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A visualized sensor for live-cell imaging of circRNA based on nucleic acid self-assembly without enzyme catalysis

A non-enzyme catalysis and self-assembly technology, applied in the field of biosensors, can solve the problems of complex instruments, limited routine applications, cumbersome operations, etc., and achieve the effect of shortening the detection time

Active Publication Date: 2021-05-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these techniques require complex instruments and cumbersome operations to extract RNA from cells and destroy cells, limiting their routine applications

Method used

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  • A visualized sensor for live-cell imaging of circRNA based on nucleic acid self-assembly without enzyme catalysis
  • A visualized sensor for live-cell imaging of circRNA based on nucleic acid self-assembly without enzyme catalysis
  • A visualized sensor for live-cell imaging of circRNA based on nucleic acid self-assembly without enzyme catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 The establishment of a visual sensor based on GO and nucleic acid self-assembly without enzyme catalysis

[0047] 1. Experimental materials

[0048] Graphene oxide GO, SYBR Gold nucleic acid dye, nucleic acid molecular weight standard ultra-low range DNAladder, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), Tris, sodium chloride, magnesium chloride, potassium chloride, ethylenediaminetetraacetic acid Disodium, sulfuric acid, tetramethylbenzidine, and urea were purchased from Thermo Scientific Life Technologies, and Beas-2B, MCF-7, H596, H1299, and L02 cells were obtained from Shanghai Institute of Biological Sciences. All experimental water was from Milli-Q pure water system.

[0049] The detection hairpin strands (HP1, HP2, H1, H2) of CHA and HCR used in the experiment and the replacement strands (I' and I) of the circRNA to be detected are as follows:

[0050] SEQ ID NO: 1 (HP1): 5'-GCTATGTTGTTTCGTCTTGAGGTAGGCCGTCCAGACAAAATCCCTCAAGACGAATTCCAGACT-3' ...

Embodiment 2

[0063] Embodiment 2 The specificity investigation of sensor

[0064] According to the biosensor constructed in Example 1, L02, H596, H1299 and Beas-2B cell lysates were added to the system for detection. The results showed that the established circRNA biosensor had better specificity ( Figure 4 ).

Embodiment 3

[0065] Example 3 Intracellular Imaging Investigation of Sensors

[0066] The biosensors constructed in Example 1 were added to Beas-2B and MCF-7 cells and incubated for 24 hours. CCK8 was used to detect the toxicity of the sensors to the cells. It was found that it hardly affected the viability of the cells, and subsequent intracellular fluorescence imaging experiments could be carried out. ( Figure 5 ), followed by adding the detection system to different concentrations of serum ( Image 6 A) and 2U DNase I to detect its stability in complex biological fluids ( Image 6 B), the detection system was found to have high stability. Finally, the biosensor was added to Beas-2B and MCF-7 cells and incubated for 7 hours, and the spatial position information of the intracellular target circRNA and the change of its content could be observed under a fluorescence microscope ( Figure 7 ).

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Abstract

The present invention provides a visual sensor for circRNA live cell imaging based on nucleic acid self-assembly without enzyme catalysis, including: (1) GO-based intracellular efficient delivery system, (2) GO-based signal switch system, ( 3) An enzyme-free nucleic acid self-assembly dual signal amplification imaging system based on the combination of CHA and HCR. Through the design of CHA and HCR hairpin probes, the present invention utilizes the biocompatibility and fluorescence quenching effect of GO to complete the detection of target circRNA in living cells. It solves the problem of cumbersome detection process of circRNA by traditional PCR, sequencing and northblot detection methods and is difficult to visualize sensitively, and realizes fast, highly sensitive and highly selective visual detection of circRNA in live cells. The detection of circRNA as a biomarker is more accurate, real-time and efficient.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a visual sensor based on nucleic acid self-assembly and non-enzyme-catalyzed circRNA imaging in living cells. Background technique [0002] Non-coding RNA is a type of RNA that is transcribed from the genome and does not have the ability to be translated into protein. It is widely involved in various physiological and pathological processes of organisms. CircRNA is a circular single-stranded non-coding RNA molecule formed by reverse splicing of the 5' and 3' ends of pre-mRNA during the splicing process. Since there are no free 5' and 3' ends of linear RNA, it is difficult to degrade circRNA through many traditional linear RNA decay pathways, which endows circRNA with strong stability in complex biological fluids. In addition, it also plays an important role in the regulation of various physiological and pathological processes because it can act as a "sponge body" for miRNAs ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6825C12Q1/02G01N21/64
CPCC12Q1/6825G01N21/64G01N21/6428G01N21/6458G01N33/5005G01N2021/6417G01N2021/6432C12Q2525/307C12Q2563/107C12Q2565/607
Inventor 许文涛黄昆仑李宏宇张博洋朱丽叶
Owner CHINA AGRI UNIV
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