Self-assembled Y-type DNA (deoxyribonucleic acid) fluorescent nano device and application of self-assembled Y-type DNA fluorescent nano device in tumor cells

Abstract

The invention belongs to the technical field of tumor identification products, and particularly relates to a self-assembled Y-type DNA fluorescent nano device and application thereof in tumor cells. The DNA fluorescent nano robot comprises a DNA A, a DNA B and a DNA C, wherein the sequence of the DNA A is as shown in SEQ ID NO: 1, the sequence of the DNA B is as shown in SEQ ID NO: 2, and the sequence of the DNA C is as shown in SEQ ID NO: 3. The fluorescent DNA nano-robot is respectively co-incubated with normal cells and tumor cells, and the fluorescence intensity of the nano-robot when the nano-robot is incubated with the normal cells is obviously higher than that of the nano-robot when the nano-robot is co-incubated with the tumor cells, so that tumor cell microenvironment response is realized, and tumor detection is further realized.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor identification products, and specifically relates to a self-assembled Y-shaped DNA fluorescent nano device and its application in tumor cells. Background Art

[0002] In the 1950s, JD Watson and FHC Crick published the double helix structure model of DNA in Nature, suggesting that DNA is the genetic template for life. In 1983, Seeman first used DNA to construct a nucleic acid nanostructure, indicating that DNA not only carries important genetic information of life, but can also be used as a component for building nanomaterials, thus giving rise to a new science - DNA nanotechnology (Seeman, 2010).

[0003] DNA nanorobots are one of the fastest-growing directions. They are nanodevices that use the accurate complementary base pairing function of DNA to controllably change the conformation of DNA by changing the order of bases under the drive of a specific form of energy, and perform some kind of mechanical movement to achieve energy transfer (Fan Chunhai and Liu Dongsheng, 2011). As we all know, DNA contains four types of bases: A, T, C, and G, which give DNA nanorobots structural diversity; at the same time, due to the variable order of DNA bases and flexible design, their design is programmable; coupled with the unique complementary base pairing principle of DNA, it has a high degree of motion controllability. DNA nanorobots can capture, store, and release target molecules like real switches, realizing the function of a "machine". At present, DNA nanorobots with various structures have been constructed one after another and play an important role in many fields, such as drug delivery (Bhatia et al., 2011; Douglas et al., 2012; Lee et al., 2012; Amir et al., 2014; Chen et al., 2017; Li et al., 2018), bioimaging (Bhatia et al., 2011; Modi et al., 2013; Jungmann et al., 2014; You et al., 2017) and biosensing (Torelli et al., 2014, 2018; He et al., 2018), and have very broad application prospects.

[0004] However, the DNA nanorobots reported in the literature currently have complex raw materials and cumbersome designs, which limit their efficiency; the fluorescent signals required to realize the characterization, tracing and imaging functions of DNA nanorobots require the DNA skeleton to be labeled with fluorescent groups, which is expensive and limits their wide application; the functions of DNA nanorobots are relatively single, which limits their wide application. Summary of the invention

[0005] The present invention provides a self-assembled Y-shaped DNA fluorescent nanodevice, which does not need to be labeled with fluorescent groups and is cheap.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a self-assembled Y-shaped DNA fluorescent nanodevice, comprising: DNAA, DNA B and DNA C; wherein the sequence of DNAA is shown in SEQ ID NO: 1, the sequence of DNA B is shown in SEQ ID NO: 2, and the sequence of DNA C is shown in SEQ ID NO: 3.

[0008] Preferably, the self-assembled Y-shaped DNA fluorescent nanodevice further comprises ascorbic acid and Cu 2+ .

[0009] Another aspect of the present invention provides a tumor detection product, which includes the self-assembled Y-shaped DNA fluorescent nanodevice of the present invention.

[0010] Preferably, the above-mentioned product includes a reagent, a kit or a microfluidic chip.

[0011] In another aspect, the present invention provides an application of a self-assembled Y-shaped DNA fluorescent nanodevice in the preparation of a product for tumor diagnosis.

[0012] Preferably, the above tumor is ovarian cancer.

[0013] In another aspect, the present invention provides a method for identifying tumor cells for non-diagnostic purposes, comprising: mixing the self-assembled Y-shaped DNA fluorescent nanodevice of the present invention with cells to be identified, and incubating them; and then identifying the tumor cells based on the fluorescence conditions.

[0014] Preferably, the above tumor cells are ovarian cancer cells.

[0015] The beneficial effects of the present invention include at least: the self-assembled Y-shaped DNA fluorescent nanodevice provided by the present invention is co-incubated with normal cells and tumor cells respectively, and the fluorescence intensity of the DNA fluorescent nanodevice when incubated with normal cells is significantly higher than the fluorescence intensity when co-incubated with tumor cells, thereby achieving tumor cell microenvironment response and further realizing tumor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of the construction principle of the fluorescent DNA nanorobot in an embodiment of the present invention;

[0017] Figure 2 The temperature response of the fluorescent DNA nanorobot in the embodiment of the present invention;

[0018] Figure 3 The response of the fluorescent DNA nanorobot to pH value in the embodiment of the present invention;

[0019] Figure 4 This is the response of the fluorescent DNA nanorobot in an embodiment of the present invention to the tumor cell microenvironment. DETAILED DESCRIPTION

[0020] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0021] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0022] (I) Self-assembled Y-shaped DNA fluorescent nanodevice

[0023] An embodiment of the present invention provides a self-assembled Y-shaped DNA fluorescent nanodevice, comprising: DNA A, DNA B and DNA C; wherein the sequence of DNAA is shown in SEQ ID NO: 1, the sequence of DNAB is shown in SEQ ID NO: 2, and the sequence of DNA C is shown in SEQ ID NO: 3.

[0024] In some specific examples, the fluorescent DNA nanorobot further comprises ascorbic acid and Cu 2+ .

[0025] Specifically, the three linear structure DNAs (DNAA, DNAB and DNAC) of different sequences in the present invention can combine with each other through chain displacement reaction to form a stable "Y-shaped" structure; metal ions, under the action of reducing agents, use this structure as a template to form metal nanoclusters, which then exhibit fluorescence.

[0026] (II) Tumor Detection Products

[0027] An embodiment of the present invention further provides a tumor detection product, which includes the self-assembled Y-shaped DNA fluorescent nanodevice of the present invention.

[0028] Specifically, the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention can generate fluorescent signals different from those of normal cells in the tumor microenvironment, so it can be prepared into a tumor detection product to realize the identification of tumors.

[0029] In some specific examples, the above-mentioned products include reagents, kits or microfluidic chips.

[0030] (III) Application of self-assembled Y-shaped DNA fluorescent nanodevices

[0031] The embodiment of the present invention also provides an application of the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention in preparing a product for tumor diagnosis.

[0032] Specifically, as described above, the fluorescent DNA nanorobot in the present invention is prepared into a tumor detection product for the identification of tumors.

[0033] In some specific examples, the tumor is ovarian cancer.

[0034] Specifically, the tumor can be any tumor in the present invention, such as ovarian cancer and the like.

[0035] (IV) Methods for identifying tumor cells

[0036] The embodiment of the present invention also provides a method for identifying tumor cells for non-diagnostic purposes, characterized in that it includes: mixing the self-assembled Y-shaped DNA fluorescent nanodevice of the present invention with cells to be identified, and incubating them; and then identifying the tumor cells based on the fluorescence conditions.

[0037] In some specific examples, the tumor is ovarian cancer cell.

[0038] Specifically, the fluorescent DNA nanorobot in the present invention presents different fluorescence intensities in different temperature environments. The higher the temperature, the weaker the fluorescence of the self-assembled Y-shaped DNA fluorescent nanodevice. In addition, the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention presents different fluorescence intensities in different pH environments. The higher the pH, the stronger the fluorescence of the Y-shaped DNA fluorescent nanorobot. Compared with normal cells, the pH in the tumor cell microenvironment is lower and the temperature is higher. Therefore, in the present invention, the self-assembled Y-shaped DNA fluorescent nanodevice is co-incubated with normal cells and tumor cells respectively. The fluorescence intensity of the self-assembled Y-shaped DNA fluorescent nanodevice when incubated with normal cells is significantly higher than that when co-incubated with tumor cells, thereby achieving tumor cell microenvironment response.

