A self-assembled Y-shaped DNA fluorescent nanodevice and its application in tumor cells

Abstract

The present invention belongs to the technical field of tumor identification products, and particularly relates to a self-assembled Y-shaped DNA fluorescent nanodevice and its application in tumor cells; the DNA fluorescent nanorobot includes: DNA A, DNA B, and DNA C; wherein, the sequence of DNA A 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. The fluorescent DNA nanorobot is co-incubated with normal cells and tumor cells respectively. When the nanorobot is incubated with normal cells, the fluorescence intensity is significantly higher than that when it is co-incubated with tumor cells, thereby realizing the response to the tumor cell microenvironment and further realizing the detection of tumors.

Description

Technical Field

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

[0002] In the 1950s, J.D. Watson and F.H.C. Crick published the double helix structure model of DNA in Nature, suggesting that DNA is the genetic template for inheriting life. In 1983, Seeman first constructed a nucleic acid nanostructure using DNA, indicating that DNA not only carries important genetic information of life but also can be used as an element for constructing nanomaterials, thus giving rise to a new science - DNA nanotechnology (Seeman, 2010).

[0003] DNA nanorobots are one of the most rapidly developing directions. It is a nanodevice that uses the accurate base complementary pairing function of DNA, driven by a specific form of energy, to controllably change the conformation of DNA by changing the base sequence and perform a certain mechanical movement to achieve energy transfer (Fan Chunhai and Liu Dongsheng, 2011). As is well known, DNA contains four types of bases, A, T, C, and G, which endow DNA nanorobots with structural diversity; at the same time, due to the variable base sequence and flexible design of DNA, it has programmability in design; in addition, the unique base complementary pairing principle of DNA gives it high motion controllability. DNA nanorobots can capture, store, and release target molecules like a real switch, realizing the function of a "machine". Currently, DNA nanorobots with various structures have been successively constructed and play important roles 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), etc., and have very broad application prospects.

[0004] However, the raw materials of DNA nanorobots reported in the current literature are complex and the design is cumbersome, which limits their usage efficiency; the fluorescence signals required for the realization of functions such as characterization, tracing, and imaging of DNA nanorobots need to label the DNA backbone with fluorescent groups, and the labeling with fluorescent groups is expensive, which 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 inexpensive.

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

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

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

[0009] On the other hand, the present invention provides a tumor detection product, which includes the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention.

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

[0011] On the other hand, the present invention provides an application of the 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] On the other hand, the present invention provides a method for identifying tumor cells for non-diagnostic purposes, including: mixing the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention with the cells to be identified and incubating; then identifying the tumor cells according to the fluorescence situation.

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

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

[0016] Figure 1 It is the schematic diagram for constructing the fluorescent DNA nanorobot in the embodiment of the present invention;

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

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

[0019] Figure 4 It is the response of the fluorescent DNA nanorobot to the tumor cell microenvironment in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

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

[0021] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having a significantly different meaning in the context, the singular form of the expression includes the plural form of the expression. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence 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 of the existence or addition of one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof. As used herein, depending on the situation, " / " can be interpreted as "and" or "or".

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

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

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

[0025] Specifically, the three linear-structured DNAs (DNA A, DNA B, and DNA C) with different sequences in the present invention enable them to bind to each other through strand displacement reactions to form a stable "Y-shaped" structure; under the action of reducing agents, metal ions use this structure as a template to form metal nanoclusters, which then exhibit fluorescence.

[0026] (II) Tumor detection products

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

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

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

[0030] (III) Applications of the self-assembled Y-shaped DNA fluorescent nanodevice

[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 the preparation of products 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 above tumor is ovarian cancer.

[0034] Specifically, the tumor can be all tumors in the present invention, such as ovarian cancer, etc.

[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, which is characterized by including: mixing the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention with the cells to be identified and incubating; then identifying the tumor cells according to the fluorescence situation.

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

[0038] Specifically, the fluorescent DNA nanorobot in the present invention exhibits different fluorescence intensities in different temperature environments. The higher the temperature, the weaker the fluorescence of the self-assembled Y-shaped DNA fluorescent nanodevice. Additionally, the self-assembled Y-shaped DNA fluorescent nanodevice in the present invention exhibits different fluorescence intensities in different pH environments. The higher the pH, the stronger the fluorescence of the Y-shaped DNA 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. When the self-assembled Y-shaped DNA fluorescent nanodevice is co-incubated with normal cells, the fluorescence intensity is significantly higher than that when co-incubated with tumor cells, thereby achieving a response to the tumor cell microenvironment.

[0039] To better understand the present invention, the content of the present invention will be further clarified below with specific examples, but the content of the present invention is not limited to the following examples.

[0040] I. Design and Preparation of Fluorescent DNA Nanorobot

[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 the 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 respectively contain partial sequences of the CuNPs template, and the sequence information is shown in Table 1 above; incubated at room temperature, the 3 DNAs 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+ is reduced to Cu 0 , and the latter uses the formed "Y-shaped" DNA as a template to generate CuNPs, producing a significantly enhanced fluorescence signal, realizing the construction of a self-assembled Y-shaped DNA fluorescent nanorobot.

[0046] II. Testing of Fluorescent DNA Nanorobot

[0047] (I) Temperature Response Test

[0048] The successfully constructed DNA fluorescent nanorobot was incubated at 37 °C and 42 °C respectively, and a fluorescence spectrophotometer was used to observe the fluorescence intensity of the nanorobot. The results are as 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] (2) pH response test

[0050] Incubate the successfully constructed DNA fluorescent nanorobot under the conditions of pH 6.2 and pH 7.4, and use a fluorescence spectrophotometer to observe the fluorescence intensity of the nanorobot. The results are as Figure 3 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] (3) Tumor cell microenvironment response test

[0052] Incubate the successfully constructed DNA fluorescent nanorobot with human ovarian cancer cells A2780 at room temperature, and use a confocal fluorescence microscope to observe the fluorescence imaging ability of the nanorobot. The results are as Figure 4 shown. The fluorescence intensity of the nanorobot is significantly higher when incubated with normal cells than when incubated with tumor cells, thus realizing the response to the tumor cell microenvironment.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all 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 with ascorbic acid and Cu 2+ ; wherein the sequence of DNA A 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.

2. Ovarian cancer tumor diagnosis product, characterized in that: It includes the self-assembled Y-shaped DNA fluorescent nanodevice as described in claim 1.

3. The ovarian cancer tumor diagnostic product according to claim 2, characterized in that: Products include reagents, kits or microfluidic chips.

4. Use of the self-assembled Y-shaped DNA fluorescent nanodevice according to claim 1 in the preparation of products for ovarian cancer diagnosis.

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

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