Composite fluorescence coding method for biomarker and application of composite fluorescence coding method

Abstract

The invention provides a composite fluorescence coding method for a biomarker and application of the composite fluorescence coding method. A composite fluorescent coding method for a biomarker comprises the following steps: a composite structure formed by connecting tetrahedral DNA frames with the same side length in series is used as a coding carrier, one of the tetrahedral DNA frames is used for loading fluorescent coding dyes with different colors to realize the distinguishing of different biological targets, and the other tetrahedral DNA frame is used for loading fluorescent coding dyes with different colors; the other tetrahedral DNA framework is used for loading quantitative dye and a recognition probe, so that quantitative analysis of the biological target is realized. Single chains respectively extend out of the same sides of the tetrahedral DNA framework, and are connected in series and assembled according to a base complementary pairing principle to obtain the coding vector. According to the invention, a composite structure formed by series connection of TDF is used as the coding vector, so that the problems of biotoxicity, easy degradation by nuclease and the like of a traditional inorganic material and a two-dimensional DNA nano-material are solved, and long-term structural stability of the coding vector in living cells is realized.

Description

Technical Field

[0001] The present application relates to the field of optical coding technology, and in particular to a composite fluorescence coding method for biomarkers and its application. Background Art

[0002] At present, traditional biological multivariate analysis methods are usually limited by the types of labeling dyes and cannot meet the needs of imaging for the detection of multiple biomarkers, making it difficult to accurately identify breast cell types. Coding is an indispensable component of information communication systems. It refers to the process of converting information from one form to another. The great advantages of coding technology in storing and interpreting large amounts of information have attracted widespread attention from researchers in other fields such as chemistry, biology, and medicine. As an emerging method dedicated to biological multiplex labeling, biocoding technology is expected to play an important role in the study of breast cancer-related biomarkers. In recent years, due to the diversity of optical materials and the easy accessibility of optical decoding instruments, optical coding has become the most attractive coding method. Among them, fluorescence coding has become one of the most common biological coding methods due to its easy decoding characteristics and is widely used in biological multiplex analysis. However, most of the existing fluorescence coding strategies have defects such as poor biocompatibility, unstable loading, small coding capacity, and inability to quantify. Therefore, it is urgent to develop biological fluorescence coding strategies with good biocompatibility, precise and stable loading, high coding capacity, and accurate quantification for the precise identification of multiple biological targets.

[0003] Fluorescence coding strategies, coding vectors, and fluorescent tags have a significant impact on the coding capacity. Fluorescence properties such as color and intensity can be easily encoded by modulating the wavelength and number of fluorescent tags. However, most traditional biological coding strategies only encode one fluorescent property and cannot meet the growing demand for multiplex analysis. Fluorescence composite coding strategies have higher multiplex labeling capabilities by integrating multiple properties and have been widely used in in vitro multiplex detection and intracellular imaging. For example, Lu et al. proposed a 3D coding strategy with extremely high coding capabilities, which achieved multiplex detection of five tumor markers by changing the size, fluorescence color, and intensity of magnetic spheres.

[0004] Inorganic materials have been widely used as carriers in fluorescent biocoding technology. For example, Wei et al. designed a composite biocoding strategy using mesoporous silica nanoparticles as carriers loaded with different ratios of Cy3 and Cy5, which enabled accurate imaging of nine breast cancer-related miRNAs. However, disadvantages such as biological toxicity and leakage of fluorescent labels severely limit the assembly efficiency of fluorescent codes. DNA nanostructures are ideal encoding carriers due to their high programmability, excellent biocompatibility and ease of chemical modification. Currently, DNA nanostructures of different shapes have been used as carriers, such as linear, framework and origami. For example, Makino et al. reported a fluorescent color-changing barcoding method using double-stranded linear DNA nanostructures as carriers. However, linear DNA nanostructures are easily degraded by nucleases, which limits their imaging applications in living cells. As one of the most commonly used DNA nanostructures, tetrahedral DNA frameworks (TDF) have higher internalization efficiency and ability to bind to cell membranes and have been widely used as encoding carriers. For example, Zhao et al. developed a fluorescence intensity encoding strategy with TDF as a carrier to achieve multiplex detection of miRNAs in living cells. Traditional bio-encoding probes only detect a certain type of biomarker in a monovalent manner and cannot accurately identify the true "identity" of the target. Therefore, an ideal bio-encoding probe should have both multiplex and multivalent detection capabilities. Bioprobes usually use signal amplification methods to accurately quantify targets. Although this improves detection sensitivity, it cannot obtain accurate positioning information of targets in living cells. At present, tandem tetrahedral nanowires assembled from multiple TDFs and linker chains (LCs) are both flexible and rigid, showing strong cell binding affinity and specificity, and showing unparalleled advantages in multivalent imaging of biomolecules and multivalent cell recognition. For example, Gao et al. designed a bead-string-shaped DNA nanowire (BS-nanow) modified by multiple sgc8 aptamers. The probe has a high binding affinity for PTK7 protein on the cell membrane surface. Summary of the invention

