Exonuclease III-driven three-dimensional DNA nanomachines and their applications

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: 2022-08-02
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Exonuclease III-driven three-dimensional DNA nanomachines and their applications
  • Exonuclease III-driven three-dimensional DNA nanomachines and their applications
  • Exonuclease III-driven three-dimensional DNA nanomachines and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of exonuclease III-driven three-dimensional DNA nanomachines

[0062] The detection probe is a polypeptide-DNA detection probe, which includes two kinds. 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 shown in Table 1:

[0064] Table 1

[0065]

[0066]

[0067] Note: The recognition site of the target in the detection probe is shown in bold. The detection probe is a polypeptide-DNA structure, wherein the amino terminus of the polypeptide is modified on the 5' end of the DNA sequence (the structure of the carboxyl and amino groups 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 step of detection of caspase activity includes 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. The cleavage of detection probes by caspase was carried out in a 20 μl reaction system containing 11.5 μl of 1× phosphate buffer solution, 8 μl of detection probe-magnetic beads and 0.5 units of Caspase-8 or / and Caspase-9, incubate for 1 hour at 37°C. After magnetic separation, the supernatant containing the cleavage product was taken for the next reaction. The reaction system of the exonuclease III-driven three-dimensional DNA walker in the second step is 20 μl, which contains 1× Cutsmart buffer (500 mmol / L potassium acetate, 200 mmol / L trihydroxyaminomethane-acetic acid) , 100 mmol / L magnesiu...

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Abstract

The present application provides an exonuclease III-driven three-dimensional DNA nanomachine and its application. The three-dimensional DNA nanomachine includes a signal probe@nano-gold structure and a detection probe, and the signal probe includes a signal probe sequence and a fluorescence Molecules, the detection probe contains a caspase recognition site and a DNA trigger strand, and the DNA trigger strand is complementary to the signal probe sequence; the detection probe is cleaved in the presence of caspase to release the DNA trigger strand containing the DNA trigger strand. structure, the DNA trigger strand in this structure hybridizes with the signal probe to form double-stranded DNA, the double-stranded DNA can be recognized and digested by exonuclease III, and the fluorescent molecules in the signal probe are released. Continue to hybridize with other signaling probes to achieve cyclic release of fluorescent molecules. The invention can realize the simultaneous detection of multiple caspase activities with high detection sensitivity, and can also be used for screening caspase inhibitors.

Description

technical field [0001] The present application relates to the technical field of biological analysis, in particular to a three-dimensional DNA nanomachine driven by exonuclease III and its application. Background technique [0002] The disclosure of information in this Background section is only for enhancement of understanding of the general background of the application and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill 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. Caspases are proteases that ultimately lead to cell disassembly by cleaving a series of downstream proteins, and their abnormal activity has been implicated in many human diseases, such as Alzheimer's disease, anemia, autoimmune dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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