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Preparation method and application of a [2]-reticular catenane dna monolayer array

A DNA chain and catenane technology, applied in the field of preparation of DNA monolayer arrays, can solve the problems of limited number of targeting ligands and limited application of DNA nanostructures, and achieve good biocompatibility and strong nuclease stability. Effect

Active Publication Date: 2021-06-29
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although different types of cells, such as healthy and diseased cells, theoretically express unique surface receptors, the limited number of targeting ligands available limits the application of DNA nanostructures in medical diagnosis and therapy

Method used

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  • Preparation method and application of a [2]-reticular catenane dna monolayer array
  • Preparation method and application of a [2]-reticular catenane dna monolayer array
  • Preparation method and application of a [2]-reticular catenane dna monolayer array

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 [2] - Preparation of a DNA monolayer array of reticulated catenane

[0024] First, with the help of ATP and PNK, the 5ʹ ends of all oligonucleotides involved in the assembly were phosphorylated by mixing 86 μL of DNA strand 1 or DNA strand 2 (10 mM) with 10 μL of 10×T4 PNK buffer, 2 μL of ATP (10mM) and 2 μL of T4 PNK enzyme (10000 U / mL) were uniformly mixed, and then placed in a constant temperature metal bath at 37°C for 1 h. Then heat inactivates the PNK enzyme to obtain the corresponding solution, which is left to stand for 4 hours before use.

[0025] The DNA monolayer array of [2]-reticulated catenanes ([2] GDA) is assembled from two types of strands of DNA with palindromic regions. The assembly process is described as follows: First, the PSa and PSb solutions were diluted with 1×T4 DNA ligase buffer to a final concentration of 1.6 μM, respectively. After annealing in a constant temperature metal bath at 90° C. for 5 minutes, the solution was slowly co...

Embodiment 2

[0028] Example 2 Cell internalization analysis and detection

[0029] HeLa cells were cultured on 12-well culture plates for 24 h, allowing the cells to grow to 70–80% of the bottom of the well plate. Then the medium was replaced with DMEM medium containing Cy5-labeled [2]GDA or 1PSb (400 μL of the medium, wherein the concentration of Cy5-labeled PSb and PSa was 50 nM). After incubation for 4 h, wash with PBS solution. Then, Hoechst 33342 (10 μg / mL) was incubated at 37°C for 15 min for nuclear staining. After washing with PBS solution again, the cells were imaged by confocal fluorescence microscopy with a laser confocal fluorescence microscope (Leica TCS SP8).

[0030] Figure 4 A is the result of confocal microscopy imaging of the internalization ability of [2] GDA cells. The three pictures show the cells of the same community. The left picture is the result of nuclear staining, the middle picture is the fluorescence imaging result of [2]GDA entering the cell, and the rig...

Embodiment 3

[0031] Example 3 Cytotoxicity Experiment

[0032] HeLa cells were seeded on 96-well culture plates and cultured for 24 h to allow the cells to grow to 70%–80% of the bottom area of ​​the wells. Then, the cells were treated with 100 μL of DMEM medium (without FBS) containing 0, 20, 80, 160, 300, 400 nM [2]GDA for 4 h. Next, replace the DMEM medium containing [2]GDA with 100 μL DMEM medium containing 10% FBS, and incubate for 20 h. Then, the cells were washed with PBS solution, and then incubated with 150 μL of MTT reagent at 37°C for 4 h. After removing the MTT reagent, wash again with PBS solution, add 100 μL DMSO to each well, and shake well at 600 rpm for 10 min. Finally, a microplate reader measures the absorbance at 450 nm.

[0033] Figure 4 B is the cytotoxicity test result of [2]GDA. The abscissa represents the concentration of [2]GDA, and the ordinate represents the cell activity. It can be seen from the figure that with the increase of the concentration of [2]GDA...

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Abstract

The invention provides a method for preparing a DNA monolayer array of [2]-reticular catenane and its application, belonging to the technical field of nanomaterials. The invention designs a DNA monolayer array on the basis of DNA nanotechnology theory, and the array structure is formed by self-assembly of two kinds of DNA chains containing palindrome fragments. In addition to good biocompatibility and nuclease stability, DNA monolayer arrays can efficiently enter different mammalian cells without co-carriers. After systemic administration to tumor-bearing mice, DNA monolayer arrays could preferentially accumulate in tumor tissue without the need for a targeting ligand. The facile and efficient assembly, unique structural features, and advantages in biological systems suggest that the DNA monolayer array is a promising DNA nanoplatform for tumor monitoring and targeted drug delivery.

Description

technical field [0001] The invention belongs to the technical field of nanomaterials, and in particular relates to a method for preparing a DNA monolayer array of [2]-reticular catenane and its application. Background technique [0002] Nucleic acid, as a versatile genetic material, can be used to construct nanoscale self-assembled structures through the Watson-Crick base pairing principle. Due to high hybridization specificity, outstanding programmability, and wide sequence diversity, scientists have synthesized various artificial DNA nanomaterials in the past 30 years, including one-dimensional nanotubes, complex two-dimensional lattices Or array, discrete three-dimensional structure, etc. These materials hold great potential in biological applications such as molecular imaging, theranostics, and drug delivery. However, although one-dimensional and three-dimensional structures, such as DNA nanowires or nanotubes, DNA origami, DNA spherical structures, and DNA polyhedral ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/713C12N15/10A61P35/00C12P19/34
CPCA61K31/713A61P35/00C12P19/34
Inventor 吴再生尹洪卫陈燕茹王伟军
Owner FUZHOU UNIV
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