A New Unnatural Base Pair System between Fluorophore and Quencher Base Analogues for Nucleic Acid-Based Imaging Technology

详细信息    查看全文

• 作者：Michiko Kimoto ; Tsuneo Mitsui ; Rie Yamashige ; Akira Sato ; Shigeyuki Yokoyama ; Ichiro Hirao
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：November 3, 2010
• 年：2010
• 卷：132
• 期：43
• 页码：15418-15426
• 全文大小：459K
• 年卷期：v.132,no.43(November 3, 2010)
• ISSN：1520-5126

文摘

In the development of orthogonal extra base pairs for expanding the genetic alphabet, we created novel, unnatural base pairs between fluorophore and quencher nucleobase analogues. We found that the nucleobase analogue, 2-nitropyrrole (denoted by Pn), and its 4-substitutions, such as 2-nitro-4-propynylpyrrole (Px) and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (NH₂-hx-Px), act as fluorescence quenchers. The Pn and Px bases specifically pair with their pairing partner, 7-(2,2′-bithien-5-yl)imidazo[4,5-b]pyridine (Dss), which is strongly fluorescent. Thus, these unnatural Dss−Pn and Dss−Px base pairs function as reporter−quencher base pairs, and are complementarily incorporated into DNA by polymerase reactions as a third base pair in combination with the natural A-T and G-C pairs. Due to the static contact quenching, the Pn and Px quencher bases significantly decreased the fluorescence intensity of Dss by the unnatural base pairings in DNA duplexes. In addition, the Dss−Px pair exhibited high efficiency and selectivity in PCR amplification. Thus, this new unnatural base pair system would be suitable for detection methods of target nucleic acid sequences, and here we demonstrated the applications of the Dss−Pn and Dss−Px pairs as molecular beacons and in real-time PCR. The genetic alphabet expansion system with the replicable, unnatural fluorophore−quencher base pair will be a useful tool for sensing and diagnostic applications, as well as an imaging tool for basic research.