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Primary Amine-Clustered DNA Aptamer for DNA–Protein Conjugation Catalyzed by Microbial Transglutaminase
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-11-20 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00594
Mari Takahara 1 , Rie Wakabayashi 1 , Kosuke Minamihata 1 , Masahiro Goto 1 , Noriho Kamiya 1
Affiliation  

DNA–protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQ-EGFP) and a primary amine-clustered DNA aptamer was enzymatically synthesized as a novel acyl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA–protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form.

中文翻译:

微生物转谷氨酰胺酶催化的初级胺簇DNA适体,用于DNA-蛋白质结合。

DNA-蛋白质结合物是有前途的生物分子,因其具有DNA和蛋白质的双重功能,因此可用于从治疗到分析的各个领域。缀合需要在不损害两个生物分子活性的情况下形成位点特异性和有效的共价键。在本文中,我们集中于微生物转谷氨酰胺酶(MTG)的使用,该酶催化谷氨酰胺残基和伯胺之间的交联反应。在模型生物缀合中,具有高MTG反应性的Gln(Q)供体肽(FYPL QMRG,FQ)与增强的绿色荧光蛋白(FQ-EGFP)融合,并酶促合成伯胺簇DNA适体,作为MTG的一种新型酰基-受体底物,其结合可有效而方便地制备DNA-蛋白结合物高纯度。通过经由DNA适体的c-Met受体识别能力对癌细胞的辨别来评估获得的DNA-EGFP缀合物的双重功能。DNA适体-EGFP缀合物仅对具有c-Met过表达的细胞显示荧光,表明以缀合物形式保留了DNA和EGFP的生化特性。
更新日期:2017-11-20
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