Isothymidine-modified thrombin nucleic-acid aptamer, and preparation method and application thereof

A technology of nucleic acid aptamer and thrombin, which is applied in the field of biomedicine, can solve problems such as difficult quality control and side effects that are difficult to overcome, and achieve the effects of improving anticoagulant effect, enzyme stability, and in vitro anticoagulant activity

Active Publication Date: 2015-01-14
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because it is a mixture of macromolecules, its quality is

Method used

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  • Isothymidine-modified thrombin nucleic-acid aptamer, and preparation method and application thereof
  • Isothymidine-modified thrombin nucleic-acid aptamer, and preparation method and application thereof
  • Isothymidine-modified thrombin nucleic-acid aptamer, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Solid phase synthesis of isothymidine-incorporated DNA

[0058] DNA was synthesized using an Appllied Biosystems model394 DNA solid-phase synthesizer. Normal deoxynucleoside phosphorylated monomers (dT,dG Ac ) 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. 5-Ethylmercapto 1H-tetrazolium solution was purchased from China Pharmaceutical Research and Development Center Co., Ltd. (Beijing).

[0059] According to the method of literature (HW Yu, LR Zhang, JC Zhuo, LT Ma, LH Zhang, Bioorg.Med.Chem., 1996,40,609-614), the isothymidine compound shown in chemical formula I and / or chemical formula II is respectively Prepare isothymidine phosphoramidite monomer shown in chemical formula III and / or chemical formula IV.

[0060]

[0061] Synthesis scale: ~1.0μmol

[0062] Preparation of nucleoside phosphorylation monomer solut...

Embodiment 2

[0073] Example 2. Isothymidine-modified TBA protein level activity test

[0074] TBA-3L: Incorporate isothymidine represented by formula I at the 3-position of TBA sense strand instead of natural isothymidine

[0075] TBA-3D: Incorporate isothymidine represented by formula II at the 3-position of TBA sense strand instead of natural isothymidine

[0076] TBA-4L: Incorporate isothymidine represented by formula I at the 4-position of TBA sense strand instead of natural isothymidine

[0077] TBA-4D: Incorporate isothymidine represented by formula II at the 4-position of TBA sense strand instead of natural isothymidine

[0078] TBA-7L: Incorporate isothymidine represented by formula I at position 7 of the sense strand of TBA instead of natural isothymidine

[0079] TBA-7D: Incorporate isothymidine represented by formula II at position 7 of the sense strand of TBA instead of natural isothymidine

[0080] TBA-9L: Incorporate isothymidine represented by formula I at position 9 of t...

Embodiment 3

[0089] Example 3. In vitro anticoagulant activity test of isothymidine-modified TBA

[0090] The in vitro anticoagulant activity of isothymidine-modified TBA was determined by PUN-2048B dual-channel coagulation instrument (Beijing Pulang Medical Technology Co., Ltd.). Referring to the requirements of experiments in relevant literature, the concentration of the thrombin nucleic acid aptamer was selected as 0.33 μM. See the result image 3 . Through experiments, it can be found that the anticoagulant effect of TBA-3L, TBA-7D, TBA-9L, and TBA-12L is significantly improved after interacting with human plasma, which is consistent with the previous experimental results on enzyme levels and verifies the validity of the experimental results. reliability. At the same experimental concentration, the anticoagulant time of TBA-12L is about twice that of natural TBA, which can greatly inhibit the activity of thrombin. In vitro experiments show that TBA-12L is a promising new high-speci...

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Abstract

The invention discloses an isothymidine-modified thrombin nucleic-acid aptamer, and a preparation method and application thereof, and belongs to the field of biomedicine. By doping isothymidine shown as a chemical formula I and/or II into synthesized thrombin nucleic-acid aptamer, the isothymidine-modified thrombin nucleic-acid aptamer is obtained. Experiments prove that the human thrombin nucleic-acid aptamer modified by employing the method is capable of relatively well recognizing thrombin, is relatively strong in bonding force and has relatively strong anticoagulation effect, and therefore the isothymidine-modified thrombin nucleic-acid aptamer is hopeful to be prepare a high-efficiency high-selectivity anticoagulation medicine with low toxic and side effects.

Description

technical field [0001] The invention relates to a nucleic acid aptamer (aptamer) and its preparation method and application, in particular to an isothymidine-modified thrombin nucleic acid aptamer (TBA) and its preparation method and application. The obtained product can be used It can be used in the treatment of diseases, especially in the preparation of anticoagulant drugs. The invention belongs to the field of biomedicine. Background technique [0002] Anticoagulant drugs are a general term for a class of drugs that inhibit blood clotting. According to their functions, they can be divided into the following three categories: (1) drugs that prevent fibrin formation, such as heparin (UFH), low molecular weight heparin (LMWH), and warfarin; (2) drugs that promote fibrinolysis, such as: Urokinase, streptokinase, defibrase, alteplase, etc.; (3) Antiplatelet drugs such as: aspirin, prostacyclin, dipyridamole, clopidogrel, anagredon, etc. The anticoagulant drugs currently ref...

Claims

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Application Information

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IPC IPC(8): C12N15/115A61K48/00A61P7/02
Inventor 杨振军蔡报彬李理宇范鑫萌武芸张礼和
Owner PEKING UNIV
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