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Targeted DNA nano-probe and preparation and application thereof

A nano-probe and targeting technology, applied in the direction of DNA preparation, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of poor tissue compatibility and metabolic difficulties, and achieve the effect of avoiding damage and good biocompatibility

Inactive Publication Date: 2016-02-17
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such substances usually have certain biological toxicity, poor tissue compatibility, and difficult metabolism

Method used

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  • Targeted DNA nano-probe and preparation and application thereof
  • Targeted DNA nano-probe and preparation and application thereof
  • Targeted DNA nano-probe and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Step 1: Take 2 μL of single-stranded DNA (50 μM) TDN-1, TDN-2, Cy5-TDN-3, N 3 - TDN-4 added to 42 μL Tris-MgCl 2 (Tris10mM, MgCl 2 50mM, pH8) solution. The mixed solution was placed in a PCR instrument at a reaction temperature of 95° C., cooled rapidly to 4° C. after 10 minutes, and continued to react for 30 minutes. A single strand of DNA can self-assemble into a three-dimensional DNA tetrahedral configuration through complementary base pairing.

[0031] Step 2: Take 180 μL of PBS solution (pH 7.3), 40 μL of CuSO 4 Aqueous solution (0.1mM), 40μL of TCEP aqueous solution (0.1mM) and 40μL of TBTA solution (10μM, dissolved in DMSO) were prepared as a reaction solution. Take 100 μL of the prepared TDN solution (2 μM) and 200 μL of the target peptide solution (2 μM) whose amino acid sequence is RGERPPR, and add them to the above reaction solution. After shaking at 37°C for 5 hours at a constant temperature of 175 rpm, the reaction is complete.

[0032] Step 3: Add the...

Embodiment 2

[0034] Step 1: Take 2 μL of single-stranded DNA (50 μM) TDN-1, TDN-2, Cy5-TDN-3, N 3 - TDN-4 added to 42 μL Tris-MgCl 2 (Tris10mM, MgCl 2 50mM, pH8) solution. The mixed solution was placed in a PCR instrument at a reaction temperature of 95° C., cooled rapidly to 4° C. after 10 minutes, and continued to react for 30 minutes. A single strand of DNA can self-assemble into a three-dimensional DNA tetrahedral configuration through complementary base pairing.

[0035] Step 2: Take 180 μL of PBS solution (pH 7.3), 40 μL of CuSO 4 Aqueous solution (0.1mM), 40μL of TCEP aqueous solution (0.1mM) and 40μL of TBTA solution (10μM, dissolved in DMSO) were prepared as a reaction solution. Take 100 μL of the prepared TDN solution (2 μM) and 200 μL of the target peptide solution (2 μM) whose amino acid sequence is RPAKPAR, and add it to the above reaction solution. After shaking at 37°C for 5 hours at a constant temperature of 175 rpm, the reaction is complete.

[0036]Step 3: Add the ab...

Embodiment 3

[0038] Step 1: Take 2 μL single-stranded DNA (50 μM) Cy5-TDN-3, N 3 -Comp.-TDN-3 (complementary chain of TDN-3 chain) added to 46μL Tris-MgCl (Tris10mM, MgCl 2 50mM, pH8) solution. The mixed solution was placed in a PCR instrument at a reaction temperature of 95° C., cooled rapidly to 4° C. after 10 minutes, and continued to react for 30 minutes. DNA single strands can self-assemble into DNA double strands through complementary base pairing.

[0039] Step 2: Take 180 μL of PBS solution (pH 7.3), 40 μL of CuSO 4 Aqueous solution (0.1mM), 40μL of TCEP aqueous solution (0.1mM) and 40μL of TBTA solution (10μM, dissolved in DMSO) were prepared as a reaction solution. Take 100 μL of the prepared TDN solution (2 μM) and 200 μL of the target peptide solution (2 μM) whose amino acid sequence is RGERPPR, and add them to the above reaction solution. After shaking at 37°C for 5 hours at a constant temperature of 175 rpm, the reaction is complete.

[0040] Step 3: Add the above reactio...

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Abstract

The invention discloses a targeted DNA tetrahedron nano-probe and preparation and application thereof. A DNA single chain marked with Cy5 and other three DNA chains with specific base sequences are added to a Tris-MgCl2 solution for mixing and reacting, and a DNA tetrahedron, namely, TDN is obtained; the DNA tetrahedron solution and a tumor penetration peptide solution with certain concentration are mixed, and through a click reaction, a targeted DNA tetrahedron, namely, targeted TDN is synthesized; thirdly, the targeted DNA tetrahedron solution is added to a concentration centrifugal tube, residual micromolecules in reaction liquid are centrifugally removed, precipitate is resuspended in TMbuffer (10mM Tris base, 50mM MgCl2, pH=8), and then the targeted DNA nano-probe can be obtained. Cy5 fluorescent molecules are modified on the DNA tetrahedron and connected with targeted polypeptide, and the probe has targeting and in-vivo near-infrared imaging functions.

Description

technical field [0001] The present invention proposes the preparation and application of near-infrared imaging targeting DNA tetrahedral nanoprobes, specifically involving the self-assembly of DNA single strands labeled with specific fluorescent molecules into DNA tetrahedrons, and further modification of tumor-penetrating peptides , so as to realize the DNA tetrahedron nanoprobe with brain tumor targeting detection. Background technique [0002] The existing nanoprobes for tumor detection mainly include the following categories: nanoprobes based on magnetic materials, such nanoprobes mainly use iron or iron compounds as the matrix; detection probes based on gold nanoparticles and their complexes. needles; and nanoprobes based on other inorganic nanomaterials, such as carbon nanotubes and their composites. Such substances usually have certain biological toxicity, poor tissue compatibility, and difficult metabolism. Therefore, it is urgent to prepare a new type of safe nano...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10A61K49/00
Inventor 何丹农王萍夏智伟刘婷金彩虹
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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