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Group of nucleic acid aptamers specifically binding to gliotoxin and application of nucleic acid aptamers

A nucleic acid aptamer and gliotoxin technology, applied in the biological field, can solve problems such as difficulty in early rapid diagnosis, limited development of detection methods, and limited detection sensitivity, and achieve high affinity, easy production and modification, and small molecular weight.

Active Publication Date: 2019-02-15
EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing detection methods (such as high performance liquid chromatography, thin layer chromatography, etc.) have been difficult to meet the needs of early and rapid diagnosis due to problems such as time-consuming, cumbersome sample preparation, and limited detection sensitivity.
At the same time, gliotoxin is a small molecule mycotoxin with a non-protein structure, and its ability to produce recognition ligands (such as antibodies) is very small, which further limits the development of corresponding detection methods.

Method used

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  • Group of nucleic acid aptamers specifically binding to gliotoxin and application of nucleic acid aptamers
  • Group of nucleic acid aptamers specifically binding to gliotoxin and application of nucleic acid aptamers
  • Group of nucleic acid aptamers specifically binding to gliotoxin and application of nucleic acid aptamers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Construction of random ssDNA library and primers thereof

[0032] 1. Construct a random ssDNA library with a length of 80 nucleotides

[0033] 5′-AGCAGCACAGAGGTCAGATG-N 40 -CCTATGCGTGCTACCGTGAA-3' (SEQ ID No.7); wherein, N represents any one of the bases A, T, C, G, N 40 Represents a random sequence of 40 nucleotides in length.

[0034] 2. Construction of primers

[0035] Upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3' (SEQ ID No.8);

[0036] Downstream primer 1: 5'-TTCACGGTAGCACGCATAGG-3' (SEQ ID No.9);

[0037] Downstream primer 2: 5'-poly(dA20)-Spacer18-TTCACGGTAGCACGCATAGG-3' (SEQ ID No. 10).

Embodiment 2

[0038] Example 2. Screening of gliotoxin nucleic acid aptamers

[0039] In order to obtain a high-affinity nucleic acid aptamer that specifically binds to gliotoxin, a total of 8 rounds of screening were carried out based on the non-specific adsorption of ssDNA by graphene oxide. Among them, reverse screening was introduced from the fifth round to further improve the efficiency and specificity of screening. The recovery rate of nucleic acid aptamer binding to gliotoxin in each round of screening is as follows: figure 1 As shown, thereby effectively monitoring the progress of the screening.

[0040] The specific screening process of graphene oxide SELEX technology is: (1) as shown in Table 2, first a certain amount of ssDNA library is dissolved in the screening buffer (containing 5mM MgCl 2, 5% DMSO in D-PBS buffer), in a 95°C water bath for 10 min, quenched in an ice bath for 5 min, and after standing at room temperature for 30 min, add 200 pmol of gliotoxin and incubate at ...

Embodiment 3

[0045] Example 3. Determination of the interaction between nucleic acid aptamers and gliotoxin

[0046] In this example, the binding affinity and specificity of the nucleic acid aptamer to gliotoxin were determined by biofilm interference technology.

[0047] (1) The biotin-labeled aptamer was dissolved in screening buffer and diluted to 2 μM, bathed in 95°C water for 10 minutes, quenched in an ice bath for 5 minutes, and left at room temperature for 30 minutes to promote its refolding into a stable three-dimensional structure.

[0048] (2) Add 200 μL of screening buffer, nucleic acid aptamer, and gliotoxin to the 96-well plate, and the streptavidin-modified biosensors are sequentially immersed in each reaction well according to the program set by the instrument, and after equilibrium 1.5 minutes, the nucleic acid aptamer was solidified for 5 minutes, rinsed for 1.5 minutes, bound for 2.5 minutes and dissociated for 2.5 minutes in five steps.

[0049] (3) The aptamer sensor i...

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Abstract

The invention relates to the field of biotechnology, in particular to a group of high-affinity nucleic acid aptamers specifically binding to gliotoxin. According to the invention, high-affinity nucleic acid aptamers that specifically recognize gliotoxin are screened and obtained by adopting an oxidized graphene SELEX technique with gliotoxin as a target, and the performance of the nucleic acid aptamers is further improved through optimizing strategies such as truncation, mutation and the like. The group of nucleic acid aptamers has broad application prospects, and can be used for separation and removal of gliotoxin in complex systems, rapid detection and early diagnosis of gliotoxin in vitro and in vivo, neutralization of gliotoxin in related diseases or the development and preparation ofantagonistic drugs and other aspects.

Description

technical field [0001] The invention relates to the field of biotechnology, specifically, a group of high-affinity nucleic acid aptamers specifically combined with gliotoxin and applications thereof. Background technique [0002] In recent years, with the popularization of organ transplantation, the increase of patients with malignant tumors, and the wide application of broad-spectrum antibiotics and glucocorticoids, the infection rate of invasive Aspergillus has increased significantly, accounting for about 15% of the immunocompromised population. %, the fatality rate can be as high as 90%. Aspergillus that causes deep infection in humans mainly includes Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus. Among them, Aspergillus fumigatus is the most common, accounting for about 95% of them. It is the most important pathogen causing severe deep Aspergillus infection and even invasive aspergillosis in immunocompromise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C07D513/20G01N33/569A61K31/711A61P31/10
CPCA61K31/711A61P31/10C07D513/20C12N15/115C12N2310/16G01N33/56961
Inventor 高顺祥吴继红郑欣胡晓波
Owner EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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