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Aptamer Met-G02 specifically combined with methamidophos and application thereof

A nucleic acid aptamer, methamidophos technology, which is applied in the field of biomedicine and can solve problems such as inability to be widely used

Inactive Publication Date: 2018-07-06
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This detection method requires specific instruments and equipment, professional operators and certain operating skills, and cannot be widely used.

Method used

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  • Aptamer Met-G02 specifically combined with methamidophos and application thereof
  • Aptamer Met-G02 specifically combined with methamidophos and application thereof
  • Aptamer Met-G02 specifically combined with methamidophos and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Screening of methamidophos nucleic acid aptamer

[0039] 1. Coupling of Ligand (Methamidophos) and Matrix (Sepharose 6B)

[0040] 1. Suspend 1 g of freeze-dried powder Sepharose 6B in 3 mL of distilled water in a sintered glass filter (porosity G3);

[0041] 2. Immediately wash the matrix with 200 mL of coupling buffer solution (0.05 M, pH9.6 carbonate buffer) in a sintered glass filter for 1 h;

[0042] 3. Dissolve 6 g of ligand with 6 mL of coupling buffer solution to make the final concentration 1 mg / mL;

[0043] 4. Mix the substrate in step 2 with the buffer solution in step 3 with a concentration of 1 mg / mL in which the ligand has been dissolved in a ratio of 1:2 by volume, and treat it for 16 h under the condition of a water bath at 35°C-40°C, and 37 h ℃ shaker overnight;

[0044] 5. At 40°C-50°C, block excess groups with 1M aminoethanol for at least 4 hours or overnight;

[0045] 6. Wash the excess ligand with coupling buffer, centrifuge at 4000...

Embodiment 2

[0090] Example 2: Cloning, isolation and sequencing of nucleic acid aptamers and prediction of secondary structure of single-stranded DNA

[0091] 1. Preparation of Escherichia coli DH5α Competent Cells

[0092] 1. Pick a single DH5α colony, inoculate it in 3 mL of LB medium without ampicillin, and incubate overnight at 37 °C. The next day, take the above bacterial solution and inoculate it in 50 mL of liquid LB medium at a ratio of 1:100, 37 Shake at ℃ for 2 min; when the OD600 value reaches 0.35, harvest the bacterial culture;

[0093] 2. Transfer the bacterial culture to a 50 mL pre-cooled sterile polypropylene tube and place it on ice for 10 min to cool the culture;

[0094] 3. Centrifuge at 4000 rpm for 10 minutes at 4°C, discard the culture medium, and invert the tube for 1 minute to drain the remaining culture medium;

[0095] 4. Add 150 μL ice-cooled 0.1 mM CaCl each 2 Solution, combine two tubes, ice bath for 10 min;

[0096] 5. Centrifuge at 4000 rpm for 10 min a...

Embodiment 3

[0110] Embodiment 3: circular dichroism to K + Research on the relationship between concentration and nucleic acid aptamer Met-G02

[0111] 1. Dilute the aptamer with sterilized water to 20 μM, denature at 94 °C for 0.5 min, and cool to 25 °C at a rate of 0.5 °C / min;

[0112] 2. Dilute the nucleic acid aptamer Met-G02 to 2.5 μM with KCl solutions of different concentrations (0, 10, 20, 40, 60, 80, 100 mM);

[0113] 3. Detect with a circular dichroism spectrometer at 25 °C and a wavelength of 220–340 nm (see the results in figure 2 ).

[0114] The results showed that the chromatograms had negative peaks between 240-250 nm and positive peaks between 275-285 nm. Because the peak at 240-250 nm is the characteristic peak of G-quadruplex and the peak at 275-285 nm is the characteristic peak of the stem-loop, the nucleic acid aptamer Met-G02 can exist stably in KCl solution without affecting its secondary structure .

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Abstract

The invention discloses an aptamer Met-G02 specifically combined with methamidophos. The aptamer Met-G02 is screened by an SELEX technology. The aptamer is single stranded DNA and is prepared from 82nucleotides, a nucleotide sequence of the aptamer is shown as SEQ ID NO:1, and a secondary structure of the aptamer contains a protruded ring and a stem; furthermore, the aptamer has a G-quadruplex structure, and the Met-G02 gibbs free energy DG is equal to -15.21. A series of character assessments on aptamer specificity, affinity, flexibility and the like are performed based on enzyme linked oligonucleotide absorption measurement, and a dissociation constant Kd of the Met-G02 is shown to be equal to 11.77+ / -2.07 nM; furthermore, the aptamer can be specifically combined with the methamidophos;thus, the aptamer has the advantages of high specificity, high affinity and quick and flexible application to methamidophos detection.

Description

technical field [0001] The invention relates to a nucleic acid aptamer Met-G02 specifically combined with methamidophos and an application thereof, belonging to the technical field of biomedicine. Background technique [0002] Nucleic acid aptamers are screened by SyStematic evolution of ligandS by exponential enrichment (SELEX) and can bind to metal ions, small molecules, biomacromolecules, and even whole cells with high specificity and affinity ssDNA or RNA molecules. Nucleic acid aptamers not only have the recognition characteristics of antibody molecules, almost all diagnostic fields involving antibodies can be replaced by nucleic acid aptamers, but also have their own unique excellent performance, which has a wide range of target molecules, small molecular weight, It has the advantages of low immunogenicity, easy chemical synthesis, transformation and labeling. Nucleic acid aptamers and related screening technology (SELEX), nucleic acid aptamers in recent years in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
CPCG01N33/5308C12N15/115G01N2430/10C12N2310/16
Inventor 韩芹芹靖乐夏雪山汪颖宋玉竹刘丽
Owner KUNMING UNIV OF SCI & TECH
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