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RNA aptamer specifically bound with CD44-hyaluronic acid binding domain protein as well as screening method and application thereof

A CD44-, hyaluronic acid technology, applied in the field of biomedicine, can solve the problems of high cost, long cycle, large labor, etc., and achieve the effects of low cost, easy storage and good stability

Active Publication Date: 2021-03-16
BIOPHARMAGEN CORP FANGZHOU SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]The traditional SELEX method has a long period of time, a large amount of labor, and high cost. It has high-affinity and high-specificity CD44-binding molecules, especially targeted binding to the HA-binding domain The molecule is still a problem to be solved

Method used

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  • RNA aptamer specifically bound with CD44-hyaluronic acid binding domain protein as well as screening method and application thereof
  • RNA aptamer specifically bound with CD44-hyaluronic acid binding domain protein as well as screening method and application thereof
  • RNA aptamer specifically bound with CD44-hyaluronic acid binding domain protein as well as screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Screening of nucleic acid aptamers.

[0047] 1. Preparation of RNA library

[0048] (1) Using the following sequences as templates and primers, a large amount of DNA library was synthesized by PCR reaction.

[0049] DNA library sequence:

[0050] 5'-CACTAATACGACTCACTATAGGGAGAGAACAATGACCTN(40)GAGTGCATTGCATCACGTCAGTAG-3', wherein, N is any one of A, T, C, G;

[0051] DNA library amplification forward primer (34bp):

[0052] 5'-CACTAATACGACTCACTATAGGGAGAGAACAATG-3'

[0053] DNA library amplification reverse primer (24bp):

[0054] 5'-CTACTGACGTGATGCAATGCACTC-3'

[0055] (2) In vitro transcription of RNA library.

[0056] Prepare 100 μL in vitro transcription system according to the following conditions:

[0057]

[0058] The composition of the 10×transcription reaction buffer is 400mM Tris-Cl, 80mM MgCl2, and 20mM spermidine.

[0059] Place in a 37°C water bath and react for 8 hours.

[0060] Put the reaction system in a water bath at 90°C for 2 minut...

Embodiment 2

[0126] Example 2 Determining the affinity between RNA and target protein.

[0127] The coupling conditions were optimized according to the operation guide of BiacoreX100 control soft, and the CD44-HABD was diluted to 10 μg / ml and then coupled to the CM5 chip. The coupling level is 500RU, and the actual coupling amount is 518.9RU.

[0128] Detection K D

[0129] Dilute the RNA series samples and negative control (RNA library transcribed from the starting DNA library) with pH 7.4 loading buffer, and dilute a series of concentrations to 0 μM, 0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM, 4.0 μM. Set the injection time to 180s, the dissociation time to 6min, and use 0.1% SDS as the regeneration buffer. Follow the operation guide of BiacoreX100control soft to carry out the computer test.

[0130] Detection of the K of each Apt by SPR D value, as shown in the table below.

[0131] Table 3. SPR results

[0132] sample name Apt-1 Apt-2 Apt-3 S2 S1 negative control D...

Embodiment 3

[0134] Example 3 Determines the specificity of RNA binding.

[0135] The CD34 protein was used as a control to coat the CM chip and perform SPR detection. The results showed that Apt-2, Apt3, and S2 had no binding and no response to the CD34 protein, indicating that the screened RNA aptamers had good specificity.

[0136] The single-stranded oligonucleotide (RNA) capable of specifically binding to human CD44-HABD was screened by optimized in vitro-SELEX technology, and the affinity coefficient of the optimal sequence binding to CD44-HABD was 13.6nM. The nucleic acid aptamer has similar functions to antibodies, but has more advantages than antibodies, such as: no immunogenicity; large-scale chemical synthesis, low cost; good stability, easy storage, etc., therefore, these nucleic acid aptamers body has broad application prospects.

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Abstract

The invention discloses an RNA aptamer specifically bound with CD44-hyaluronic acid binding domain protein and a screening method thereof, the CD44 hyaluronic acid binding domain (CD44-HABD) is used as a target, a nitrocellulose membrane is used as a screening medium, an in vitro SELEX screening step is optimized, after eight rounds of screening, three groups of RNA sequences (Apt-1, Apt-2 and Apt3) are enriched, and the affinity and dissociation constants of the RNA sequences and target protein through SPR (Surface Plasmon Resonance) are determined by SPR to obtain an RNA aptamer sequence which is highly specifically combined with CD44-HABD.

Description

technical field [0001] The invention belongs to the field of biomedicine, and more specifically relates to an RNA aptamer specifically binding to a CD44-hyaluronic acid binding domain protein and a screening method and application thereof. Background technique [0002] At present, cancer has become one of the important threats to human health. The traditional method of treating cancer is to remove the tumor tissue through surgery, chemotherapy, radiotherapy, etc., but one of the serious problems after conventional treatment is the recurrence and metastasis of cancer. An important reason for this phenomenon is that although tumor stem cells with strong proliferative potential account for a small part of the entire tumor, they have the self-protection properties of normal stem cells, making it difficult to be completely eliminated, leading to tumor recurrence and metastasis. Therefore, one of the current cutting-edge ideas in cancer treatment is to locate or even purify tumor...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10G01N33/68G01N33/574
CPCC12N15/115G01N33/68G01N33/57492G01N33/574C12N2310/16C12N2330/31C12N2320/13G01N2333/70525
Inventor 蒋永平褚畅王含璐
Owner BIOPHARMAGEN CORP FANGZHOU SUZHOU
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