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Integrated synchronized dna nanodevice and its live-cell multi-target imaging application and imaging method

A nano-device and living cell technology, applied in the field of live cell imaging, can solve the problems of limited reaction rate, loss of intracellular stoichiometric information, poor interference ability, etc.

Active Publication Date: 2020-10-27
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of these devices are composed of multiple separate modules, and different modules have different cell entry efficiencies, resulting in loss of intracellular stoichiometric information, resulting in signal differences, interfering with accurate monitoring imaging, and causing misjudgment
In addition, the researchers found that there are orders of magnitude differences in the diffusion coefficients of macromolecules and nanoscale structures in the cytoplasm of mammalian cells and in other solutions
Therefore, the joint entry of multiple modules, exogenous energy substances or nucleic acid probes into living cells will complicate the reaction system and limit the reaction rate
At the same time, these DNA nanodevices lack signal calibration modules, cannot respond to the heterogeneity between single cells, and have poor ability to resist interference from complex cytoplasmic environments, resulting in unreliable imaging results, which limits the research application in the field of single cells

Method used

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  • Integrated synchronized dna nanodevice and its live-cell multi-target imaging application and imaging method
  • Integrated synchronized dna nanodevice and its live-cell multi-target imaging application and imaging method
  • Integrated synchronized dna nanodevice and its live-cell multi-target imaging application and imaging method

Examples

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Embodiment 1

[0036] Example 1: Imaging of single target miR-21 in living cells by using integrated synchronous DNA nanodevices of different designs.

[0037] Using the HeLa cell line of cervical cancer cells as the basic model, 20 μL of integrated synchronous DNA nanodevice probes were added to HeLa cells pre-cultured in eight-well confocal culture dishes (0.2 mL, 1×10 6 mL -1), incubated at 37°C for 4 hours. After washing with PBS, imaging was performed using a confocal laser fluorescence microscope. In the target miR-21-positive cells, miR-21 replaces the Block chain that seals the R-SD and releases the single-stranded R-SD, whose 5' end 14 bases are connected to the loop corresponding to the R-HD chain The 14 bases in the shape part are complementary to form a double strand, and the damaged base oG site on the R-HD chain can be acted on by the enzyme hOGG1 to form an apurinic / apyrimidinic AP site, which is then digested by APE 1 to release FAM fluorescence.

[0038] In addition, the...

Embodiment 2

[0040] Example 2: Using an integrated synchronous DNA nano-device to simultaneously image multiple targets in different living cells.

[0041] Using the HeLa cell line of cervical cancer cells as the basic model, 20 μL of integrated synchronous DNA nanodevice probes were added to HeLa cells pre-cultured in eight-well confocal culture dishes (0.2 mL, 1×10 6 mL -1 ), incubated at 37°C for 4 hours. After washing with PBS, imaging was performed using a confocal laser fluorescence microscope. In the presence of the target miR-21, the strand displacement reaction and the Block DNA strand complement each other to form a double strand, releasing the R-SD strand; the T-SD-S strand, the primer at the 5' end is extended in the presence of the target telomerase T-SD chain. The 14 bases at the 5' end of the R-SD chain, T-SD chain and C-SD chain are complementary to the 14 bases of the ring part of the corresponding R-HD chain, T-HD chain and C-HD chain to form a double chain; After the...

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Abstract

The invention discloses an integrated synchronous deoxyribonucleic acid (DNA) nano device, application of integrated synchronous DNA nano device to living cell multi-target imaging, and a living cellmulti-target imaging method of the integrated synchronous DNA nano device, and belongs to the technical field of living cell imaging. The integrated synchronous DNA nano device is constituted in the mode that Au nanoparticles (AuNP) are loaded with three sets of DNA chains, the integrated synchronous DNA nano device is triggered by microRNA and / or telomerase in living cells and driven by human 8-oxoguanine DNA glycosylase (hOGG1) and apurinic-apyrimidinic (AP) endonuclease I APE1 (APE1) to release fluorescence signals, and simultaneous imaging of living cell multiple targets is achieved. The integrated synchronous DNA nano device integrates all the reaction modules, different in-to-cell efficiency of a separation module and motion differences in cell pulp in diffusion limitation can be avoided, cell exogenous supplying of any substances is not needed, and precise imaging analysis of the multiple targets in the living cells can be simultaneously achieved.

Description

technical field [0001] The invention belongs to the technical field of living cell imaging, and in particular relates to an integrated synchronous DNA nanometer device and its live cell multi-target imaging application and imaging method. Background technique [0002] A variety of biomolecules and corresponding interaction networks in living cells coordinately regulate various metabolic processes in organisms. Therefore, it is of great significance to simultaneously monitor multiple biological targets in the cell and obtain comprehensive information in practical applications such as biochemical analysis and clinical diagnosis. Deoxyribose nucleic acid (Deoxyribonucleic acids, DNA) has become a dark horse among probe construction materials due to its outstanding advantages of following the principle of complementary base pairing and being precise and programmable. DNA-based nanodevices have flexible and diverse structures and are widely used in multi-target monitoring and im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵永席薛静陈锋黄平
Owner XI AN JIAOTONG UNIV
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