Covalent fluorescence coding assembly for multiple analysis of target RNA, DNA fluorescence bar code and application of covalent fluorescence coding assembly and DNA fluorescence bar code

Abstract

The invention discloses a covalent fluorescence coding assembly for multiple analysis of target RNA, a DNA fluorescence bar code and application, and belongs to the field of nucleic acid detection. The covalent fluorescence coding assembly comprises a report sequence, an enrichment sequence complementarily combined with the report sequence and a binding chain used for binding a target, the enrichment sequence and the binding chain are complementarily paired, the target is RNA, and the report sequence is connected with a fluorophore. A DNA fluorescent bar code is constructed based on the covalent fluorescent coding assembly in combination with magnetic beads, the DNA fluorescent bar code can detect multiple targets at the same time, the flux of the targets capable of being detected at the same time is greatly expanded, and based on the specificity and predictability of the DNA fluorescent bar code, the DNA fluorescent bar code can be used for detecting multiple targets at the same time. The method has important application value in the fields of target multiple detection, omics analysis and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a covalent fluorescent coding assembly, a DNA fluorescent barcode and applications thereof for multiplex analysis of target RNA. Background Art

[0002] DNA fluorescent probe is a technology that combines fluorescent probes with targets based on the principle of complementary base pairing. A designed DNA sequence can cross-link different colors of fluorescence to mark different targets, thus being used as a tool to indicate internal information of molecules. Since the fluorescent molecules carried by a single sequence have low brightness and weak signals, most of the fluorescent probes currently used are combined with different signal amplification strategies, such as polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid-dependent amplification (NASBA), rolling circle amplification (RCA), etc. On the one hand, these technologies require the participation of enzymes and have high requirements for the reaction environment. On the other hand, these technologies are generally relatively cumbersome and expensive. Limited by the influence of detection equipment, it is difficult to quickly and cheaply perform high-throughput single-molecule level detection, and it is not easy to promote them in laboratories. Summary of the invention

[0003] The purpose of the present invention is to provide a covalent fluorescent encoding assembly, a DNA fluorescent barcode and an application for multiple analysis of target RNA to solve the problems existing in the above-mentioned prior art. By constructing a new type of DNA fluorescent barcode combined with magnetic beads of different particle sizes, the flux of targets that can be detected simultaneously is greatly expanded, which is of great significance for the simultaneous realization of multiple target detection.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a covalent fluorescent coding assembly for multiple analysis of target RNA. The covalent fluorescent coding assembly comprises a reporter sequence, an enrichment sequence complementary to the reporter sequence, and a binding chain for binding to the target. The enrichment sequence is complementary to the binding chain, and both the binding chain and the reporter sequence are modified with a photocrosslinker for generating a covalent structure between the assembly to ensure the stability of the assembly in a complex environment and the reliability of the coding system. The target is RNA, and a fluorescent group is connected to the reporter sequence.

[0006] In the embodiments of the present invention, the target RNA is described by taking miRNA associated with breast cancer as an example, but the target RNA of the present invention is not limited thereto and can be any form of RNA.

[0007] Preferably, the nucleotide sequence of the reporter molecule is as shown in SEQ ID NO.50, the nucleotide sequence of the enrichment sequence is as shown in SEQ ID NO.49, and the nucleotide sequence of the binding chain is as shown in any one of SEQ ID NO.37-48. The sequences of the three chains of the present invention are not limited to the above-listed sequences. The nucleotide sequence of the reporter molecule preferably has a length interval of 15-23 nt and a GC content of 40-60%. The sequences shown are for reference only. The enrichment sequence is determined according to the reporter molecule sequence, and its unique design site is the part that binds to the binding chain. The part preferably has a length of 15-25 nt and a GC content of 40-60%. The tail of the binding chain sequence is determined according to the binding site of the enrichment chain, and the head is complementary to the target RNA, so that it can bind to the target RNA. The reporter molecule, enrichment sequence and binding chain that meet the requirements of the above-mentioned design principle are all within the scope of protection of the present invention, and can achieve the same technical effects as those recorded in the present invention.

