Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nuclelic acids
Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nuclelic acids
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DOI：
10.1073/pnas.0801707105
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发表时间：
2008-08-12
影响因子：
11.1
通讯作者：
Lilley, David M. J.
中科院分区：
文献类型：
--
作者：
Iqbal, Asif;Arslan, Sinan;Lilley, David M. J.
我们发现，在DNA双链末端连接的Cy3和Cy5之间的荧光共振能量转移效率受到两个荧光团的相对取向的显著影响。花青素荧光团主要以附加碱基对的方式堆叠在螺旋的末端，因此它们的相对取向取决于螺旋的长度。观察到的荧光共振能量转移（FRET）效率取决于螺旋的长度，以及它的螺旋周期性。通过改变螺旋几何结构从B形式的双链DNA到A形式的杂交RNA/DNA， FRET效率随螺旋长度的调制发生了明显的相移。这两条曲线都可以很好地解释为B和A形成螺旋的标准几何形状。对这两种聚合物所观察到的调制小于对荧光团的完全刚性附着所计算的调制。然而，一个涉及螺旋末端荧光团横向迁移的模型解释了观察到的实验数据。这已被进一步修改，以考虑到通过荧光寿命测量观察到的非堆叠荧光团的一小部分。我们的数据明确地表明，福斯特转移服从取向依赖，如预期的偶极子-偶极子相互作用。
We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5' ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Forster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.