Primary Amine-Clustered DNA Aptamer for DNA-Protein Conjugation Catalyzed by Microbial Transglutaminase
Primary Amine-Clustered DNA Aptamer for DNA-Protein Conjugation Catalyzed by Microbial Transglutaminase
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DOI：
10.1021/acs.bioconjchem.7b00594
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发表时间：
2017-12-01
影响因子：
4.7
通讯作者：
Kamiya, Noriho
中科院分区：
文献类型：
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作者：
Takahara, Mad;Wakabayashi, Rie;Kamiya, Noriho
由于DNA和蛋白质的双重功能，DNA-蛋白质偶联物有望在从治疗到分析等领域得到广泛的应用。结合需要在不损害两个生物分子的活性的情况下，形成特定的和有效的共价键。在这里，我们重点介绍了一种微生物谷氨酰胺转氨酶(MTG)的使用，它催化谷氨酰胺残基和伯胺之间的交联反应。在生物偶联模型中，将高活性谷氨酰胺供体多肽(FYPLQMRG，FQ)与增强型绿色荧光蛋白(FQEGFP)融合，酶促合成伯胺类DNA适配子作为MTG的新型芳基受体底物，从而高效、简便地制备高纯度的DNA-蛋白质偶联物。通过DNA适配子的c-Met受体识别能力对癌细胞的识别来评价所获得的DNA-EGFP结合物的双重功能性。DNA适体-EGFP结合物只对c-Met过表达的细胞显示荧光，表明DNA和EGFP在结合物形式下的生化性质保持不变。
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 (FQEGFP) and a primary amine clustered DNA aptamer was enzymatically synthesized as a novel aryl-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.