A genome-inspired, reverse selection approach to aptamer discovery.
A genome-inspired, reverse selection approach to aptamer discovery.
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一种受基因组启发的反向选择适体发现方法。
DOI：
10.1016/j.talanta.2017.08.093
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发表时间：
2018
期刊：
影响因子：
6.1
通讯作者：
McGown,LindaB
中科院分区：
文献类型：
--
作者：
Albanese,ChristinaM;Suttapitugsakul,Suttipong;Perati,Shruthi;McGown,LindaB
指数富集配体系统进化（SELEX）和依赖于组合寡核苷酸文库的相关方法的局限性阻碍了该领域的进展。我们的实验室引入了一种新的适体发现方法，该方法使用具有来自人类基因组的序列的寡核苷酸来捕获生物样品中的蛋白质。具体而言，我们专注于使用G-四链体（G4）形成基因组序列捕获来自人类细胞系的核提取物中的蛋白质。先前的研究证实了ERBB 2启动子区域的Pu 28-mer序列在体外和活细胞中捕获几种蛋白质。在这里，我们提供了一个更全面的研究蛋白捕获从BT474和MCF 7人乳腺癌细胞使用G4形成序列的CMYC，RB，VEGF和ERBB 2人癌基因启动子区域。质谱分析和蛋白质印迹分析的蛋白质捕获在修饰的表面显示捕获核仁素的所有三个寡核苷酸在BT474和MCF 7细胞，以及核糖体蛋白L19（RPL 19）在BT474细胞。染色质免疫沉淀（ChIP）分析证实了核仁素与MCF 7细胞中的所有三个启动子序列以及与BT474细胞中的RBin的相互作用。在BT474细胞中，RPL 19与CMYC相互作用，RPL 19和核糖体蛋白L14（RPL 14）与ERBB 2相互作用。这些结果为开发基于CMYC、RB、VEGF和ERBB 2启动子的G4序列的新适体提供了基础，这些启动子针对包括核仁素、RPL 19和RPL 14在内的蛋白质。这些相互作用也可能具有生物学和治疗意义。
Limitations of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and related methods that depend upon combinatorial oligonucleotide libraries have hindered progress in this area. Our laboratory has introduced a new approach to aptamer discovery that uses oligonucleotides with sequences drawn from the human genome to capture proteins from biological samples. Specifically, we have focused on capture of proteins in nuclear extracts from human cell lines using G-quadruplex (G4) forming genomic sequences. Previous studies identified capture of several proteins bothin vitroand in live cells by the Pu28-mer sequence from theERBB2promoter region. Here we provide a more comprehensive study of protein capture from BT474 and MCF7 human breast cancer cells using G4-forming sequences from theCMYC, RB,VEGFandERBB2human oncogene promoter regions. Mass spectrometric analysis and Western blot analysis of protein capture at oligonucleotide-modified surfaces revealed capture of nucleolin by all three of the oligonucleotides in BT474 and MCF7 cells, and also of ribosomal protein L19 (RPL19) in BT474 cells. Chromatin immunoprecipitation (ChIP) analysis confirmed the interaction of nucleolin with all three promoter sequences in MCF7 cells and withRBin BT474 cells. ChIP also revealed interactions of RPL19 withCMYCin BT474 cells and of both RPL19 and ribosomal protein L14 (RPL14) withERBB2in BT474 cells. These results offer the basis for development of new aptamers based on the G4 sequences from theCMYC,RB,VEGF, andERBB2promoters toward proteins including nucleolin, RPL19 and RPL14. These interactions also may have biological and therapeutic significance.