A self-assembled four-arm DNA fluorescent nanoprobe and its construction method and application in temperature detection

Abstract

The present invention discloses a self-assembled four-arm DNA fluorescent nanoprobe and a construction method thereof and an application in temperature detection, which belong to the field of genetic engineering in molecular biology. The four single-stranded DNA sequences of the self-assembled four-arm DNA fluorescent nanoprobe are shown in the sequence table SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. The self-assembled four-arm DNA fluorescent nanoprobe provided by the present invention presents different fluorescence intensities in different temperature environments, and the temperature and the maximum fluorescence intensity present a significant linear relationship between 40-80°C. The present invention utilizes genetic engineering technology to construct an enzyme-free, label-free, and modification-free self-assembled four-arm DNA fluorescent nanoprobe, which has important practical application value in environmental temperature detection and fluorescence imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a self-assembled four-arm DNA fluorescent nanoprobe and a construction method thereof and an application thereof in temperature detection. Background Art

[0002] Temperature, as the most basic parameter of living organisms, affects every biochemical reaction, enzyme activity, material transport, cell division and energy metabolism in living cells. Accurately measuring the temperature of a tiny system is of great significance for understanding life processes at the cellular level. Therefore, the development of nanothermometers is of great value.

[0003] In the 1950s, JD Watson and FHC Crick published the structural model of the DNA double helix in Nature, suggesting that DNA is the genetic material that inherits life. In 1983, Seeman used DNA to construct a nucleic acid nanostructure for the first time, 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. DNA nanomachines are one of the fastest-growing directions. They are nanodevices that use the accurate base complementary 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. As we all know, DNA contains four types of bases, A, T, C, and G, which give DNA nanomachines structural diversity; at the same time, because the order of DNA bases can be changed and the design is flexible, its design is programmable; coupled with the unique base complementary pairing principle of DNA, it has a high degree of motion controllability. DNA nanomachines can capture, store and release target molecules like real switches, realizing the function of "machines". At present, DNA nanomachines with various structures have been constructed one after another and play an important role in many aspects, such as drug delivery, bioimaging and biosensing, and have very broad application prospects.

[0004] At present, the main method of constructing nanomachines is to design multiple linear DNA sequences and form DNA nanomachines through complex self-assembly, and fluorescent groups are needed to label them to give the nanomachines fluorescent properties. At the same time, most of the reported nanomachines are used in single fields such as drug delivery, imaging, and molecular sensing.

[0005] In summary, the problems existing in the prior art are:

[0006] (1) The currently reported methods for constructing DNA nanomachines are complex and have high sequence requirements, which limits their widespread application;

[0007] (2) The fluorescent signals required for the characterization, tracing, and imaging of DNA nanomachines reported in the literature require the DNA backbone to be labeled with fluorescent groups, which is expensive and limits its widespread application.

[0008] (3) The functions of DNA nanomachines are relatively single, which limits their wide application. That is, the application scope of DNA nanomachines needs to be expanded;

[0009] (4) The development of a DNA nanomachine that can respond to temperature changes remains to be solved.

[0010] The difficulty in solving the above technical problems lies in:

[0011] How to design a new type of nanomachine so that its sequence design is easy and its assembly is simple; how to design a new type of fluorescent nanomachine so that its fluorescent signal can be obtained without fluorescent group labeling and modification, and the intensity of the fluorescent signal can be regulated by the target; how to design a new type of fluorescent nanomachine so that it has new functions, such as temperature response. Summary of the invention

[0012] In view of this, the main purpose of the present invention is to construct an enzyme-free, label-free, and modification-free self-assembled four-arm DNA fluorescent nanoprobe.

