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Entropy-driven DNA nano loop and application thereof

A circuit and nanotechnology, applied in the field of live cell imaging, achieves the effect of strong operability and simple design

Active Publication Date: 2020-07-14
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the complexity of the design of these DNA nanostructures, it is difficult to use them to analyze the differential gene expression and enzyme activity changes in living cells.

Method used

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  • Entropy-driven DNA nano loop and application thereof
  • Entropy-driven DNA nano loop and application thereof
  • Entropy-driven DNA nano loop and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Combining Figure 1~3 , to prepare an entropy-driven DNA nanocircuit, and monitor the signal amplification process in real time through the LightCycler 96 instrument.

[0045] In 10 μL of TEMg reaction buffer (10 mM Tris-HCl, 2 mM EDTA, 1 mM Mg 2+ , pH=8.0), add 0.4mM multivalent DNA nano-substrate, 0.5mM amplification probe, and different concentrations of target probes, incubate at 37°C for 60min, and monitor the signal amplification process in real time through LightCycler 96 instrument.

[0046] figure 1 Contains monovalent, bivalent and trivalent DNA nanocircuits. Input telomerase, miRNA 21 and miRNA31 respectively, through strand displacement reaction and target recycling reaction, the target signal is significantly amplified, and detected with three output fluorescent signals (FAM, TAMRA, Cy5).

[0047] figure 2 The shape of the fully assembled trivalent DNA nanostructure presents an obvious "T" or "Y" shape, and its size is about 20 nanometers, whi...

Embodiment 2

[0055] Example 2: Combining Figure 4~6 , Entropy-driven multivalent DNA nanocircuits for combined analysis of RNA and enzyme catalysis in living cells in different cell types.

[0056] First, the multivalent DNA nanosubstrates (SPs) were prepared, and 2 μM initiation probe (Bott module), 1.6 μM P21 probe (P21 module), 1.6 μM telomerase probe (Ptelo module) and P31 probe (P31 module) was reacted at 95°C for 10 min, hybridized at 45°C for 3 h, and the prepared SPs were stored at -20°C until use.

[0057] Three different cell lines of human normal mammary gland epithelial cell MCF-10A, human breast cancer cell MCF-7 and human breast cancer cell MDA-MB-231 were selected for the following operations. Cells were seeded in eight-well plates at a density of 50,000 cells per well and grown overnight in a 37°C incubator. 100nM polyvalent DNA nano-substrates (SPs), 125nM amplifying probes (AMPs) and 100nM telomerase recognition primer (TR primer) were transfected into living cells by ...

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Abstract

The invention discloses an entropy-driven DNA nano-loop and application thereof, belongs to the technical field of living cell imaging, and aims to realize signal amplification by utilizing the entropy-driven DNA nano-loop through strand displacement reaction and target circulating reaction and finally realize RNA and enzyme catalysis combined imaging analysis in living cells.

Description

technical field [0001] The invention belongs to the technical field of living cell imaging, and in particular relates to an entropy-driven DNA nano circuit and an application thereof. Background technique [0002] Gene expression and enzyme catalysis are dynamically changed in different cell states and cell types, and changes in genes are closely related to human diseases. For example, non-coding miRNA expression and telomerase activity differ between cancer and normal tissues. Therefore, monitoring this dynamic change can determine the state or type of cells, with applications in biological research and clinical applications. However, conventional multiplex RNA detection methods including reverse transcription PCR and sequencing are not suitable for enzyme activity analysis, and they cannot observe the dynamic changes of RNA and enzyme catalysis in living cells, and the spatiotemporal information will be ignored. Furthermore, how to translate these multigene expression an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12Q1/682
CPCC12Q1/682G01N21/6486C12Q2531/119C12Q2565/629C12Q2521/113C12Q2563/107
Inventor 赵永席陈锋白敏
Owner XI AN JIAOTONG UNIV
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