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Probe based on G-quadruplex-chlorine heme DNA enzyme and application of probe

A DNA enzyme and quadruplex technology, applied in the field of molecular bioinformatics, can solve the problems of unrealistic detection, unrealizable protein detection, poor sensitivity, etc., and achieve good long-term storage stability, large-scale rapid preparation, and simple preparation.

Inactive Publication Date: 2015-11-04
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] (1) Large background noise and poor sensitivity
Specifically, in the reaction system, the hairpin structure and the straight chain that does not form a hairpin structure maintain a dynamic balance, and the straight chain does not form a closed loop and is protected, resulting in that the G-quadruplex sequence is not all target molecules It is formed by opening the first hairpin and then hybridizing with the second hairpin, and some straight-chain sequences directly hybridize with the second hairpin to form a free G-quadruplex sequence, so it cannot Achieving more sensitive detection
[0004] (2) Detection of target molecules by hybridization and coordination of bases, in fact, only DNA detection can be realized, but protein detection cannot be realized
Although these methods can improve the sensitivity of protein detection, they usually need to chemically modify the aptamer with fluorescein and quencher, or use exonuclease, endonuclease, nanoparticles, etc., so that the preparation The cost is greatly increased, and the difficulty of preparation is increased

Method used

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  • Probe based on G-quadruplex-chlorine heme DNA enzyme and application of probe
  • Probe based on G-quadruplex-chlorine heme DNA enzyme and application of probe
  • Probe based on G-quadruplex-chlorine heme DNA enzyme and application of probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Probe design based on G-quadruplex-hemin DNase for detection of thrombin

[0040] In this embodiment, the probes based on G-quadruplex-hemin DNase include: first probe P1, second probe P2, first hairpin H1 and second hairpin H2, each The composition sequence is shown in the table below:

[0041] Table 1 Probe sequences based on G-quadruplex-hemin DNase

[0042]

[0043] The first probe P1 includes: the first aptamer region I that can specifically recognize thrombin, which is Apt29, and can bind to the heparin binding site of thrombin; poly T sequence II; and the second probe A first base complementary to region III that forms a duplex; a first DNase that can hybridize to the first hairpin portion amplifies region IV.

[0044] The second probe P2 includes: the second aptamer region I' that can specifically recognize thrombin, which is Apt15, and can bind to the fibrinogen binding site of thrombin; poly T sequence II; The first probe forms a second base c...

Embodiment 2

[0054] Embodiment 2: The detection of the thrombin of different concentrations based on the probe of G-quadruplex-hemin DNase

[0055] Human α-thrombin (Tb) used in this example was purchased from Sigma-Aldrich Company (St. Louis, Missouri, U.S.), hemin, tris(hydroxymethyl)aminomethane (Tris), N-2- Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS), peroxide Hydrogen was purchased from Aladdin Reagent Company (Shanghai, China), and there was no difference in the effect of the above-mentioned materials among the commercially available models. All solutions were prepared using redistilled water prepared by Milli-Q Purification System (Billerica, Massachusetts, USA).

[0056] The detection of thrombin involves the following steps:

[0057] First, take the solution of the first hairpin and the second hairpin, heat it to 95° C., keep it for 5 minutes, then slowly lower it to room temperature, and se...

Embodiment 3

[0063] Embodiment 3: Detection of thrombin in human serum based on the thrombin probe of G-quadruplex-hemin DNase

[0064] The human serum used in this example was from Gangwon Hospital (with ethics approval). Take the human serum diluted 10 times, take human α-thrombin and dilute it with the above human serum diluent until the concentration of thrombin is 10pM, 100pM, 1000pM, numbered 1, 2, 3 respectively, as the solution to be tested. The same method as in Example 2 was used for detection.

[0065] The above detection has further verified the feasibility of the method of the present invention in a real biological environment. When detecting actual biological samples (such as proteins in diluted human serum), accurate quantitative detection can be achieved, as shown in Table 2. The detection result of the recovery rate is in the range of 97.63-103.60%.

[0066] Therefore, the method of the present invention has better practicability and can be applied to the detection of pr...

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Abstract

The invention provides a probe based on a G-quadruplex-chlorine heme DNA enzyme and a method for detecting protein. The method for detecting the protein facilitates detecting operation and is low in cost and free of marking. The probe has the advantages of being high in sensitivity and low in background noise. When the protein is detected, the detecting operation is easy, consumed time is short, cost is low, and the problems that a method for detecting the protein in the prior art is long in consumed time, high in cost and complex in detecting probe manufacturing are solved.

Description

technical field [0001] The invention belongs to the field of molecular bioinformatics, and in particular relates to a probe based on G-quadruplex-hemin DNase and a method for detecting protein thereof. Background technique [0002] Deoxyribozyme (DNA enzyme) is a kind of artificial nucleic acid, which is isolated by in vitro selection, has high catalytic activity for specific substrates, good thermal stability, and can be denatured and refolded many times without losing catalytic activity, etc. Advantages, it is an ideal biocatalyst for signal amplification in biological applications. In the field of DNase, the discovery of G-quadruplex-hemin DNase has attracted widespread attention. The G-quadruplex sequence can combine with the cofactor—hemin to form a peroxidase-like DNA enzyme, and then convert H 2 o 2 mediated 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS 2- ) catalytic oxidation to green ABTS ·- , or improve the chemiluminescence of the luminol-hydrog...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 吴昊邹霈刘娅灵王洪勇吴军诸飞帆
Owner JIANGSU INST OF NUCLEAR MEDICINE
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