targets leukemia cell lines CEM and JURKAT mixed cells to perform logic operation and distinguish cell types.

[0068] CEM and JURKAT 2x10 5 cells are taken in 200ul 1xPBS solution, Hoechst cell nucleus dye is added in CEM first, incubated at room temperature for 10min, mixed with JURKAT after washing with PBS for 3 times, then 50ul 0.5um probe is added, incubated at 25°C for 1.5h, washed with PBS for 3 times, finally 200ul PBS is used to resuspend the cells and placed in an imaging dish for white light and fluorescence confocal imaging, as shown in Figure 15The results show that CEM incubated with Hoechst cell nucleus dye all present strong logical calculation fluorescent signals, and the research results prove that the probe can distinguish leukemia cell types by recognizing specific cell membrane proteins and performing logical calculation.

[0069] Figure 16 The probe is verified to target leukemia cells in clinical samples and logical operation is verified.

[0070] Clinical blood samples of acute leukemia patients (in acute phase and after chemotherapy) and normal people are collected, lymphocytes are separated and extracted, 5x10 5 cells are taken in 200ul 1xPBS solution, 50ul 0.5um probe is added, incubated at 25℃ for 1.5h, washed with PBS for 3 times, Hoechst cell nucleus dye is added, incubated at room temperature for 10min, washed with PBS for 3 times, finally 200ul PBS is used to resuspend the cells in an imaging dish for fluorescence confocal imaging, as shown in Figure 16 The results show that normal people have no logical calculation fluorescent signal output, acute leukemia cells in acute phase present strong R1, R2 and R3 logical calculation fluorescent signals, and the positive cells of R1, R2 and R3 logical calculation fluorescent signals significantly decrease after chemotherapy. The clinical research results prove that the probe targeting leukemia cells and logical operation results have a hinting effect on leukemia diagnosis and prognosis evaluation.

[0071] The multi-specific leukemia cell membrane protein logic circuit analysis DNA nanocage probe of the scheme can effectively perform operation function, and any two short chains in CF, CS and CQ can effectively trigger the operation of the corresponding logic component and output fluorescent signals. The probe has good effect in intracellular and extracellular function verification and detection, can be used for single leukemia cell membrane protein expression in situ imaging analysis, can perform relative quantification on leukemia cell specific membrane protein expression through fluorescence intensity, and can also be used for cell type analysis. The logical calculation detection results of the probe applied to clinical leukemia lymphocyte membrane protein analysis also prove that the detection results of the probe of the scheme have a hinting effect on disease diagnosis and prognosis evaluation.

[0072] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation of the present application based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A DNA nanocage probe for multispecific membrane protein logic circuit analysis of leukemia cells, characterized in that: The probe comprises a cross-sectional octahedral DNA nanocage and a DNA molecule logic computation sequence designed based on nucleic acid aptamers; The cross-sectional octahedral DNA nanocage is formed by the self-assembly of 8 chains as the main backbone to form a cross-sectional octahedron. Among them, the 3' ends of 3 backbone chains extend leukemia tumor cell DNA aptamer sequences, and the DNA aptamer sequences also have 3 complementary short DNA strands, as well as the linking sequences of DNA molecular logic calculation sequences extended at the 3' ends of 3 backbone chains. The DNA molecular logic computing sequence is assembled from 9 sequences, forming 3 groups of logic computing sequences with different fluorescent luminescent groups and quenching groups, named R1, R2 and R3 respectively, and connected to the linking sequence of the DNA molecular logic computing sequence at the 3' end of the cross-sectional octahedral DNA nanocage. When the three aptamers of the probe bind to the specific membrane protein of leukemia cells, the short DNA strands that are partially complementary to the aptamers are detached. The release of any two or more short strands will further trigger the logic calculations of R1, R2, and R3, resulting in the generation of corresponding fluorescent signals. The logical computation sequence of the DNA molecule is shown in SEQ ID NO. 9 to EQ ID NO. 17; The main backbone sequence of the cross-sectional octahedral DNA nanocage is composed of bases 1 to 75 of SEQ ID NO.1 to SEQ ID NO.8; The leukemia tumor cell DNA aptamer is at least one of Sgc4f, TC01 and Sgc8c; The sequences of the three short DNA strands are shown in SEQ ID NO.18 to SEQ ID NO.

20.

2. Application of the DNA nanocage probe for analyzing multispecific membrane proteins in leukemia cells as described in claim 1 in the preparation of a leukemia membrane protein in situ imaging analysis kit.

3. A kit containing the probe of claim 1.

4. The reagent kit according to claim 3, characterized in that: The kit also includes an assembly solution, which is a TAM buffer with the following component concentrations: pH 7.0, 40 mM Tris acetic acid, and 12.6 mM magnesium acetate.

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