CN112345507B - Biosensor for targeting cancer cells based on DNA triangular prism structure conformational change - Google Patents

Abstract

The invention belongs to the technical field of biosensors, and relates to a biosensor for targeting cancer cells based on DNA triangular prism structure conformational change. Before detection, the DNA triangular prism nano-structure is constructed. The pH of the reaction solution is then changed to change the conformation of the triangular prism nanostructure. Then adding a cancer marker ATP, enabling two fluorescent groups to be close to generate FRET through a proximity reaction, and detecting through fluorescence spectroscopy. The sensor has the advantages of high detection speed, low detection limit, high specificity and the like, can make up for the defects and shortcomings of the existing detection method, and realizes quick and accurate quantitative detection of the sensor.

Description

A biosensor targeting cancer cells based on conformational changes of DNA triangular prism structure

technical field

The invention belongs to the technical field of biosensors, and relates to a biosensor targeting cancer cells based on the change of DNA triangular prism structure and conformation.

Background technique

An excellent nanostructure should have both good imaging and drug delivery capabilities in order to exert the greatest application value in tumor diagnosis and treatment. Compared with exogenous materials, DNA has good bioaffinity and is the main material for biotherapy and biomedical applications. With the development of DNA nanotechnology, many DNA nanoassemblies such as DNA origami, DNA tetrahedra and DNA cages have been successfully constructed and applied to cancer cell targeted imaging, but these nanostructures are too stable and have low space utilization. Therefore, there is an urgent need to develop a specific and structurally controllable DNA nanostructure for target cell imaging.

SUMMARY OF THE INVENTION

In order to provide a DNA nanostructure with strong specificity and controllable structure, the present invention utilizes the controllable feature of the triangular prism structure to specifically form an i-motif structure in the acidic environment of tumor cells, and reasonably modify the fluorescent group, The triangular prism nanostructures have the function of imaging target cells.

In order to achieve the above objects, the present invention adopts the following technical solutions.

A biosensor for imaging cancer cells, comprising 6 pieces of Oligo DNA: T1, T2, S1, S2, S3, S4, the sequences of which are shown in SEQ ID NOs: 1-6; wherein, 5' and 3 of S4 The ' ends are modified with FRET fluorescent labeling group pairs respectively.

The FRET fluorescent labeling group pair is selected from CY3 and CY5 or FITC and Rhodamine.

A preparation method of the above biosensor, comprising the following steps:

(1) Synthesize T1, T2, S1, S2, S3, S4;

(2) Mix the solutions of T1, T2, S1, S2, S3, and S4 in an equimolar equimolar solution in an alkaline buffer containing Mg ²⁺ , and anneal to form triangular prism nanostructures.

The annealing step is as follows: holding at 95° C. for 10 min, and gradually cooling to room temperature.

The pH of the buffer is 8.0-8.3; the final concentration of Mg ²⁺ is 10 mM; the buffer is selected from TAE buffer or TBE buffer.

An application of the above biosensor in the preparation of cancer cell imaging reagents.

A kit containing the above biosensor.

The detection principle of the present invention is as follows:

The present invention uses a total of 6 DNA strands, and their sequences (5'-3') are:

T1:

GGACCCACATTTGCAATAACACATATCGGTTCAGTACTCTTCATCGTATTCGTGACTGC

T2: CTCAGCGCATTGCCTATATCTCAAGTCTTACGTATACAGTTAATGCTTACACCTCGAC

S1:CCCATT TTAAGGAGGAGGC TTTTTAT GTGGGTCCGCAGTCACGA TTTCCCTATCCCTATCCCTATCCCTTT GCGCTGAGGTCGAGGTGT TTTTT GAGGGGGTCTTAA TCTACC

S2: CCCATT TTAAGGAGGAGGC TTTTT TACGATGAAGAGTACTGA TTTCCCTATCCCTATCCCTATCCCTT AGACTTGAGATATAGGCA TTTTT GAGGGGGTCTTAA TCTACC

S3:CCCATT TTAAGGAGGAGGC TTTTT CCGATATGTGTTATTGCA TTTCCCTATCCCTATCCCTATCCCTT AGCATTAACTGTATACGT TTTTT GAGGGGGTCTTAA TCTACC

S4: cy5-AATGGGATAGGGATAGGGATAGGGTAGA-cy3

Among them, T1 and T2 constitute the two bases of the triangular prism, which are respectively related to the "bases" of S1/S2/S3. "and" ” sequence binding; the italic sequence of S1/S2/S3 is an aptamer cleaved by ATP, and the bold part can specifically bind to the bold sequence of S4; the two ends of the S4 sequence are modified with FRET fluorescent labeling group pairs respectively. When FRET fluorescent labeling group pairs, such as cy5 and cy3, will undergo fluorescence resonance energy transfer when they are adjacent.

When the DNA triangular prism nanostructure is in an acidic environment, the edges of the triangular prism can form an i-motif structure and release the S4 chain. Both ends of S1, S2, and S3 can recognize ATP to form a clip "I"-shaped structure, which can specifically bind to the sequences at both ends of the S4 chain. .

