Determination of DNA helical handedness by fluorescence resonance energy transfer
Determination of DNA helical handedness by fluorescence resonance energy transfer
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DOI：
10.1006/jmbi.1996.0188
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发表时间：
1996-04-05
影响因子：
5.6
通讯作者：
Jovin, TM
中科院分区：
文献类型：
--
作者：
JaresErijman, EA;Jovin, TM
荧光共振能量转移（FRET）已被用来确定不同的DNA构象的螺旋旋向，扭曲和上升。该方法是基于一组分子组成的两个融合的螺旋片段，其中之一是在一个已知的参考螺旋形式的建设。双链体在一端用供体共价标记，在另一端用受体共价标记。通过系统地改变连接点的位置，同时保持碱基对的总长度不变，能量传递效率的变化可以被证明主要和敏感地取决于两个组成部分的螺旋扭曲和上升的差异。如果后者具有相同的螺旋意义，则预测FRET信号是连接位置的单调函数。相反，当两个线段具有相反的手性时，出现周期函数。形式主义包括明确考虑染料的方向（偶极偶极取向因子κ）和实施有效的单螺旋分子，并介绍了新的功能的测量荧光信号建立FRET效率;该方法已被应用到一个家庭的寡核苷酸形成发夹双链含有反平行链（aps）d（m（5）C。G）（m）片段在5'末端用荧光素（供体）标记，第二个平行链d（A . T）（N-m）片段（ps（AT）-DNA）在发夹环处用磺基吲哚菁染料Cy 3标记。片段长度在4至12的范围内，但总长度N保持恒定在16。d（m（5）C .选择G）序列是因为其分别在低和高盐浓度下采用B或Z构象的能力。平行链的d（A . T）序列作为第二段，以确定ps（AT）-DNA的螺旋上升和扭曲，从分子建模和拓扑约束DNA的先前研究中推测为右旋。通过在d（m（5）C中诱导B-Z转变，在两个片段之间产生Z-DNA/ps-DNA连接。G）（m），用MgCl 2。通过圆二色性测量确定所需盐浓度的范围。对于具有d（m（5）C的寡核苷酸获得0.38至0.41的FRET效率值。G）处于B构象的片段。相反，在诱导B-Z跃迁时，FRET效率是d（m（5）C的递减函数。G）（m）含量（对于m = 6至12，0.38至0.28）。螺旋参数由数据的函数拟合估计，并且与B-和Z-DNA的已知性质一致，并且与ps（AT）-DNA具有接近于B-DNA的螺旋上升和扭曲的结论一致。这里概述的方法不限于DNA，也可以应用于其他螺旋结构，例如RNA、蛋白质和蛋白质-核酸复合物。(C)1996年学术出版社
Fluorescence resonance energy transfer (FRET) has been used to determine the helical handedness, twist and rise of different DNA conformations. The approach is based on the construction of a set of molecules consisting of two fused helical segments, one of which is in a known reference helical form. The duplexes are covalently labeled at one end with a donor and at the other with an acceptor. By systematically shifting the position of the junction while maintaining constant the total length in base-pairs, the variation in the efficiency of energy transfer can be shown to depend primarily and sensitively on the differences in helical twist and rise of the two constituent segments. If the latter have the same helical sense, one predicts a FRET signal that is a monotonic function of the junctional position. In contrast, a periodic function arises when two segments are of opposite handedness. The formalism includes explicit consideration of dye orientation (the dipole-dipole orientation factor kappa) and an implementation valid for single helix molecules, and introduces new functions of measured fluorescence signals for establishing the FRET efficiency; The method has been applied to a family of oligonucleotides forming hairpin duplexes containing an antiparallel-stranded (aps) d(m(5)C . G)(m) segment labeled at the 5' end with fluorescein (donor) and a second parallel-stranded d(A . T)(N-m) segment (ps(AT)-DNA) labeled at the hairpin loop with the sulfoindocyanine dye Cy3. The segment lengths were in the range 4 to 12, but the total length N was maintained constant at 16. The d(m(5)C . G) sequence was chosen due to its capacity for adopting a B or a Z conformation at low and high concentrations of salt, respectively. The parallel-stranded d(A . T) sequence served as the second segment in order to determine the helical rise and twist of ps(AT)-DNA, presumed to be right-handed from molecular modeling and a prior study of topologically constrained DNA. A Z-DNA/ps-DNA junction was created between the two segments by inducing a B-Z transition in d(m(5)C . G)(m), with MgCl2. The range of required salt concentration was established by circular dichroism measurements. FRET efficiency values of 0.38 to 0.41 were obtained for the oligonucleotides with the d(m(5)C . G) segment in the B conformation. In contrast, upon induction of the B-Z transition the FRET efficiency was a decreasing function of the d(m(5)C . G)(m) content (0.38 to 0.28 for m = 6 to 12). Helical parameters were estimated from functional fits of the data, and were consistent with the known properties of B- and Z-DNAs and with the conclusion that ps(AT)-DNA has a helical rise and twist close to that of B-DNA. The approach outlined here is not restricted to DNA but can be applied to other helical structures, e.g. RNA, proteins and protein-nucleic acid complexes. (C) 1996 Academic Press Limited