[0008] Preferably, the fluorescent group includes but is not limited to FAM, Cy3, Cy5 and / or Texas Red.

[0009] The present invention also provides a method for constructing a DNA fluorescent barcode based on the covalent fluorescent coding assembly, comprising the following steps: mixing a biotin-modified capture chain with a streptavidin-modified magnetic bead to connect the capture chain to the streptavidin-modified magnetic bead; then, mixing with a probe chain, the covalent fluorescent coding assembly and a sample to be tested; when the sample to be tested contains a target RNA, a DNA fluorescent barcode with different color combinations and color ratios is formed.

[0010] The principle of forming the above-mentioned DNA fluorescent barcode is as follows: when the sample to be tested contains miRNA, according to the principle of base complementary pairing, the capture chain specifically binds to the probe chain, the probe chain specifically binds to one end of the miRNA, and the other end of the miRNA binds to the covalent fluorescent coding assembly, and fluorescent barcodes with different color combinations and color ratios are formed through the fluorescent groups on the reporter molecules of the covalent fluorescent coding assembly.

[0011] In an embodiment of the present invention, the final concentration of the biotin-modified capture chain is 250-300 nM, the final concentration of the streptavidin-modified magnetic beads is 0.05-0.1 mg / mL; and / or the final concentration of the probe chain is 10-20 nM, the final concentration of the covalent fluorescent coding assembly is 10-20 nM; and / or the concentration ratio of the binding chain and the reporter sequence in the covalent fluorescent coding assembly is 1:20; and / or the conditions of the mixed reaction are: 37°C for 1-2 hours.

[0012] Preferably, the nucleotide sequence of the capture strand is any one of the sequences shown in SEQ ID NOs. 13-24; and / or the nucleotide sequence of the probe strand is any one of the sequences shown in SEQ ID NOs. 25-36.

[0013] The invention also provides a DNA fluorescent barcode, which is constructed by using the method described.

[0014] Preferably, the method comprises the following steps: performing fluorescence imaging on the DNA fluorescent barcode using a fluorescence imaging method, analyzing various fluorescence intensities of different DNA fluorescent barcodes, screening fluorescence types, and distinguishing different colors; and then screening fluorescence ratios, distinguishing different color ratios, thereby completing fluorescence decoding.

[0015] The present invention also provides the use of the DNA fluorescent barcode in any of the following:

[0016] (1) Application in the preparation of products for screening RNA related to breast cancer;

[0017] (2) Application in the preparation of products for simultaneous detection of multiple RNAs;

[0018] The present invention discloses the following technical effects:

[0019] The present invention uses a stable DNA assembly as a fluorescent carrier, and constructs a DNA fluorescent barcode by connecting different ratios of fluorescence to the assembly through base complementary pairing. While amplifying the signal of the target, it can also increase the number of analyzed targets, thereby achieving the purpose of high-throughput analysis. After excluding fluorescent barcode groups with similar colors, a single assembly can analyze at least 33 targets, and by combining it with magnetic beads of different particle sizes, at least x*33 targets (x is the particle size type) can be distinguished, realizing a codable high-throughput fluorescent barcode, and the subsequent decoding process can also correspond to the target one by one, realizing accurate encoding and decoding, which will be of great significance in the fields of multi-target analysis, omics analysis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1Schematic diagram of the complex reaction formed by DNA assembly; based on the principle of base complementary pairing, the fluorescent short chain III will bind to the secondary enrichment chain II, and the secondary enrichment chain can bind to the primary target binding chain I, thereby achieving the ability to amplify the signal of a single target;

[0022] Figure 2 The structural formulas of four fluorescent groups emitting at different excitation wavelengths and the schematic diagram of their modification on the fluorescent short chain III;

