DNA fiber bar code probe for multiple miRNA detection and application of DNA fiber bar code probe

Abstract

The invention belongs to the technical field of biomedical detection, and particularly relates to a DNA fiber bar code probe for multiple miRNA detection and application of the DNA fiber bar code probe. In order to overcome the defects that existing miRNA detection is low in sensitivity, small in flux, serious in cross interference and the like, the probe comprises a DNA fiber and a fluorescence signal probe, and the DNA fiber is formed by self-assembly of DNA tile monomers; a recognition sequence complementary with target miRNA extends from the 3'end of the DNA tile monomer, a fluorescent signal probe with a hairpin structure is matched, a fluorescent signal is released when the target miRNA exists, the signal intensity is in direct proportion to the miRNA concentration, different target miRNAs trigger the hairpin signal probe with the corresponding fluorescent color, a decodable bar code is formed through fluorescent color combination, multiple miRNA detection is achieved, and the miRNA detection efficiency is improved. Further, the tumor cell types are accurately distinguished.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a DNA fiber barcode probe for multiplex miRNA detection and its applications. This DNA fiber is constructed using programmed DNA self-assembly technology to create functional DNA fibers. Combined with specific recognition units and a fluorescence signal switching strategy, it achieves high-throughput, high-sensitivity detection of multiple miRNAs, providing a new method for early tumor diagnosis and precise subtyping. Background Technology

[0002] Tumor cell typing has significant clinical value in clarifying tumor origin, assisting diagnosis, guiding treatment, and assessing prognosis. Currently used membrane protein receptor typing methods are easily affected by cellular metabolic state and microenvironment, limiting typing accuracy. In contrast, microRNAs (miRNAs), as key regulators of gene expression, exhibit characteristic abnormal expression profiles in the early stages of tumor development and possess higher species diversity and sequence specificity, making them more promising tumor typing biomarkers. However, existing miRNA detection technologies have significant limitations: single miRNA detection methods lack specificity and have limited information content; while in multiplex miRNA combined analysis techniques, qPCR suffers from primer cross-interference, and high-throughput sequencing faces application bottlenecks such as high cost and operational complexity. Furthermore, the limited number of fluorescence detection channels severely restricts their widespread clinical application. To address these technical challenges, this invention, based on the programmability and sequence specificity advantages of DNA nanotechnology, innovatively develops a DNA fiber barcode probe. Through modular design and a fluorescence encoding strategy, it achieves efficient simultaneous detection of multiplex miRNAs, providing a new technical solution for accurate tumor cell typing. Summary of the Invention

[0003] This invention addresses the aforementioned problems by providing a DNA fiber barcode probe for multiplex miRNA detection and its applications. This probe constructs DNA fibers through programmed self-assembly, integrating a specific recognition unit and a fluorescence signal switching module to achieve efficient detection of multiple miRNAs and tumor cell typing, overcoming the shortcomings of existing technologies such as low sensitivity, low throughput, and severe cross-interference.

[0004] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a DNA fiber barcoding probe for multiplex miRNA detection, comprising DNA fibers and a fluorescent signal probe, wherein: DNA fibers are formed by the self-assembly of DNA tile monomers, with a recognition sequence complementary to the target miRNA extending from the 3' end of each DNA tile monomer. This recognition sequence can be designed to be complementary to the target miRNA sequence, and is not limited to the complementary sequences of miR-21, miR-105, or miR-155. The fluorescent signal probe has a hairpin structure with a fluorescent group / quencher group pair labeled at the end. After binding to the target miRNA, it triggers a conformational change. In the presence of the target miRNA, the hairpin structure is opened through a strand displacement reaction and a fluorescent signal is released. The signal intensity is proportional to the miRNA concentration. Different target miRNAs trigger hairpin signal probes with corresponding fluorescent colors. By combining the fluorescent colors, a decodeable fluorescent barcode is formed, enabling single-tube multiplex miRNA detection.

[0005] The DNA tile monomers mentioned in this invention refer to basic DNA assembly units with sticky ends formed by base complementary pairing of five single-stranded DNA molecules, DT1-DT5; the fluorescent barcodes refer to the characteristic fluorescent spectra formed by the combination of fluorescent colors triggered by signal probes of different target miRNAs; the strand displacement reaction refers to the nucleic acid reaction process in which the hairpin structure of the fluorescent signal probe is opened after the target miRNA binds to the recognition sequence, causing the fluorescent group to separate from the quenching group.

[0006] Furthermore, the DNA tile monomers are formed by annealing five single-stranded DNA strands (DT1-DT5) and self-assembled into DNA fibers through complementary base pairing at the sticky ends between the DNA tile monomers.

