Nakamura Mitsunobu, Fukunaga Yudai, Sasa Kazuhiro, Ohtoshi Yukinori, Kanaori Kenji, Hayashi Haruhisa, Nakano Hidehiko, Yamana Kazushige
Department of Materials Science and Chemistry, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan.
Nucleic Acids Res. 2005 Oct 19;33(18):5887-95. doi: 10.1093/nar/gki889. Print 2005.
Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene-DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. (1)H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene-RNA/RNA and the pyrene-DNA/DNA duplexes.
通过对寡核苷酸的结合和荧光研究，这些寡核苷酸在糖残基处通过一个碳连接子与芘基团共价连接，我们先前发现芘修饰的RNA寡核苷酸在单链形式下发光不佳，但在与互补RNA链结合时，连接的芘以显著高的量子产率发光。与之形成鲜明对比的是，类似修饰的芘-DNA探针在双链和单链形式下均表现出非常微弱的荧光。因此，芘修饰的RNA寡核苷酸为监测RNA杂交提供了一种有用的工具。本文的目的是阐述芘修饰的RNA/RNA和芘修饰的DNA/DNA双链体不同荧光特性的结构基础。吸收、荧光各向异性和圆二色性研究的结果均一致表明，连接到RNA双链体上的芘位于双链体之外，而掺入DNA双链体中的芘则插入双螺旋中。核磁共振测量明确证实，连接到DNA双链体上的芘确实插入到双链体的碱基对之间。分子动力学模拟支持了芘-RNA/RNA和芘-DNA/DNA双链体中芘周围局部结构元件的这些差异。
