Pham Ha H, Murphy Connor T, Sureshkumar Gopalsamy, Ly Danith H, Opresko Patricia L, Armitage Bruce A
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213.
Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213.
Org Biomol Chem. 2014 Oct 7;12(37):7345-7354. doi: 10.1039/c4ob00953c.
GammaPNA oligomers having one or two repeats of the sequence AATCCC were designed to hybridize to DNA having one or more repeats of the complementary TTAGGG sequence found in the human telomere. UV melting curves and surface plasmon resonance experiments demonstrate high affinity and cooperativity for hybridization of these miniprobes to DNA having multiple complementary repeats. Fluorescence spectroscopy for Cy3-labeled miniprobes demonstrate increases in fluorescence intensity for assembling multiple short probes on a DNA target compared with fewer longer probes. The fluorescent γPNA miniprobes were then used to stain telomeres in metaphase chromosomes derived from U2OS cells possessing heterogeneous long telomeres and Jurkat cells harboring homogenous short telomeres. The miniprobes yielded comparable fluorescence intensity to a commercially available PNA 18mer probe in U2OS cells, but significantly brighter fluorescence was observed for telomeres in Jurkat cells. These results suggest that γPNA miniprobes can be effective telomere-staining reagents with applications toward analysis of critically short telomeres, which have been implicated in a range of human diseases.
设计了具有一个或两个AATCCC序列重复片段的γPNA寡聚物，使其与在人类端粒中发现的具有一个或多个互补TTAGGG序列重复片段的DNA杂交。紫外熔解曲线和表面等离子体共振实验表明，这些微型探针与具有多个互补重复片段的DNA杂交时具有高亲和力和协同性。对Cy3标记的微型探针进行荧光光谱分析表明，与较少的较长探针相比，在DNA靶标上组装多个短探针时荧光强度会增加。然后，使用荧光γPNA微型探针来染色来自具有异质性长端粒的U2OS细胞和具有均一性短端粒的Jurkat细胞的中期染色体中的端粒。在U2OS细胞中，微型探针产生的荧光强度与市售的PNA 18聚体探针相当，但在Jurkat细胞的端粒中观察到明显更亮的荧光。这些结果表明，γPNA微型探针可以成为有效的端粒染色试剂，用于分析与一系列人类疾病有关的极短端粒。