[0005] The purpose of the present application is to provide a composite fluorescence encoding method for biomarkers and its application to solve the above-mentioned technical problems and achieve multiple identification and accurate quantification.

[0006] This application adopts the following technical solutions: A composite fluorescence encoding method for biomarkers, comprising the following steps: A composite structure formed by connecting two tetrahedral DNA frames with the same side length in series is used as a coding carrier, wherein one of the tetrahedral DNA frames is used to load fluorescent coding dyes of different colors to distinguish different biological targets, and the other tetrahedral DNA frame is used to load quantitative dyes and recognition probes to achieve quantitative analysis of the biological targets.

[0007] Preferably, single strands are extended from the same sides of the tetrahedral DNA framework and assembled in series through the principle of complementary base pairing to obtain the encoding vector.

[0008] Preferably, the side length of the tetrahedral DNA framework is 7 bp to 17 bp, and the distance between two tetrahedral DNA frameworks is 5 bp to 25 bp.

[0009] Preferably, the number of the tetrahedral DNA frameworks is ≥2.

[0010] Preferably, the fluorescent coding dye is fixed to the vertices of the tetrahedral DNA framework by chemical modification.

[0011] Preferably, each of the tetrahedral DNA frameworks contains 4 binding sites.

[0012] Preferably, the fluorescent coding dye is selected from Alexa Fluor 405, FITC and Cy5 and combinations thereof.

[0013] Preferably, the quantitative dye is Cy3.

[0014] Preferably, the recognition probe comprises a molecular beacon or a nucleic acid aptamer.

[0015] Application of any of the above-mentioned composite fluorescence encoding methods for biomarkers in the detection of nucleic acids, cell membrane surface proteins, and cell secretions.

[0016] In the prior art, firstly, most of the existing fluorescence coding strategies use single intensity coding. Although multiple identification can be achieved, the distinction between different coding signals is poor, which makes the accuracy of target multiple identification poor. Secondly, in most fluorescence multiplex analysis work, in order to increase the coding capacity, the fluorescence intensity of the coding dye is used as a standard for measuring the target expression. However, compared with the method of using a certain dye as a quantitative dye, the accuracy of this quantitative method is poor and it is easy to be confused with the coding signal.

[0017] The present application has the following advantages over the prior art: 1. This application uses the composite structure formed by TDF series connection as the coding vector, which solves the problems of biological toxicity and easy degradation by nucleases existing in traditional inorganic materials and two-dimensional DNA nanomaterials, and realizes the long-term structural stability of the coding vector in living cells.

[0018] 2. This application is based on tandem TDF and adjusts the type and quantity of fluorescent molecules loaded on the coding carrier to construct a new type of composite coding strategy of fluorescent color and intensity with higher coding capacity to achieve multiple recognition of biological targets, which can not only improve the coding ability, but also have better distinguishability between different coding signals. In addition, this application combines multiple quantitative dyes and recognition units (molecular beacons or nucleic acid aptamers) to achieve multivalent and accurate quantification of multiple biological targets.

[0019] 3. This application can detect multiple different targets at one time, can achieve multiple identification and accurate quantification, and can be widely used in research and practice in the fields of chemistry, biology, and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the encoding vector of Example 1.

[0021] Figure 2 This is a structural diagram of the Vwf nucleic acid aptamer (recognition probe) of Example 1.

