Bavi Rohit, Hang Zhang, Banerjee Parikshit, Aquib Md, Jadhao Mahendra, Rane Niraj, Bavi Sneha, Bhosale Raghunath, Kodam Kisan, Jeon Byong-Hun, Gu Yueqing
State Key Laboratory of Natural Medicines, Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing, Gulou District 210009, China; School of Chemical Sciences, Punyashlok Ahilyadevi Holkar Solapur University, Solapur 413255, Maharashtra, India.
State Key Laboratory of Natural Medicines, Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing, Gulou District 210009, China.
Mol Ther Nucleic Acids. 2020 Sep 4;21:1074-1086. doi: 10.1016/j.omtn.2020.07.038. Epub 2020 Jul 31.
Aptamers are small, functional single-stranded DNA or RNA oligonucleotides that bind to their targets with high affinity and specificity. Experimentally, aptamers are selected by the systematic evolution of ligands by exponential enrichment (SELEX) method. Here, we have used rational drug designing and bioinformatics methods to design the aptamers, which involves three different steps. First, finding a probable aptamer-binding site, and second, designing the recognition and structural parts of the aptamers by generating a virtual library of sequences, selection of specific sequence via molecular docking, molecular dynamics (MD) simulation, binding energy calculations, and finally evaluating the experimental affinity. Following this strategy, a 16-mer DNA aptamer was designed for Annexin A1 (ANXA1). In a direct binding assay, DNA1 aptamer bound to the ANXA1 with dissociation constants value of 83 nM. Flow cytometry and fluorescence microscopy results also showed that DNA1 aptamer binds specifically to A549, HepG2, U-87 MG cancer cells that overexpress ANXA1 protein, but not to MCF7 and L-02, which are ANXA1 negative cells. We further developed a novel system by conjugating DNA1 aptamer with doxorubicin and its efficacy was studied by cellular uptake and cell viability assay. Also, anti-tumor analysis showed that conjugation of doxorubicin with aptamer significantly enhances targeted therapy against tumors while minimizing overall adverse effects on mice health.
适体是小型功能性单链DNA或RNA寡核苷酸，它们以高亲和力和特异性与其靶标结合。在实验中，适体通过指数富集配体系统进化（SELEX）方法进行筛选。在此，我们使用合理药物设计和生物信息学方法来设计适体，这涉及三个不同步骤。首先，找到一个可能的适体结合位点，其次，通过生成虚拟序列文库、经由分子对接选择特定序列、分子动力学（MD）模拟、结合能计算来设计适体的识别和结构部分，最后评估实验亲和力。按照此策略，设计了一种针对膜联蛋白A1（ANXA1）的16聚体DNA适体。在直接结合试验中，DNA1适体与ANXA1结合，解离常数为83 nM。流式细胞术和荧光显微镜结果还表明，DNA1适体特异性结合过表达ANXA1蛋白的A549、HepG2、U-87 MG癌细胞，但不结合ANXA1阴性的MCF7和L-02细胞。我们进一步通过将DNA1适体与阿霉素偶联开发了一种新系统，并通过细胞摄取和细胞活力试验研究了其疗效。此外，抗肿瘤分析表明，阿霉素与适体偶联显著增强了对肿瘤的靶向治疗，同时将对小鼠健康的总体不良影响降至最低。