[0007] Furthermore, the self-assembly method of the DNA fibers is as follows: An equimolar mixture of 5 single-stranded DNA strands and TAE-Mg 2+ Add buffer and DEPC-free water to nuclease-free microcentrifuge tubes. The final concentration of each single-stranded DNA is 300 nM. The mixture was subjected to thermal annealing: initial denaturation at 94°C for 5 minutes, followed by gradual cooling at a gradient of -1 °C / min until 4°C was reached.

[0008] Furthermore, the fluorescent signal probe is selected from any one or more of FAM-BHQ1, TAMRA-BHQ2, and AMCA-Dabcyl, and the 3' end sequence of the fluorescent signal probe is complementary to the target miRNA sequence. The final concentration of the fluorescent signal probe used is 100 nM.

[0009] Furthermore, when the fluorescent signal probe does not bind to the target miRNA, it hybridizes with the complementary quenching group and no fluorescence is detected; when it encounters a region complementary to the target miRNA, the fluorescent group separates from the quenching group and fluorescence is detected.

[0010] This invention also provides an application of a DNA fiber barcode probe for multiplex miRNA detection in tumor cell typing.

[0011] Tumor cell subtype differentiation: MCF-10A, MCF-7, MDA-MB-231 and HeLa cells were identified by miR-21, miR-105 and miR-155 expression profiles; Early diagnosis: Detecting abnormal expression of miRNAs in precancerous lesions.

[0012] Efficacy monitoring: Analyze the dynamic changes of miRNAs before and after treatment.

[0013] This invention also provides a multiplex miRNA detection kit for tumor cell typing, containing DNA fiber barcode probes for the detection of the multiplex miRNAs. The kit specifically includes: DNA fiber solution (final concentration of each monomer 300 nM), fluorescent signal probe solution (final concentration of each probe 100 nM), and 10×TAE-Mg... 2+ Buffer solution, negative control (DEPC water without miRNA), positive control (miR-21 / 105 / 155 standard solution); the kit should be stored at -20℃ protected from light and has a shelf life of 6 months.

[0014] This invention also provides a method for detecting multiplex miRNAs, characterized by comprising the following steps: a. Extract total RNA from the cells to be tested; the OD260 / OD280 ratio of the extracted total RNA should be 1.8~2.0 to ensure that the RNA is free from protein / DNA contamination; b. Incubate 1 μL of total RNA stock solution with DNA fiber barcode probe and hairpin signal probe at room temperature in the dark for 1 h; c. Acquire multi-channel fluorescence images using a laser confocal scanning microscope. The detection parameters are 40× objective lens, 1024-pixel resolution, and sequential scanning mode. d. Decode miRNA types based on fluorescent color combinations; e. The fluorescence intensity can be directly calculated from the target concentration using the linear regression equation of each target miRNA.

[0015] Compared with the prior art, the present invention has the following advantages: High versatility: It can be extended to any miRNA target simply by changing the recognition sequence at the end of the tile; High-throughput: Single-system expansion to 8 fluorescent coding combinations, simultaneously detecting at least 3 miRNA targets; Amplification-free: No reverse transcription and PCR are required, avoiding cross-interference; Low cost: DNA fiber annealing assembly eliminates the need for expensive instruments; compared to enzyme-dependent detection methods, detection costs are reduced. Small sample size: Multiplex detection can be achieved with just 1 μL of total RNA, suitable for puncture cells and liquid biopsy samples. Attached Figure Description

[0016] Figure 1 This diagram illustrates multiple miRNA analysis for cell typing based on DNA fiber barcoding; where a represents the sensing mechanism of multiple miRNA analysis, b represents the principle of simultaneous multi-target analysis based on functional DNA fibers, and c represents a schematic diagram of cell typing based on multiple miRNA analysis.

[0017] Figure 2 The results show the characterization of DNA fibers; where a is a schematic diagram of fiber assembly, b is a transmission electron microscope (TEM) image, c is a laser confocal microscopy (CLSM) image, d is a time-resolved fluorescence image, and e is the result of polyacrylamide gel electrophoresis (PAGE).

[0018] Figure 3 CLSM image of functionalized DNA fibers.

[0019] Figure 4 CLSM image for multiple miRNA logical recognition and barcode output.

[0020] Figure 5 The standard curves are for the three targets.