[0022] Figure 3 The excitation and emission spectra of four fluorophores are shown in Figure 2.

[0023] Figure 4 These are the fluorescence images of B1G1R1-TDF2 (Cy3) in HUVEC cells under different fluorescence channels in Example 1.

[0024] Figure 5 This is the structural diagram of the integrin Αvβ3 nucleic acid aptamer (recognition probe) of Example 2.

[0025] Figure 6 These are the fluorescence images of B1G1-TDF2 (Cy3) in HUVEC cells under different fluorescence channels in Example 2.

[0026] Figure 7 This is a structural diagram of the VEGF nucleic acid aptamer (recognition probe) of Example 3.

[0027] Figure 8 These are the fluorescence images of B1R1-TDF2 (Cy3) in HUVEC cells under different fluorescence channels in Example 3.

[0028] Fig. 9 These are the fluorescence images of B1G1R1-TDF2 (Alexa Fluor 546) in HUVEC cells under different fluorescence channels in Example 4. DETAILED DESCRIPTION

[0029] The technical scheme of the present application will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of the present application embodiments, rather than all embodiments, and are only used to illustrate the present application, and should not be considered as limiting the scope of the present application. Those who do not specify specific conditions in the embodiments are carried out according to normal conditions or conditions recommended by the manufacturer. Those who do not specify the manufacturer for reagents or instruments used are all conventional products that can be purchased commercially.

[0030] A composite fluorescence encoding method for biomarkers, comprising the following steps: 1. Use two TDFs with the same side length in series to form a composite structure as the encoding vector. The same sides of the TDFs extend out single strands and assemble in series through the principle of base complementary pairing, and maintain a certain spacing to obtain the encoding vector. The side length of TDF is 7bp~17bp, which can be 7bp, 10bp, 17bp, etc. The spacing between the two TDFs is 5bp~25bp, which can be 5bp, 15bp, 25bp, etc. The setting of the side length and spacing in the encoding vector is very important for constructing an ideal encoding vector. The setting of the spacing has an important influence on the construction of subsequent encoding probes and the multiple recognition and precise quantification of targets.

[0031] 2. One TDF is used to load fluorescent coding dyes of different colors to distinguish different biological targets, and the other TDF is used to load quantitative dyes and identification probes to achieve quantitative analysis of biological targets. Fluorescent coding dyes are fixed to the vertices of TDF through chemical modification. Each TDF can contain 1 to 4 binding sites, and selecting a variety of fluorescent dyes can minimize spectral crosstalk between fluorophores.

[0032] Fluorescent coding dyes are dyes that can distinguish fluorescence emission spectra, and may be Alexa Flour dyes, Atto dyes, Di dyes, Cy series dyes, FITC, etc., among which Alexa Fluor 405, FITC and Cy5 and combinations thereof are preferred.

[0033] The quantitative dye may be a Cy series dye, Alexa Flour dye, etc., wherein Cy3 is preferred. The number of the quantitative dyes is 1 to 3, preferably 2 or 3.

[0034] Recognition probes include molecular beacons or nucleic acid aptamers, which are easy to chemically modify. Both molecular beacons and aptamers are composed of DNA or RNA molecules, with low synthesis costs, and both can achieve target detection in living cells. For example: Vascular endothelial growth factor (VEGF), integrin αvβ3, factor VIII antigen or von Willebrand factor (Vwf), E / P-selectin, etc.

[0035] The naming rule of the fluorescent code is a combination of the type (letter) and quantity (number) of loaded fluorescent coding dyes. Specifically, the fluorescent coding strategy includes the following situations: when TDF-1 is loaded with only one fluorescent coding dye, there are three situations: only loading Alexa Fluor 405, only loading FITC, and only loading Cy5. Since TDF-1 contains 4 binding sites, intensity coding can also be achieved by changing the number of fluorescent coding dyes. For example, TDF-1 is loaded with 1, 2 or 3 Alexa Fluor 405 (B1, B2, B3). When TDF-1 is loaded with two fluorescent coding dyes, there are three situations: loading Alexa Fluor 405 and FITC at the same time, loading FITC and Cy5 at the same time, and loading FITC and Cy5 at the same time. At the same time, by changing the number of loaded fluorescent coding dyes, multiple intensity codes can be assembled. For example, TDF-1 is loaded with 2 Alexa Fluor 405 and 1 FITC at the same time (B2G1), and TDF-1 is loaded with 1 FITC and 2 Cy5 at the same time (G1R2). When TDF-1 is loaded with three fluorescent coding dyes, B, G and R are loaded at the same time. Similarly, intensity coding can also be achieved by changing the number of fluorescent coding dyes. For example, TDF-1 is loaded with 2 Alexa Fluor405, 1 FITC and 1 Cy5 (B2G1R1) at the same time.