Before detection, DNA triangular prism nanostructures were constructed. Then, the pH value of the reaction solution was changed to change the conformation of the triangular prism nanostructures. Then, the cancer marker ATP is added, and the proximity reaction brings the two fluorophores close to each other to generate FRET, which is detected by fluorescence spectroscopy.

The present invention has the following advantages:

The biosensor of the invention utilizes the variability and operability of the DNA triangular prism nanostructure to realize the imaging and treatment of target cells; utilizes the unique acidic environment of cancer cells and high concentration of ATP to specifically target the target cells; utilizes two fluorescence The group undergoes FRET, the properties are stable, and the background signal is low; the reaction conditions of the sensor are mild and the reaction speed is fast; due to the use of the fluorescence method, the detection method is easy to operate and the detection period is short; the main process of the detection principle is realized in a homogeneous phase It improves the reaction speed, reduces the complexity of the operation, and realizes the fast, simple and sensitive detection of the target; the preparation method is simple, the performance is stable, and the repeatability of the fluorescence detection is good, which is suitable for cell-targeted imaging and biosensors The practical application of industrialization; the process cost of making the biosensor is low, and it is suitable for the requirement of low price in industrialization.

Based on the conformational change of the DNA triangular prism nanostructure, the invention targets cancer cells for cell imaging and drug release. The sensor has the advantages of fast detection speed, low detection limit and high specificity, can make up for the defects and deficiencies of the existing detection methods, and realize the fast and accurate quantitative detection of the sensor.

Description of drawings

1 is a schematic diagram of the biosensor of the present invention;

Fig. 2 is pH optimization detection result graph;

Fig. 3 is a graph of ATP concentration optimization detection result;

Figure 4 shows the specificity of the sensor to ATP;

Figure 5 is a graph of the detection results under the four conditions.

Detailed ways

The present invention will be further described below with reference to the embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments.

Example 1 Preparation of triangular prism nanostructures

(1) Synthesize T1, T2, S1, S2, S3, and S4 according to the sequence of SEQ ID NOs: 1-6, wherein the 5' and 3' ends of S4 are modified with CY3 and CY5 respectively;

1×TAE solution (pH 8.0, containing 10 mM Mg ²⁺ ) was used to prepare dilutions with a concentration of 100 μM;

(2) Take sterilized EP tubes, add 5 μL of T1, T2, S1, S2, S3, and S4 solutions respectively, make up to 50 μL of sterile water, keep at 95°C for 10 min, gradually cool to room temperature, and anneal to obtain a solution containing three A solution of prismatic nanostructures (10 µM).

Example 2 Screening for detecting pH conditions

(1) Prepare buffer containing MgAc ₂ (10 mM), KAc ₂ (140 nM), EDTA (0.1 mM), and adjust the pH of the solution with acetic acid or potassium hydroxide to 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8.

(2) Add 5 µL of buffers with different pH into 9 sterilized EP tubes, respectively, add 5 µL of the solution containing triangular prism nanostructures in Example 1, and shake for 30 s;

(3) Add 5 μL of 10 mM ATP solution, put the centrifuge tube in a water bath at 37 °C for 2 h; after the reaction, use F-4600 fluorescence spectrum detection at room temperature (excitation wavelength 525 nm, fluorescence emission spectrum scanning range 550 -750 nm, slit 1 nm);

Taking the fluorescence intensity of pH 4.5 buffer at 667nm as 1, the normalized fluorescence intensity was calculated. The results are shown in Figure 2. It can be seen from the figure that the detected fluorescence signal is in the range of 8-5.5 with the concentration of pH. When the pH reaches 5, the fluorescence signal tends to be stable, so the optimal concentration of pH is 5.

Example 3 ATP concentration screening

(1) Add 5 µL of the pH 5 buffer prepared in Example 2 into 12 sterilized EP tubes, respectively, add 5 µL of the solution containing triangular prism nanostructures in Example 1, and shake for 30 s;

(2) Add 5 μL of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10 mM ATP solution, and place the centrifuge tube in a water bath at 37 °C for 2 h; At the end of room temperature, use F-4600 fluorescence spectrum detection (excitation wavelength 525 nm, fluorescence emission spectrum scanning range 550-750 nm, slit 1 nm);

The fluorescence intensity at 667 nm of the test solution with an ATP concentration of 10 mM is 1, and the normalized fluorescence intensity is calculated. The results are shown in Figure 3. It can be seen from the figure that the detected fluorescence signal increases with the concentration of ATP at 0. When the concentration reaches 10 mM, the fluorescence signal reaches the maximum value, so the optimal concentration of ATP is 10 mM.