[0021] Figure 6 A comparison of miRNA expression profiles for four cell lines (MCF-10A, MCF-7, MDA-MB-231, and HeLa). Detailed Implementation

[0022] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0023] like Figure 1 As shown, a DNA fiber barcoding probe for multiplex miRNA detection includes DNA fibers and a fluorescent signal probe, wherein: DNA fibers are formed by the self-assembly of DNA tile monomers, wherein the 3' end of the DNA tile monomer extends a recognition sequence complementary to the target miRNA; the DNA tile monomer is formed by annealing 5 single-stranded DNA molecules and self-assembling into DNA fibers through complementary base pairing at the sticky ends of the DNA tile monomers.

[0024] The fluorescent signal probe has a hairpin structure with a fluorescent group and a quencher group labeled at the end. After binding to the target miRNA, it triggers a conformational change. In the presence of the target miRNA, the hairpin structure is opened through a strand displacement reaction and a fluorescent signal is released. The signal intensity is proportional to the miRNA concentration. The fluorescent signal probe can be selected from any one or more of FAM-BHQ1, TAMRA-BHQ2, and AMCA-Dabcyl. The 3' end sequence of the fluorescent signal probe is complementary to the partial sequence of the target miRNA.

[0025] Different target miRNAs trigger hairpin signal probes with corresponding fluorescent colors. The combination of fluorescent colors forms a decodeable barcode, enabling multiplex miRNA detection.

[0026] When the fluorescent signal probe does not bind to the target miRNA, it hybridizes with a complementary quencher group, resulting in no fluorescence detection. However, when it encounters a region complementary to the target miRNA, the fluorescent group separates from the quencher group, and fluorescence is detected. Tumor cell typing is achieved by quantifying the types and abundance of miRNAs in different cells. Example 1

[0027] Fiber assembly: In a 10 μL system, add an equimolar mixture of DT1, DT2, DT3, DT4, and DT5 (final concentration 300 nM), and 1 μL of 10 × TAE-M. 2+ Add the above mixture (120 mM MgCl2, 10 mM Tris-HCl, pH 8.0) and 4 μL DEPC water to a nuclease-free microcentrifuge tube. To ensure controlled and efficient self-assembly, the mixture was subjected to a thermal annealing program in a PCR thermal cycler: initial denaturation at 94°C for 5 min, followed by a gradient decrease to 4°C at -1°C / min to obtain DNA fibers.

[0028] Figure 2 The TEM, CLSM, time-resolved confocal microscopy, and PAGE assays show that the assembly was successful, revealing the DNA fiber morphology and molecular weight. Figure 2 The TEM image of a shows the topological morphology of the fiber surface; Figure 2 The CLSM plot in b shows that the components exhibit continuous linear red fluorescence with an average length of 14 μm, thus confirming the structural integrity of the DNA fibers. Figure 2 In the middle c, the fluorescence image is time-resolved, showing multiple molecular fluorescent dots forming a rigid linear fiber structure. Figure 2 In the figure, d represents the PAGE result. The migration rate of the fully assembled product (lane 5) indicates that its molecular weight is >500 bp. Example 2

[0029] Functional fiber assembly: Three functional fibers were assembled by annealing DT3-H1-TAMRA, DT3-H2-FAM, DT3-H3-AMCA, and four other single-stranded DNA molecules (DT1, DT2, DT4, DT5). The final concentration of each single-stranded DNA was 300 nM. H1, H2, and H3 were recognition sequences complementary to the target miRNA extended from the 3' end of the DNA tile monomers. TAMRA, FAM, and AMCA were fluorescent groups. Confocal imaging, excitation / emission: FAM (488 ex / 499-551 em); TAMRA (561 / 590 nm); TAMRA (561 ex / 571-625 em); AMCA (405 ex / 430-475 em). Detection parameters were 40× objective lens, 1024-pixel resolution, and sequential scanning mode.

[0030] Figure 3 The CLSM images shown demonstrate that the identified sequences and fluorescent groups have no effect on fiber synthesis. Specifically, four single chains (DT1, DT2, DT4, DT5) assemble with the core chain DT3 to form tiles of corresponding colors. Tiles of different colors assemble into fibers of different corresponding colors. Single tiles self-assemble to form monochromatic fibers; double-tile assembly produces mixed colors (red + green = yellow, red + blue = magenta, blue + green = cyan); and the co-assembly of three tiles (red + green + blue) produces white fluorescence, confirming the successful assembly of the multi-component structure. Example 3

