Colorimetric Gene assay Using DNA-Modified Nanoparticles

1.寡核苷酸修饰纳米粒的制备及其聚集性的初步研究用水溶性碳二亚胺(EDAC)将氨基末端的寡核苷酸修饰为羧基修饰的纳米粒。互补ODN在合适的条件下使颗粒聚集。聚集体的尺寸达到数十μm，使用普通荧光显微镜和相应的滤光片可以很容易地观察到它们。以红绿二元系统中的两个45mer ODN为模型基因样本，其中一个是野生型p53基因的一部分，另一个是含有一个碱基替换的突变体。将两个25聚体ODN分别固定在红色和绿色纳米颗粒上，每个ODN含有与野生型45聚体末端互补的15个核苷酸序列。野生型将两个球体聚集在一起，在适当的条件下产生发出黄色光(黄色=红色+绿色)的聚集体。另一方面，突变体几乎不影响这两个球体的分布。有趣的是，野生型聚集体还表现出基于绿色颗粒到紧密接触的红色颗粒(从绿色到红色)的荧光共振能量转移(FRET)的特征发射。将上述三元体系(红-绿-蓝)体系中的聚集体聚集实验推广到三元体系(R/G/B)，采用不同颜色(R/G/B)三种tbe ODN修饰纳米粒子的混合溶液。野生型和突变型45MERS得到的聚集体基本上分别为黄色(Y=R+G)和洋红(M=R+B)，改进对ODN的修饰方法有助于获得更明显的结果。本研究提出的SNP比色分析方法将为高通量基因诊断和分析提供一种新的选择。

1. Preparation of the ODN (oligonudeotide) -modified nanoparticles and preliminary study of their aggregationAmmo-terminated ODNs were modified onto carboxylates-modified nanoparticles using water soluble carbodiimide (EDAC). Complementary ODN made the particle aggregate under appropriate conditions. The dimension of the aggregates reached tens ofμm and we could easily observe them using ordinary fluorescence microscopy with corresponding optical filters.2. Aggregates in binary system (red-green)Two 45 mer ODNs, one of which is a part of wild type p53 gene and the other is the mutant containing one base substitution, were used as model gene samples. Two 25 mer ODNs, each of which contains the 15 nudeotide sequence complementary to each of the terminus of fhe wild type 45 mer, were immobilized onto red and green nanoparticles, respectively. The wild type gathered the both spheres to produce aggregate that emits yellow light (yellow=red+green) under the appropriate conditions. On the other hand, the mutant scarcely affects the distribution of the both spheres. Interestingly the aggregates with the wild type also showed characteristic emission based on the FRET (fluorescence resonance energy transfer) from green particles to closely contacted red particles (from green to red).3. Aggregates in ternary system (red-green-blue)Aggregation experiments in binary system mentioned above were extended to the ternary system, in which mixed solution of differently colored (R/G/B) three kinds of tbe ODN-modified nanoparticles were adopted. The wild type and the mutant 45 mers gave the aggregates with yellow (Y=R+G) and magenta (M=R+B) on the whole, respectively.Improvement of the method for ODN modification onto the particles should help to obtain more distinct results. Colorimetric SNP analysis proposed in this study would be an alternative for high-throughput gene diagnosis and analysis.