Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology
Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology
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DOI：
10.1002/chem.200600197
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发表时间：
2006-10-16
影响因子：
4.3
通讯作者：
Burgess, Kevin
中科院分区：
文献类型：
--
作者：
Jiao, Guan-Sheng;Thoresen, Lars H.;Burgess, Kevin
我们设计了荧光“通过键合能量转移盒”，可以收集相对较短波长（例如 490 nm）的能量，并以明显更长的波长发射能量，而不会显着损失强度。这种类型的探针在生物技术中的多重实验中特别有用，在多重实验中，要从单个激发源观察到几种不同的输出。盒1-4作为模型系统被设计、准备和研究以实现该目的。它们是通过收敛途径合成的，其特征是特别制备的荧光素和罗丹明衍生片段的偶联。四个盒体均表现出强烈的发射能力，并且具有高效的能量传输。它们的发射最大值覆盖了广泛的波长范围（比目前用于大多数高通量 DNA 测序的四种染料盒更宽），并且它们表现出比类似的空间能量传输盒更快的能量传输速率。具体来说，这些盒中的能量传输速率约为 6-7 ps，而类似的穿越空间能量传输系统的衰减时间约为 35 ps。此外，这些盒对于光漂白比荧光素更稳定，尽管它们各自含有荧光素衍生的供体。这已通过大量荧光测量和单分子检测研究得到证实。对商业自动化 DNA 测序仪进行改造以检测这四个能量转移盒的发射，从而实现单色染料引物测序。
We have designed fluorescent "through-bond energy-transfer cassettes" that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1-4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein- and rhodamine-derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high-throughput DNA sequencing), and they exhibit faster energy-transfer rates than a similar through-space energy-transfer cassette. Specifically, energy-transfer rates in these cassettes is around 6-7 ps, in contrast to a similar through-space energy-transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein-derived donors. This was confirmed by bulk fluorescent measurements, and in single-molecule-detection studies. Modification of a commercial automated DNA-sequencing apparatus to detect the emissions of these four energy-transfer cassettes enabled single-color dye-primer sequencing.