[0031] Multiplex miRNA analysis: Further, DT3-H1, DT3-H2, DT3-H3 (100 nM each) and four other single-stranded DNA molecules (DT1, DT2, DT4, DT5, 300 nM each) were annealed and assembled into a functional fiber. In a 10 μL reaction system, the following were added: 8 μL of DNA fiber, a mixture of three fluorescent hairpin signal probes (P1, P2, P3) (100 nM each), and 1 μL of target miRNA. The mixture was incubated at room temperature in the dark for 1 h. Confocal imaging was performed with the following excitation / emission parameters: FAM (488 ex / 499-551 em; TAMRA 561 / 590 nm; TAMRA (561 ex / 571-625 em; AMCA (405 ex / 430-475 em)). Detection parameters were 40× objective lens, 1024-pixel resolution, and sequential scanning mode. The results are shown below. Figure 4As shown, the functional fiber is colorless when there is no target; it is red when containing only miR-21; green when containing only miR-105; and blue when containing only miR-155. When two targets coexist, the fluorescence superimposes to produce a mixed color: miR-21 and miR-105 together produce yellow, miR-21 and miR-155 together produce magenta, and miR-105 and miR-155 together produce cyan. When all three targets are present, the red, green, and blue colors superimpose, and the fiber appears white. Therefore, single-target, dual-target, and triple-target combinations all produce corresponding color barcodes. Example 4

[0032] Quantitative analysis of miRNAs: Synthetic standard miRNAs at different concentrations (10 pM-70 nM) were analyzed using a constructed sensing system. Three gradient concentrations of target miRNAs (miR-21, miR-105, miR-155) were analyzed, and the relationship between target abundance and fluorescence output was investigated. Figure 5 The logarithmic concentrations of the three miRNAs were linearly proportional to the fluorescence intensity (R0). 2 ≥0.987). For miR-21, the linear equation is y=478.04 lg c-51.44, with a detection limit of 2.4 pM. For miR-105, the linear fit is y=447.25 lg c-146.80, with a LOD of 5.0 pM. The linear relationship for miR-155 is y=268.03 lg c+3.81, with a LOD of 3.4 pM. The target concentration can be directly calculated from the linear equation. Figure 5 (a, b, c). Compared to the traditional qPCR method, this method does not require reverse transcription amplification, thus shortening the detection time. Example 5

[0033] Cell typing analysis: Total RNA was extracted from four cell types (MCF-10A, MCF-7, MDA-MB-231, and HeLa) using the Trizol method. The OD260 / OD280 ratio of the extracted total RNA was 1.8–2.0 to ensure that the RNA was free of protein / DNA contamination. 1 μL of the RNA was used directly for detection. Comparison of DNA fibrillation and qPCR results was performed. Figure 6Figures a, b, and c show the expression profiles of miRNA-21, miRNA-105, and miRNA-155 in four cell lines, respectively. It can be seen that the expression trends of miRNA-21, miRNA-105, and miRNA-155 are consistent. The expression level of miR-155 in MDA-MB-231 cells was 6.5 times that in HeLa cells and 2.2 times that in MCF-7 cells; the expression level of miR-21 was 2.6 times that in HeLa cells and 1.7 times that in MCF-7 cells; and the expression level of miR-105 was 3.2 times that in HeLa cells and 1.4 times that in MCF-7 cells. This clearly distinguishes normal cells from cancer cells and further differentiates tumor cell subtypes.