[0036] The method for constructing the above encoding vector comprises: 1. Use NanoDrop to quantify the four oligonucleotides of TDF-1 (or TDF-2); 2. Disperse the four oligonucleotides in a buffer according to a certain ratio and place them in a PCR instrument. Heat them at 95°C for 2 minutes, then quickly cool them to 60°C, and finally cool them to 4°C. The purpose of placing them in the PCR instrument is to fold the oligonucleotides into a specific tetrahedral structure through complementary base pairing of DNA molecules; 3. The synthesized TDF-1 and TDF-2 were mixed in the same proportion and placed in a PCR instrument under the same conditions as in step 2. Finally, a coding vector of TDF-1 and TDF-2 in series was obtained. The purpose of placing in the PCR instrument is to connect the two TDFs in series through complementary base pairing of DNA molecules.

[0037] Example 1 This embodiment provides a composite fluorescence encoding method for biomarkers, comprising the following steps: A composite structure formed by connecting two TDFs with the same side length in series is used as the encoding vector. The sequences of the four chains of TDF are shown in Table 1. The side length is 7 bp, and the distance between the two TDFs is 25 bp. Figure 1 As shown, the two TDFs are TDF-1 and TDF-2. Figure 1 On the left is a coding vector composed of two TDFs of the same size connected in series. Each TDF is composed of 4 oligonucleotide chains. Figure 1 The right side is a simplified form of the encoding vector, in order to better reflect the modification position and quantity of the dye in the encoding strategy and the construction of the subsequent probe.

[0038] Table 1

[0039] TDF-1 is used to load fluorescent coding dyes of different colors, 1 Alexa Fluor 405, 1 FITC and 1 Cy5 (B1G1R1), to distinguish different biological targets. TDF-2 is used to load quantitative dye Cy3 and recognition probe Vwf. The recognition probe structure is as follows Figure 2 As shown in Figure 2, quantitative analysis of biological targets is achieved. The excitation spectra (dashed lines) and emission spectra (filled lines) of the above dyes are shown in Figure 2. Figure 3 The dye information is shown in Table 2.

[0040] Table 2

[0041] HUVEC cells were tested using the encoding vector of Example 1.

[0042] HUVEC cells were cultured until they were completely attached, and the culture medium used to culture HUVEC cells was removed. The B1G1R1-TDF2 structure was dispersed in DMEM culture medium at a final concentration of 50 nM. The B1G1R1-TDF2 solution was added to the attached HUVEC cells, placed in a cell culture incubator and incubated for 4 hours, and the fluorescence images were taken. Figure 4As shown, Alexa Fluor 405, FITC, Cy3 and Cy5 were mixed to obtain a Merge image, and each cell in the field of view showed four pseudo colors of blue, green, orange and red, indicating that the B1G1R1-TDF2 (Cy3) encoded sample had entered the HUVEC cells, and different encoding signals were well distinguishable, achieving multivalent recognition, increasing the affinity for binding to the target, and improving the detection sensitivity.

[0043] Example 2 This embodiment provides a composite fluorescence encoding method for biomarkers, comprising the following steps: A composite structure formed by connecting two TDFs with the same side length (same as in Example 1) in series is used as the encoding vector, with a side length of 7 bp and a spacing of 25 bp between the two TDFs. TDF-1 is used to load fluorescent coding dyes of different colors, 1 Alexa Fluor405 and 1 FITC (B1G1), and TDF-2 is used to load the quantitative dye Cy3 and the recognition probe integrin Αvβ3. The recognition probe structure is as follows Figure 5 shown.

