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Nanoprobe applied to intracellular imaging based on DNA tetrahedron

A tetrahedron probe and tetrahedron technology are applied in preparations for in vivo tests, microbe measurement/inspection, biochemical equipment and methods, etc. It can solve problems such as loose and soft structures, unfavorable imaging, and decomposition, and achieve operational Simple, Possibility-enhancing, Low-Cost Effects

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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  • Nanoprobe applied to intracellular imaging based on DNA tetrahedron
  • Nanoprobe applied to intracellular imaging based on DNA tetrahedron
  • Nanoprobe applied to intracellular imaging based on DNA tetrahedron

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Materials and preparation

[0037] TM buffer (20mM Tris, 50mM MgCl 2 , pH 8.0) and other reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., SSMCC was purchased from Sigma Biotechnology Co., Ltd., and hepatitis B surface antigen protein was provided by other research groups. All reagents were prepared using 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.

[0038] The equipment used in this embodiment includes: refrigerated high-speed centrifuge (Hitachi), Nanodrop One, ice maker (Shanghai Anting, ZBS-20), gel developer (Tanon, 1600), vortex shaker (IKA LAB DANCER S25), polyacrylamide gel electrophoresis instrument (BIO-RAD, USA), PCR instrument (BIO-RAD, T100Thermal Cycler), analytical balance (METTLER TOLEDO, ME204E), shaker (Kylin-Bell, TS-8), Ultrasonic cleaning machine (Ningbo Xinzhi, SB-5200DT), STORM super-resolution microscope (Nikon);

...

Embodiment 2

[0042] (1) DNA tetrahedron is connected with hepatitis B antigen

[0043] 1) Dissolve SSMCC and three NH2-modified DNA tetrahedrons at a ratio of 1:10 in 10mM pBbuffer (reaction system 200uL), and shake at 25°C for 2-3h.

[0044] 2) Purify by filtration with a 10kd amicon filter ultrafiltration tube, 14000g, 15min, centrifuge and wash 3-4 times, and finally dissolve in 0.1M PBS for later use. So far, the connection between NH2-DNA tetrahedron and SSMCC is completed.

[0045] 3) Add 30 μL HBsAg and 90 μL SSMCC-modified DNA tetrahedron according to the final concentration ratio of 1:4, and shake at 25°C for 2-3 hours.

[0046] 4) Ultrafiltration with a 30kd amicon filter ultrafiltration tube, 5000g, 15min, 4-5 times, the final washing liquid 1*TMbuffer. So far, SSMCC has successfully linked the DNA tetrahedron with three NH2 modifications to HBV surface antigen. like image 3 , The results of polyacrylamide gel electrophoresis showed that the band position of the DNA tetrahe...

Embodiment 3

[0048] (1) DNA tetrahedrons induce antigens into cells

[0049] 1) Cultivate macrophage RAW264.7 to 60%-70% of the culture dish, add 200mM DNA tetrahedron and protein connection product.

[0050] 2) Incubate overnight in a 25°C incubator.

[0051] 3) Aspirate excess medium and fix cells with 4% paraformaldehyde.

[0052] 4) The intracellular fluorescence was observed under a STORM super-resolution microscope.

[0053] like Figure 4 , the three-dimensional results of STORM showed that the DNA tetrahedron linked with hepatitis B surface antigen can enter the cell and distribute evenly in the cytoplasm.

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Abstract

The invention discloses a nanoprobe applied to intracellular imaging based on a DNA tetrahedron and preparation and applications thereof. The novel nanoprobe of the DNA tetrahedron is prepared via a following method comprising the steps of annealing and hybridizing four DNA single-strands used for building the DNA tetrahedron, wherein the nucleotide sequences of the four DNA single-strands are asfollows: SED ID NO. 1-4; modifying one of the four DNA single-strands with fluorochrome, and modifying the other three ones with NH2 groups, wherein the fluorochrome is Alexa647; and connecting the NH2 on the DNA tetrahedron with SSMCC, and then connecting with a disulfide bond in a hepatitis B surface antigen protein via the SSMCC. According to the nanoprobe provided by the invention, stating from the design of the DNA single-strand sequence, the four DNA single-strands with different marks are hybridized to form the DNA tetrahedron structure by using a biological method; the DNA single-strand structure is provided with the Alexa647 fluorochrome and the three hepatitis B surface antigen proteins, and can be actively taken by cells, and the structure of the DNA single-strand structure canbe completely preserved for 48h; the nanoprobe is a super-resolution microimaging probe with no need to transfect and with 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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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12Q1/02
CPCA61K49/0052A61K49/0056A61K49/0089G01N21/6428
Inventor 宓现强刘馨馨张欢
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI