Method for achieving efficient delivery of nucleic acid and ultra-sensitive detection of miRNA through DNA nanotetrads

A sensitive detection and detection method technology, applied in the field of biochemistry, can solve the problems of low efficiency of nucleic acid and drug delivery, low sensitivity of miRNA detection, etc., and achieve rapid delivery and effective escape ability, high sensitivity and spatial resolution, and high selectivity Effect

Inactive Publication Date: 2018-10-09
HUNAN UNIV
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  • Application Information

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Problems solved by technology

[0004] In view of this, the present invention aims to propose DNA nano-tetrads to realize the efficient delivery of nucleic acid and the ultra-sensitive detection method of miRNA, so as to solve the problems of low delivery efficiency of nucleic acid and drugs in cells and low detection sensitivity of miRNA in cells.

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  • Method for achieving efficient delivery of nucleic acid and ultra-sensitive detection of miRNA through DNA nanotetrads
  • Method for achieving efficient delivery of nucleic acid and ultra-sensitive detection of miRNA through DNA nanotetrads
  • Method for achieving efficient delivery of nucleic acid and ultra-sensitive detection of miRNA through DNA nanotetrads

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Embodiment 1

[0033] In this example, the target miRNA is miRNA-21 as an example.

[0034] The DNA nano-tetrad realizes the efficient delivery of nucleic acid and the ultra-sensitive detection method of miRNA. The detection method includes the following steps:

[0035] (1) Design H1 nanotetrads and H2 nanotetrads according to the target miRNA, and the H1 nanotetrads and H2 nanotetrads are respectively composed of a streptavidin molecule and four biotinylated fluorescent Formed by the combination of clip probes H1 and H2, H1 labeled Cy3 as a fluorescent donor, H2 labeled Cy5 as a fluorescent acceptor

[0036] (2) cHCR reaction, the trigger chain of cHCR reaction is miRNA;

[0037] (3) In vitro fluorescence quantitative analysis;

[0038] (4) Intracellular high-sensitivity imaging analysis.

[0039] The preparation method of the step (1) H1 nanometer tetrad and H2 nanometer tetrad is as follows: the hairpin probes H1 and H2 are first subjected to quenching pretreatment, and the final conce...

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Abstract

The invention provides a detection method for achieving efficient delivery of nucleic acid and ultra-sensitive detection of miRNA through DNA nanotetrads. The detection method comprises the followingsteps: (1) designing an H1 nanotetrad and an H2 nanotetrad according to target miRNA, wherein each of the H1 nanotetrad and the H2 nanotetrad is formed by combining one streptavidin molecule with fourbiotinylated fluorescent hairpin probes H1 and H2; (2) carrying out Crosslinking Hybridization Chain Reaction (cHCR), wherein a triggering chain of the cHCR is miRNA; (3) carrying out in-vitro fluorescent quantitative analysis; and (4) carrying out intracellular high-sensitivity imaging analysis. The detection method is high in delivery efficiency of nucleic acid; the target miRNA can be detectedby triggering the cHCR; the cHCR specifically triggered by the target miRNA can form a three-dimensional cross-linked hydrogel network, so that the reaction sensitivity is high, the detection specificity is excellent, and the selectivity is high; high sensitivity and spatial resolution are provided for intracellular imaging.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a DNA nanometer tetrad to realize efficient delivery of nucleic acid and an ultrasensitive detection method of miRNA. Background technique [0002] Nucleic acids play crucial roles in many fundamental cellular processes, such as genetic information carriers, intracellular regulatory molecules, and catalytic enzymes. Because nucleic acid not only has the ability of standard base pairing, but also can bind with various molecules, it has become a valuable regulation and analysis tool with unique specificity in cell biology and medicine. The application of nucleic acid in biomedicine is mainly through the transfection system, so that it can be effectively delivered to different cells. In recent years, DNA / RNA nanostructures have emerged as an attractive method for the intracellular delivery of nucleic acids, most notably spherical nucleic acids (SNAs) and self-assembled nucle...

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Application Information

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IPC IPC(8): C12N15/87A61K48/00C12Q1/6886C12Q1/682
CPCA61K48/0025C12N15/87C12Q1/682C12Q1/6886C12Q2600/178
Inventor蒋健晖吴振坤黄杜娟黄志梅唐丽娟
OwnerHUNAN UNIV