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Nucleic acid aptamer of aristolochic acid A as well as screening method and application of nucleic acid aptamer

A nucleic acid aptamer and aristolochic acid technology, which is applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of difficult screening of immobilized small molecules, and achieve stable structure, easy synthesis, and simple operation Effect

Active Publication Date: 2021-11-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to use a capture-SELEX technology to solve the difficult screening problem of immobilizing small molecules by immobilizing the oligonucleotide library on a solid substrate instead of immobilizing the target, and to provide a specificity and high affinity The nucleic acid aptamer of aristolochic acid A also provides the screening method and application of said aptamer accordingly, and further improves the performance of the nucleic acid aptamer through truncation and construction of a stable secondary structure, so as to provide a basis for subsequent detection of horses Laying the foundation for the establishment of methods for Chinese medicines belonging to the family Paolochiliaceae, Chinese medicines that may contain aristolochic acid A and their products

Method used

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  • Nucleic acid aptamer of aristolochic acid A as well as screening method and application of nucleic acid aptamer
  • Nucleic acid aptamer of aristolochic acid A as well as screening method and application of nucleic acid aptamer
  • Nucleic acid aptamer of aristolochic acid A as well as screening method and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Screening of nucleic acid fittings

[0048] The SSDNA library consists of 30 base random regions in the middle, and both sides are co-consisting of 43 base primers binding regions 5'-cgcataggcagACGAGTCATTC-3 ', N represents A or G or C or T.

[0049] CDNA-BIO (5'-Bio-TTTTTGTCGTAAGTTCTGCCATTT-3 ')

[0050] PCR primer sequence:

[0051] PRIMER FP (5'- Cgagcata GGCAGAACTC-3 ')

[0052] PRIMER RP-BIO (5'-Bio-GaatgactcgctctTtacgac-3 ')

[0053] First round screening:

[0054] S1.SSDNA library fixation

[0055] Heat 600ml of water in the beaker to boiling, such as microwave heating, using a 1000pmol library, library and capture cDNA press 1: 5 ratio, mixed with 5 × buffer (Tris-HCl 50mm, NaCl 100mm, MGCL 2 2.5 mM) Solution Sterilization Sterilization Sterilization Mixed by 250 μl of 1 × Buffer solution, boiling water heats the library, captures the cDNA mixture for 10 min, and removes cooling to room temperature, so that the library is hybridized with capture cDNA. 250 ...

Embodiment 2

[0079] Example 2 nucleic acid and aristolochic acid aptamer A binding ability and analysis of specific embodiments

[0080] To further validate the aptamer affinity and specificity aristolochic acid A, using the MST solutions detecting the dissociation constant of the aptamer, chemically synthesized 5 'end of the Cy5-labeled aptamer sequence SEQ1, SEQ2 and SEQ3, by microcalorimetry thermophoresis understanding of its target was measured dissociation constant. Measurement procedure is as follows:

[0081] S1 formulated mother liquor to 1 × buffer Buffer (Tris-HCL 50mM, NaCL 100mM, MgCL22.5mM, 0.005% Tween-20,5% DMSO) as a solvent was prepared at a concentration of 200nM aptamer liquor, 95 ℃ 10min, ice-water bath cooling; stock concentration of 100μM are prepared aristolochic acid a, nuciferine, bile acid, berberine, dehydrogenation corydaline target solution; 1 × buffer buffer.

[0082] S2 gradient solution prepared formulation concentration gradient 16, the numbers 1-16,2-16 were ...

Embodiment 3

[0088] Example 3 Dye displacement detection results detected aptamer method embodiment of aristolochic acid A

[0089] Diethylsulfosuccinate tricarbocyanine (Cy7) is a small molecule dyes to balance between the monomeric and dimeric forms, their absorption peaks at 760 and 670 nm. Studies have shown that, Cy7 hydrophobic monomer may be incorporated into a target aptamer binding domain, which results in a strongly enhanced absorbance at 760nm. However, the target substance bound to the aptamer can be displaced from a monomer Cy7 binding domain, which results in dimerization dye in an aqueous solution, resulting in decrease of absorbance at 760 nm, absorbance at 670 nm is enhanced. Therefore, this method can be used as a small molecule indicator colorimetric detection agent.

[0090] The mixture was first 70μL SEQ 2 aptamer (final concentration = 15μM total body adapted) (2 uM final concentration) in the reaction with 2μL Cy7 buffer (final concentration 10mM Tris-HCl, 0.5mM magnesiu...

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Abstract

The invention discloses a nucleic acid aptamer capable of specifically recognizing aristolochic acid A and application of the nucleic acid aptamer for the first time and belongs to the field of bioengineering. According to the nucleic acid aptamer and the application thereof, the aristolochic acid A is adopted as a target, a cpature-SELEX technology is adopted to screen aptamers having the specific recognition ability on the aristolochic acid A, the aptamer having the highest occurrence frequency in a high-throughput sequencing result is selected, and the performance of the nucleic acid aptamer is further improved through truncation and construction of a stable secondary structure. The nucleic acid aptamer has the characteristics of being easy to synthesize and modify, good in stability and the like, and can be applied to construction of a rapid detection method for aristolochiaceae traditional Chinese medicines, traditional Chinese medicines possibly containing the aristolochic acid A and products of the traditional Chinese medicines.

Description

Technical field [0001] The present invention belongs to the screening and application of nucleic acid fittings, and more particularly to nucleic acid fittings and screening methods thereof in a taponic acid A. Background technique [0002] Aristolochic Acids, AAS is also known as the gapper, Tutong Pyrin, is a strong carcinogenic and nephrotoxic nitridic carboxylic acid, wherein the ariste is a natural existence. One of the largest, highest, nephoxicity, and carcinogenic mutation risk. At present, different countries have adopted different measures to address the toxicity of Maotactapolic acid. For example, most countries prohibit the use of AAS-containing products, but the world does not regularly have cases of renal injury, At the same time, there is a relatively low in Maotic Acids in a rattles of Maotae, and the formulations are not banned, so the contents of content and strict quality control standards containing or may contain AAS Chinese medicine and their formulations are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N21/78
CPCC12N15/115C12N15/1048G01N21/78C12N2310/16Y02A50/30
Inventor 余伯阳刘秀峰陈美琪田蒋为李纪伟
Owner CHINA PHARM UNIV
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