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Preparation method and application of dendritic DNA assembly

An assembly and dendritic technology, applied in the field of biomedicine, can solve problems such as cumbersome process, inapplicability to industrialized preparation, inapplicability to flexible structures, etc., and achieve the effect of high flexibility

Active Publication Date: 2018-10-02
SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]1. The basic unit (Y structure) needs to be prepared first, and then assembled into DNA dendrimer step by step. The process is cumbersome and not suitable for industrial production;
[0005]2. The final product prepared is a rigid molecule with a specific three-dimensional structure, so it is not suitable for applications requiring flexible structures

Method used

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  • Preparation method and application of dendritic DNA assembly
  • Preparation method and application of dendritic DNA assembly
  • Preparation method and application of dendritic DNA assembly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] This embodiment provides a method for preparing a dendritic DNA assembly, which specifically includes the following steps:

[0032] Mix in TAE / Mg by the following DNA sequence in a certain ratio 2+ (40mM Tris, 20mM acetic acid, 1mM EDTA, 12.5mM MgCl 2 ) buffer solution, the temperature was raised to 90°C, and the temperature was lowered to 4°C at a cooling rate of 2min / °C to prepare D1-3, D2-3, and D3-3. figure 2 It is a schematic diagram of the structure of the prepared dendritic assembly. image 3 Sequence maps of the two dendrimers used in this example. Figure 4 Taking D2-3 as an example, the sequence map and structural composition of the dendritic DNA assembly in this example are shown.

[0033] Its short-strand DNA sequence is

[0034] Core 1-1 5'-ATCGTTAGGACTCTGACGGC-3'

[0035] Core 2-1 5'-ATCGTTAGGAACTGTATCGGCAGTATAATACTCTGACGGC-3'

[0036] Core 2-2 5'-ATCGTTAGGAAATTATACTGCCGATACAGACTCTGACGGC-3'

[0037] Core 3-1 5'-ATCGTTAGGAAGCTCACGCCAGATGGTGCGGACTCTGAC...

Embodiment 2

[0055] Prepare the sequence of D2-3 according to Example 1, combined with the following aptamer (AptamerAS1411) with tumor targeting function and model drug siRNA, AptamerAS1411 5'-GGTGGTGGTGGTTGTGGTGGTGGTGGATACCATGGC-3'siRNA

[0056] 5’-CGATGGCCArArCrCrArCrArUrArUrGrArArArCrCrArGrCrUrUrCrCrUrGrArA-3’

[0057] 5'-Alex647-rCrArG rGrArArGrCrU rGrGrU rUrUrC rArUrArUrGrG rUrGG T-3'

[0058] With Core2-1 at a concentration of 200nM, mix the desired DNA in TAE / Mg 2+ (40mM Tris, 20mM acetic acid, 1mM EDTA, 12.5mM MgCl 2 ) buffer solution, the temperature was raised to 90° C., and the temperature was lowered to 4° C. at a cooling rate of 2 min / ° C. to prepare a complex with tumor-targeted drug delivery function.

[0059] Table 1. Short-chain DNA combinations and their quantity ratios required to prepare different dendritic DNA assemblies.

[0060]

[0061] For the convenience of statistics, in the gene sequence list, each DNA and RNA sequence in Example 1 and Example 2 is named ...

Embodiment 3

[0076] Figure 7 As one of the application examples, dendritic DNA assembly D2-3 is loaded with tumor targeting factor AS1411 and model drug siRNA. It successfully delivered Alexa647 fluorescently labeled siRNA to cancer cells. The application of this assembly is a substrate and a carrier, especially a carrier for drug delivery, and its loaded substances include small molecule drugs, nanoparticles, polypeptides, proteins, siRNA / miRNA, and combinations used for gene editing (CRISPR-protein and nucleoside acid complex). These dendritic DNA assemblies loaded with therapeutic, targeting, imaging and gene editing functions have broad application prospects in biomedicine and biotechnology.

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Abstract

The invention provides a preparation method and application of a dendritic DNA assembly. The preparation method of the dendritic DNA assembly includes the steps of: mixing several single stranded DNAin a buffer solution, performing heating to 90DEG C, and then conducting cooling to 4DEG C at a rate not exceeding 3.6DEG C / min, thus obtaining the dendritic DNA assembly. The invention also providesapplication of the dendritic DNA assembly as a molecular carrier in imaging, diagnosis and treatment. The invention has the advantages that: through one-step quenching treatment of a single stranded DNA mixture, the dendritic DNA assembly with precisely controllable structure can be obtained.

Description

technical field [0001] The invention relates to a preparation method and application of a dendritic DNA assembly, belonging to the technical field of biomedicine. Background technique [0002] In 2004, Dan Luo's research group first reported the assembly structure and method of dendritic DNA (NatureMaterials 2004, 3, 38), and then underwent a series of improvements (Biomacromolecules 2015, 16, 1095; AcsNano 2014, 8, 6171; Angew. Chem.Int.Ed.2012,124,11433), a method for forming an existing dendritic DNA assembly (DNAdendrimer). The existing technology heats up three single-stranded DNAs (20mM each strand) in a phosphate buffer solution (50mM, pH 8.0, with NaCl 100mM) to 95°C, and then cools down to 4°C at a cooling rate of 1°C / min. , thus self-assembling ( figure 1 A) into the basic structural unit Y 0 , Y 1 , Y 2 , Y 3 . Afterwards, after a step-by-step assembly method (step-by-step), Y was mixed separately at room temperature 0 and Y 1 (quantity ratio 1:3) 1 hour,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K47/26
CPCA61K47/26C12N15/11C12N2310/10
Inventor 鲁敬雄曹玲燕
Owner SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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