[0023] Figure 3 Schematic diagram of fluorescent barcodes constructed with four fluorescent groups in different combinations and proportions; there are 33 fluorescent barcodes in total, with blank positions marked as 0, namely 1: FFFFF, 2: 33333, 3: 55555, 4: TTTTT, 5: F3333, 6: FF333, 7: FFF33, 8: FFFF3, 9: F5555, 10: FF555, 11: FFF55, 12: FFFF5, 13: FTTTT, 14: FFTTT, 15 :FFFTT, 16:FFFFT, 17:35555, 18:33555, 19:33355, 20:33335, 21:5TTTT, 22:55TTT, 23:555TT, 24:55 55T, 25:3TTTT, 26:33TTT, 27:333TT, 28:3333T, 29:F3355, 30:3355T, 31:F55TT, 32:F33TT, 33:F35T0;

[0024] Figure 4 This is a confocal characterization image of four-fluorescence four-combination DNA fluorescent barcodes on the magnetic bead interface;

[0025] Figure 5 The bar graph is obtained by analyzing the fluorescence intensity of the dual-color coding;

[0026] Figure 6 The bar graph is obtained by analyzing the fluorescence intensity of the three-color coding;

[0027] Figure 7 This is a schematic diagram of the structure of the target-binding DNA assembly captured by magnetic beads; only when the target miRNA is present can the assembly be bound to the magnetic bead interface to generate a stable fluorescence signal;

[0028] Figure 8 This is a confocal image of the integrated detection of 12 breast cancer-related miRNAs using fluorescence encoding technology.

[0029] Fig. 9 The integrated detection results of 12 breast cancer-related miRNAs using magnetic beads of different particle sizes and the same DNA-encoded fluorescence. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Encoding DNA assemblies and step-by-step decoding of assemblies (see Figure 1-Figure 2): First, generate a specific assembly structure, and combine multiple reporter sequences with a length of 15-20nt and cross-linked fluorescent molecules (called chain III) with a length of 60-80nt (called chain II), and finally combine chain II with the binding chain used to bind to the target (called chain I). For a probe chain that binds to a target, it can bind to multiple enrichment chains for enriching fluorescent chains. The fluorescent short chains on the enrichment chains can be modified with four different fluorescent groups, namely FAM, Cy3, Cy5, and Texas Red. The excitation wavelengths are 488nm, 570nm, 600nm, and 633nm, respectively, and appear green, yellow, red, and orange under a confocal microscope. When the fluorescent chains modified with different fluorescence are simultaneously combined with the enrichment chain through complementary pairing, the large complex formed will show fluorescence with different color combinations and fluorescent barcodes with different color ratios.

[0036] After successfully constructing 33 different fluorescent coded assemblies, they need to be decoded and analyzed. Specifically, the fluorescent barcodes are first screened for fluorescence types to distinguish different colors; then the fluorescence ratios are screened to distinguish different color ratios to achieve accurate decoding analysis. In order to prevent the pre-generated DNA assembly structure from unwinding and reorganizing, a photocrosslinker is used to modify chain III, so that it can generate a stable covalent structure under light conditions, ensuring the stability of the assembly during the reaction.

[0037] Application of this assembly encoding technology in multiple miRNA detection technology:

[0038] Using the specific binding of biotin and streptavidin, the biotin-modified C chain (abbreviation of Capture, i.e., capture chain) is connected to the surface of the streptavidin-modified magnetic beads; then, based on the principle of base complementary pairing, this capture chain can specifically capture the p chain (abbreviation of probe), and the probe chain can capture the target (i.e., miRNA). On the other hand, the designed assembly structure can be captured through the other end of the target, which means that each target can correspond to a specific assembly (see Figure 7 ), after the reaction is completed, the fluorescence on the magnetic beads is characterized by fluorescence imaging, and then the fluorescence types are analyzed, so that high-throughput analysis of multiple targets can be performed simultaneously. The specific steps of this method are:

[0039] A. Design 12 sets of sequences specific to 12 breast cancer-related miRNAs, and simultaneously design 12 sets of sequences for binding the DNA assembly to the target miRNAs. The specific sequences are shown in Table 1;

[0040] B. reacting the biotin-modified capture chains, i.e., different groups of C chains, with magnetic beads modified with streptavidin in TE buffer. Due to the specific binding of biotin and streptavidin, the magnetic beads can capture the C chains, thereby obtaining magnetic beads that capture different groups of C chains;

[0041] C. The chains I, II and III required for the assembly are simultaneously placed in TE buffer for reaction to obtain a stable assembly structure (called complex IV), which is then irradiated with ultraviolet light for 1-2 minutes; the concentration of chain I is 200 nM, the concentration of chain II is 800 nM, and the concentration of chain III is 4 μM, that is, the concentration ratio is chain I:chain II:chain III=1:4:20.

[0042] D. reacting the magnetic beads obtained in step B, the probe strand P, 12 target miRNAs, and 12 different complexes IV in TE buffer;

[0043] E. The magnetic beads obtained in step D were photographed by fluorescence imaging and the fluorescence signals of each group of assembly products were analyzed using image J image analysis software.

[0044] Table 1 Fluorescent barcode element sequences and group modifications for screening breast cancer-related microRNAs

[0045]

[0046]

[0047]

[0048] The specific operation steps of the above step B are: adding streptavidin-modified magnetic beads and 12 biotin-modified capture chains C-Biotin to TE buffer respectively, so that the final concentrations of streptavidin-modified magnetic beads and C-Biotin are: 0.05-0.1 mg / mL, 250-300 nM respectively; after oscillation mixing, placing in an oscillating constant temperature metal bath, reacting at 37°C for 1-2 hours; after the reaction is completed, washing the magnetic beads, and obtaining 12 types of streptavidin-modified magnetic beads bound with different C-Biotin.

[0049] The specific operation steps of the above-mentioned step D are: adding 12 complexes IV, 12 simulated target miRNAs, 12 magnetic beads bound to the C chain and 12 probe chains P chain to TE buffer at the same time for reaction, so that the final concentration of the simulated target miRNA is 1nM~10nM, the concentration of complex IV is 10nM~20nM, and the final concentration of the probe chain P is 10nM~20nM; placing in an oscillating constant temperature metal bath, reacting at 37°C for 1~2h; washing the magnetic beads after the reaction is completed.

[0050] The images obtained by fluorescence imaging were then analyzed, and based on this, high-throughput simultaneous analysis of twelve different simulated targets was completed. Compared with multiple independent analyses, the detection of multiple targets in the same space and time helps to achieve real-time and high-precision marker analysis.

[0051] In order to more specifically illustrate how to construct the DNA fluorescent barcode, decode it, and use it in breast cancer miRNA detection, the following examples are used to further illustrate the details.

[0052] Example 13 Confocal characterization images of 3 kinds of DNA fluorescent barcodes on magnetic beads