Example 4 Influence of triangular prism nanoconfiguration on detection

(1) Add 5 µL of the pH 5 buffer prepared in Example 2 into 5 sterilized EP tubes, respectively, and then add 5 µL of the solution containing triangular prism nanostructures in Example 1, respectively, and shake for 30 s;

(2) Add 5 μL of 10 mM ATP solution or UTP, CTP, GTP with a concentration of 1 M, and place the centrifuge tube in a water bath at 37 °C for 2 h; after the reaction is completed, use F-4600 fluorescence spectrum detection (excitation) at room temperature. Wavelength 525 nm, fluorescence emission spectrum scanning range 550-750 nm, slit 1 nm);

Taking the fluorescence intensity of the test solution containing the target ATP at 667 nm as 1, the normalized fluorescence intensity was calculated. The results are shown in Figure 4. It can be seen from the figure that only in the presence of ATP will it produce obvious fluorescence Signal.

Example 5 Influence of triangular prism nanoconfiguration on detection

(1) Add 5 µL of pH 5 and pH 8 buffers prepared in Example 2 into sterilized EP tubes, 2 of each, and then add 5 µL of the solution containing triangular prism nanostructures in Example 1. Shake for 30 s;

(2) Add 5 μL of 10 mM ATP solution, put the centrifuge tube in a water bath at 37 °C for 2 h; after the reaction, use F-4600 fluorescence spectrum detection at room temperature (excitation wavelength 525 nm, fluorescence emission spectrum scanning range 550 -750 nm, slit 1 nm);

Taking the fluorescence intensity of the test solution containing 10 mM ATP at pH 8 as 1 at 667 nm, the normalized fluorescence intensity was calculated. The results are shown in Figure 5. It can be seen from the figure that the system containing ATP at pH 5 has obvious The FRET of the three systems with ATP pH 8 and without ATP was low at 667 nm, and no FRET occurred, indicating that only in the presence of ATP in an acidic environment, the nanostructure of the triangular prism can change accordingly. FRET fluorescence signals are detected and target cells are specifically imaged.

sequence listing

<110> Jinan University

<120> A biosensor targeting cancer cells based on the conformational change of DNA triangular prism structure

<130> 20201009

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> T1

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ggacccacat ttgcaataac acatatcggt tcagtactct tcatcgtatt cgtgactgc 59

<210> 2

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<212> DNA

<213> Artificial Sequence

<220>

<223> T2

<400> 2

ctcagcgcat tgcctatatc tcaagtctta cgtatacagt taatgcttac acctcgac 58

<210> 3

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<213> Artificial Sequence

<220>

<223> S1

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cccattttaa ggaggaggct ttttatgtgg gtccgcagtc acgatttccc tatccctatc 60

cctatccctt tgcgctgagg tcgaggtgtt ttttgagggg gtcttaatct acc 113

<210> 4

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<212> DNA

<213> Artificial Sequence

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cccattttaa ggaggaggct tttttacgat gaagagtact gatttcccta tccctatccc 60

tatcccttag acttgagata taggcatttt tgagggggtc ttaatctacc 110

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<212> DNA

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cccattttaa ggaggaggct ttttccgata tgtgttattg catttcccta tccctatccc 60

tatcccttag cattaactgt atacgttttt tgagggggtc ttaatctacc 110

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<212> DNA

<213> Artificial Sequence

<220>

<223> S4

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aatgggatag ggatagggat agggtaga 28

Claims (7)

1. a biosensor targeting cancer cell imaging, is characterized in that, comprises 6 Oligo DNAs: T1, T2, S1, S2, S3, S4, and its sequence is as shown in SEQ ID NO: 1-6; Wherein, The 5' and 3' ends of S4 are respectively modified with FRET fluorescent labeling group pair; The T1, T2, S1, S2, S3, and S4 form a triangular prism nanostructure; T1 and T2 constitute the two bottoms of the triangular prism, which are respectively combined with the GTGGGTCCGCAGTCACGA and GCGCTGAGGTCGAGGTGT sequences of S1, the TACGATGAAGAGTACTGA and AGACTTGAGATATAGGCA sequences of S2, and the CCGATATGTGTTATTGCA and AGCATTAACTGTATACGT sequences of S3; The sequences TTAAGGAGGAGGC and GAGGGGGTCTTAA in S1, S2, and S3 are ATP-cleaving aptamers; the sequences CCCATT and TCTACC in S1, S2, and S3 specifically bind to the sequences AATGGG and GGTAGA in S4, respectively. 2 . The biosensor according to claim 1 , wherein the FRET fluorescent labeling group pair is selected from CY3 and CY5 or FITC and Rhodamine. 3 . 3. a preparation method of the described biosensor of claim 1 or 2, is characterized in that, comprises the following steps: (1) Synthesize T1, T2, S1, S2, S3, S4; (2) Mix the solutions of T1, T2, S1, S2, S3, and S4 in an equimolar equimolar solution in an alkaline buffer containing Mg ²⁺ , and anneal to form triangular prism nanostructures. 4 . The preparation method according to claim 3 , wherein the annealing step is: keeping the temperature at 95° C. for 10 min, and gradually cooling to room temperature. 5 . 5. The preparation method according to claim 3, wherein the pH of the buffer is 8.0-8.3; the final concentration of the ^Mg is 10 mM; the buffer is selected from TAE buffer or TBE buffer. 6. An application of the biosensor of claim 1 or 2 in the preparation of cancer cell imaging reagents. 7. A kit containing the biosensor of claim 1 or 2.

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