[0044] HUVEC cells were tested using the coding vector of Example 2. The experimental process was the same as that of Example 1, and the test results were as follows: Figure 6 As shown, Alexa Fluor 405, FITC and Cy3 were mixed to obtain a Merge image, and each cell in the field of view showed three pseudo colors of blue, green and orange, indicating that the B1G1-TDF2 (Cy3) encoded sample had entered the HUVEC cells, achieving multivalent recognition, increasing the affinity for binding to the target, and improving the detection sensitivity.

[0045] Example 3 This embodiment provides a composite fluorescence encoding method for biomarkers, comprising the following steps: A composite structure formed by connecting two TDFs with the same side length (same as in Example 1) in series is used as the encoding vector. The side length is 7 bp, and the distance between the two TDFs is 25 bp. TDF-1 is used to load fluorescent coding dyes of different colors, 1 Alexa Fluor405 and 1 Cy5 (B1R1), and TDF-2 is used to load the quantitative dye Cy3 and the recognition probe VEGF. The recognition probe structure is as follows Figure 7 shown.

[0046] The coding vector of Example 3 was used to detect HUVEC cells. The experimental process was the same as that of Example 1, and the detection results were as follows: Figure 8As shown, Alexa Fluor 405, Cy3 and Cy5 were mixed to obtain a Merge image, and each cell in the field of view showed three pseudo colors of blue, orange and red, indicating that the B1R1-TDF2 (Cy3) encoded sample had entered the HUVEC cells, achieving multivalent recognition, increasing the affinity for binding to the target, and improving the detection sensitivity.

[0047] Example 4 This embodiment provides a composite fluorescence encoding method for biomarkers, comprising the following steps: The composite structure formed by connecting two TDFs with the same side length (same as in Example 1) in series was used as the encoding vector. The side length was 7 bp, and the distance between the two TDFs was 25 bp. TDF-1 was used to load fluorescent coding dyes of different colors, 1 Alexa Fluor 405, 1 FITC and 1 Cy5 (B1G1R1), and TDF-2 was used to load the quantitative dye Alexa Fluor 546 and the recognition probe Vwf.

[0048] The coding vector of Example 3 was used to detect HUVEC cells. The experimental process was the same as that of Example 1, and the detection results were as follows: Fig. 9 As shown, Alexa 405, FITC, Cy5 and Alexa Fluor 546 were mixed to obtain a Merge image, and each cell in the field of view showed four pseudo colors of blue, green, orange and red, indicating that the B1G1R1-TDF2 (Alexa Fluor 546) encoded sample had entered the HUVEC cells, and different encoding signals were well distinguishable, achieving multivalent recognition, increasing the affinity for binding to the target, and improving the detection sensitivity.

[0049] Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A composite fluorescence encoding method for biomarkers, characterized in that: The following steps are involved: A composite structure formed by connecting tetrahedral DNA frames with the same side length in series is used as a coding carrier, wherein one of the tetrahedral DNA frames is used to load fluorescent coding dyes of different colors to distinguish different biological targets, and the other tetrahedral DNA frame is used to load quantitative dyes and recognition probes to achieve quantitative analysis of the biological targets.

2. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: Single strands are extended from the same sides of the tetrahedral DNA framework and assembled in series through the principle of complementary base pairing to obtain the encoding vector.

3. The composite fluorescence encoding method for biomarkers according to claim 2, characterized in that: The side length of the tetrahedral DNA framework is 7 bp to 17 bp, and the distance between two tetrahedral DNA frameworks is 5 bp to 25 bp.

4. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: The number of the tetrahedral DNA frameworks is ≥2.

5. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: The fluorescent coding dye is fixed to the vertices of the tetrahedral DNA framework by chemical modification.

6. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: Each of the tetrahedral DNA frameworks contains four binding sites.

7. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: The fluorescent coding dye is selected from Alexa Fluor 405, FITC and Cy5 and combinations thereof.

8. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: The quantitative dye is Cy3.

9. The composite fluorescence encoding method for biomarkers according to claim 1, characterized in that: The recognition probe includes a molecular beacon or a nucleic acid aptamer.

10. Use of the composite fluorescence encoding method for biomarkers according to any one of claims 1 to 9 in the detection of nucleic acids, cell membrane surface proteins, and cell secretions.

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