Location of cyanine-3 on double-stranded DNA:: Importance for fluorescence resonance energy transfer studies
Location of cyanine-3 on double-stranded DNA:: Importance for fluorescence resonance energy transfer studies
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DOI：
10.1021/bi992944a
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发表时间：
2000-05-30
期刊：
影响因子：
2.9
通讯作者：
Lilley, DMJ
中科院分区：
文献类型：
--
作者：
Norman, DG;Grainger, RJ;Lilley, DMJ
荧光共振能量转移提供了关于溶液中大分子的有价值的远程信息。荧光素和Cy3是一对重要的荧光团供体-受体对，其在DNA上的特征Forster长度为56埃，因此可用于较长距离的研究。用荧光素和Cy3末端标记的一系列DNA双链的FRET效率的测量表明，Cy3靠近DNA的螺旋轴。用Cy3标记的自互补DNA双链的核磁共振分析表明，荧光团以类似于额外碱基对的方式堆积在螺旋的末端。这提供了一个已知的点，从该点开始测量由FRET测量计算出的距离。使用一系列DNA双链的FRET效率作为限制条件，我们已经确定了荧光素的有效位置，该位置最大限度地从螺旋横向延伸。现在可以利用荧光团位置的知识来更精确地解释来自核酸的FRET数据。
Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Forster length for this pair on DNA is 56 Angstrom, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.