A New Unnatural Base Pair System between Fluorophore and Quencher Base Analogues for Nucleic Acid-Based Imaging Technology
A New Unnatural Base Pair System between Fluorophore and Quencher Base Analogues for Nucleic Acid-Based Imaging Technology
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DOI：
10.1021/ja1072383
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发表时间：
2010-11-03
影响因子：
15
通讯作者：
Hirao, Ichiro
中科院分区：
文献类型：
--
作者：
Kimoto, Michiko;Mitsui, Tsuneo;Hirao, Ichiro
在开发用于扩展遗传字母表的正交额外碱基对时，我们在荧光团和猝灭剂核碱基类似物之间创建了新颖的非天然碱基对。我们发现，核苷碱基类似物2-硝基吡咯（Pn）及其4位取代基如2-硝基-4-丙炔基吡咯（Px）和4-[3-（6-氨基己酰胺基）-1-丙炔基]-2-硝基吡咯（NH_2-hx-Px）都具有荧光猝灭作用。Pn和Px碱基与它们的配对配偶体7-（2，2 '-联噻吩-5-基）咪唑并[4，5-B]吡啶（Dss）特异性配对，Dss具有强荧光。因此，这些非天然的Dss Pn和Dss Px碱基对起到报告猝灭剂碱基对的作用，并且通过聚合酶反应作为第三碱基对与天然A-T和G-C对组合互补地掺入DNA中。由于静态接触猝灭，Pn和Px猝灭碱基通过DNA双链体中的非天然碱基配对显著降低Dss的荧光强度。此外，Dss Px对在PCR扩增中表现出高效率和高选择性。因此，这种新的非天然碱基对系统将适用于目标核酸序列的检测方法，并且在这里，我们展示了Dss Pn和Dss Px对作为分子信标和在实时PCR中的应用。具有可复制的非天然荧光团猝灭剂碱基对的遗传字母扩展系统将是传感和诊断应用的有用工具，以及基础研究的成像工具。
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.