[0013] To achieve the above object, the technical solution of the present invention is as follows:

[0014] In a first aspect, the present invention provides a self-assembled four-arm DNA fluorescent nanoprobe, wherein the nanosensor comprises a DNA self-assembly and a metal nanocluster, wherein the DNA self-assembly is self-assembled by four linear single-stranded DNA molecules through base pairing to generate a four-arm structure self-assembly, wherein the sequence of the first single-stranded DNA is shown in SEQ ID NO: 1, the sequence of the second single-stranded DNA is shown in SEQ ID NO: 2, the sequence of the third single-stranded DNA is shown in SEQ ID NO: 3, and the sequence of the fourth single-stranded DNA is shown in SEQ ID NO: 4.

[0015] Among them, starting from the 5' end, the 26th and 27th bases of the first single-stranded DNA are the bending bases, the 27th and 28th bases of the second single-stranded DNA are the bending bases, the 27th and 28th bases of the third single-stranded DNA are the bending bases, and the 27th and 28th bases of the fourth single-stranded DNA are the bending bases, thereby forming a four-arm structure self-assembly.

[0016] Furthermore, the metal nanoclusters in the self-assembled four-arm DNA fluorescent nanoprobe are located in the base region from 1 to 15 of SEQ ID NO: 2 and the base region from 38 to 52 of SEQ ID NO: 4.

[0017] Furthermore, the metal nanoclusters in the self-assembled four-arm DNA fluorescent nanoprobe are copper nanoclusters.

[0018] In a second aspect, the present invention provides a method for preparing the self-assembled four-arm DNA fluorescent nanoprobe, the method comprising the following steps:

[0019] Incubate four single-stranded DNA molecules at room temperature to self-assemble into a stable four-arm structure self-assembly;

[0020] 2) Adding metal ions and a reducing agent to obtain a nanoprobe containing metal nanoclusters.

[0021] Wherein, in step 1), the incubation time is 30 min and the incubation temperature is 37°C.

[0022] In step 2), the metal ion concentration is 1 mM and the reducing agent concentration is 6 mM.

[0023] Furthermore, the reducing agent is sodium ascorbate, and the metal ion is Cu 2+ .

[0024] In a third aspect, the present invention also provides the application of the self-assembled four-arm DNA fluorescent nanoprobe in temperature detection.

[0025] Furthermore, the temperature range that the self-assembled four-arm DNA fluorescent nanoprobe can detect is 40-80°C.

[0026] That is, in the present invention, the self-assembled four-arm DNA fluorescent nanosensor includes four linear structure DNAs of different sequences (i.e., four single-stranded DNA molecules), and the template DNA single-stranded sequence for synthesizing metal nanoclusters is rationally designed into the linear structure DNA, so that they can combine with each other through base complementary pairing to form a four-arm DNA structure. Under the action of a reducing agent, metal ions use this structure as a template to form metal nanoclusters, which then exhibit fluorescence.

[0027] Based on the temperature-responsive application of fluorescent DNA nanoprobes, the successfully constructed nanosensor was incubated at different temperatures. The results showed that the four-arm DNA fluorescent nanoprobe exhibited different fluorescence intensities at different temperatures. The higher the temperature, the weaker the fluorescence of the four-arm DNA fluorescent nanoprobe; and the temperature and the maximum fluorescence intensity showed a significant linear relationship in the range of 40-80°C.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) The present invention designs a self-assembling four-arm DNA nanomachine for the first time, that is, the four-arm DNA nanomachine is self-assembled through complementary base pairing of four DNA chains, and the method is simple and easy to design;

[0030] (2) The present invention constructs a four-arm fluorescent nanoprobe, which realizes fluorescence labeling-free, provides a new idea for the construction of functional DNA nanomachines, and provides a new tool for fluorescence imaging technology;

[0031] (3) The present invention is the first to use DNA fluorescent nanoprobes to respond to temperature changes. The application of this nanoprobe is relatively novel and provides a new idea for the construction of multifunctional nanomachines. It has important scientific significance for the innovation and development of multifunctional DNA nanomachines. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of a method for constructing a self-assembled four-arm DNA fluorescent nanoprobe provided in an embodiment of the present invention.

[0033] Figure 2 This is a self-assembled four-arm DNA fluorescent nanoprobe metal nanoparticle identification diagram provided by an embodiment of the present invention.

[0034] Figure 3 This is a temperature response spectrum of the self-assembled four-arm DNA fluorescent nanoprobe provided in an embodiment of the present invention.

[0035] Figure 4 This is a temperature response standard curve of the self-assembled four-arm DNA fluorescent nanoprobe provided in an embodiment of the present invention.

[0036] Figure 5 This is a linear relationship diagram between the maximum fluorescence intensity and temperature of the self-assembled four-arm DNA fluorescent nanoprobe provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0037] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] Example 1 Preparation of self-assembled four-arm DNA fluorescent nanoprobe

[0040] Through rational design and simulation with DNAMAN software, the construction principle of an enzyme-free, label-free, and modification-free DNA nanoprobe is shown in Figure 1 .

[0041] In this system, the sequence information of linear A, linear B, linear C, and linear D is shown in Table 1, where the single-stranded sequence of the CuNPs template is ATGAACGTATGAGCG. After annealing at 95°C and slow cooling, the four DNA strands self-assembled to form a stable "four-arm" DNA structure ABCD. At this point, the formed "four-arm" DNA contains a complete CuNPs template. Finally, through the reduction of sodium ascorbate (6mM), 1mM Cu 2+ Reduced to Cu 0 The latter uses the formed "four-arm" DNA as a template to generate CuNPs, thereby realizing the construction of a self-assembled four-arm DNA fluorescent nanoprobe.

[0042] The nanoprobe prepared in this example was identified, and the results are as follows: Figure 2 As shown, it indicates that the nanoparticles are well dispersed and spherical in aqueous solution.

[0043] Table 1 Oligonucleotide sequences used

[0044]

[0045] Example 2 Application of self-assembled four-arm DNA fluorescent nanoprobe

[0046] The self-assembled four-arm DNA fluorescent nanoprobe successfully constructed in Example 1 was incubated at 20-80°C (5°C interval) for 30 min, and the fluorescence intensity under different temperature conditions was detected using a fluorescence spectrophotometer with the excitation light wavelength set to 345 nm and the emission light wavelength set to 610 nm.

[0047] The results are as follows Figure 3 and 4 As shown, the self-assembled four-arm DNA fluorescent nanoprobe exhibits different fluorescence intensities in a temperature environment of 20-80°C. The higher the temperature, the weaker the fluorescence of the self-assembled four-arm DNA fluorescent nanoprobe.

[0048] like Figure 5 As shown, there is a significant linear relationship between the maximum fluorescence intensity and the temperature in the range of 40-80°C.

[0049] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0050] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of a self-assembled four-arm DNA fluorescent nanoprobe in temperature detection, characterized in that: The nanoprobe comprises a DNA self-assembly and a metal nanocluster, wherein the DNA self-assembly is generated by self-assembly of four single-stranded DNA molecules through base pairing, wherein the sequence of the first single-stranded DNA is shown in SEQ ID NO: 1, the sequence of the second single-stranded DNA is shown in SEQ ID NO: 2, the sequence of the third single-stranded DNA is shown in SEQ ID NO: 3, and the sequence of the fourth single-stranded DNA is shown in SEQ ID NO: 4; The metal nanoclusters are copper nanoclusters; The method for preparing the self-assembled four-arm DNA fluorescent nanoprobe comprises the following steps: 1) After annealing at 95°C and slowly cooling down, four single-stranded DNA molecules self-assemble to form a stable four-arm structure self-assembly; 2) adding metal ions and reducing agents to obtain nanosensors containing metal nanoclusters; Wherein, the reducing agent is sodium ascorbate, and the metal ion is Cu 2+ ; The temperature detection range is 40-80°C.

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