Method for screening aptamer via high performance liquid chromatography, aptamer and application

A high-performance liquid chromatography and nucleic acid aptamer technology, which can be used in pharmaceutical formulations, medical preparations with inactive ingredients, and medical preparations containing active ingredients, etc., and can solve the problems of difficulty in automation, high cost, and low efficiency. , to achieve the effect of low instrument requirements, low cost and strong universality

Active Publication Date: 2015-04-01
HUNAN UNIV
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Problems solved by technology

[0004] In the current conventional nucleic acid aptamer screening method, it is necessary to sequence the pre-enriched nucleic acid library, and then select nucleic acid a

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  • Method for screening aptamer via high performance liquid chromatography, aptamer and application
  • Method for screening aptamer via high performance liquid chromatography, aptamer and application
  • Method for screening aptamer via high performance liquid chromatography, aptamer and application

Examples

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Example Embodiment

[0048] Example 1:

[0049] A method for HPLC-assisted screening of nucleic acid aptamers, mainly using microfluidic chips for screening, and the specific screening process includes the following steps:

[0050] (a) Optimize the nucleic acid library: The structure of the DNA library is divided into five parts: the middle is a 26-base fixed sequence for complementary hybridization with the fixed probe (BDNA); the fixed sequence is flanked by two DNA arms, Both include a random sequence of 18 bases and a sequence necessary for PCR amplification of 19 bases. The immobilized probe is a DNA strand that is labeled with biotin at one end and is complementary to the immobilized sequence of the library, and is used for immobilization of the library.

[0051] The random library RS36 has the nucleotide sequence described in SEQ ID NO. 1, specifically:

[0052] 5'-CCGCTTCGCCGTCTCCTAC-NNNNNNNNNNNNNNNNNN-AAAAGTGGGTAGGGCGGGTTGGAAAA-NNNNNNNNNNNNNNNNNNNN-CGCTCGTCACCCTTCTCCT-3' (Note: N represents any ...

Example Embodiment

[0070] Example 2

[0071] A nucleic acid aptamer 1 of docetaxel obtained by the method of Example 1. The nucleotide sequence of the nucleic acid aptamer 1 has the nucleotide sequence described in SEQ ID NO. 2, specifically 5'- TTGTTTCTCTGTCGATTA-3'.

Example Embodiment

[0072] Example 3

[0073] A nucleic acid aptamer 2 of docetaxel screened by the method of Example 1. The nucleotide sequence of the nucleic acid aptamer 2 has the nucleotide sequence described in SEQ ID NO. 3, specifically 5'- ATTAGCTGTCTCTTTGTT-3'.

[0074] The nucleotide sequences of the nucleic acid aptamers of Example 2 and Example 3 are selected from naturally occurring or artificially synthesized sequences, or the same sequences from any other source.

[0075] Examples 2 and 3 are only the preferred nucleic acid aptamers screened in the present invention. According to the screening method of the present invention, nucleic acid aptamers with the following nucleotide sequences can also be screened:

[0076] (1) The homology with the nucleotide sequence of the nucleic acid aptamer listed in Example 2 or 3 is above 60% (for example, the sequence of the nucleic acid aptamer can be deleted or partially complementary nucleotides can be added);

[0077] (2) A sequence that hybridizes wit...

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Abstract

The invention discloses a method for screening an aptamer via a high performance liquid chromatography, the aptamer and an application. The method for screening aptamers via high performance liquid chromatography comprises the following steps: screening a nucleic acid library of aptamers combined with docetaxel specificity from a random library RS36; screening the nucleic acid library by the high performance liquid chromatography to obtain the aptamer. In the high performance liquid chromatography, the stationary phase is an octadecyl bonded silica gel chromatographic column, the moving phase adopts water-acetonitrile gradient elution, the detection wavelength is 260nm and the detector is a diode array detector. The aptamer screened according to the high performance liquid chromatography has the advantages of being bonded with high specificity and high appetency of docetaxel, being free of immunogenicity, being capable of carrying out chemical synthesis and being good in biocompatibility, small in molecular weight, stable and easy to store, and can be applied to drug carriers, drug separation and purification, and preparation of docetaxel detection probes or target probes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for screening nucleic acid aptamers by high performance liquid chromatography, and also relates to the nucleic acid aptamers obtained by screening by high performance liquid chromatography and the application of the nucleic acid aptamers. Background technique [0002] Docetaxel (Docetaxel) belongs to paclitaxel antineoplastic drugs, which mainly acts on the tubulin of cells to interfere with cell mitosis and has antitumor effects. Its adverse reactions are mainly allergic reactions, thrombocytopenia, tachycardia, and hypotension , neurotoxicity, gastrointestinal tract and skin toxicity. Docetaxel has low water solubility, and the commercially available drug Taxotere is solubilized by Tween-80 and dissolved by ethanol, which will increase the burden on the liver for metabolism and may cause side effects such as allergic reactions and hemolytic reactions. Therefore, how to re...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10G01N30/02A61K47/48A61K47/36A61K31/337A61P35/00
Inventor 羊小海陈南迪王柯敏王青朱莹
Owner HUNAN UNIV
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