[0053] The biotin-modified C chain (Capture-biotin) reacts with the streptavidin-modified magnetic beads in TE buffer. Due to the specific binding of biotin and streptavidin, the magnetic beads can capture the C chain. The specific operation is as follows: prepare 33 1.5mL centrifuge tubes, marked as 1-33. (The final concentration of the C chain in this process is 200nM). Add 100μL of streptavidin-modified magnetic beads with an initial concentration of 10mg / mL to all centrifuge tubes, discard the supernatant, wash with TE 3 times, and add 500μL of 33 different C chain solutions with a concentration of 300μM. After oscillation and mixing, place in an oscillating constant temperature metal bath and react at 37℃ for 1.5h. After the reaction, wash the magnetic beads 2-3 times. After washing, add 1mL TE buffer, mix well, and take 10μL of magnetic bead suspension from each of the 33 tubes and add it to the EP tube. At this time, the concentration of the magnetic beads is 1mg / mL. At this time, 10 μL of the pre-prepared assembly complex IV was added to each of the 1-33 groups. At this time, chain I in the assembly complex IV is used as the main structure of the assembly, and its concentration ratio with the reporter sequence chain III is 1:20, and its concentration is 200 nM. The 20 reporter sequences are divided into 5 equal groups, and each group is named with a labeled fluorescent group. 1: All five groups are FAM, recorded as FFFFF, 2: All are Cy3, recorded as 33333, 3: All are Cy5, recorded as 55555, 4: All are Texas Red, recorded as TTTTT, 5:F3333, 6:FF333, 7:FFF33, 8:FFFF3, 9:F5555, 10:FF555, 11:FFF55, 12:FFFF5, 13:FTTTT, 14:FFTTT, 15:FFFTT, 16:FFFFT, 17:35555, 18:33555, 1 9:33355, 20:33335, 21:5TTTT, 22:55TTT, 23:555TT, 24:5555T, 25:3TTTT, 26:33 TTT, 27:333TT, 28:3333T, 29:F3355, 30:3355T, 31:F55TT, 32:F33TT, 33:F35T0, see Figure 3; Then use 1× concentration of TE to make up each centrifuge tube system to 100μL, at this time the concentration of backbone chain I is 20nM. Place in an oscillating constant temperature metal bath and react at 37°C for 0.5h. After the reaction, wash the magnetic beads 2-3 times, then resuspend the magnetic beads in 50μTE buffer, aspirate 10μL of the solution and drop it on the glass slide, cover with a coverslip and place it at room temperature away from light for half an hour to dry the moisture to prevent the coverslip from moving, and place it under a 20x confocal microscope to capture images. The four different fluorescence excitation channels are FAM-488, Cy5-633, Cy3-570, and Texas Red-600, and the same fluorescence shooting settings are the same for all images. The specific results are as follows Figure 4-Figure 6 shown.

[0054] Depend on Figure 4 From the analysis, it can be seen that the HCR product without modified FAM fluorescence cannot be observed on the magnetic bead interface, and the same is true for other fluorescence. This shows that the 33 DNA fluorescent barcodes can be screened by fluorescence type first. Then, the DNA fluorescent barcodes with only FAM and Cy5 were analyzed separately, as shown in Figure 2. Figure 5 As shown in the figure, the FAM fluorescence becomes weaker as the hairpin chain modified with the FAM fluorescent group decreases, and the Cy5 fluorescence becomes stronger as the hairpin chain modified with the Cy5 fluorescent group increases. The ratio of the strongest fluorescence to the weakest fluorescence is approximately 8:3:3:8, which is almost consistent with the ratio of the fluorescent signal chain added when preparing the assembly. Finally, the DNA fluorescent barcodes with only Texas Red, FAM, and Cy3 fluorescence were analyzed. Figure 6 As shown, it is found that the result obtained by dividing the strongest fluorescence by the weakest fluorescence is approximately 2:1:2, which is consistent with the result obtained by analyzing the barcode of dual fluorescence encoding. Therefore, the DNA fluorescent barcode in the invention can be successfully encoded, and decoding requires two steps, the order of which is first to determine the type of fluorescence, then analyze its fluorescence ratio, and finally successfully decode.