[0034] The relevant primer sequences involved in the above embodiments are shown in Table 1 below: Table 1 Primer sequences Name Sequence (5-3) Corresponding Sequence DT1 5-CTCAGTGGACAGCCGTTCTGGAGCGTTGGACGAAACT-3 SEQ.ID.NO.1 DT2 5-GTCTGGTAGAGCACCACTGAGAGGTA-3 SEQ.ID.NO.2 DT3 5-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGC-3 SEQ.ID.NO.3 DT4 5-CAGACAGTTTCGTGGTCATCGTACCT-3 SEQ.ID.NO.4 DT5 5-CGATGACCTGCTTCGGTTACTGTTTAGCCTGCTCTAC-3 SEQ.ID.NO.5 DT3-H1-TAMRA 5-TAMRA-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCTTTTTTCAACATCAGT-3 SEQ.ID.NO.6 DT3-H2-FAM 5-FAM-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCTTTTTACCACAGGAGT-3 SEQ.ID.NO.7 DT3-H3-AMCA 5-AMCA-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCTTTTTACCCCTATCACG-3 SEQ.ID.NO.8 DT3-H1 5-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCTTTTTTCAACATCAGT-3 SEQ.ID.NO.9 DT3-H2 5-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCTTTTTACCACAGGAGT-3 SEQ.ID.NO.10 DT3-H3 5-TCCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCTTTTTACCCCTATCACG-3 SEQ.ID.NO.11 P1 5-TAMRA-CTGATAAGCTATAGTCGACCATTTATCAGC-BHQ2-3 SEQ.ID.NO.12 P2 5-FAM-TCAAATCTAGGACTGACTGAGCATTTGA-BHQ1-3 SEQ.ID.NO.13 P3 5-AMCA-ATTAGCATTAACACTCACCATCTGCTAAT-Dabcy1-3 SEQ.ID.NO.14 miRNA-105 5-UCAAAUGCUCAGACCUCUGUGGU-3 SEQ.ID.NO.15 miRNA-155 5-UUAAUGCUAUCGUGAUAGGGGU-3 SEQ.ID.NO.16 miRNA-21 5-UAGCUAUCAGACUGAUGUUGA-3 SEQ.ID.NO.17 miR-105 F 5-GCGCGTCAAATGCTCAGACTCC-3 SEQ.ID.NO.18 miR-105 R 5-AGTGCAGGGTCCGAGGTATT-3 SEQ.ID.NO.19 miR-105 RT 5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCCGCACTGGATACGACACCACA-3 SEQ.ID.NO.20 miR-155 F 5-GCGCGTTAATGCTAATCGGTGAT-3 SEQ.ID.NO.21 miR-155 R 5-GTGCAGGGTCCGAGGTATTC-3 SEQ.ID.NO.22 miR-155 RT 5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCCGCACTGGATACGACACCCCT-3 SEQ.ID.NO.23 miR-21F 5-GCGCGTAGCTTATCAGACTGA-3 SEQ.ID.NO.24 miR-21 R 5-AGTGCAGGGTCCGAGGTATT-3 SEQ.ID.NO.25 miR-21 RT 5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCCGCACTGGATACGACTCAACA-3 SEQ.ID.NO.26 The foregoing has shown and described the main features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0035] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A DNA fiber barcode probe for multiplex miRNA detection, characterized in that, Includes DNA fibers and fluorescent signal probes, among which: DNA fibers are formed by the self-assembly of DNA tile monomers, wherein the 3' end of the DNA tile monomer extends a recognition sequence complementary to the target miRNA; The fluorescent signal probe has a hairpin structure with fluorescent and quenching groups labeled at the ends. After binding to the target miRNA, it triggers a conformational change. In the presence of the target miRNA, the hairpin structure is opened through a strand displacement reaction and a fluorescent signal is released. The signal intensity is proportional to the miRNA concentration. Different target miRNAs trigger hairpin signal probes with corresponding fluorescent colors. The combination of fluorescent colors forms a decodeable barcode, enabling multiplex miRNA detection.

2. The DNA fiber barcode probe for multiplex miRNA detection according to claim 1, characterized in that, The DNA tile monomers are formed by annealing five single-stranded DNA molecules and then self-assemble into DNA fibers through complementary base pairing at the sticky ends of the DNA tile monomers.

3. The DNA fiber barcode probe for multiplex miRNA detection according to claim 2, characterized in that, The self-assembly method of the DNA fibers is as follows: An equimolar mixture of 5 single-stranded DNA strands and TAE-Mg 2+ Add buffer and DEPC water to a nuclease-free microcentrifuge tube; The mixture was subjected to thermal annealing: initial denaturation at 94°C for 5 minutes, followed by gradual cooling at a gradient of -1 °C / min until 4°C was reached.

4. The DNA fiber barcode probe for multiplex miRNA detection according to claim 1, characterized in that, The fluorescent signal probe is selected from any one or more of FAM-BHQ1, TAMRA-BHQ2, and AMCA-Dabcyl, and the 3' end sequence of the fluorescent signal probe is complementary to the target miRNA sequence.

5. The DNA fiber barcode probe for multiplex miRNA detection according to claim 1, characterized in that, When the fluorescent signal probe does not bind to the target miRNA, it hybridizes with the complementary quenching group, and no fluorescence is detected; when it encounters a region complementary to the target miRNA, the fluorescent group separates from the quenching group, and fluorescence is detected.

6. The application of a DNA fiber barcode probe for multiplex miRNA detection according to any one of claims 1-5, characterized in that, Application in the preparation of tumor cell typing products.

7. A multiplex miRNA detection kit, characterized in that, A DNA fiber barcode probe containing the multiplex miRNA detection according to any one of claims 1-5.

8. The application of the multiplex miRNA detection kit according to claim 7, characterized in that, Application in tumor cell typing.

9. A method for detecting multiplex miRNAs, characterized in that, Includes the following steps: Total RNA was extracted from the cells to be tested. The RNA was co-incubated with the DNA fiber barcode probe according to any one of claims 1-5; Multichannel fluorescence images were acquired using a laser confocal scanning microscope; Decoding miRNA types based on fluorescent color combinations; The fluorescence intensity was used to directly calculate the target concentration using linear regression equations for each target miRNA.

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