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Preparation and application of nano-probe for intracellular imaging based on DNA tetrahedron

A tetrahedral probe and tetrahedral technology, applied in the biological field, can solve the problems of loose and soft structure, unfavorable imaging, decomposition, etc., and achieve the effects of simple operation, improved possibility and low cost

Inactive Publication Date: 2019-10-01
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional single-stranded DNA probes are easily decomposed by intracellular nucleases due to their loose and soft structure, which is not conducive to imaging
And due to the random excitation principle of super-resolution, it is not suitable for super-resolution microscopes such as STROM

Method used

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  • Preparation and application of nano-probe for intracellular imaging based on DNA tetrahedron
  • Preparation and application of nano-probe for intracellular imaging based on DNA tetrahedron
  • Preparation and application of nano-probe for intracellular imaging based on DNA tetrahedron

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] (1) Materials and preparation

[0035] TM buffer (20mM Tris, 50mM MgCl 2 ,pH 8.0) and other reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and all reagents were prepared with DEPC water. DNA oligo was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and purified by HPLC. The sequence is shown in SEQ ID NO.1-4,

[0036] The equipment used in this embodiment includes: fluorescence spectrophotometer (F-900, Edinburg), refrigerated high-speed centrifuge (Hitachi), Nanodrop One, ice machine (Shanghai Anting, ZBS-20), gel developer (Tanon , 1600), vortex shaker (IKA LAB DANCER S25), polyacrylamide gel electrophoresis (BIO-RAD, USA), PCR instrument (BIO-RAD, T100 Thermal Cycler), analytical balance (METTLER TOLEDO, ME204E), Shaker (Kylin-Bell, TS-8), ultrasonic cleaning machine (Ningbo Xinzhi, SB-5200DT);

[0037] (2) Preparation and characterization of DNA tetrahedron

[0038] Dissolve the single-stranded DNA first, and measure the absor...

Embodiment 2

[0040] Verification of FRET Effect of DNA Tetrahedron Probe

[0041] Configure a 200nM, 100μL system, set the excitation light to 555nm, and set the scanning wavelength range to 545-700nm. The FRET effect of the above-designed DNA tetrahedral probes was measured using a spectrofluorometer. And compare this curve with the fluorescence curve of Alexa555-labeled ssDNA alone. The results were processed and plotted with Origin software. Such as image 3 , there are two obvious emission peaks, proving that the FRET pair was constructed successfully.

Embodiment 3

[0043] (1) Application of DNA tetrahedron probe

[0044] 1) Cultivate macrophage RAW264.7 and HepG2 cells to 60%-70% confluence of the culture dish, add 200mM DNA tetrahedron and protein connection product.

[0045] 2) Incubate overnight in a 25°C incubator.

[0046] 3) Aspirate excess medium and fix cells with 4% paraformaldehyde.

[0047] 4) Observe the intracellular fluorescence under a confocal microscope.

[0048] In order to examine the ability of DNA tetrahedrons to enter cells, we used RAW264.7 macrophages and HepG2 cells simultaneously endowed with Alexa555 and Alexa647 double-labeled DNA tetrahedrons. The imaging result is a three-channel imaging stack: bright field, DAPI and Alexa555 / 647. The results showed that Alexa555 / 647-labeled DNA tetrahedron successfully entered RAW264.7 and HepG2 cells. Overall, the DNA tetrahedron content in RAW264.7 cells was higher than that in HepG2 cells. ( Figure 4 , Figure 5 )

[0049] Shanghai Advanced Research Institute, ...

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Abstract

The invention discloses a novel nano-probe design based on DNA tetrahedron and its preparation and application. The novel nano-probe of the DNA tetrahedron is obtained by the following method that four DNA single strands for construction of the DNA tetrahedron are annealing-hybridized; the nucleotide sequence of the four DNA single strands is shown as SEQ ID NO.1-4; the aforementioned four DNA single strands are modified with different fluorescent dyes, which are Alexa555 and Alexa647, respectively. The invention starts from designing a single-stranded DNA sequence, and uses a synthetic biological method to hybridize four DNA single strands with different fluorescent labels into a DNA tetrahedral structure. The DNA tetrahedral mechanism, having a FRET effect and can be actively taken up bycells and completely preserved for 48 hours, is a microscopic imaging probe for cells that does not require transfection and has no cytotoxicity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the design, preparation and application of a novel nano-probe based on DNA tetrahedron. Background technique [0002] With the in-depth research on the function of biological macromolecules, the demand for imaging the biochemical reactions occurring in its morphology and structure is increasing day by day. Because biomacromolecules form complex multimolecular complexes in cells, common biochemical methods lack functional research and observation of the complete complexes in the natural state; The impact of changes in the intracellular microenvironment on the interaction between molecules can only be studied in cells; therefore, it is necessary to study various biochemical reactions and dynamic processes of biological macromolecules in cells with living cells. [0003] At present, the molecular detection and related imaging technologies in living cells mainly include fluorescence mic...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/64
Inventor 宓现强刘馨馨张欢
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI