Preparation method of [2]-reticular catenane DNA monolayer array, and application of [2]-reticular catenane DNA monolayer array

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

Active Publication Date: 2020-09-04
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 of [2]-reticular catenane DNA monolayer array, and application of [2]-reticular catenane DNA monolayer array
  • Preparation method of [2]-reticular catenane DNA monolayer array, and application of [2]-reticular catenane DNA monolayer array
  • Preparation method of [2]-reticular catenane DNA monolayer array, and application of [2]-reticular catenane DNA monolayer array

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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 was evenly mixed with 2 μL of ATP (10 mM) and 2 μL of T4 PNK enzyme (10000 U / mL), 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 ...

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 preparation method of a [2]-reticular catenane DNA monolayer array, and an application of the [2]-reticular catenane DNA monolayer array, and belongs to the technical field ofnanometer materials. A DNA monolayer array is designed on the theoretical basis of a DNA nanometer technique, and the structure of the array is formed through self-assembly of two kinds of DNA chainscontaining palindrome segments. The DNA monolayer array has favorable biocompatibility and favorable nucleases stability, can also efficiently enter different lactation animal cells under the condition that co-vectors do not exist. After the whole body of a mouse having tumors is subjected to medicine administration, the DNA monolayer array can preferentially accumulate in tumor tissue, and ligands do not need to be targeted. Simple and efficient assembly, unique structural features and advantages in a biosystem indicate that the DNA monolayer array is an up-and-coming DNA nanometer platformand can be used for tumor monitoring and targeted medicine administration.

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 Applications(China)
IPC IPC(8): A61K31/713A61P35/00C12P19/34
CPCA61K31/713A61P35/00C12P19/34
Inventor 吴再生尹洪卫陈燕茹王伟军
Owner FUZHOU UNIV
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