[0055] Example 2 Integrated detection of 12 breast cancer-related miRNAs using DNA fluorescent barcodes

[0056] The biotin-modified C chain (Capture-biotin) reacts with the streptavidin-modified magnetic beads in TE buffer. Since biotin and streptavidin bind specifically, the magnetic beads can capture the C chain. The specific operation is as follows: prepare 12 1.5mL centrifuge tubes, marked as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. (In this process, the final concentration of the C chain and the P chain is 200nM). Add 10μL of the streptavidin-modified magnetic beads with an initial concentration of 1mg / mL to all centrifuge tubes. Then, 3 μL of each of G1-C-biotin, G2-C-biotin, G3-C-biotin, G4-C-biotin, G5-C-biotin, G6-C-biotin, G7-C-biotin, G8-C-biotin, G9-C-biotin, G10-C-biotin, G11-C-biotin, and G12-C-biotin at a concentration of 10 μM was added to each tube at a time, and then each centrifuge tube system was supplemented to 100 μL with 1× concentration TE buffer, and after oscillation and mixing, placed in an oscillating constant temperature metal bath, and reacted at 37°C for 1.5 hours. After the reaction, the magnetic beads were washed 2-3 times. After washing, they were redissolved in 100 μL of 1× concentration TE buffer, and then 12 centrifuge tubes were mixed in one centrifuge tube, which was numbered 13. Add 0.5 μL of target miRNA and P chain corresponding to G1-G12, respectively, with an initial concentration of 10 μM, and then add 10 μL of all assembly structures corresponding to G1-G12, with an initial concentration of 200 nM, and then use 1× concentration of TE to make up each centrifuge tube system to 200 μL, place it in an oscillating constant temperature metal bath, and react at 37°C for 1.5 hours. After the reaction, wash the magnetic beads 2-3 times, resuspend the magnetic beads in 50 μL TE buffer, draw 10 μL of solution and drop it on the slide, cover it with a coverslip, place it at room temperature away from light for half an hour to dry the moisture, prevent the coverslip from moving, and place it under a 20x microscope to take images with confocal microscope.

[0057] The results are as follows Figure 8As shown in the figure, there are 12 different magnetic beads, and separate channel analysis is performed on them. G1 has only Cy5 fluorescence, G2 has Cy3 and FAM fluorescence, G3 has only Cy3 fluorescence, G4 has Cy5 and FAM fluorescence, G5 has Cy5 and CY3 fluorescence, G6 has TexasRed and Cy5 fluorescence, G7 has Cy5, Texas Red and CY3 fluorescence, G8 has Cy3, Cy5, FAM and Texas Red fluorescence, G9 has FAM, Cy5 and Texas Red fluorescence, G10 has FAM and Texas Red fluorescence, G11 has FAM fluorescence, and G12 has Texas Red fluorescence. It shows that when the number of targets is small, several fluorescent barcodes in the invention can be selected to analyze the targets, and they can be imaged once and for all.

[0058] Example 3 Integrated detection of 12 breast cancer-related miRNAs using magnetic beads of different particle sizes under the same DNA-encoded fluorescence

[0059] The process of magnetic bead capture of C chain is the same as that in Example 2, except that three different sizes of magnetic beads and four pure color assembly structures of C chain are prepared. The specific operation is as follows: prepare 12 1.5mL centrifuge tubes, marked as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. (In this process, the final concentration of C chain and P chain is 200nM). Magnetic beads with a diameter of 5μm are added to tubes 1, 2, 3, and 4, magnetic beads with a diameter of 2.8μm are added to tubes 5, 6, 7, and 8, and magnetic beads with a diameter of 1μm are added to tubes 9, 10, 11, and 12, and 10μL of each is added. Then, 3 μL of each of G1-C-biotin, G2-C-biotin, G3-C-biotin, G4-C-biotin, G5-C-biotin, G6-C-biotin, G7-C-biotin, G8-C-biotin, G9-C-biotin, G10-C-biotin, G11-C-biotin, and G12-C-biotin at a concentration of 10 μM was added to each tube at a time, and then each centrifuge tube system was supplemented to 100 μL with 1× concentration TE buffer, and after oscillation and mixing, placed in an oscillating constant temperature metal bath, and reacted at 37°C for 1.5 hours. After the reaction, the magnetic beads were washed 2-3 times. After washing, they were redissolved in 100 μL of 1× concentration TE buffer, and then 12 centrifuge tubes were mixed in one centrifuge tube, which was numbered 13. Add 0.5 μL of target miRNA and P chain corresponding to G1-G12, respectively, with an initial concentration of 10 μM, and then add 10 μL of all assembly structures corresponding to G1-G12, with an initial concentration of 200 nM, and then use 1× concentration of TE to make up each centrifuge tube system to 200 μL, place it in an oscillating constant temperature metal bath, and react at 37°C for 1.5 hours. After the reaction, wash the magnetic beads 2-3 times, resuspend the magnetic beads in 50 μL TE buffer, draw 10 μL of solution and drop it on the slide, cover it with a coverslip, place it at room temperature away from light for half an hour to dry the moisture, prevent the coverslip from moving, and place it under a 20x microscope to take images with confocal microscope.

