Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA.
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA.
Talanta. 2018 Jan 15;177:150-156. doi: 10.1016/j.talanta.2017.08.093. Epub 2017 Sep 1.
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 both in vitro and in live cells by the Pu28-mer sequence from the ERBB2 promoter region. Here we provide a more comprehensive study of protein capture from BT474 and MCF7 human breast cancer cells using G4-forming sequences from the CMYC, RB, VEGF and ERBB2 human 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 with RB in BT474 cells. ChIP also revealed interactions of RPL19 with CMYC in BT474 cells and of both RPL19 and ribosomal protein L14 (RPL14) with ERBB2 in BT474 cells. These results offer the basis for development of new aptamers based on the G4 sequences from the CMYC, RB, VEGF, and ERBB2 promoters toward proteins including nucleolin, RPL19 and RPL14. These interactions also may have biological and therapeutic significance.
基于指数富集的配体系统进化（SELEX）和其他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。我们实验室引入了一种新的适体发现方法，该方法使用源自人类基因组的序列的寡核苷酸来从生物样品中捕获蛋白质。具体而言，我们专注于使用来自人类基因启动子区域的 G-四链体（G4）形成的基因组序列从核提取物中捕获蛋白质。先前的研究表明，通过 ERBB2 启动子区域的 Pu28-mer 序列，在体外和活细胞中都可以捕获几种蛋白质。在这里，我们提供了更全面的研究，使用来自 CMYC、RB、VEGF 和 ERBB2 人类癌基因启动子区域的 G4 形成序列，从 BT474 和 MCF7 人乳腺癌细胞中捕获蛋白质。寡核苷酸修饰表面的蛋白质捕获的质谱分析和 Western blot 分析显示，所有三种寡核苷酸都可以在 BT474 和 MCF7 细胞中捕获核仁素，并且在 BT474 细胞中还可以捕获核糖体蛋白 L19（RPL19）。染色质免疫沉淀（ChIP）分析证实核仁素与 MCF7 细胞中的所有三个启动子序列以及 BT474 细胞中的 RB 相互作用。ChIP 还显示了 RPL19 在 BT474 细胞中与 CMYC 的相互作用以及 RPL19 和核糖体蛋白 L14（RPL14）在 BT474 细胞中与 ERBB2 的相互作用。这些结果为开发基于 CMYC、RB、VEGF 和 ERBB2 启动子的 G4 序列的新适体提供了基础，这些适体可以针对包括核仁素、RPL19 和 RPL14 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。
