Choi Siyoung, Murphy William L
Department of Biomedical Engineering, University of Wisconsin, Madison, WI 53706, USA.
Langmuir. 2008 Jun 1;24(13):6873-80. doi: 10.1021/la800553p. Epub 2008 May 29.
The ability of DNA strands to influence cellular gene expression directly and to bind with high affinity and specificity to other biological molecules (e.g., proteins and target DNA strands) makes them a potentially attractive component of cell culture substrates. On the basis of the potential importance of immobilized DNA in cell culture and the well-defined characteristics of alkanethiol self-assembled monolayers (SAMs), the current study was designed to create multifunctional SAMs upon which cell adhesion and DNA immobilization can be independently modulated. The approach immobilizes the fibronectin-derived cell adhesion ligand Arg-Gly-Asp-Ser-Pro (RGDSP) using carbodiimide activation chemistry and immobilizes DNA strands on the same surface via cDNA-DNA interactions. The surface density of hexanethiol-terminated DNA strands on alkanethiol monolayers (30.2-69.2 pmol/cm2) was controlled using a backfill method, and specific target DNA binding on cDNA-containing SAMs was regulated by varying the soluble target DNA concentration and buffer characteristics. The fibronectin-derived cell adhesion ligand GGRGDSP was covalently linked to carboxylate groups on DNA-containing SAM substrates, and peptide density was proportional to the amount of carboxylate present during SAM preparation. C166-GFP endothelial cells attached and spread on mixed SAM substrates and cell adhesion and spreading were specifically mediated by the immobilized GGRGDSP peptide. The ability to control the characteristics of noncovalent DNA immobilization and cell adhesion on a cell culture substrate suggests that these mixed SAMs could be a useful platform for studying the interaction between cells and DNA.
DNA链直接影响细胞基因表达以及与其他生物分子（如蛋白质和靶DNA链）以高亲和力和特异性结合的能力，使其成为细胞培养基质中潜在有吸引力的成分。基于固定化DNA在细胞培养中的潜在重要性以及烷硫醇自组装单分子层（SAMs）明确的特性，本研究旨在创建多功能SAMs，在其上细胞黏附和DNA固定可独立调节。该方法利用碳二亚胺活化化学固定源自纤连蛋白的细胞黏附配体Arg-Gly-Asp-Ser-Pro（RGDSP），并通过cDNA-DNA相互作用将DNA链固定在同一表面。使用回填法控制烷硫醇单分子层上己硫醇封端的DNA链的表面密度（30.2 - 69.2 pmol/cm²），通过改变可溶性靶DNA浓度和缓冲液特性来调节含cDNA的SAMs上的特异性靶DNA结合。源自纤连蛋白的细胞黏附配体GGRGDSP共价连接到含DNA的SAM底物上的羧基，肽密度与SAM制备过程中存在的羧基量成正比。C166-GFP内皮细胞在混合SAM底物上附着并铺展，细胞黏附和铺展由固定化的GGRGDSP肽特异性介导。在细胞培养基质上控制非共价DNA固定和细胞黏附特性的能力表明，这些混合SAMs可能是研究细胞与DNA相互作用的有用平台。
