Isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 as well as preparation method and application thereof

A nucleic acid aptamer and tenascin technology, applied in the field of biomedicine, can solve the problems of shortened action distance, increased action distance, and reduced activity, and achieve the effects of improved inhibition, high synthesis efficiency, and good biological activity

Active Publication Date: 2015-11-18
PEKING UNIV
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  • Summary
  • 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 ...

Method used

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  • Isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 as well as preparation method and application thereof
  • Isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 as well as preparation method and application thereof
  • Isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 as well as preparation method and application thereof

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 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 (HWYu, LRZhang, JCZhuo, LTMa, LHZhang, Bioorg.Med.Chem., 1996,40,609-614), the isonucleoside compounds shown in Chemical Formula I / Chemical Formula II are prepared into Chemical Formula IV / Chemical Formula V respectively The isonucleoside phosphoramidite monomer shown. That is: vacuum-dry the monomer compound I / II, add 3.5 times the equ...

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 TN-C protein to 100 μL, dissolve it in a solution with pH 9.6, 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: ssDNAAptamer dry powder (1nM) centrifuged at 36,000 rpm for 10min, diluted ...

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Abstract

The invention relates to an isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 as well as a preparation method and an application thereof and belongs to the field of biological medicines. Nucleotides in different positions of a tenascin-C aptamer GBI-10 are substituted with isonucleoside or isonucleoside and 2'-deoxyinosine, local spatial conformation of the aptamer is changed, the space structure is optimized, and accordingly, the isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 is obtained. An experiment proves that the isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 has higher affinity and a high-specificity target identification function on target protein as well as higher biological activity. Accordingly, efficient, high-selectivity and low-toxic anti-tumor medicines can be prepared from the isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 potentially, and the isonucleoside or isonucleoside and 2'-deoxyinosine modified tenascin-C aptamer GBI-10 can also be applied to reagents for early detection of tumor.

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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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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