[0039] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0040] 1. Design and preparation of fluorescent DNA nanorobots

[0041] Example 1

[0042] In the following examples, the oligonucleotide sequences used are shown in Table 1 below.

[0043] Table 1 Oligonucleotide sequences used

[0044]

[0045] Through rational design and simulation with DNAMAN software, the construction principle of an enzyme-free, label-free, and modification-free DNA nanorobot is shown in Figure 1 ; In this system, linear A, linear B, and linear C contain partial sequences of the CuNPs template, respectively. The sequence information is shown in Table 1 above. Under room temperature conditions, the three DNA strands self-assemble to form a stable "Y-shaped" DNA structure ABC. At this time, the formed "Y-shaped" DNA contains a complete CuNPs template. Finally, through the reduction of sodium ascorbate, Cu 2+ Reduced to Cu 0 The latter uses the formed "Y-shaped" DNA as a template to generate CuNPs, producing a significantly enhanced fluorescence signal, thereby realizing the construction of a self-assembled Y-shaped DNA fluorescent nanorobot.

[0046] 2. Fluorescent DNA Nanorobot Test

[0047] (I) Temperature response test

[0048] The successfully constructed DNA fluorescent nanorobots were incubated at 37°C and 42°C, respectively, and the fluorescence intensity of the nanorobots was observed using a fluorescence spectrophotometer. Figure 2As shown, the Y-shaped DNA fluorescent nanorobot exhibits different fluorescence intensities in different temperature environments. The higher the temperature, the weaker the fluorescence of the Y-shaped DNA fluorescent nanorobot.

[0049] (II) pH response test

[0050] The successfully constructed DNA fluorescent nanorobots were incubated at pH 6.2 and pH 7.4, and the fluorescence intensity of the nanorobots was observed using a fluorescence spectrophotometer. Figure 3 As shown, the Y-shaped DNA fluorescent nanorobot exhibits different fluorescence intensities in different pH environments. The higher the pH, the stronger the fluorescence of the Y-shaped DNA fluorescent nanorobot.

[0051] (III) Tumor cell microenvironment response test

[0052] The successfully constructed DNA fluorescent nanorobot was co-incubated with human ovarian cancer cell A2780 at room temperature, and the fluorescence imaging ability of the nanorobot was observed using a confocal fluorescence microscope. Figure 4 As shown, the fluorescence intensity of the nanorobots when incubated with normal cells was significantly higher than that when co-incubated with tumor cells, thereby achieving a tumor cell microenvironment response.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A self-assembled Y-shaped DNA fluorescent nanodevice, characterized in that: include: DNA A, DNA B and DNA C; wherein the sequence of DNAA is shown as SEQ ID NO: 1, the sequence of DNA B is shown as SEQ ID NO: 2, and the sequence of DNAC is shown as SEQ ID NO:

3.

2. The self-assembled Y-shaped DNA fluorescent nanodevice according to claim 1, characterized in that: Also includes ascorbic acid and Cu 2+ .

3. Tumor detection product, characterized in that: It comprises the self-assembled Y-shaped DNA fluorescent nanodevice as described in claim 1 or 2.

4. The tumor detection product according to claim 3, characterized in that: Products include reagents, kits or microfluidic chips.

5. Use of the self-assembled Y-shaped DNA fluorescent nanodevice according to claim 1 or 2 in the preparation of products for tumor diagnosis.

6. The use according to claim 5, characterized in that: Tumors include ovarian cancer.

7. A method for identifying tumor cells for non-diagnostic purposes, characterized in that: include: The self-assembled Y-shaped DNA fluorescent nanodevice according to claim 1 or 2 is mixed with cells to be identified, and incubated; then the tumor cells are identified according to the fluorescence conditions.

8. The method according to claim 7, characterized in that Tumor cells include ovarian cancer cells.

Images