Luy B, Marino J P
Center for Advanced Research in Biotechnology of the University of Maryland, Biotechnology Institute and the National Institute for Standards and Technology, Rockville 20850, USA.
J Biomol NMR. 2001 May;20(1):39-47. doi: 10.1023/a:1011210307947.
Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range 1H-19F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring 1H-19F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2'-hydroxyl position of three ribose sugars. Experimentally measured 1H-19F dipolar couplings for the 2'-deoxy-2'-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR.
剩余偶极耦合可为确定和优化大分子溶液结构提供有力的限制条件。这些耦合在核酸结构解析中的应用可能会产生特别显著的影响，因为它们提供了长程限制条件，而这在NOE和J耦合测量中通常是不存在的。在此，我们描述了灵敏的X滤波-E.COSY型方法，用于测量在液晶介质中定向的选择性氟标记RNA寡核苷酸中远程1H-19F偶极耦合的符号和大小。在一个21聚体RNA发夹上展示了测量1H-19F偶极耦合的技术，该发夹在三个核糖糖的2'-羟基位置特意标记了氟。发现位于RNA发夹螺旋区域的2'-脱氧-2'-氟糖的实验测量1H-19F偶极耦合与使用标准A-型螺旋几何结构预测的值非常吻合，这表明这些耦合可为通过NMR确定的RNA结构的优化提供准确的限制条件。