[0060] The results are as follows Fig. 9 As shown in the figure, there are 12 different pure color magnetic beads, which are analyzed by separate fluorescence channels. Among them, G1, G5, and G9 only contain FAM fluorescence, and the difference lies in the directly observed particle size difference. G2, G6, and G10 only contain Cy3 fluorescence, G3, G7, and G11 only contain Texas Red fluorescence, and G4, G8, and G12 only contain Cy5 fluorescence. It shows that when the number of targets is small, several fluorescent barcodes in the invention can be selected to analyze the targets in combination with magnetic beads of different particle sizes, and they can be easily imaged once.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A covalently fluorescently encoded assembly for multiplex analysis of target RNA, characterized in that: The covalent fluorescent coding assembly includes a reporter sequence, an enrichment sequence complementary to the reporter sequence, and a binding chain for binding to a target. The enrichment sequence is complementary to the binding chain, and both the binding chain and the reporter sequence are modified with a photocrosslinker to generate a covalent structure between the assembly to ensure the stability of the assembly in a complex environment and the reliability of the coding system. The target is RNA, and a fluorescent group is connected to the reporter sequence.

2. The covalent fluorescently encoded assembly according to claim 1, characterized in that: The nucleotide sequence of the reporter molecule is shown as SEQ ID NO.50, the nucleotide sequence of the enrichment sequence is shown as SEQ ID NO.49, and the nucleotide sequence of the binding chain is any one of the sequences shown as SEQ ID NO.37-48.

3. The covalent fluorescently encoded assembly according to claim 1, characterized in that: The fluorescent groups include FAM, Cy3, Cy5 and / or Texas Red.

4. A method for constructing a DNA fluorescent barcode based on the covalent fluorescent coding assembly according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing and reacting the capture chain modified with biotin and the magnetic beads modified with streptavidin so that the capture chain is connected to the magnetic beads modified with streptavidin; then, mixing and reacting with the probe chain, the covalent fluorescent coding assembly and the sample to be tested; when the sample to be tested contains target RNA, forming DNA fluorescent barcodes with different color combinations and color ratios.

5. The method according to claim 4, characterized in that The nucleotide sequence of the capture strand is any one of the sequences shown in SEQ ID NOs. 13-24; and / or the nucleotide sequence of the probe strand is any one of the sequences shown in SEQ ID NOs. 25-36.

6. A DNA fluorescent barcode, characterized in that: The method is constructed by any one of claims 4 to 5.

7. The method for decoding a DNA fluorescent barcode according to claim 6, wherein: The method comprises the following steps: using a fluorescent imaging system to image the DNA fluorescent barcode, analyzing the various fluorescence intensities of different DNA fluorescent barcodes, screening the types of fluorescence, and distinguishing different colors; Then the fluorescence ratio is screened and different color ratios are distinguished to complete the fluorescence decoding.

8. Use of the DNA fluorescent barcode according to claim 6 in any of the following: (1) Application in the preparation of products for screening RNA related to breast cancer; (2) Application in the preparation of products for simultaneous detection of multiple RNAs.

Images