Tenascin c nucleic acid aptamer gbi-10 modified by isonucleoside or isonucleoside combined with 2'-deoxyinosine and its preparation method and application

A nucleic acid aptamer and tenascin technology, which is applied in the field of biomedicine, can solve the problems of shortening the action distance, increasing the action distance, and increasing the activity, and achieves the effects of improving the inhibitory effect, high synthesis efficiency and good biological activity.

Active Publication Date: 2018-04-24
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the incorporation site of the isonucleoside is the interaction site between the nucleic acid aptamer and the target, the change in the spatial conformation of the base will lead to a change in the spatial distance from the target. When incorporated into the binding site between the nucleic acid and the target, it will lead to an increase in the action distance with the target and a decrease in activity, while the action distance between the nucleic acid aptamer incorporated in another configuration isonucleoside and the target will be shortened, and the activity will be reduced. Increase

Method used

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  • Tenascin c nucleic acid aptamer gbi-10 modified by isonucleoside or isonucleoside combined with 2'-deoxyinosine and its preparation method and application
  • Tenascin c nucleic acid aptamer gbi-10 modified by isonucleoside or isonucleoside combined with 2'-deoxyinosine and its preparation method and application
  • Tenascin c nucleic acid aptamer gbi-10 modified by isonucleoside or isonucleoside combined with 2'-deoxyinosine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Solid phase synthesis of GBI-10 incorporated by isonucleoside or isonucleoside combined with 2'-deoxyinosine

[0062] DNA was synthesized using an Appllied Biosystems model 394 DNA solid-phase synthesizer.

[0063]Normal deoxynucleoside phosphorylated monomers (dT, dGAc, dABz, dCAc) were purchased from Shanghai Gemma Pharmaceutical Technology Co., Ltd.; CPG (CPG-dG), CAP-A and CAP-B, oxidation I 2 Liquid, Cl 3 CCOOH was purchased from Beijing Aoke Biotechnology Company; 0.25M 5-Ethylmercapto 1H-tetrazolium solution was purchased from Shanghai Zhiyan Technology Co., Ltd. (Shanghai).

[0064] According to the method of literature (HW Yu, LR Zhang, JC Zhuo, LT Ma, LH Zhang, Bioorg.Med.Chem., 1996,40,609-614), the isonucleoside compound shown in chemical formula I / chemical formula II is prepared respectively The isonucleoside phosphoramidite monomer shown in chemical formula IV / chemical formula V. That is: vacuum-dry the monomer compound I / II, add 3.5 times the...

Embodiment 2

[0081] Research on the Basic Properties of the Modified GBI-10 Sequence in Example 2

[0082] 1. Sample name: The 18th, 21st, 26th or 32nd position of the GBI-10 sequence is mixed with the isonucleoside shown in the chemical formula I or the chemical formula II or the 2'-deoxyinosine shown in the chemical formula III (wherein Base is selected from The modified GBI-10 sequence obtained by coupling thymine T) instead of the natural nucleoside at the corresponding position was prepared according to the method in Example 1.

[0083] GBI-10-18A L : Incorporate the isonucleoside shown in the chemical formula I at the 18th position of the sense chain of GBI-10 to replace the natural adenine nucleoside;

[0084] GBI-10-21T D : The 21st position of the sense chain of GBI-10 incorporates the isonucleoside shown in chemical formula II instead of thymidine;

[0085] GBI-10-32dI: 2'-deoxyinosine represented by chemical formula III is incorporated into the 32nd position of the sense stra...

Embodiment 3E

[0091] Example 3 ELISA Determination of the Affinity of GBI-10 Sequence Modified by Ionucleosides and Modified Ionucleosides Combined with Deoxyinosine and Tenascin C

[0092] 1. Sample name: GBI-10-18A L 、GBI-10-21T D , GBI-10-32dI, GBI-10-18A L / 26T L and GBI-10-18A L / 26T L / 32dI, prepared according to the method of Example 1.

[0093] 2. Method

[0094] (1) Dissolve 50 pmol of TN-C protein to 100 μL, dissolve it in a pH9.6 solution, add it to a 96-well plate, and incubate overnight at 4°C to fully bind the protein to the plate. Note: Dilute the sample in the sample tank, and inject the gun into each well to ensure that the three replicate wells of the same sample are parallel.

[0095] (2) Aspirate the protein solution, wash once with blocking solution, then fill each well with blocking solution, and block for one hour. Blocking solution: 1% BSA in PBST.

[0096] (3) Sample preparation: ssDNA Aptamer dry powder (1nM) centrifuged at 36,000 rpm for 10min, diluted wi...

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Abstract

The invention relates to a tenascin C nucleic acid aptamer GBI-10 modified with isonucleosides or isonucleosides combined with 2'-deoxyinosine, and a preparation method and applications thereof, belonging to the field of biomedicine. In the present invention, isonucleosides or isonucleosides combined with 2'-deoxyinosine are used to replace nucleotides in different positions of tenascin C nucleic acid aptamers GBI-10, so as to change the local spatial conformation of the nucleic acid aptamers, and optimize the Its spatial structure is obtained, thereby obtaining an isonucleoside or isonucleoside combined with 2'-deoxyinosine modified tenascin C nucleic acid aptamer GBI-10. Experiments show that the tenascin C nucleic acid aptamer GBI-10 modified by this method has stronger affinity with the target protein, more specific target recognition, and higher biological activity. Therefore, it is hoped that the isonucleosides or isonucleosides combined with 2'-deoxyinosine-modified tenascin C nucleic acid aptamer GBI-10 can be prepared into anti-tumor drugs with high efficiency, high selectivity, and low toxic and side effects. It is used in tumor early detection reagents.

Description

technical field [0001] The present invention relates to a nucleic acid aptamer (aptamer) and its preparation method and application method, in particular to a tenascin C nucleic acid aptamer GBI- 10 and its preparation method and application. The invention belongs to the field of biomedicine. Background technique [0002] Aptamer, nucleic acid aptamer, derived from the Latin word "aptus", is a single-stranded oligonucleotide (RNA) or single-stranded oligodeoxynucleotide (DNA) composed of 20-60 bases. It can specifically bind various target molecules such as proteins, small molecules, ions and cells. Nucleic acid aptamers were invented by Nobel Prize winners Szostak and Gold in 1990, and they have many advantages that antibodies cannot match. The nucleic acid aptamer is screened by SELEX technology, which can be used to screen out the nucleic acid aptamer (Aptamer) that specifically binds to the target with high affinity from a random single-stranded nucleic acid sequence ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C07H19/20C07H19/173C07H1/00A61K31/7115A61P35/00G01N33/574
Inventor 杨振军李昆峰邓家荔
Owner PEKING UNIV
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