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Excision enzyme III-driven three-dimensional DNA nano machine and application thereof

A nanomachine and exonuclease technology, applied in the field of biological analysis, can solve the problems of poor sensitivity, complicated operation and high background signal, and achieve the effect of high sensitivity and improved detection sensitivity.

Active Publication Date: 2021-07-13
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing detection methods have limitations such as cumbersome operation, high background signal, and poor sensitivity.

Method used

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  • Excision enzyme III-driven three-dimensional DNA nano machine and application thereof
  • Excision enzyme III-driven three-dimensional DNA nano machine and application thereof
  • Excision enzyme III-driven three-dimensional DNA nano machine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of a three-dimensional DNA nanomachine driven by exonuclease III

[0062] The detection probe is a polypeptide-DNA detection probe, which includes two types. The polypeptide chain of the detection probe 1 contains the recognition site Ile-Glu-Thr-Asp (IETD) of caspase-8, and its DNA part It can be complementary to signal probe 1; the polypeptide chain of detection probe 2 contains the recognition site Leu-Glu-His-Asp (LEHD) of caspase-9, and its DNA part can be complementary to signal probe 2 .

[0063] The sequence information involved in the exemplary embodiment of the present invention is as shown in Table 1:

[0064] Table 1

[0065]

[0066]

[0067] Note: The recognition site of the target in the detection probe is indicated in bold. The detection probe is a polypeptide-DNA structure, wherein the amino terminal of the polypeptide is modified on the 5' end of the DNA sequence (the structure of carboxyl and amino in the probe struct...

Embodiment 2

[0071] Example 2 Detection of caspase activity and detection of caspase under activity inhibition

[0072] 1. Detection of caspase activity: the detection of caspase activity consists of two steps, which are the cleavage of the detection probe by caspase and the fluorescence caused by the three-dimensional DNA nanomachine driven by exonuclease III. Cyclic release of molecules. Cleavage of detection probes by caspases was performed in a 20-microliter reaction system containing 11.5 microliters of 1× phosphate buffer solution, 8 microliters of detection probe-magnetic beads and 0.5 units of For caspase-8 or / and caspase-9, incubate at 37°C for 1 hour. After magnetic separation, the supernatant containing the lysed product was taken for the next reaction. In the second step, the reaction system of the three-dimensional DNA walker driven by exonuclease III is 20 μl, which contains 1×Cutsmart buffer (500 mmol per liter of potassium acetate, 200 mmol per liter of tris-acetic acid...

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Abstract

The invention provides an excision enzyme III-driven three-dimensional DNA nano machine and application thereof. The three-dimensional DNA nano machine comprises a signal probe and nanogold structure and a detection probe, the signal probe comprises a signal probe sequence and fluorescent molecules, the detection probe comprises a caspase recognition site and a DNA trigger chain, and the DNA trigger chain and the signal probe sequence are complementary; the detection probe is split in the presence of caspase to release a structure containing a DNA trigger chain, the DNA trigger chain in the structure is hybridized with the signal probe to form double-chain DNA, the double-chain DNA can be recognized and digested by excision enzyme III and releases fluorescent molecules in the signal probe, and the DNA trigger chain immediately recovers a single chain and continues to be hybridized with other signal probes, therefore, the cyclic release of the fluorescent molecules is realized. The machine can be used for simultaneously detecting the activities of various caspase, is high in detection sensitivity, and can also be used for screening caspase inhibitors.

Description

technical field [0001] This application relates to the technical field of bioanalysis, in particular to a three-dimensional DNA nanomachine driven by exonuclease III and its application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the application, and is not necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Apoptosis is a manifestation of programmed cell death, which can lead to cell death without triggering any inflammation, and caspases play a key regulatory role in the process of apoptosis. Abnormal activity of caspases, proteases that cleave a series of downstream proteins ultimately leading to cell disintegration, has been implicated in many human diseases, such as Alzheimer's disease, anemia, autoimmune diseases and cancer. [0004] Among t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6823C12Q1/37C12N15/11
CPCC12Q1/6823C12Q1/37C12Q2521/319C12Q2521/537C12Q2563/107C12Q2563/137
Inventor 张春阳刘萌徐瑞
Owner SHANDONG NORMAL UNIV
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